Abstract
Purpose :
Uveitic glaucoma is a common complication in approximately 20% of patients who have uveitis. Recently, the role of immune-mediated neurodegeneration has been determined to be critical in the pathogenesis of primary open angle glaucoma.To this end, we aim to utilize single B cell sorting technology to develop immune modulating bispecific antibodies.
Methods :
Peptides were synthesized for various immune surface targets and BALB/C mice were subsequently immunized with a vaccine schedule consisting of 3 immunizations at Day 0, Day 14, Day 21. Spleens from immunized mice were harvested at Day 28 for single B cell sorting to generate murine chimeric monoclonal antibodies for our various immune targets. Blood was drawn from two volunteers and PBMC isolation was performed. Human PBMCs were cultured for 5 days in the presence of apoptotic agents or media only. T-cell apoptosis was assessed by binding of Annexin V via Fluorescently Activated Cell Sorting (FACS). Percent increase of apoptosis over negative control was determined for total human PBMCs, CD4+, and CD8+ T cells. GraphPad Prism 9 software was used for statistical analysis.
Results :
Spleens from CD25 peptide immunized mice demonstrated positive reactivity toward biotinylated CD25 peptide compared to unimmunized control mice spleens using FACS analysis. Incubation of agent AQZ-1 with human PBMCs for 24 hours demonstrated an increase in total PBMC apoptosis (31.3%, 25.49%), CD4+( 62.67%,75.98%),CD8+(55.33%,46.52%) at 250 nM and 125 nM respectively. Apoptosis was compared to Brefeldin A which had total human PBMC apoptosis (45.3%, 21.5%), CD4+( 28%,1.47%) CD8+(45.33%,28.33%) at 500 µM and 250 µM respectively.
Conclusions :
Mice were successfully immunized with CD25 target confirmed by FACS. Single B cells were sorted and mAbs will be generated for further bispecific antibody engineering. AQZ-1 is a chimeric protein-inducer hypothesized to be an apoptosis-related immune target. AQZ-1 provided a proof-of-concept for potent apoptosis induction as compared to Brefeldin A (positive control). Combinations of immune checkpoint targets provide a viable therapeutic approach to modulate inflammation in the setting of T cell-mediated uveitis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.