Anti‐angiogenic therapies using biological molecules that neutralize vascular endothelial growth factor‐A (VEGF‐A) have revolutionized treatment of retinal vascular diseases including age‐related macular degeneration (AMD). This study reports preclinical assessment of a strategy to enhance anti‐VEGF‐A monotherapy efficacy by targeting both VEGF‐A and angiopoietin‐2 (ANG‐2), a factor strongly upregulated in vitreous fluids of patients with retinal vascular disease and exerting some of its activities in concert with VEGF‐A. Simultaneous VEGF‐A and ANG‐2 inhibition was found to reduce vessel lesion number, permeability, retinal edema, and neuron loss more effectively than either agent alone in a spontaneous choroidal neovascularization (CNV) model. We describe the generation of a bispecific domain‐exchanged (crossed) monoclonal antibody (CrossMAb; RG7716) capable of binding, neutralizing, and depleting VEGF‐A and ANG‐2. RG7716 showed greater efficacy than anti‐VEGF‐A alone in a non‐human primate laser‐induced CNV model after intravitreal delivery. Modification of RG7716's FcRn and FcγR binding sites disabled the antibodies' Fc‐mediated effector functions. This resulted in increased systemic, but not ocular, clearance. These properties make RG7716 a potential next‐generation therapy for neovascular indications of the eye.
We have cloned the first CD8α gene from an ectothermic source using a degenerate primer for Ig superfamily V domains. Similar to homologues in higher vertebrates, the rainbow trout CD8α gene encodes a 204-aa mature protein composed of two extracellular domains including an Ig superfamily V domain and hinge region. Differing from mammalian CD8α V domains, lower vertebrate (trout and chicken) sequences do not contain the extra cysteine residue (C strand) involved in the abnormal intrachain disulfide bridging within the CD8α V domain of mice and rats. The trout membrane proximal hinge region contains the two essential cysteine residues involved in CD8 dimerization (αα or αβ) and threonine, serine, and proline residues which may be involved in multiple O-linked glycosylation events. Although the transmembrane region is well conserved in all CD8α sequences analyzed to date, the putative trout cytoplasmic region differs and, in fact, lacks the consensus p56lck motif common to other CD8α sequences. We then determined that the trout CD8α genomic structure is similar to that of humans (six exons) but differs from that of mice (five exons). Additionally, Northern blotting and RT-PCR demonstrate that trout CD8α is expressed at high levels within the thymus and at weaker levels in the spleen, kidney, intestine, and peripheral blood leukocytes. Finally, we show that trout CD8α can be expressed on the surface of cells via transfection. Together, our results demonstrate that the basic structure and expression of CD8α has been maintained for more than 400 million years of evolution.
The blood–retina barrier and blood–brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-β pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-β, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.
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