Abstract
Purpose :
Secretogranin III (Scg3) was recently discovered as a disease-restricted angiogenic factor. We previously generated and characterized anti-Scg3 ML49.3 monoclonal antibody (mAb) that was converted into EBP2 humanized antibody Fab fragment (hFab) with therapeutic potential. To achieve optimal therapeutic efficacy, we further generated anti-Scg3 mT4 mAb and related EBP3 hFab. This study aimed to characterize all these mAbs and hFabs for their binding, neutralizing, and therapeutic activities in vitro and in vivo, so that an optimal hFab can be selected to develop anti-Scg3 therapy.
Methods :
hFabs were generated from cognate mAbs with binding affinity maturation. The binding activity of anti-Scg3 mAbs and hFabs to Scg3 was detected by ELISA and Western blot. Individual antibodies were also characterized for their binding to retinal Scg3 by immunohistochemistry. Binding affinity to biotinylated Scg3 was quantified using an Octet instrument. Endothelial proliferation and transwell migration assays were performed to detect the neutralizing activity of anti-Scg3 antibodies using human umbilical vein endothelial cells (HUVECs). Therapeutic efficacy was analyzed in diabetic mice by intravitreally injecting individual antibodies or mock control. After 24 h, the Evans Blue assay was performed to quantify retinal vascular leakage.
Results :
ELISA and Western blot confirmed the binding of all anti-Scg3 mAbs and hFabs to Scg3. EBP3 but not EBP2 hFab detected Scg3 in mouse retinal sections by immunohistochemistry. Octet binding kinetics analyses revealed that ML49.3 mAb, EBP2 hFab, mT4 mAb, and EBP3 hFab have Scg3-binding affinities of 35, 8.7, 0.859, and 0.116 nM, respectively. Anti-Scg3 EBP2 and EBP3 hFabs significantly inhibited Scg3-induced endothelial proliferation and migration of HUVECs. Both hFabs alleviated DR vascular leakage in diabetic mice. To compare their therapeutic efficacy, we intravitreally injected EBP2 hFab into one eye of DR mice with EBP3 hFab for the fellow eye. Paired t-test revealed that EBP3 hFab is 26.4% more effective in ameliorating DR leakage than EBP2 hFab (p<0.0001).
Conclusions :
We compared two sets of mAb/hFab, including ML49.3/EBP2 and mT4/EBP3 antibodies, and found that EPB3 has the best binding affinity. More importantly, EBP3 hFab also has better therapeutic efficacy than ML49.3-derived EBP2 hFab and, therefore, will be selected to develop anti-Scg3 therapy.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.