Abstract 6745: Generation and validation of a recombinant monoclonal antibody to PAX8: a multi-assay approach

Abstract Introduction: Paired box 8 (PAX8) is a nuclear protein involved in thyroid follicular cell development and expression of thyroid-specific genes. While primarily associated with thyroid follicular carcinomas and atypical follicular thyroid adenomas, recent research has indicated that this marker is prognostic in small cell lung cancer and positively correlated with Ki67 expression (Tao et al., 2024) -- perhaps opening PAX8 up as a future therapeutic target. Studies such as these, though, require the use of extremely reliable antibodies to generate reproducible and trustworthy data. In congruence with the International Working Group for Antibody Validation’s (IWGAV) pillars of antibody validation, a program has been implemented to enhance our existing validation principles including the recombinant monoclonal PAX8. Methods: Four unique PAX8 peptides were synthesized, purified, and used for rabbit immunization and B-cell sorting. Clones were screened for baseline activity and assessed microscopically for specificity using variable tissue and cell microarrays. Specificity was further assessed via reciprocal IPs, western blot, and flow cytometry. Finally, multiplex immunofluorescence staining was conducted utilizing two separate approaches. First, staining and imaging was done using the Lunaphore COMET™ followed by traditional TSA sequential staining methods and imaged with the Akoya PhenoImager® HT system. Results: Over 40 clones were screened against OVCAR3 cell lysate using ProteinSimple Simple Western™ technology and stained in tandem across target-specific microarrays using chromogenic immunohistochemistry. This dual screen approach allowed for the narrowing of the substantial clone set down to 8, which was reduced further after in-depth CTWB/IP studies eliminated an additional 5 clones. The remaining 3 clones were examined through expanded chromogenic testing on large (80+ cores) human, mouse, and rat tissue and cell microarrays. Throughout the various assays, PAX8 specificity was assessed utilizing other external antibodies, PAX8-overexpressing cells, and known target localization via literary sources. Conclusions: In utilizing a multi-assay approach, we were able to narrow the original 40+ clone set to a single, highly-specific monoclonal antibody. This approach is one that the International Working Group for Antibody Validation has been emphasizing as a superior way to produce antibodies that foster reproducibility across research. In future studies, multiplex immunofluorescence staining will be conducted using PAX8 in combination with PAX5 and Ki67 across small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) to evaluate expression rates. References: 1. Tao, Fengyun et al. Prognostic value of PAX8 in small cell lung cancer. Heliyon. 2024. 10(7): e28251. Citation Format: Amber Miller, Danielle Fails, Jessica Tracy, Hailey Wooten, Michael Spencer. Generation and validation of a recombinant monoclonal antibody to PAX8: a multi-assay approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6745.