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CD33 and FLT3 are validated targets for myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). These malignancies have poor prognosis and high clinical unmet need, and current targeted therapies present ample limitations. One challenge is that untargeted leukemic stem cells (LSCs) can contribute to eventual relapse; however, targeting the LSC markers FLT3 and CD33 can contribute to off-tumor toxicities against normal hematopoietic stem cells and progenitor cells (HSCs/HPCs), leading to bone marrow toxicities such as thrombocytopenia, neutropenia, and infectious complications. SENTI-202 is designed to address these challenges and provide a broader therapeutic window. SENTI-202 is a preclinical CAR-NK cell product candidate engineered with an OR and NOT logic gated gene circuit onto allogeneic healthy adult peripheral blood NK cells along with multi-arming via expression of calibrated release IL-15 (crIL-15) to enhance therapeutic activity and tolerability. The OR gate is implemented using a bivalent CAR (aCAR) that binds both CD33 and FLT3 to target both AML blasts and LSCs. Additionally, the NOT gate is implemented using an inhibitory CAR (iCAR) that recognizes the healthy cell protective antigen endomucin (EMCN) and targets endomucin which is designed to prevent CAR-mediated cytotoxicity against healthy cells, such as HSCs and early HPCs, potentially reducing on-target/off-tumor toxicities. Finally, crIL-15 provides NK cell activation and persistence via simultaneous autocrine, juxtracrine, and paracrine activities. Preclinical studies demonstrated the [antitumor] activity and tolerability of SENTI-202. All SENTI-202 components are stably expressed via a single g-retroviral vector. SENTI-202 preclinical pharmacokinetics and secondary pharmacodynamics demonstrated CAR target engagement, function, as well as mechanism of action for all components in different AML models. SENTI-202 CAR-NK cells demonstrated cytotoxic activity when co-cultured with AML target cells and primary patient-derived samples and had increased antitumor capacity compared to non-engineered NK cells. SENTI-202 induced the production of cytotoxic cytokines and increased expression of activation and cytotoxicity markers consistent with CAR-mediated activation of the NK cells. The presence of EMCN iCAR reduced cytotoxicity in an EMCN-dependent manner in target cells and primary HSCs. crIL-15 expression yielded functional pSTAT5 signaling and increased persistence of SENTI-202 in vitro and in vivo. These results demonstrate the antitumor activity and tolerability of SENTI-202 and warrant further clinical development as a novel therapeutic option for patients with CD33+ and/or FLT3+ tumors. Citation Format: Alba Gonzalez, Enping Hong, Gozde Yucel, Elizabeth Leitner, Pearley Chinta, Han Deng, Ian Li, Alice Lam, Abla Bakir, Brandon Lee, Papia Chakraborty, Carmina Blanco, Chen-Ting Lee, Niran Almudhfar, Mengxi Tian, Wenqi Song, Andrew Banicki, Otto Contreras, Martin Gieldin, Brian Garrison, Timothy K. Lu, Kanya Rajangam. Preclinical development of SENTI-202, an off-the-shelf logic gated CAR-NK cell therapy, for the treatment of CD33/FLT3+ hematologic malignancies including AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3195.

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.

