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PDF - 53KB, Demonstration of benign immunogenicity profile of PRS-110 in ex vivo PBMC model

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Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be “fit for purpose” for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients. Mol Cancer Ther; 12(11); 2459–71. ©2013 AACR.

PDF - 31KB, Characterisation of cell line panel for MET and HGF expression

PDF - 136KB, Demonstration of PRS-110 dependent MET trafficking in HT29 cells

Figure S1: PRS-343 specificity for 4-1BB within TNF receptor superfamily. Seven recombinant human TNF receptor superfamily proteins were purchased from Sino Biological (4-1BB: 10041-H08H, RANK: 16078-H08H, GITR: 13643-H08H, Ox40: 10481-H08H) or R&D Systems (CD30: 6126-CD, TNF-RII: 1089-R2, TNF-RI: 636-R1) and used for determination of PRS-343 selectivity for 4-1BB. Proteins were coated to an ELISA plate, PRS-343 was added in a dilution series and detected via HRP-labeled anti-human IgG Fc antibody. Within the set of tested TNF receptor family proteins, PRS-343 binds exclusively to 4-1BB. Figure S2: Characterization of multiple bispecific formats of a 4-1BB targeting Anticalin protein recombinantly fused to an anti-HER2 antibody (A). ELISA based binding properties of all formats were compared to parental building blocks (C) while the ability to activate T-cells (IL2 induction) was assessed in a co-culture assay. Figure S3: Cytokine release assay with PRS-343. PBMC were isolated from the blood of twelve healthy donors and incubated for 72 hours with PRS-343 either air dried, in soluble form, or wet coated. Four concentrations of PRS-343 in a volume of 50 µl were tested in each setting as indicated in the figure. The anti-CD3 monoclonal antibody OKT3 at three different concentrations served as the positive control, and an IgG4 isotype antibody was the negative control. Supernatant levels of ten cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, GM-CSF, IFN-γ and TNF-α) were analyzed. The figure shows the average response for the ten donors that displayed a significant response to OKT3, and for a selection of the most relevant cytokines. Figure S4: Dose-dependent 4-1BB activation of a 4-1BB over-expressing Jurkat Nf-kB reporter cell line induced by PRS-343 with ON preincubation of the drug in the presence of NCI-N87 (HER2 high), MKN45 (HER2 low) and HepG2 (HER2 null) cell lines, or without tumor cells. Briefly, cancer cells were seeded onto tissue culture plates with PRS-343 (at 10, 1, 0.1 and 0,01 nM) and incubated ON. All plates were then washed twice with PBS. 4-1BB over-expressing Jurkat Nf-kB reporter (at 3:1 ratio) were added to each well. Following a 6 hours incubation, Bio-Glow luciferase reagent was added to each well and luminescence was measured. Figure S5: Fold increase changes of a panel of cytokines induced by human T-cells co-stimulated by PRS-343 in the presence of SKBR-3 (HER2 high) or MCF-7 (HER2 low). Figure S6: h-4-1BB expression in T-cells during co-culture assay. Using a similar set up as described in M&M, purified Pan T-cells (from healthy donors) and SK-BR3 high Her2 expressing cells were co-incubated in the presence of coated anti-CD3 antibody. After 24, 48 and 72 hours of co-incubation, Pan T-cells were collected and 4-1BB expression on CD3 positive cells was assessed by flow cytometry. (A) shows the % of 4-1BB positive T-cells for two donors and (B) shows histogram of h-4-1BB expression on CD3 positive T-cells from two donors (red 24 hours; blue 48 hours; brown 72 hours). Table S1: Relative HER2 cell surface expression on a panel of cell lines. Expression was experimentally determined using a specific anti-HER2-antibody binding capacity (sABC [HER2]) and quantitative indirect immunofluorescence in flow cytometry (QIFIKIT). HER2 surface levels are also provided relative to the level on SKBR3 cells which were chosen as a reference cell line.

