Screening Of New CD20 Nucleic Acid Aptamer And Evaluation Of Targeting Binding Characteristics Of Lymphoma

Objective:Non-Hodgkin’s lymphoma (NHL) is a major threat to human health and accounts for5.1%of all cancer cases. CD20is expressed in most NHLs and represents an important therapeutic target. At present, CD20-targeted therapy was mainly achieved by the monoclonal antibody Rituximab. However, most patients treated with the antibody will relapse with therapy-resistant disease and become refractory to further Rituximab treatment. Therefore, it is necessary to develop novel CD20-targeted therapeutic strategies against NHL. Targeted tumor therapy requires ligands that can recognize and bind with the cancer cells. Aptamers possess certain advantages as tumor-targeting ligand compared with antibodies, including high affinity, superior specificity, low immunogenicity, and rapid penetration into tumor tissue. So far, however, CD20aptamer has not been developed due to technical difficulties. In this study, we selected the first aptamer that can recognize the CD20-expressing lymphoma cells and may potentially function as a tumor-binding ligand in CD20-targeted therapies.Methods:Using the SELEX technique and a peptide epitope of CD20as the selection target, we selected a57-base DNA aptamer (termed Z17). Flow cytometry was employed to monitor the bindings of the aptamer to albumin, IgG, trypsin, CD20-positive cells (Raji and Ramos), and CD20-negative cells (Jurkat). To enaluate whether Dox was incorporated into Z17and formed an Apt-Dox complex, we measured the fluorescence spectrum of the complex. Confocal microscope and flow cytometry were used to evaluate the uptake of Apt-Dox complex by either CD20-positive cells (Raji) or CD20-negative cells (Jurkat). MTS was used to evaluate the cytotoxicity of Apt-Dox complex against CD20-positive cells (Raji) or CD20-negative cells (Jurkat).Results:(1) The aptamer was a57-base single-strand DNA, which could recognize CD20structure with a Kd of100.7nM.(2) The aptamer demonstrated relatively specific binding to the CD20structure, with minimal cross-reactivity to albumin, IgQ or trypsin. (3) Moreover, the aptamer bound strongly with the CD20-expressing lymphoma cells (Raji and Ramos), but did not bind with the CD20-negative cells (Jurkat).(4) An aptamer-doxorubicin (Apt-Dox) complex was constructed by inserting doxorubicin into the DNA structure of the aptamer.(5) The Apt-Dox complex made a targeted delivery of doxorubicin to the CD20-expressing lymphoma cells.(6) Furthermore, the Apt-Dox complex achieved a CD20-targeted therapeutic efficacy against lymphoma cells in vitro.Conclusion:In summary, a DNA aptamer against CD20was selected in this study. The aptamer could recognize CD20-expressing lymphoma cells, with minimal cross-reactivity to CD20-negative cells. Moreover, the Apt-Dox complex made a targeted delivery of doxorubicin to CD20-positive lymphoma cells in vitro, and induced selective cytotoxicity to these cancer cells. The results suggest that CD20aptamers may have application potential in targeted therapy against CD20-expressing tumors.