Background Agonist antibodies and recombinant cytokines have had limited success in the clinic due to three factors: severe toxicity leading to a narrow therapeutic index, the diminished activity of an agonistic antibody compared with natural ligand, and the lack of multiple signals needed to effectively activate most cell types. To address these limitations, Rubius Therapeutics has developed RTX-240, an allogeneic cellular therapy using red blood cells genetically engineered to express 4-1BBL and IL-15/IL-15Ra fusion (IL-15TP) in their natural conformation on the cell surface. RTX-240 is designed to recapitulate human biology by broadly stimulating adaptive and innate immunity to generate an anti-tumor response and provide improved safety due to the restricted biodistribution of red blood cells to the vasculature. Here we demonstrate that RTX-240 is highly active in preclinical models. Methods PBMCs or NK cells were treated with RTX-240 in vitro. mRBC-240 was used for in vivo studies. Results Treatment of either PBMCs or isolated NK cells with RTX-240 induced a dose-dependent increase in NK cell activation, proliferation and functionality. These effects were further enhanced with increased 4-1BBL and IL-15TP expression on the surface of RTX-240. NK cell counts, NKp44 and Trail expression were increased 150, 4.6 and 6-fold over media control, respectively. Activation of NK cells with RTX-240, followed by incubation with K562 targets enhanced NK cell cytotoxicity (1.3-2.8 over control), that was accompanied by increased NK cell activation (CD69) and degranulation (CD107a) (3.1-fold and 1.9-fold, respectively). RTX-240-activated NK cells showed higher frequency of CD56dim/CD16+ NK cells, which have been reported to induce natural and ADCC-dependent cytotoxicity. Correspondingly, RTX-240 promoted enhanced ADCC-induced killing of Raji cells when combined with anti-CD20 mAb (1.4-fold over control). Intravenous administration of mRBC-240 to a B16F10 intravenous lung metastases model led to NK cell expansion on Day 4 (3.8-fold over control). These NK cells were cytotoxic (Granzyme B+) and highly proliferative (Ki67+) (1.4-fold and 18.8-fold over control, respectively). Treatment with mRBC-240 increased the frequency of terminally differentiated NK cells (NK1.1+/CD11b+/CD27-/KLRG1+) in the tumor (2.1-fold increase over control). Terminally differentiated NK cells are highly cytotoxic and their frequency in the tumor was strongly correlated with efficacy in this model (p=0.0001). Conclusions Taken together, these data indicate that RTX-240 promotes NK cell activity and functionality in preclinical models. RTX-240 has now entered a first-in-human Phase 1 trial for the treatment of patients with relapsed/refractory or locally advanced solid tumors, with a planned arm evaluating RTX-240 in relapsed/refractory acute myeloid leukemia.

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.

BackgroundTLR agonists can elicit anti-tumor activity by activating innate immune cells and promoting a proinflammatory microenvironment. Local delivery of TLR agonists has shown encouraging preclinical and clinical anti-tumor activity. However, intratumoral (IT) delivery of naked TLR agonists such as resiquimod, a TLR7/8 agonist, can lead to rapid efflux from the tumor, resulting in acute high systemic drug exposure and transient but high level of peripheral proinflammatory cytokines, thus limiting anti-tumor benefit and increasing risk of cytokine-driven adverse effects.MethodsTransCon™ TLR7/8 Agonist was designed to elicit a sustained and local release of resiquimod following IT administration of a hydrogel depot. In the syngeneic murine CT26 tumor model, a single IT injection of TransCon TLR7/8 Agonist monotherapy was sufficient to induce potent tumor growth inhibition. Following treatment, the induction of key cytokines and chemokines associated with innate immunity was determined.ResultsProinflammatory cytokines (IL-1b, IL-6, and TNFα) were induced following IT TransCon TLR7/8 Agonist treatment, but in contrast to free resiquimod, peak levels were more than 10-fold lower than those observed with an equimolar dose of free resiquimod. The circulating levels of these cytokines were sustained above control alone through Day 21. TH1-associated IFNγ was induced with levels increased at Day 1 and maintained at Day 7. Additionally, expression of myeloid-associated chemokines (KC/GROa/CXCL1, MCP-1/CCL2, IP-10/CXCL10, and MIP-1a/CCL3) were induced and sustained in a largely dose-dependent manner through Day 21. The sustained increase in cytokines was consistent with an increase in circulating innate immune cells, such as NK and myeloid cells. Furthermore, evidence of adaptive immune cell activation was observed as indicated by expression of Ly6C, ICOS and Ki67, which were increased on CD8+ T cells, CD4+ T cells (Ki67, ICOS), and B cells (Ly6C).ConclusionsThese data show that a single IT injection of TransCon TLR7/8 Agonist can elicit sustained expression of key cytokines and chemokines, promote innate immune cell mobilization, activate adaptive immune cells, and mediate robust anti-tumor activity. The levels of the cytokines remained relatively low through the observation period of 21 days, suggesting a low risk of systemic cytokine-associated adverse events. The increase in activated B, T, and NK cells in blood was associated with induction of a potent anti-tumor response, further supporting TransCon TLR7/8 Agonist as a novel and potentially efficacious PRRA therapy. A clinical trial to evaluate its safety and efficacy in cancer patients is currently underway (transcendIT-101; NCT04799054).Ethics ApprovalThe animal studies described were performed in accordance with the ‘Guide for the Care and Use of Laboratory Animals: Eighth Edition’ and approved by the institutional animal care and use committee (IACUC).