Supplementary Legend

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 (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, 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 toxicologic findings.Conclusions: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 with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule.See related commentary by Su et al., p. 5732

Background and Purpose Anaemia of chronic disease (ACD) has been linked to iron-restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte-derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin-derived, small protein-based anticalin PRS-080 hepcidin antagonist with high affinity and selectivity. Experimental Approach Anticalin- and hepcidin-specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t1/2 extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS-080-PEG30 administration in vivo. Key Results PRS-080 had a hepcidin-binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS-080-PEG30), a t1/2 of 43 h in cynomolgus monkeys. Dose-dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS-080-PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment-free interval, all iron parameters returned to pre-dose values. Conclusions and Implications In conclusion, we developed a dose-dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron-restricted erythropoiesis that can address the root cause of ACD. PRS-080-PEG30 is currently in early clinical development.

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 (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, 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 toxicologic findings. Conclusions: 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 with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule. See related commentary by Su et al., p. 5732

BACKGROUND AND PURPOSE: Anaemia of chronic disease (ACD) has been linked to iron‐restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte‐derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin‐derived, small protein‐based anticalin PRS‐080 hepcidin antagonist with high affinity and selectivity. EXPERIMENTAL APPROACH: Anticalin‐ and hepcidin‐specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t (1/2) extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS‐080‐PEG30 administration in vivo. KEY RESULTS: PRS‐080 had a hepcidin‐binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS‐080‐PEG30), a t (1/2) of 43 h in cynomolgus monkeys. Dose‐dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS‐080‐PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment‐free interval, all iron parameters returned to pre‐dose values. CONCLUSIONS AND IMPLICATIONS: In conclusion, we developed a dose‐dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron‐restricted erythropoiesis that can address the root cause of ACD. PRS‐080‐PEG30 is currently in early clinical development.

Anticalins are a novel class of biopharmaceuticals, displaying highly desirable attributes as imaging agents. The anticalin PRS-110 was rationally engineered to target the oncogene MET with high affinity and specificity. The aim of this study was to visualize MET expression and analyze biodistribution of Zr-89-labeled PRS-110 in human tumor-bearing mice. Methods: Zr-89-PRS-110 was generated. For biodistribution studies (96 h after injection of tracer) 10 mu g of Zr-89-PRS-110 (with 0-490 mu g of unlabeled PRS-110) were injected into BALB/c mice bearing high MET-expressing H441 non-small cell lung cancer xenografts. Further characterization with PET imaging was performed at 6, 24, 48, and 96 h after injection of 50 mu g of Zr-89-PRS-110 into mice bearing H441, primary glioblastoma U87-MG (intermediate MET), or ovarian cancer A2780 (low MET) xenografts. Drug distribution was also analyzed ex vivo using fluorescently labeled PRS-110. Results: Biodistribution analyses showed a dose-dependent tumor uptake of Zr-89-PRS-110, with the highest fractional tumor uptake at 10 mu g of Zr-89-PRS-110, with no unlabeled PRS-110. Small-animal PET imaging supported by biodistribution data revealed specific tumor uptake of Zr-89-PRS-110 in the METexpressing H441 and U87-MG tumors whereas the MET-negative A2780 tumor model showed a lower uptake similar to a non-MET binder anticalin control. Tumor uptake increased up to 24 h after tracer injection and remained high, whereas uptake in other organs decreased over time. Ex vivo fluorescence revealed intracellular presence of PRS-110. Conclusion: Zr-89-PRS-110 specifically accumulates in MET-expressing tumors in a receptor density-dependent manner. PET imaging provides real-time noninvasive information about PRS-110 distribution and tumor accumulation in preclinical models.

