Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.

Targeted IgMs agonize ocular targets with extended vitreal exposure.

Treatment of ocular disease is hindered by the presence of the blood-retinal barrier, which restricts access of systemic drugs to the eye. Intravitreal injections bypass this barrier, delivering high concentrations of drug to the targeted tissue. However, the recommended dosing interval for approved biologics is typically 6-12 weeks, and frequent travel to the physician's office poses a substantial burden for elderly patients with poor vision. Real-world data suggest that many patients are under-treated. Here, we investigate IgMs as a novel platform for treating ocular disease. We show that IgMs are well-suited to ocular administration due to moderate viscosity, long ocular exposure, and rapid systemic clearance. The complement-dependent cytotoxicity of IgMs can be readily removed with a P436G mutation, reducing safety liabilities. Furthermore, dodecavalent binding of IgM hexamers can potently activate pathways implicated in the treatment of progressive blindness, including the Tie2 receptor tyrosine kinase signaling pathway for the treatment of diabetic macular edema, or the death receptor 4 tumor necrosis family receptor pathway for the treatment of wet age-related macular degeneration. Collectively, these data demonstrate the promise of IgMs as therapeutic agonists for treating progressive blindness.

Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics.

PURPOSE: To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. METHODS: Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. RESULTS: After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. CONCLUSIONS: We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.

Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies.

Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the ß-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway.

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.

Evaluation of heterophilic antibody blocking agents in reducing false positive interference in immunoassays for IL-17AA, IL-17FF, and IL-17AF.

IL-17AA, IL-17FF, and IL-17AF are proinflammatory cytokines that have been implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). In order to measure the levels of these cytokines in synovial fluid and serum samples from RA patients, immunoassays specific for IL-17AA, FF, and AF were developed. Although these assays could tolerate up to 50% pooled normal human serum, false positive reactivity was problematic in patient samples suggesting interference from heterophilic antibodies. We therefore evaluated the ability of several commercially available heterophilic antibody blocking agents to reduce false positive reactivity by testing them against samples that were confirmed as false positives in the IL-17AA, FF, and AF assays. Several of the blockers performed well, including HBR-1, HBR-9, HBR-11, HBR-Plus, Serum Cytokine Assay Diluent, and IIR. We chose to move forward using IIR blocker for sample analysis and verified that IIR had no effect on the assay standard curves and did not affect IL-17 quantitation in plasma from ex vivo stimulated human whole blood. IL-17FF and IL-17AF were below the limits of quantitation of the assays (12.3 and 10.5pg/ml, respectively) in synovial fluid and serum samples from patients with RA and osteoarthritis (OA). For the more sensitive IL-17AA assay (1.6pg/ml limit of quantitation), low levels of IL-17AA were measurable in 48% of RA synovial fluid samples (mean, 7.9pg/ml; median, <1.6pg/ml; range, <1.6-29.7pg/ml; n=23) but not in synovial fluid from patients with OA (n=33). For serum samples, however, IL-17AA was below the limit of detection for both RA and OA patients. When these same serum samples were analyzed in the absence of a heterophilic antibody blocker, false positive reactivity yielded apparent mean IL-17AA levels of 43.3pg/ml (28% positive; n=50) and 14.8pg/ml (12% positive; n=50) for RA and OA patients, respectively, results that could potentially be interpreted as consistent with disease biology. These studies demonstrate the importance of ensuring that HAb interference is well controlled, particularly when measuring low concentrations of cytokines in samples from patients with autoimmune disease.

ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha vbeta 3 and induces blood vessel formation in vivo.

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.