Abstract 6415: CD25-targeted aptamer-drug conjugate (CD25-ApDC) depletes and blocks regulatory T cells selectivel

Abstract

Abstract Background: Regulatory T cells (Tregs) have a strong immunosuppressive function, inhibit anti-tumor immunity, and promote the occurrence and development of tumors. In addition, a high infiltration of Treg cells is associated with poor survival in various types of cancer. Since CD25 is highly expressed on Treg cells, it is a potential target protein for Treg depletion. Methods: The binding affinity(Kd) of CD25 aptamer to rhCD25 protein was calculated through bio-layer interferometry(BLI). The cell binding of Cy5-labelled CD25 aptamer was detected by flow cytometry, and the internalization was visualized by confocal microscopy. The cell internalization rate was determined using the MFI value of flow cytometry. Competitive ELISA, western blot, and qPCR were performed to investigate the effect of CD25 aptamer on CD25/IL-2 binding. In vitro cytotoxicity of prototype-ApDC was determined by CCK-8 assay. Moreover, CD25-dependent cytotoxicity of prototype-ApDC was evaluated by Karpas299(Treg-like):HuT78(Teff-like) 1:1 co-culture model using FACS analysis. Results: CD25 aptamer binds human CD25 protein with 4.64nM affinity. CD25 aptamer showed selective binding and internalization to CD25-positive Karpas299 and had no interaction with CD25-negative Daudi. The internalization half-time of CD25 aptamer in karpas299 was determined to 9.9 min (95% CI 8.2~12.6min). Interestingly, CD25 aptamer was identified as IL-2R antagonist by evaluating competitive ELISA and the level of phosphor-STAT5 and downstream mRNA expression of TGF-beta. The IC50 value of prototype-ApDC was 24nM to Karpas299 while >1uM to Daudi. These results show that prototype-ApDC has selective cytotoxicity against CD25-expressing cells. Furthermore, in a study of an in vitro co-culture model mimicking the Treg-rich intratumoral T cell environment, prototype-ApDC treatment induced CD25-specific depletion resulting in escalating the ratio of HuT89/Karpas299 with increasing exposure time. Conclusions: Our CD25-targeted aptamer and its drug-conjugated ApDC have demonstrated the selective blocking of IL-2R-STAT5 signaling and induction of CD25-positive cell-specific depletion, respectively. Moreover, in the co-culture system of the Treg-like and Teff-like cells, CD25-ApDC also proved its cytotoxic selectivity. Therefore, one potential strategy in cancer patients is to use CD25-ApDC with excellent tumor permeability as an anticancer agent with a mechanism of Treg cell depletion through specific targeting of CD25 overexpressed in tumor-infiltrating Treg cells. Citation Format: Daekyun Lee, Ju-hyung Kang, Inu Song. CD25-targeted aptamer-drug conjugate (CD25-ApDC) depletes and blocks regulatory T cells selectivel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6415.