Background. 4-1BB (CD137) is a key costimulatory immunoreceptor and a highly promising therapeutic target in cancer. To overcome toxicity and efficacy limitations of current 4-1BB targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific based on Anticalin® technology. We have previously reported on the generation and characterization of PRS-343 with regard to preclinical proof-of-concept and basic drug-like properties.1 Here, we describe the preclinical dataset supporting initiation of a first-in-patient trial. Methods and Results. The pharmacology of PRS-343 was investigated by ex vivo assays based on mixed culture of human PBMC and tumor cell lines. We find that 4-1BB costimulated T cells prominently increase production of IL-2, GM-CSF, TNF-α and IFN-γ. Using a set of immortal cancer cell lines spanning a range of HER2 surface copy numbers, we identified a copy number threshold above which PRS-343 reliably elicited T cell costimulation with a high potency and an EC50 in the subnanomolar range. PRS-343 was well tolerated in a repeat-dose study in cynomolgus monkeys, with no overt toxicity and no significant drug-related toxicological findings. Pharmacokinetic assessment confirmed dose-proportional exposure of the animals during the course of the study. In a mouse model of human PBMC-induced xenograft-vs-host disease (xGvHD), PRS-343 did not show an acceleration of xGvHD development, in contrast to a 4-1BB targeting benchmark. Again utilizing ex vivo assays, we found no PRS-343 induced T cell costimulation in a panel of primary cells, showing that physiological levels of HER2 are insufficient for activation. In a cytokine release assay, proinflammatory cytokine induction by PRS-343 in the absence of a primary TCR stimulation was negligible. Conclusion. The ex vivo experiments described indicate that HER2 expression level is expected to be a reliable marker for patient stratification for PRS-343. The toxicology assessment of PRS-343 indicates that the benign toxicity profile of trastuzumab is retained in PRS-343 with regard to HER2 targeting, and that PRS-343 is expected to elicit its costimulatory effects strictly on T cells also receiving a primary TCR signal and strictly localized to HER2-positive tumors. This is in agreement with in vivo mouse model data showing PRS-343 leads to tumor-localized CD8+ T cell expansion,1 and supports the potential of PRS-343 as an efficacious yet well tolerable 4-1BB costimulating agent. The reported data is the basis for the trial design of a first in patient Phase 1 trial with PRS-343. 1Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B016. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Thomas Jaquin, Sven Berger, Manuela Dürr, Corinna Schlosser, Andrea Allersdorfer, Christine Rothe, Louis A. Matis, Shane A. Olwill. Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343: A first-in-class costimulatory T cell engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2017-3673

To the Editor: Anticalin® proteins are human lipocalins that are engineered to bind relevant targets with high affinity and specificity (1). By applying a mutation and selection process, it is possible to select Anticalin proteins that can specifically bind to very diverse protein targets in a monovalent fashion. Anticalin proteins are also suited for engaging small and compact ligands owing to their cuplike binding pocket. Compared with monoclonal antibodies, Anticalin proteins are about 8 times smaller and can be recombinantly produced in bacterial cells in large amounts. The hepatic peptide hormone hepcidin is a highly conserved molecule of only 25 amino acids with 4 disulfide bridges that plays a central role in body iron metabolism (2). Therefore, it could become a useful biomarker. Numerous hepcidin assays, by use of either mass spectrometry (MS)-based1 techniques or traditional immunochemical assays, have been described to quantify hepcidin in biological fluids (2). However, both MS and immunochemical approaches have disadvantages, including costly and sophisticated instrumentation, low throughput, need for highly specialized personnel, and difficulties in obtaining high-quality antibodies. The latter is ascribed to the small size of hepcidin, its folded nature, and its conservation throughout evolution, which complicate the generation of hepcidin antibodies for immunochemical assays such as ELISA. Thus, there is a need and opportunity for alternative approaches. We developed …

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be “fit for purpose” for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients. Mol Cancer Ther; 12(11); 2459–71. ©2013 AACR . This article is featured in Highlights of This Issue, [p. 2283][1] [1]: /lookup/volpage/12/2283?iss=11

