Introduction

The concept of immune checkpoint blockade has revolutionized anti-cancer strategies in nearly all cancer fields.¹ Monoclonal antibodies targeting the cytotoxic T lymphocyte antigen-4 (CTLA-4) and programed cell death protein 1 (PD)-1/programmed death-ligand 1 (PD-L1) axes are now part of routine practice.^2–5 Although widely adopted clinically, monoclonal antibody (mAb)-based therapy still consists of several inevitable drawbacks that need to be made aware of. First of all, the manufacturing process of mAb requires a cell-based production system. This may influence production capacity and cause variation between batches.^6,7 Second, mAbs are protein molecules and are prone to induce T cell-dependent neutralizing antibodies after repetitive administrations. Although the humanization process has significantly reduced such a possibility, the presence of a neutralizing antibody that reduces the therapeutic efficacy has been documented even in whole humanized mAbs.^6–8 Third, although a relatively long half-life of mAbs (days to weeks) can be an advantage in terms of drug dosing, its large molecular size (∼150 kDa) may limit its tissue-penetration efficiency.⁹ Most important of all, because severe immune-related adverse effects are not infrequently observed in patients receiving mAb-based immune checkpoint blockades, a long serum half-life of mAbs can be a disadvantage in this regard.⁴

Aptamers are small DNA or RNA molecules that form complex three-dimensional (3D) structures.¹⁰ Since its first emergence in 1990, the aptamer has been considered as an antibody surrogate due to its specific binding affinity and low immunogenicity. Aptamers are chemically synthesized. This potentiates aptamers to be produced in large scale with controllable batch-to-batch variations and be customized and modified for specific purposes.^11–17 Over the past years, aptamers have been shown to serve as successful guiding molecules for targeted delivery, with good tissue penetration efficiency.^13,18–20 Recent aptamer-related research has further extended the field into functional aptamer therapeutics, especially in the field of immuno-oncology, an area full of new hope and uncertainty.^10,21 Because the immune checkpoint landscape encompasses multiple positive and negative regulators, aptamers may have several advantages over mAbs in cancer immunotherapy per se. For example, it is possible to engineer aptamers with dual desirable functions, either being a dual antagonist or an agonist-antagonist integrative.^10,22 In addition, the small size of the aptamer (6–30 kDa) facilitates its rapid renal clearance, with a half-life ranging from hours to 2 days.^23,24 This allows for better managing in case of side effects, which are now recognized an an important issue in cancer immunotherapy.²¹

CTLA-4 is expressed on T cells. It is a homolog of CD28, which binds to the surface antigens B7-1 (CD80) and B7-2 (CD86).²⁵ CTLA-4 expression is initiated upon T cell activation, which attenuates CD28 co-stimulation and inhibits signaling by competing for B7 binding.²⁶ Blocking of CTLA-4 and B7 conjugation reshapes the host immune response and exerts a sustained anti-tumor effect in some cancer subpopulations.^27–29 CTLA-4 and PD-1/PD-L1 axes blockade constitutes the backbone of the current cancer immunotherapy.⁵ As potential advantages of the aptamer are recognized, it is worth developing a CTLA-4-antagonizing aptamer. Although a tetrameric CTLA-4 RNA aptamer had been reported, it does not function as an effective antagonist in a monomeric form.³⁰ It indeed, however, can serve as a carrier for targeting delivery, such as the case of the aptamer-siSTAT3 chimera against T-regulatory cells.³¹ Based on this unmet need, we developed a novel CTLA-4-antagonizing DNA aptamer, aptCTLA-4, by the integration of two high-throughput platforms: SELEX (systematic evolution of ligands by exponential enrichment) and next-generation sequencing (NGS). We showed that aptCTLA-4 is biologically functional and binds to CTLA-4 with high affinity and promotes T cell activity in the tumor microenvironment.