Selecting a DNA Aptamer Against CTLA-4 for Immune Checkpoint Therapy

Abstract

Immune checkpoint therapies for cancer treatment leverage the body’s immune system by preventing its inhibition through cancer cell signaling. Currently available immune checkpoint therapies are antibody-based, leading to adverse side effects in the majority of patients. Aptamers—short, single stranded nucleic acids—have been explored as alternatives to antibodies for immune checkpoint therapy. In this project, we utilized bead-based SELEX to begin selection for aptamers with a specific affinity to the immune checkpoint protein CTLA-4. CTLA-4 with a histidine tag was immobilized on Ni-NTA magnetic beads and incubated with a GC-rich DNA library. Bound sequences were amplified with PCR, cleaned, and quantified before digestion with lambda exonuclease to generate ssDNA to be used in further rounds of selection. Ideal lambda exonuclease concentrations and incubation times for the generation of ssDNA after symmetric PCR were determined for use within the project, and potential aptamer candidates were amplified after the first round of selection. In the future, we will continue this project by running more rounds of selection, completing sequencing of our aptamers, and performing binding affinity assays for aptamer candidates.