Targeted IgMs agonize ocular targets with extended vitreal exposure

Abstract

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.

Keywords: IgM; agonism; antibody engineering; long-acting delivery; ocular therapeutics.

Figure 1.

Characterization of recombinant IgMs. Structural representations of IgM ‘monomers’, pentamers, and hexamers. Black lines joining HC represent disulfide bonds (a). 30 ml of Expi293 cells were transiently transfected with various ratios of hIgM HC:LC:JC incubated for 7 d and partially purified using CaptoL resin. Samples were analyzed for total protein by A280 using trastuzumab (Tras) as a positive control (b) and for homogeneity using SEC (c). Human IgM hexamers were purified via two-step purifications. Purified hexamer was reduced and deglycosylated prior to analysis by LC-MS/MS. Extracted ion chromatograms are shown for LC (top) and heavy chain (bottom) (d). Negative Staining TEM characterization of hIgM hexamer reveals the anticipated six-fold symmetry (e).

Figure 2.

Rheology measurements of IgMs. Viscosity of a non-targeting rabbit IgM was assessed by rotary rheology.

Figure 3.

Vitreal pharmacokinetic analysis of IgMs. A non-binding Fab and IgM were nonspecifically labeled with Alexa Fluor-488. Following administration of 0.5 or 1.2 mg respectively, vitreal concentrations were assessed via in vivo fluorophotometric measurements as previously described. Data is shown as observed concentrations (symbols) and noncompartmental projections (lines) in vitreous humor following intravitreal injection of untargeted Fab or untargeted IgM in New Zealand White rabbits.

Figure 4.

Systemic pharmacokinetic analysis of IgMs. Non-binding recombinant hIgM pentamers and hexamers along with IgM isolated from human serum injected intravenously into female SCID mice (a). At indicated time points, serum samples were taken and analyzed for the presence of human IgM. The dotted line is the minimum quantifiable of IgM (0.156 µg/ml). Glycans were removed from the IgMs using PNGase F, and global N-linked glycan profiles were assessed using LC-MS (b).

Figure 5.

Characterization and attenuation of hIgM CDC activity. Rituximab and the corresponding anti-CD20 hIgM hexamer were incubated with Wil-2S cells in the presence of 20% human serum for 2 h. Extent of cell death was evaluated by addition of Alamar blue (a). The P436G mutant shows attenuated CDC compared to the anti-CD20 IgM hexamer (b). Alignment of hIgG1 with hIgM highlighting the prolines critical for effective engagement with complement (c).

Figure 6.

Phospho-(p)AKT signaling downstream of Tie-2 clustering. Serial dilutions of Tie-2 or CD20 targeted IgMs were incubated with rat-aortic endothelial cells for 15 minutes prior to lysis and analysis of pAKT levels using the CisBio Ser473 pAKT kit.

Figure 7.

Death receptor activation downstream of clustering. Serial dilutions of death receptor targeted therapeutics were added to COLO 205 cells in the presence or absence of cross-linker. Viability was determined by Cell-titer glo after 24 h (a). Caspase-8 activity was determined by Caspase-glo 8 4 h following addition of therapeutics (b). The legend is shared for both graphs.