Recent strides in immunotherapy have illuminated the crucial role of CTLA-4 and PD-1/PD-L1 pathways in contemporary oncology, presenting both promises and challenges in response rates and adverse effects. This study employs a computational biology tool (in silico approach) to craft aptamers capable of binding to dual receptors, namely, inhibitory CTLA4 and NKG2A, thereby unleashing both T and NK cells and enhancing CD8⁺ T and NK cell functions for tumor cell lysis. Computational analysis highlighted AYA22T-R2-13 with HADDOCK scores of −78.2 ± 10.2 (with CTLA4), −60.0 ± 4.2 (with NKG2A), and −77.5 ± 5.6 (with CD94/NKG2A). Confirmation of aptamer binding to targeted proteins was attained via ELISA and flow cytometry methods. In vitro biological functionality was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. Direct and competitive assays using ELISA and flow cytometry demonstrated the selective binding of AYA22T-R2-13 to CTLA4 and NKG2A proteins, as well as to the cell surface receptors of IL-2-stimulated T cells and NK cells. This binding was inhibited in the presence of competition from CTLA4 or NKG2A proteins. Remarkably, the blockade of CTLA4 or NKG2A by AYA22T-R2-13 augmented human CD8 T cell- and NK cell-mediated tumor cell lysis in vitro. Our findings highlight the precise binding specificity of AYA22T-R2-13 for CTLA4-B7-1/B7-2 (CD80/CD86) or CD94/NKG2A-HLA-E interactions, positioning it as a valuable tool for immune checkpoint blockade aptamer research in murine tumor models. These in vitro studies establish a promising foundation for further enhancing binding capacity and establishing efficacy and safety in animal models. Consequently, our results underscore the potential of AYA22T-R2-13 in cancer immunotherapy, offering high specificity, low toxicity, and the potential for cost-effective production.

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.

The nasal microenvironment plays a crucial role in the transmission, modulation, and clinical progression of COVID-19; however, the immune responses at the site of viral entry remain poorly understood. We deciphered the link between nasopharyngeal (NP) immune and inflammatory response that triggers cytokine/chemokine storms in the nasal route of COVID-19-positive patients. We used RT-PCR, multiplex ELISA, flow cytometry, and LC-MS/MS to decipher nasopharyngeal immune perturbations associated with severe COVID-19. In addition, we performed in vitro assays using cultured human monocytes-derived macrophages trained both in the presence and absence of SARS-CoV-2 trimeric spike protein(s) and co-cultured with and without autologous human peripheral blood mononuclear cells (hPBMCs)/total T-cells/CD8 T-cells. In vitro immune perturbations were examined by flow cytometry and LC-MS/MS assays. Our findings confirm that macrophages orchestrate NP immune inflammatory responses and highlight the cytokine/chemokine storms associated with the increased CD8${}^{+}$T-cells along with Tregs, Th1, and Th17.1 T-helper cells. We observed a correlation between in vitro and nasal findings that trained macrophages, profoundly M2c, differentially promote the inflammatory surfactome on CD8 T-cells, including ITGAM, LGALS3, CD38, TKT, LRPAP1, and SSBP1. The findings of this study conclude that inflammatory lymphocyte perturbations within the nasopharynx of COVID-19 patients may enforce immune homeostasis during SARS-CoV-2-infection and contribute to COVID-19 pathology. This study explored the therapeutic target proteins that could facilitate the development of new medications, which could allow for immediate treatment of possible emerging viral infections.

Food allergy, particularly peanut allergy (PA), is a growing health concern affecting millions globally. PA can lead to severe reactions, including fatal anaphylaxis. Despite the availability of FDA-approved therapies like Palforzia, a cure remains elusive. Current immunotherapies show promise but lack a definitive cure. This study applies an established computational biology tool to design aptamers targeting Ara h1 and Ara h2. The in silico design aims to streamline the selection process, enabling cost-effective and rapid identification of aptamer candidates. The developed aptamers (AYA22A, including AYA22AR321, AYA22AR211, and AYA22AR524), demonstrated efficacy in inhibiting degranulation of RBL-2H3 cells (rat basophilic leukemia cell line) in vitro. They showed promise in neutralizing peanut allergen-induced immune responses. The selected aptamers inhibited degranulation in RBL-2H3 cells, addressing concerns in raw peanuts. Moreover, these aptamers demonstrated stability and effectiveness in peanut plant seeds and commercial products. Our aptamers exhibited potential in modulating immune responses associated with peanut allergy. They influenced Th1/Th2 balance, indicating a role in cytokine regulation. In vitro studies also showed the aptamers’ impact on immune cell expression and cytokine production, resembling responses observed with established immunotherapies. The findings suggest AYA22A aptamers as a potential therapeutic option for peanut allergy, providing a basis for further in vivo investigations.

Peanut allergies, driven by sensitization to key allergens Ara h1, Ara h2, and Ara h3, present significant health risks, particularly in food processing and consumer settings where accidental exposure is frequent. To mitigate this risk, we developed AYA22AR321, a novel aptamer with selective, high-affinity binding to these allergens (Kd values: 0.5 nM for Ara h1, 14.5 nM for Ara h2, and 6.6 nM for crude peanut extract). Functional assays using RBL-2H3 (rat basophilic leukemia cell line) cells showed that AYA22AR321 significantly reduces IgE-mediated degranulation, indicating its potential to attenuate allergic responses. To translate these findings into practical use, we formulated an allergen-neutralizing spray, FISTOQ, containing AYA22AR321, which effectively neutralized peanut allergens on peanut-butter-contaminated surfaces. Stability tests confirmed that FISTOQ, comprising eco-friendly surfactant and preservative, maintains its allergen-neutralizing efficacy over time. Comprehensive safety assessments, including immunogenicity, cytotoxicity in human PBMCs, and mutagenicity via the Ames test, demonstrated that AYA22AR321 is non-immunogenic, non-cytotoxic, and non-mutagenic. This study establishes AYA22AR321 as a promising, targeted strategy for allergen control, providing a significant advancement in allergen mitigation and food safety for high-risk environments.

Background: Thrombin is a central mediator of coagulation and platelet activation and a key anticoagulant target. Direct thrombin inhibitors broadly suppress thrombin proteolytic activity, potentially disrupting procoagulant and anticoagulant signaling. Exosite-targeting aptamers may enable selective modulation and programmable reversal. Methods: We characterize AYA1809002, a 40-nt DNA aptamer targeting thrombin Exosite I. Results: AYA1809002 bound thrombin with nanomolar affinity and inhibited fibrin formation with an IC₅₀ of 25.7 nM and K_i of 10.6 nM. Reduced $\gamma$-thrombin binding supported an Exosite I-dependent mechanism. AYA1809002 preserved thrombin activity toward small peptide substrates and thrombomodulin-dependent protein C activation. It showed no measurable activity against tested coagulation, anticoagulant, fibrinolytic, and control enzymes and inhibited thrombin-mediated platelet activation while preserving responses to non-thrombin agonists. AYA1809002 showed greater functional stability than thrombin-binding aptamer NU172 following serum exposure and remained active under thermal, pH, oxidative, and photostability stress. Anticoagulant activity was rapidly and sequence-specifically reversed by a reverse-complement oligonucleotide, while lipid conjugation preserved function. AYA1809002 also retained high-affinity binding to rat thrombin and anticoagulant activity in rat plasma. Conclusions: These findings identify AYA1809002 as a selective, stable, reversible Exosite I-targeting thrombin aptamer that suppresses key procoagulant functions while preserving thrombomodulin-dependent protein C activation.