The CD3-TCR complex is the primary receptor responsible for T cell antigen-specificity, which triggers the activation cascade in T lymphocytes. CD3 agonistic antibodies are often utilized to expand T lymphocytes as adoptive cell therapy in cancer patients. Using these antibodies is problematic because they may activate or expand T cells independently of the TCR's specific reactivity. Therefore, there is a need to develop novel therapeutic agents that can decrease the threshold of T activation while maintaining antigen specificity for adoptive cell therapy expansion.

To identify CD3 T cell stimulating aptamers we perform six rounds of SELEX against the heterodimeric CD3DE recombinant protein. After high throughput sequencing and binding experiments, only four aptamer candidates were chosen for further characterization. These aptamers displayed specific binding to the target CD3 protein, as well as binding to a CD3 expressing cell line and primary murine lymphocytes. Binding was confirmed using CD3-/- cells generated via CRISPR/Cas9. When dimerized, these aptamers increased the proliferation of T cells that were suboptimally stimulated with antigenic low affinity p-MHC stimulus in vitro and in vivo. To evaluate the efficacy and persistence of adoptive cell therapy, low affinity TCR-reactive lymphocytes were expanded using limiting amount of antigen with the CD3 aptamer and were tested in a melanoma tumor-bearing mice showing a strong antitumor response.

The results of these studies indicate that CD3 targeting oligonucleotide based-therapeutic agent can facilitate the expansion of tumor-reactive lymphocytes while retaining tumor specificity.  CD3 aptamers selected herein may have potential for further translation in the future.