Development of single chain antibodies for targeting Ewing sarcoma surface marker CD99

Recombinant antibodies have become an important class of therapeutic agents in cancer treatment. CD99, a transmembrane glycoprotein, is upregulated in several malignancies, including Ewing sarcoma (ES). The current therapy for ES comes with significant toxicity and a substantial risk of life-threatening late complications. Hence, there is a growing need for new and more effective treatments. Thus, we developed CD99-targeting single-chain antibodies by isolating the variable regions from a hybridoma-derived anti-CD99 antibody and engineering two scFvs with distinct linkers. Both scFvs were expressed in Escherichia coli, purified, and characterized. Comparative in vitro binding analyses demonstrated that the scFv containing the shorter 15-mer linker exhibits significantly enhanced affinity and specificity toward CD99-overexpressing Ewing sarcoma cells relative to the 18-mer variant, highlighting linker length as a critical determinant of scFv performance. These findings establish a rational design principle for optimizing CD99-targeting scFvs and support the potential application of the 15-mer scFv in CAR-T cell development and in targeting other CD99-overexpressing carcinomas.