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Nucleic acid nanoparticles, or NANPs, are rationally designed to communicate with the human immune system and can offer innovative therapeutic strategies to overcome the limitations of traditional nucleic acid therapies. Each set of NANPs is unique in their architectural parameters and physicochemical properties, which together with the type of delivery vehicles determine the kind and the magnitude of their immune response. Currently, there are no predictive tools that would reliably guide NANPs’ design to the desired immunological outcome, a step crucial for the success of personalized therapies. Through a systematic approach investigating physicochemical and immunological profiles of a comprehensive panel of various NANPs, our research team has developed a computational model based on the transformer architecture able to predict the immune activities of NANPs via construction of so-called artificial immune cell, or AI-cell. The AI-cell will aid addressing in timely manner the current critical public health challenges related to overdose and safety criteria of nucleic acid therapies and promote the development of novel biomedical tools.

Cancer is a complex systemic disorder that affects many organs and tissues and arises from the altered function of multiple cellular and molecular mechanisms. One of the systems malfunctioning in cancer is the immune system. Restoring and improving the ability of the immune system to effectively recognize and eradicate cancer is the main focus of immunotherapy, a topic which has garnered recent and significant interest. The initial excitement about immunotherapy, however, has been challenged by its limited efficacy in certain patient populations and the development of adverse effects such as therapeutic resistance and autoimmunity. At the same time, a number of advances in the field of nanotechnology have sought to address the challenges faced by modern immunotherapeutics and allow these therapeutic strategies to realize their full potential. This endeavour requires an understanding of not only the immunological barriers in cancer but also the mechanisms by which modern technologies and immunotherapeutics modulate the function of the immune system. Herein, we summarize the major barriers relevant to cancer immunotherapy and review current progress in addressing these obstacles using various approaches and clinically approved therapies. We then discuss the remaining challenges and how they can be addressed by nanotechnology. We lay out translational considerations relevant to the therapies described and propose a framework for the development of next-generation nanotechnology-enabled immunotherapies.

Nucleic acid–based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible ‘smart’ devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA–DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-021-03001-7.

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Infusion reactions (IRs) create a translational hurdle for many novel therapeutics, including those utilizing nanotechnology. Nucleic acid nanoparticles (NANPs) are a novel class of therapeutics prepared by rational design of relatively short oligonucleotides to self-assemble into various programmable geometric shapes. While cytokine storm, a common type of IR, has halted clinical development of several therapeutic oligonucleotides, NANP technologies hold tremendous potential to bring these reactions under control by tuning the particle's physicochemical properties to the desired type and magnitude of the immune response. Recently, we reported the very first comprehensive study of the structure⁻activity relationship between NANPs' shape, size, composition, and their immunorecognition in human cells, and identified the phagolysosomal pathway as the major route for the NANPs' uptake and subsequent immunostimulation. Here, we explore the molecular mechanism of NANPs' recognition by primary immune cells, and particularly the contributing role of the Toll-like receptors. Our current study expands the understanding of the immune recognition of engineered nucleic acid-based therapeutics and contributes to the improvement of the nanomedicine safety profile.