Recombinant IL-12 is a potent cytokine that has held significant promise as an immunotherapeutic. In preclinical studies, recombinant IL-12 has impressive anti-tumor activity; however toxicity and a narrow therapeutic window halted development in humans. To address these limitations, Rubius Therapeutics has developed genetically engineered red cell therapeutics (RCTs) with cell surface expression of IL-12 alone (RTX-IL-12) or in combination with co-stimulatory or other cytokine molecules. These cells present ligands in their native form that potently activate and expand both T and NK cells through potent cell-cell interactions. On-target, off-tissue toxicity of RCTs may be limited due to their biodistribution, which is restricted to the vascular system. Based on its reported role in promoting a TH1 response and proliferation of cytotoxic NK and CD8 T cells, RTX-IL-12 activity was evaluated in vitro and demonstrated proliferation of human CD8 T cells (2-3 fold), Th1 differentiation of naïve CD4 T cells, IFNγproduction (6-11 fold increase over control), as well as NK cell activation and cytotoxicity against K562 targets (2-5 fold increase over control). To evaluate in vivoimmune response, anti-tumor activity and safety, a mouse surrogate red cell therapeutic, mRBC-IL-12, was developed by chemically conjugating recombinant Fc-IL-12 to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Administration of mRBC-IL-12 (1X109cells) to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 52% decrease in the number of lung metastases and was associated with increased proliferating and cytotoxic CD8 and NK cells in the lungs (p=0.0002). Administration of mRBC-IL-12 (1X109cells) to mice bearing B16F10 and MC38 subcutaneous models exhibited 82% and 80% tumor growth inhibition, respectively. When combined with anti-PD1 treatment, mRBC-IL-12 (1X109cells) efficacy was improved in these two models and showed 86% and 85% tumor growth inhibition, respectively. Efficacy was accompanied by an increase in survival (p=0.0004 B16F10, p=0.0003 MC38 ) and the infiltration of M1 macrophages (p=0.007) into the tumor. Mice treated with the highest feasible dose of mRBC-IL-12 displayed no significant body weight loss and 3-10-fold lower serum IFNg levels compared to soluble rec. Fc-IL-12. By combining IL-12 with IL-15TP or 4-1BBL on the same red cell, tumor growth was strongly inhibited and in some cases improved over mRBC-IL-12 alone (TGI 46% and 63% in B16F10 SC, TGI 83%, 77% in MC38 and 78%, 70% in B16F10 IV). In summary, the data demonstrate that sequestering IL-12 in the vasculature through expression on red cells drives significant reductions in tumor growth, while improving the tolerability profile of the cytokine,supporting further testing in cancer patients. Citation Format: Anne-Sophie Dugast, Enping Hong, Maegan Hoover, Arjun Bollampalli, Douglas C. McLaughlin, Omkar Bhate, Timothy J. Lyford, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. RTX-IL-12, an allogeneic red cell therapeutic expressing IL-12, exhibits potent in vitro and in vivo activity and favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3256.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical activity perhaps due to 1) toxicity; 2) a need for coordinated activation of co-stimulatory and cytokine pathways; or 3) the failure of agonist antibodies to recapitulate signaling by endogenous ligands. To address these limitations, Rubius Therapeutics developed genetically engineered red cells with cell surface expression of both co-stimulatory and cytokine ligands, which present ligands in their native form through cell-cell contact to potently activate and expand both T and NK cells. On-target, off-tissue toxicity may be limited due their biodistribution, which is restricted to the vascular system. IL-15 is known to promote NK survival and CD8 T cell memory and 4-1BB agonists are known to promote T cell prolioferation and survival. Rubius Therapeutics has developed RTX-212, an allogeneic red cell therapeutic genetically engineered to co-express 4-1BBL and an IL-15/IL-15Rαfusion (IL-15TP). In vitro assays demonstrated that the combination of both ligands on RTX-212 expanded both memory CD8 T cells (2.5-fold) and NK cells (10-15-fold), in the absence of TCR stimulation. RTX-212 further induced dramatic proliferation of CD8 T cells and CD4 T cells in the presence of TCR stimulation with increased IFNγsecretion. In addition to the synergistic effects of 4-1BBL and IL-15TP, each molecule provided complementary functions that expanded the activity of RTX-212 beyond RTX-4-1BBL or RTX-IL-15TP alone. IL-15TP uniquely activated NK cytotoxicity and ADCC, while 4-1BBL uniquely stimulated CD4 and CD8 T cell proliferation and production of IFNγ. To evaluate in vivo immune responses, anti-tumor activity and safety, a mouse surrogate therapeutic, mRBC-212, was developed where recombinant Fc-IL-15-sushi and m4-1BBL were chemically conjugated to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Intravanous administration of mRBC-212 to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 66% decrease in the number of lung metastases compared to control mice (p=0.0001) and was associated with a significant increase in NK cell infiltration into the lungs (p=0.02). In a CT26 tumor model, mRBC-212 treated mice exhibited 55% tumor growth inhibition, which was accompanied by a 1.7-fold increase in the tumor infiltration of proliferating and cytotoxic CD8 T cells. Mice treated with the highest feasible dose of mRBC-212 showed no change in serum transaminases, infiltration of CD8 T cells and macrophages to the liver or liver inflammation score compared to agonistic 4-1BB antibodies treated mice. Taken together, these data indicate that RTX-212 has the potential to be an effective therapy with an improved safety profile compared to 4-1BB agonist antibodies and IL-15 agonists, supporting its clinical development. Citation Format: Anne-Sophie Dugast, Shannon McArdel, Maegan Hoover, Enping Hong, Shannon Curtis Leonard, Arjun Bollampalli, Douglas C. McLaughlin, Jennifer Mellen, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. RTX-212, an allogeneic red cell therapeutic expressing 4-1BBL and IL-15TP, exhibits potent in vitro and in vivo activity and a favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3272.

