ICB is an important strategy for cancer immunotherapy. To further improve the efficacy of ICB therapy, it appears advantageous to construct a tumor-targeting bispecific agent that can also realize the function of ICB. Bispecific agent can recruit T lymphocytes to tumor tissue, thereby enhancing antitumor immunity (Li et al. 2019; Suurs et al. 2019). Both ICB and bispecific agent can boost anticancer response and have generated remarkable clinical results (Brahmer et al. 2015; Topp et al. 2014). It is reasonable to hypothesize that implementing the two strategies with one nanostructure may further improve the therapeutic efficacy. On the one hand, such a design can recruit more lymphocytes to the vicinity of cancer cells, forming immune synapses and promoting T cell-mediated lysis of cancer cells (Fucà et al. 2021). On the other hand, targeted delivery of ICB agent to tumor tissue may facilitate anticancer immunity while mitigating the undesirable side effects of ICB therapy. Hence, a bispecific agent made of an ICB agent and a tumor-homing ligand appears a promising strategy worthy of further exploration.
In this study, we designed a novel bispecific agent (BiApt) based on a PD-1 aptamer and a nucleolin aptamer (AS1411). The BiApt had an average diameter of 11.7 nm, which was above the threshold of renal clearance (Fig. 3). Phosphorothioate modification of BiApt increased its serum stability (Fig. 4). The BiApt maintained good affinity to both PD-1 expressing T cells and nucleolin-positive tumor cells (Fig. 5). Moreover, in the presence of BiApt, more T cells were recruited to the vicinity of nucleolin-positive cancer cells (Fig. 6). Furthermore, compared with free PD-1 aptamers, BiApt significantly enhanced the PBMC-mediated antitumor cytotoxicity in vitro (Fig. 7). Importantly, in tumor-bearing mice, BiApt also significantly improved the anticancer efficacy vs. PD-1 aptamer (Fig. 8).
Although most ICB agents currently in clinical use are antibodies, aptamers can also fulfill such function with certain technical fortes. First, aptamers for a certain molecular target can be cost-effectively screened in vitro. Second, aptamers can be easily synthesized and manufactured in large-scale (Thomas et al. 2022; Zhu and Chen 2018). Third, the stable structure of aptamers affords them a long storage period and a relatively high resistance to heat, for their tertiary structures can be quickly restored following heat denaturation (Morita et al. 2018). Another advantageous property of aptamer is that it can be easily modified site-specifically and conjugate with various functional agents for biomedical applications (Thomas et al. 2022; Zhao et al. 2015). Because of these technical advantages, aptamers have also been developed for cancer immunotherapy. For example, the PD-1 aptamer used in this study could bind with both human and murine PD-1 proteins, and inhibited tumor growth in CT26-bearing mice (Gao and Pei 2020). Huang et al. selected a CTLA-4-antagonizing DNA aptamer, which could promote lymphocyte proliferation and suppress tumor growth in animal models (Huang et al. 2017). These studies demonstrate that, aside from antibodies, aptamers can also be used for cancer immunotherapy with potential for clinical applications.
Aptamers have also been employed to construct bispecific agents for boosting anticancer immune response. Pastor et al. showed a bispecific agent with a bivalent 4-1BB agonistic aptamer and a PMSA-binding tumor-targeting aptamer, which could deliver the 4-1BB agonist to PSMA-expressing cancer cells in situ and improve antitumor immunity (Pastor et al. 2011). Soldevilla et al. engineered a MRP1-CD28 bispecific aptamer, which could target CD28 co-stimulation to drug-resistant MRP1-positive melanoma stem cells and thereby boost immune response (Soldevilla et al. 2016). Boltz et al. designed a bispecific agent based on CD16 aptamer and tumor-homing c-Met aptamer, which could specifically recruit CD16-expressing NK cells to c-Met-overexpressing tumors, and induce ADCC-mediated tumor lysis (Boltz et al. 2011). Li et al. constructed a bispecific agent with two CD16 aptamers and two MUC1 aptamers, which enhanced the immune cytotoxicity against MUC1-expressing tumor cells by CD16-positive immunocytes (Li et al. 2019). These developments indicate that aptamer-based bispecific agents have application potential in cancer immunotherapy.
So far, however, no bispecific aptamer that combines a tumor-homing ligand with a PD-1 aptamer has been reported in literature. Since PD-1 protein is expressed on activated T cells (Aksoylar and Boussiotis 2020), a bispecific agent made of PD-1 aptamer can not only block inhibitory immune checkpoints, but also recruit T cells into tumor tissue. We speculated that such a design could further improve the anticancer efficacy of PD-1 blockade, and constructed a BiApt based on a PD-1 aptamer and a tumor-homing AS1411 aptamer in this study. As shown in Fig. 8, BiApt indeed significantly enhanced antitumor efficacy in CT-26 bearing mice.
The mechanisms by which BiApt further improves therapeutic efficacy may involve several aspects. First, conjugating PD-1 aptamer with tumor-homing AS1411 aptamer may enrich the BiApt in tumor tissue, thereby strengthening the ICB effect around the tumor cells. Second, in addition to blocking PD-1 pathway, BiApt can also recruit activated T cells to nucleolin-expressing tumor cells, form a bridge between the two, and facilitate T cell-mediated antitumor response. Third, although AS1411 aptamer is mostly used as a tumor-targeting ligand, the aptamer also has a moderate tumor inhibitory effect (Soundararajan et al. 2008; Yazdian-Robati et al. 2020). However, at the dosage used in this study, the antitumor effect by AS1411 was probably quite weak (Fig. 8C). Nevertheless, the tumor inhibitory effect of AS1411 may have contributed to the overall therapeutic efficacy generated by BiApt. Fourth, a free aptamer generally has a very small size and is rapidly eliminated via renal filtration, whereas BiApt has a size of 11.7 nm, which is above the renal clearance threshold, allowing a longer circulation time and prolonged functions. Taken together, all these mechanisms may collectively contribute to the enhanced antitumor efficacy of BiApt.