Background: CD137 (4-1BB) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. While multiple lines of evidence show that CD137 is a highly promising therapeutic target in cancer, current mAb-based approaches are not designed to achieve a tumor-target driven activation and may display toxicity and a limited therapeutic window due to peripheral T cell and NK cell activation. To overcome this limitation, we generated PRS-343, a CD137/HER2 bispecific that is designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells. Methods: Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. PRS-343 was obtained by genetic fusion of the CD137-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. We have shown previously that the bispecific fusion PRS-343 targets CD137 and HER2 in a bispecific manner and efficiently activates T cells ex vivo in the presence of HER2-positive cells. Here, in vivo proof of concept data is presented utilizing a humanized mouse model in immunocompromised mice and the SK-OV-3 cell line as a HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC via an intravenous route and weekly intraperitoneal injections of PRS-343 for three weeks. An IgG4 isotype antibody served as the negative control, while a CD137-targeting benchmark antibody and trastuzumab with an engineered IgG4 backbone (“tras-IgG4”) served as controls for monospecific targeting of CD137 and HER2, respectively. Results: PRS-343 activity was investigated at four different weekly doses of PRS-343 (4μg, 20μg, 100μg and 200μg). We found that PRS-343 dose-dependently led to strong tumor growth inhibition compared to treatment with the isotype control, and that the tumor response was accompanied by a significantly higher tumor infiltration with human lymphocytes (hCD45+). Interestingly, the anti-CD137 benchmark neither displayed tumor growth inhibition nor enhanced lymphocyte infiltration into tumors compared to isotype. The tras-IgG4 control was also devoid of lymphocyte infiltration into the tumor, but displayed a tumor growth inhibition comparable to PRS-343. Taken together, these data show that PRS-343 provided dual activity by both increasing the frequency of tumor-infiltrating lymphocytes by bispecific targeting of CD137 and HER2 as well as mediating direct tumor growth inhibition by the direct, monospecific targeting of 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 tras-IgG4 control lack the ability to interact with Fc-gamma receptors on NK cells that ADCC would require. The animal model also allowed investigating the potential safety of PRS-343: While the anti-CD137 benchmark accelerated the onset of graft-versus-host-disease and led to stronger expansion of CD8+ T cells in the peripheral blood compared to the isotype control group, both of these effects were absent for PRS-343. The data therefore support the envisaged mode of action of selective, tumor-localized costimulatory T cell activation, as well as the concept that such an approach may lead to higher efficacy and reduced systemic toxicity compared to conventional anti-CD137 mAbs. Conclusion: We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the CD137/HER2 bispecific PRS-343. In a humanized mouse model, PRS-343 displays dual activity based on monospecific HER2-targeting and bispecific, tumor-localized costimulation of CD137. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The direct, monospecific HER2-targeting activity may provide an additional therapeutic benefit and work in synergy with local CD137 costimulation. The positive functional ex vivo and in vivo data of PRS-343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Thomas Jaquin, Andrea Allersdorfer, Sven Berger, Alexander Wiedenmann, Gabriele Matschiner, Julia Schüler, Ulrich Moebius, Christine Rothe, Shane A. Olwill. Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in humanized mouse model [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B016.

Background. CD137 (4-1BB) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. While multiple lines of evidence show that CD137 is a highly promising therapeutic target in cancer, current mAb-based approaches are not designed to achieve a tumor-target driven activation and may display toxicity and a limited therapeutic window due to peripheral T cell and NK cell activation. To overcome this limitation, we generated PRS-343, a HER2/CD137 bispecific that is designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells. Methods. Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. PRS-343 was obtained by genetic fusion of the CD137-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. Results. The bispecific fusion PRS-343 targets CD137 and HER2 with nearly identical affinities compared to the parental building blocks, and is capable of binding both targets simultaneously. We show ex vivo that T cells are efficiently activated when incubated with PRS-343 and HER2-positive cells, and that the activation is HER2-dependent. The in vivo activity of PRS-343 was investigated utilizing a humanized mouse model with a tumor cell-line-derived, HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC via an intravenous route and weekly intraperitoneal injections of PRS-343 or controls for three weeks. We found that PRS-343 led to strong tumor growth inhibition and a significantly better response compared to either isotype control or anti-CD137 benchmark mAbs. The data, which include phenotyping of peripheral and intra-tumoral lymphocytes, support the envisaged mode of action of tumor-localized costimulatory T cell activation. Conclusion. We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the HER2/CD137 bispecific PRS-343. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The positive functional ex vivo and in vivo data of PRS-343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Alexander Wiedenmann, Andrea Allersdorfer, Gabriele Matschiner, Sven Berger, Ulrich Moebius, Christine Rothe, Shane A. Olwill. Costimulatory T-cell engagement by the HER2/CD137 bispecific PRS-343 leads to strong antitumor effect in humanized mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 556.

