Abstract
Purpose :
BI-X is an intravitreal anti-semaphorin 3A (Sema3A) agent under investigation in patients with laser-treated proliferative diabetic retinopathy and diabetic macular ischemia. We tested 4 hypotheses: H1) BI-X specifically binds to human Sema3A in an antibody-capture assay; in human retinal microvascular endothelial cell (HRMEC) assays, BI-X H2) prevents Sema3A- but not vascular endothelial growth factor-A (VEGF-A)-induced endothelial cell permeability and H3) prevents Sema3A-induced cytoskeletal collapse; H4) BI-X reduces ischemic avascular area and increases tip cell density in a mouse model of oxygen-induced retinopathy (OIR).
Methods :
H1) Recombinant human, cynomolgus, mouse, rat and rabbit Sema3A were injected (40 μL/min for 600 s) over captured BI-X (0.5 μg/mL) and allowed to dissociate for 7200 s. H2) HRMECs were treated with either recombinant VEGF-A (100 ng/mL) or Sema3A (500 ng/mL), with and without BI-X (1 μg/mL) or anti-TNP (1 μg/mL) overnight. Permeability was determined based on the passage of FITC-coupled dextran through the endothelial cell layer. H3) Potency was determined with a concentration–response curve of recombinant human Sema3A. Cytoskeletal collapse was measured as reduction of cellular impedance. For specificity determination, HRMECs were stimulated with 0.5 μg/mL recombinant human Sema3A, 3C or 3E or 2 μg/mL recombinant mouse Sema3B or 3F with and without 2 μg/mL BI-X. H4) Newborn mice (n=23) were exposed to a 75% oxygen atmosphere from P7 to P12 and then returned to normoxia. The animals received a single 10 μg intravitreal injection of either BI-X or an IgG control antibody in each eye at P12. Tip cell density and avascular area were determined using retinal flatmounts prepared from eyes enucleated at P17.
Results :
H1) BI-X binds to human, cynomolgus, mouse, rat and rabbit Sema3A with a KD of 29 pM, 28 pM, 27 pM, 27 pM and 42 pM, respectively. H2) BI-X completely prevented the permeability induced by Sema3A, but not VEGF-A. H3) BI-X prevented cytoskeletal collapse induced by Sema3A (potency: 69 pM), but not Sema3B, 3C, 3E or 3F. H4) Tip cell density increased by 33% (p<0.001) and avascular area numerically decreased by 12% in BI-X-treated eyes compared with IgG control.
Conclusions :
BI-X binds to human Sema3A with pM affinity, and specifically inhibits Sema3A function, preventing cytoskeletal collapse and endothelial cell permeability in vitro. BI-X also improved ischemia in a mouse model of OIR.
This is a 2021 ARVO Annual Meeting abstract.