(T1030-01-07) Development of an Immunoassay to Evaluate Transgene Product Expression from an Investigational Gene Therapy to Treat Dry Age-Related Macular Degeneration (dryAMD) in Various Non-clinical Studies

Purpose: Administration of complement factor inhibitors in the eye is a viable strategy to preserve vision in patients with dry AMD. Investigational gene therapy is expected to produce the anti-human C5 (hC5) single chain fragment variable (scFv) as a transgene product (TP) to treat dry AMD. Here, we have developed a ligand binding assay to measure the expression of TP in cynomolgus monkey ocular matrices dosed with proprietary NAV® capsid carrying anti-hC5-scFv expression cassette.

Methods: An electrohemiluminescence (ECL) based immunoassay was developed to measure TP in various monkey ocular matrices. Briefly, TP from ocular matrices was captured using recombinant C5 complement protein. Captured TP was detected and quantified using SULFO-TAG labeled rabbit polyclonal antibodies (against recombinant scFv) on an MSD platform. To increase the quantification range, biotinylated rabbit monoclonal antibodies (purified from polyclonal antibodies) were tested to capture TP.

Results: Non-clinical studies demonstrated that increase in expression of TP is dose-dependent at 4 Weeks post AAV injection. The use of rabbit monoclonal antibody as capture reagent improved quantification range by 5-fold at the upper limit of calibration curve, compared to C5. The calibration curve range for anti-hC5-scFv was 25.0 - 1000 ng/mL. The Precision and Accuracy of calibrators and QCs were within 20%. Dilution linearity suggests that up to 100,000 ng/mL of TP can be diluted within the range using assay matrix. Selectivity was demonstrated in aqueous humor, vitreous humor and other monkey ocular matrices.

Conclusion: We have developed and qualified an ECL based immunoassay for the quantitation of anti-hC5-scFv in monkey aqueous humor, vitreous humor, and tissue samples to support non-clinical studies. The qualification assessments, such as precision, accuracy, linearity, target interference, selectivity, and stability, demonstrated that this method is fit-for-purpose in measuring TP in various monkey ocular matrices and is suitable for use in non-clinical studies.