Extracorporeal Photochemotherapy (ECP) is a widely applied anti-cancer immunotherapy for patients with cutaneous T cell lymphoma (CTCL). By using apoptotic malignant cells as a source of patient-specific tumor antigen, it enables clinically relevant and curative anti-CTCL immunity, with potential efficacy in other tumors. Currentmethods to track patient-specific responses are tedious, and new methods are needed to assess putative global immunity. We developed a clinically practical method to assess antigen-specific T cell activation that does not rely on knowledge of the particular antigen, thereby eliminating the requirement for patient-specific reagents. In the OT-I transgenic murine system, we quantified calcium flux to reveal early T cell engagement by antigen presenting cells constitutively displaying a model antigenic peptide, ovalbumin (OVA)-derived SIINFEKL. We detected calcium flux in OVA-specific T cells, triggered by specific T cell receptor engagement by SIINFEKL peptide-loaded DC. This approach led to sensitive detection of antigen-specific calcium flux (ACF) down to a peptide-loading concentration of ∼10-3uM and at a frequency of ∼0.1% OT-I cells among wild-type (WT), non-responding cells. Antigen-specific T cells were detected in spleen, lymph nodes, and peripheral blood after adoptive transfer into control recipient mice. Methods like this for assessing therapeutic response are lacking in patients currently on immune-based therapies, such as ECP, where assessment of clinical response is made by delayed measurement of the size of the malignant clone. These findings suggest an early, practical way to measure therapeutically-induced anti-tumor responses in ECP-treated patients that have been immunized against their malignant cells.

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.

Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.

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

Nature Nanotechnology 9, 639–647 (2014); published online 3 August 2014; corrected after print 14 August 2014. In the version of this Article previously published, the name of the co-author Kevan C. Herold was spelt incorrectly. This has now been corrected in the online versions of the Article.