Abstract B049: Bispecific Aptamers for Enhanced Binding, Internalization, and Therapeutic Delivery in Burkitt Lymphoma

Abstract Burkitt lymphoma is an aggressive cancer that accounts for 30% of all pediatric lymphomas. Due to its aggressive clinical course, patients are immediately started on a high-intensity chemo-immunotherapy protocol with central nervous system (CNS) prophylaxis to prevent relapse or CNS involvement. The Magrath regimen (CODOX-M/IVAC – cyclophosphamide, vincristine, doxorubicin, high-dose methotrexate, ifosfamide, cytarabine, etoposide, and intrathecal methotrexate) is most commonly used for medical management, yielding two-year event-free survival rates as high as 90%. However, this drug regimen consists of nonspecific, broadly toxic medications and has frequently led to off-target toxicity. To prevent these unwanted effects, it is necessary to design a more targeted therapeutic that utilizes tumor-specific markers. Burkitt lymphoma cells (Ramos) have been shown to inappropriately display a component of the spliceosomal complex (hnRNP U) on their cell surface and many human tumors have been shown to overexpress the transferrin receptor (TfR) relative to native tissues, making these exciting targets. We aimed to produce dual-targeting aptamers and to characterize their binding to, internalization by, and therapeutic deliver to Ramos cells. Anti-hnRNP U DNA aptamer (c10.36) and an anti-TfR RNA aptamer (E3) each containing a 3’-terminal 21-nucleotide antitail or tail, respectively, were synthesized and annealed together using sense:antisense complexation. For fluorescent experiments, monospecific aptamers were annealed to a cyanine 3 (Cy3)-labelled antitail and bispecific aptamers were constructed using DNA aptamers with 3’ cy3 modification. Ramos cells were treated with bispecific, monospecific, or control aptamers for 1 hour prior to flow cytometric analysis or fixation, staining, and imaging by a confocal microscope. Ramos cells treated with c10.36-E3 bispecific aptamers demonstrated 9-fold higher fluorescence by flow cytometry relative to monospecific controls. Confocal microscopy showed greater fluorescent intensity and diffuse punctate staining pattern in Ramos cells treated with c10.36-E3 bispecific aptamers relative to their monospecific controls. In Ramos cells stained with LysoTracker dye, co-localization of c10.36-E3 bispecific aptamers with lysosomes was observed whereas this was not observed with control aptamers. When Ramos cells were pre-treated with endocytic inhibitors or transferrin, internalization of c10.36-E3 was found to be reduced by treatment with sucrose, low temperature, or human transferrin. Finally, Ramos cells were treated with on- or off-target bispecific aptamers containing 3’-Val-Cit-PABA-MMAF and viability was assessed using live-dead stain HelixNP via flow cytometry. Over 48 hours, c10.36-E3 led to dramatic reductions in viability relative to appropriate control products. In conclusion, the c10.36-E3 bispecific aptamer is internalized through TfR-mediated endocytosis with superior binding and trafficking relative to controls making it an exciting vehicle capable of targeted therapeutic delivery to Ramos cells. Citation Format: Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery. Bispecific Aptamers for Enhanced Binding, Internalization, and Therapeutic Delivery in Burkitt Lymphoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B049.