Background: CD137 is a potent costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. The receptor, also known as 4-1BB, is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells. While multiple lines of evidence show that CD137 is a highly promising therapeutic target, current approaches are not designed to achieve a tumor-target driven activation, which may reduce the available therapeutic window via peripheral T cell activation and toxicity. To overcome this limitation, we applied Anticalin® technology to generate a bispecific protein therapeutic binding to CD137 and a differentially expressed tumor target, HER2. Methods: Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins which enable straight-forward multimeric drug targeting across several formats. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. The CD137-specific Anticalin protein was genetically fused to a Trastuzumab variant, yielding four different constructs covering a range of distances between the binding sites of the T cell-target and the tumor cell target. To minimize Fc-receptor interaction of the resulting bispecific and concomitant potential toxicity towards CD137-positive cells, the backbone of Trastuzumab was switched from IgG1 to an engineered IgG4 isotype. Results: All four bispecific constructs bound the targets CD137 and HER2 with a nearly identical affinity compared to the parental building blocks, and both targets could be simultaneously bound. Compared to non-engineered Trastuzumab, binding to human receptors FcγRI and FcγRIII was significantly reduced, while binding to the neonatal Fc receptor (FcRn) was retained. Functional activity was demonstrated in human T cell activation assays, and shown to be tumor target (HER2) dependent. Conclusion: We report the first bispecific therapeutic protein that targets the potent costimulatory immunoreceptor CD137 in a tumor-target dependent manner, utilizing HER2 as the tumor target. Compared to currently existing CD137-targeting antibodies, this approach has the potential to provide a more localized activation of the immune system with reduced peripheral toxicity. Bispecific T cell engagers based on CD137 and HER2 may have utility in HER2-positive cancers where there is a significant unmet medical need, such as bladder, ovarian and gastric cancer. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Alexander Wiedenmann, Corinna Schlosser, Andrea Allersdorfer, Gabriele Matschiner, Christine Rothe, Ulrich Moebius, Shane A. Olwill. Costimulatory T-cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein based on Anticalin® technology. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B023.

CD137 is a potent costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. The receptor, also known as 4-1BB, is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells. While multiple lines of evidence show that CD137 is a highly promising therapeutic target, current approaches using monospecific antibodies may display a limited therapeutic window due to peripheral T cell and NK cell activation, leading to unwanted toxicity. To overcome this limitation, we have generated a bispecific protein therapeutic designed to achieve a tumor-target driven activation of immune cells via binding to CD137 and to a differentially expressed tumor target, HER2. Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. Using phage display technology a CD137-specific Anticalin was identified. The Anticalin was recombinantly fused to a trastuzumab variant at either the C or N terminus of the antibody´s heavy or light chain, yielding four different constructs covering a range of distances between the binding sites of the T cell-target and the tumor cell target. To minimize Fcγ-receptor interaction of the resulting bispecific and concomitant potential toxicity towards CD137-positive cells, the backbone of trastuzumab was switched from IgG1 to an engineered IgG4 isotype. Using ELISA or cell-based assays it was shown that all bispecific constructs bound their targets CD137 and HER2 with similar affinity compared to the parental building blocks, and both targets could be simultaneously bound. Binding to human receptors FcγRI and FcγRIII was significantly reduced in the bispecific constructs compared to non-engineered trastuzumab, while binding to the neonatal Fc receptor (FcRn) was retained. All constructs were shown to have excellent drug-like properties including thermal stability and plasma stability. HER2-dependent agonistic engagement of CD137 was demonstrated in ex-vivo T-cell activation assays utilizing HER2-positive human cell lines. The functional activity of the bispecific constructs was found to be dependent on their geometry. In conclusion, we report the first bispecific therapeutic protein that targets the potent costimulatory immunoreceptor CD137 in a tumor-target dependent manner, utilizing HER2 as the tumor target. Compared to currently existing CD137-targeting antibodies, this approach has the potential to provide a more controlled activation of the immune system in the tumor microenvironment with reduced peripheral toxicity. Bispecific T-cell engagers based on CD137 and HER2 have potential utility in HER2-positive cancers where there is a significant unmet medical need. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Alexander Wiedenmann, Corinna Schlosser, Andrea Allersdorfer, Gabriele Matschiner, Christine Rothe, Ulrich Moebius, Holbrook E. Kohrt, Shane A. Olwill. Costimulatory T-cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C205.

