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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

T2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16kD protein abundant in human periciliary fluid, has a robust drug-like structure well-suited to protein engineering, but has never been used to make an inhaled "Anticalin" protein therapeutic.To re-engineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile.Lcn1 was systematically modified by directed protein mutagenesis yielding a high affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated 'PRS-060' with properties analogous to dupilumab, competitively antagonizing IL-4Ra dependent cell proliferation, mucus induction and eotaxin expression in vitro. As PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting hIL-4Ra, hIL-4, and hIL-13 at their correct syntenic murine loci to model clinical dosing.Inhaled PRS-060 strongly suppressed acute allergic inflammation indices in triple humanized mice with a duration of action longer than its bulk clearance suggesting it may act locally in the lung.Lcn1 can be re-engineered into the Anticalin antagonist PRS-060, exemplifying a new class of inhaled topical, long-acting therapeutic with potential to treat T2 endotype asthma.