Bioanalytical solutions and immunogenicity challenges of species-matched antibodies in preclinical studies while investigating protein-loaded long-acting intravitreal delivery technologies

Abstract

Purpose :
Species-matched antibodies, e.g., rabbit Fab (RabFab), have been developed to enable improved nonclinical pharmacokinetic (PK) and safety assessments of protein-loaded ocular long-acting delivery (LAD) technologies by reducing immunogenicity relative to humanized proteins. However, distinguishing the species-matched antibody from endogenous rabbit antibodies requires specialized bioanalytical methods; furthermore, factors beyond species may conspire against immune evasion. This study compares bioanalytical solutions for measuring RabFab LADs and demonstrates the challenge of immunogenic species-matched LAD molecules.

Methods : Specialized species-matched analyte measurements of rabbit ocular fluids require either a labeled Fab or a specific assay, such as mass spectrometry (MS) or enzyme-linked immunosorbent assay (ELISA). For the latter, we used anti-idiotype antibodies for the RabFab ELISA. A liquid chromatography (LC)-MS/MS approach involved detection using a signature peptide derived from a tryptic digest. To investigate immunogenicity resulting from intravitreal injections of RabFab LADs in rabbits, anti-drug-antibody (ADA) assays were performed on serum by ELISA.

Results : Agreement of ELISA versus gamma count radiolabelled RabFab concentrations differed depending on RabFab LAD conjugate and matrix. For example, both methods agreed for HA-RabFab in aqueous humor but disagreed in serum, while for TSG6-RabFab the two methods disagreed in all tested matrices. Serum under-recovery by ELISA was seen for all tested RabFab LAD molecules as well as unconjugated RabFab. TCA precipitates in serum showed a closer agreement to the RabFab ELISA results, indicating the serum contained free label. A LC-MS/MS approach demonstrated LAD-agnostic recovery of the Fab analyte in the ocular matrix. ADA assays revealed that RabFab can be immunogenic in rabbit serum when paired with certain LAD technologies.

Conclusions : The use of a specialized mass spectrometry assay overcame bioanalytical challenges associated with the quantification of Fab LADs in ocular matrices. Although RabFab has very low immunogenicity on its own, when coupled with LAD technologies, some LAD factors may increase ADA incidence including conjugation other than hydrogel, higher amounts, and smaller multimeric arrays.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.