# A Novel FLI1 Monoclonal Antibody Which Recognizes EWS::FLI1 with High Affinity Is Useful for Detecting Ewing Sarcoma

## TL;DR

A new high-affinity monoclonal antibody for EWS::FLI1 fusion protein is developed, offering improved detection and study of Ewing sarcoma.

## Contribution

Development of a novel monoclonal antibody with high specificity and affinity for EWS::FLI1 fusion oncoprotein.

## Key findings

- The FLI1 1.2 monoclonal antibody reliably detects EWS::FLI1 in various molecular biology techniques.
- The antibody has a high affinity (0.3 nM KD) and is specific to EWS::FLI1 with minor cross-reactivity to ERG.

## Abstract

Background: Ewing sarcoma (ES) is a rare tumor that affects children, adolescents, and young adults. ES is associated with high morbidity in all patients and high mortality for those who present with metastatic disease. A chromosomal translocation, either t(11;22)(q24;p12) or t(21;22)(q22;q12) leads to the fusion oncoproteins EWS::FLI1 or EWS::ERG in 95% of ES patients. We recognized a critical need for a stably sourced high-affinity antibody that recognizes EWS::FLI1 with maximal specificity. Understanding EWS::FLI1 protein complexes is a pivotal gap in ES knowledge that necessitates the development of antibodies capable of identifying native proteins in solution. Further, variable epitope sequencing of a monoclonal antibody enables the construction of degraders and nanobody identifiers. Methods: Monoclonal antibodies were produced following informed peptide synthesis, injection, and hybridoma creation. Hybridoma antibodies were validated for specificity and function. Results: Our results indicate that the FLI1 1.2 monoclonal antibody, which recognizes the EWS::FLI1 fusion oncoprotein, can be reliably applied to multiple molecular biology applications like immunoblot, immunoprecipitation, immunofluorescence, and immunohistochemistry. This FLI1 1.2 monoclonal antibody has a high affinity of 0.3 nM KD to EWS::FLI1. In terms of specificity, this antibody is highly specific to EWS::FLI1 and some cross reactivity with ERG. Conclusions: This reagent will provide the research community with valuable tools for further biochemical and genomic interrogation of the oncogenic activity of EWS::FLI1 in ES.