Enhanced therapeutic efficacy of ApoA1-Anti CD99 antibody fusion protein for treatment of Ewing sarcoma

Most conventional anticancer therapies suffer from poor target specificity, frequently resulting in significant off-target toxicity and limited therapeutic indices. To address these shortcomings, advanced drug delivery systems such as targetable hybrid polymer-lipid nanoparticles (tHPLNs) have been developed for improved tumor specificity. To date, tHPLNs have been formulated to enable covalent attachment of tumor-specific antibodies on their surfaces, with the aim of enhancing selective accumulation at tumor sites. However, current chemical conjugation strategies are often complex and rely on multi-step bioconjugation chemistry that can produce particles with misoriented or nonfunctional antibodies or antibody fragments, compromising targeting efficiency and reproducibility. By genetically fusing apolipoprotein A1 (ApoA1) to the Fc domain of human anti-CD99 antibodies, we generated a fusion protein that spontaneously associates with the HPLN surface, enabling effective antibody targeting of irinotecan-loaded nanoparticles (NV105) - all without modifying the core nanoparticle chemistry or performing additional antibody conjugation steps. Importantly, compared to chemical linkage methods, the ApoA1 attachment technology yields 5-fold higher affinity binding of our nanoparticles to tumor cells. In Ewing Sarcoma xenograft models, NV105 exhibited substantially greater antitumor efficacy than NV103, which was targeted via conventional chemical conjugation of human anti-CD99 antibodies. Much better tumor reduction has been observed at doses as low as 0.5 mg/kg irinotecan, treated twice per week, which was superior to 1 mg/kg NV103 treated biweekly. Complete tumor ablation has been observed at doses as low as 5 mg/kg NV105, treated twice per week. In conclusion, HPLN/Ir formulated with anti-CD99 antibodies fused with ApoA1 demonstrated substantially greater efficacy against Ewing Sarcoma. We anticipate that this approach will enhance tumor cell binding and drug targeting broadly for other antibodies and nanoparticle formulations.