Meeting abstracts CD137 is a potent costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. The receptor, also known as 4-1BB, is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells. While multiple lines of evidence show

Abstract 687 Rationale: Anticalins® have been developed as a new class of therapeutic proteins based on human lipocalins. We reasoned that given the diversity of compounds that these molecules naturally bind, it should be possible to engineer and identify Anticalins with specificity for small, biologically active compounds, including constrained peptides. To test this hypothesis, we asked whether it would be possible to identify an engineered lipocalin that could specifically block hepcidin, a 25 amino acid hepatic hormone. The central role of hepcidin in regulating iron homeostasis through its interaction with the only known cellular iron exporter ferroportin is supported by human genetics and preclinical data and suggests that sub-population of patients with anemia, exhibiting elevated serum levels of hepcidin, may be responsive to therapies directed at blocking hepcidin. Hepcidin-specific antagonistic Anticalins with different pharmacokinetic properties were generated to develop Anticalin drug candidates with different PK/PD relationships as different clearance rates of the Anticalin-hepcidin complex may be desirable when used as a therapeutic approach in different patient populations. Methods: State of the art phage display technology and high throughput screening were used to isolate a human lipocalin-derived Anticalin to specifically bind and antagonize hepcidin. Randomization of Anticalin binding loops and affinity-based phage display selection were used for affinity maturation and optimization of drug-like properties. Anticalins (MW ∼21 kDa) were produced in E.coli and subjected to site-directed PEGylation with different size PEG molecules. Affinity constants for hepcidin from different species were determined using ELISA-based assays and surface plasmon resonance. We then examined the ability of these Anticalin drug candidates to neutralize human hepcidin activities in cellular and in vivo assays. In addition, PK properties were determined in different animal species to predict PK properties in humans by allometric scaling. Results and Discussion: By using 2 different assay formats the lead candidate displayed high affinity (sub-nM) against human hepcidin and the extension of its plasma half-life by site directed PEGylation did not impact target binding. For example, in an SPR kinetic assay where the PEGylated Anticalin was immobilized and human hepcidin used as analyte, a dissociation constant of Kd = 50±3 pM (n=3) was determined for an Anticalin conjugated with a 40 kDa branched PEG molecule. A stable cell line expressing ferroportin fused to green fluorescent protein was established to determine blockade of hepcidin in vitro by measuring hepcidin-induced ferroportin (FPN) internalization and degradation. Hepcidin bioactivity was completely inhibited by the PEGylated Anticalin at concentrations at or above ∼40 nM (n=6), thus demonstrating the ability of the Anticalin to neutralize hepcidin's principal biological activity on the iron exporter FPN. Furthermore, the ability of PRS-080 to neutralize short-term hypoferremic effects was evaluated in mice after stimulation via a single intraperitoneal injection of 1 mg/kg synthetic human hepcidin. The PEGylated version of PRS-080, administered intravenously several hours prior to hepcidin injection, completely prevented the hypoferremic response at a dose of 95 mg/kg and showed partial prevention at 30 mg/kg (59%) and 9.5 mg/kg (23%). Tunable PK properties were demonstrated with an additional set of PEGylated molecules, tested in rats at a dose of 10 mg/kg, and exhibited terminal half-lives of 9.2, 14.2, 20.4, and 40 hours for Anticalins conjugated with 12, 20, 30 kDa linear and 40 kDa branched PEG, respectively. Conclusions: Our data describe the discovery and characterization of a hepcidin-antagonistic Anticalin for the treatment of anemia. PRS-080 displays numerous differentiating features posited to be relevant for the proposed therapeutic concept of inhibiting hepcidin-mediated hypoferremic effects, including: binding of its relatively small target with high affinity and specificity (pocket binding), favourable safety and tolerability (human scaffold, lack of immune effector cell interactions), tunable half-life and low production costs (bacterial expression). Anticalins provide a powerful novel therapeutic approach to develop antagonists against hepcidin and bioactive peptides in general. Disclosures: Hohlbaum: Pieris AG: Employment, Patents & Royalties. Trentman:Pieris AG: Employment, Patents & Royalties. Gille:Pieris AG: Employment, Patents & Royalties. Allersdorfer:Pieris AG: Employment. Belaiba:Pieris AG: Employment. Huelsmeyer:Pieris AG: Employment, Patents & Royalties. Christian:Pieris AG: Employment, Patents & Royalties. Sandal:Pieris AG: Employment. Matschiner:Pieris AG: Employment, Patents & Royalties. Jensen:Pieris AG: Employment, Patents & Royalties. Skerra:Pieris AG: Consultancy, Patents & Royalties, Research Funding. Audoly:Pieris AG: Employment.