Here we report a "configuration-dependent" mechanism of action for IL-15:IL-15Rα (heterodimeric IL-15 or hetIL-15) where the manner by which IL-15:IL-15Rα molecules are presented to target cells significantly affects its function as a vaccine adjuvant. Although the cellular mechanism of IL-15 trans-presentation via IL-15Rα and its importance for IL-15 function have been described, the full effect of the IL-15:IL-15Rα configuration on responding cells is not yet known. We found that trans-presenting IL-15:IL-15Rα in a multivalent fashion on the surface of antigen-encapsulating nanoparticles enhanced the ability of nanoparticle-treated dendritic cells (DCs) to stimulate antigen-specific CD8(+) T cell responses. Localization of multivalent IL-15:IL-15Rα and encapsulated antigen to the same DC led to maximal T cell responses. Strikingly, DCs incubated with IL-15:IL-15Rα-coated nanoparticles displayed higher levels of functional IL-15 on the cell surface, implicating a mechanism for nanoparticle-mediated transfer of IL-15 to the DC surface. Using artificial antigen-presenting cells to highlight the effect of IL-15 configuration on DCs, we showed that artificial antigen-presenting cells presenting IL-15:IL-15Rα increased the sensitivity and magnitude of the T cell response, whereas IL-2 enhanced the T cell response only when delivered in a paracrine fashion. Therefore, the mode of cytokine presentation (configuration) is important for optimal immune responses. We tested the effect of configuration dependence in an aggressive model of murine melanoma and demonstrated significantly delayed tumor progression induced by IL-15:IL-15Rα-coated nanoparticles in comparison with monovalent IL-15:IL-15Rα. The novel mechanism of IL-15 transfer to the surface of antigen-processing DCs may explain the enhanced potency of IL-15:IL-15Rα-coated nanoparticles for antigen delivery.

In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential to understand the immunostimulatory aptitude of such tools and identify potential complications. This paper presents a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. It is demonstrated that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance the current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, quantitative structure-activity relationship modeling is conducted. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predicts the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.

Clinical translation of cell therapies requires strategies that can manufacture cells efficiently and economically. One promising way to reproducibly expand T cells for cancer therapy is by attaching the stimuli for T cells onto artificial substrates with high surface area. Here, we show that a carbon nanotube-polymer composite can act as an artificial antigen-presenting cell to efficiently expand the number of T cells isolated from mice. We attach antigens onto bundled carbon nanotubes and combined this complex with polymer nanoparticles containing magnetite and the T-cell growth factor interleukin-2 (IL-2). The number of T cells obtained was comparable to clinical standards using a thousand-fold less soluble IL-2. T cells obtained from this expansion were able to delay tumour growth in a murine model for melanoma. Our results show that this composite is a useful platform for generating large numbers of cytotoxic T cells for cancer immunotherapy.

Sandeep S Saluja,1 Douglas J Hanlon,1 Fiona A Sharp,2 Enping Hong,2 David Khalil,1Eve Robinson,1 Robert Tigelaar,1 Tarek M Fahmy,2,3 Richard L Edelson1 1Department of Dermatology, Yale University School of Medicine, 2Department of Biomedical Engineering, Yale University, 3Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Abstract: Targeting antigen to dendritic cells (DCs) is a powerful and novel strategy for vaccination. Priming or loading DCs with antigen controls whether subsequent immunity will develop and hence whether effective vaccination can be achieved. The goal of our present work was to increase the potency of DC-based antitumor vaccines by overcoming inherent limitations associated with antigen stability and cross-presentation. Nanoparticles prepared from the biodegradable polymer poly(lactic-co-glycolic acid) have been extensively used in clinical settings for drug delivery and are currently the subject of intensive investigation as antigen delivery vehicles for vaccine applications. Here we describe a nanoparticulate delivery system with the ability to simultaneously carry a high density of protein-based antigen while displaying a DC targeting ligand on its surface. Utilizing a targeting motif specific for the DC-associated surface ligand DEC-205, we show that targeted nanoparticles encapsulating a MART-127–35 peptide are both internalized and cross-presented with significantly higher efficiency than isotype control-coated nanoparticles in human cells. In addition, the DEC-205-labeled nanoparticles rapidly escape from the DC endosomal compartment and do not colocalize with markers of early (EEA-1) or late endosome/lysosome (LAMP-1). This indicates that encapsulated antigens delivered by nanoparticles may have direct access to the class I cytoplasmic major histocompatibility complex loading machinery, overcoming the need for “classical” cross-presentation and facilitating heightened DC stimulation of anti-tumor CD8+ T-cells. These results indicate that this delivery system provides a flexible and versatile methodology to deliver melanoma-associated antigen to DCs, with both high efficiency and heightened potency. Keywords: dendritic cells, DEC-205, PLGA nanoparticles, cross-presentation, melanoma-associated antigen