Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation

Abstract

CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.

Development of nucleic acid aptamer-based lateral flow assays: A robust platform for cost-effective point-of-care diagnosis

“…The rapid progress in aptamer (chemical antibodies) technologies over the past three decades provide a valuable opportunity to solve many of the obstacles faced by the conventional antibody-based LFAs 53 , 134 . Aptamers are identified via in vitro procedures which do not require the use of animals, and compatible with chemical modifications 11 .…”

Improving Isolation of Extracellular Vesicles by Utilizing Nanomaterials

“…Song et al immobilized two DN tamers with high affinity and specific to CD63 proteins onto the magnetic beads. found that CD63 aptamers not only presented a comparable diagnostic efficacy for positive breast cancer with commercial antibodies but also could release the capture via a simple 0.5 M NaCl elution step [57].…”

“…Song et al immobilized two DNA aptamers with high affinity and specific to CD63 proteins onto the magnetic beads. They found that CD63 aptamers not only presented a comparable diagnostic efficacy for CD63-positive breast cancer with commercial antibodies but also could release the captured EVs via a simple 0.5 M NaCl elution step [57]. In addition to the classic antigen antibody interaction, aptamers represent an native approach for antibodies due to their high binding affinity, low/no immunoge ease of synthesis and accessibility for different chemical modifications [8].…”

Application of engineered extracellular vesicles for targeted tumor therapy

“…To date, immunoaffinity chromatography has been used to isolate EVs derived from T cells and melanoma cells. Using CD63 aptamers, Song et al demonstrated that this technology might increase the efficiency of EVs isolation and lead to improved cancer diagnosis [ 60 , 61 ]. Additionally, the affinity of antibodies for their ligands enables them to access EVs-specific populations.…”