A recombinant CD99-targeted antibody-drug conjugate for Ewing sarcoma therapy

Ewing sarcoma (ES) is an aggressive paediatric malignancy characterised by high CD99 expression and limited targeted therapeutic options. In this study, we developed a recombinant anti-CD99 Cancer Institute Antibody (CIAb-anti-CD99) by isotype switching a hybridoma-derived IgM/κ antibody to an IgG2a/κ format to improve manufacturability and therapeutic applicability. Recombinant CIAb-anti-CD99 was expressed in CHO-K1 cells and retained specific recognition of CD99, as demonstrated by immunofluorescence, flow cytometry, Western blotting, and immunohistochemistry. Functionally, CIAb-anti-CD99 increased the Annexin V/PI-positive cell population and inhibited proliferation and clonogenic growth of CD99-positive ES cells in vitro. To evaluate the potential of CIAb-anti-CD99 for targeted drug delivery, CIAb-anti-CD99 was conjugated to doxorubicin to generate a CD99-targeted antibody-drug conjugate (CIAb-Dox). In vivo biodistribution studies demonstrated tumour localisation and prolonged retention of CIAb-anti-CD99 in A673 xenograft tumours. Therapeutic evaluation in an A673 cell line-derived xenograft (CDX) model showed that CIAb-Dox significantly suppressed tumour growth compared with vehicle and antibody monotherapy. Histopathological analysis demonstrated lower viable tumour content and greater tumour necrosis in CIAb-Dox-treated tumours. Quantitative immunohistochemical analysis further showed a significant reduction in the Ki67-positive proliferative index and increased cleaved PARP staining, consistent with apoptosis-associated cellular changes. CIAb-Dox demonstrated enhanced antitumour activity while maintaining a favourable tolerability profile. Collectively, these findings provide preclinical evidence supporting further investigation of CD99-directed antibody-drug conjugates. CIAb-anti-CD99 represents a functionally active CD99-targeting antibody, while CIAb-Dox warrants further evaluation in additional Ewing sarcoma models, together with mechanistic, pharmacokinetic, and comprehensive analytical studies.