(1123-D) Implementation of automation to overcome matrix interference in a PK assay for a targeted immunotherapy, UGN-301

Abstract: Systemic immune checkpoint inhibitors effectively treat cancer by harnessing the immune system to target tumor cells. However, there are significant risks to immunotherapies administered systemically, with the potential to induce adverse effects due to widespread activation of the immune system. UGN-301 is an intravesical formulation of zalifrelimab (anti-CTLA-4) administered as an admixture with proprietary reverse thermal hydrogel technology. Zalifrelimab blocks the inhibitory signaling cascade between CTLA-4 on T cells and CD80 and CD86 on antigen presenting cells, resulting in an enhanced T cell response. The reverse thermal properties of hydrogel allow for administration of zalifrelimab as a liquid in a cooled state, with subsequent conversion to a semi-solid gel in the bladder. The gel slowly disintegrates and is excreted by normal urine flow, facilitating sustained intravesical release of zalifrelimab over time. Thus, the effectiveness of zalifrelimab + hyrdrogel may be increased compared with administration as a liquid formulation. Given the localized delivery of UGN-301, the development of a pharmacokinetic assay that measures zalifrelimab in urine was needed. A traditional immunoassay was designed in which the drug target, CTLA-4, was used to capture zalifrelimab, and ruthenylated anti-human IgG was used as a detection antibody. While this assay format was able to accurately detect the drug in serum, there were consistent issues in recovering the drug when in urine. This was hypothesized to be due to absorption of the drug to the dilution plates in the absence of buffering levels of protein found in serum. Therefore, to prevent the drug from binding to the plates, the assay implemented the MRD of 1:10,000 in deep-well low bind plates via automation on the Hamilton Starlet system. This presentation will detail the steps involved in taking the assay standards, QCs, and samples from the initial stocks through multiple dilution steps and incubations into the final MSD plate. Use of automation improved the speed of the sample dilution process and robustness of the assay allowing successful validation and sample testing per regulatory guidelines.