Relevance of controlled cooling and freezing phases in T-cell cryopreservation.

When cells are cryopreserved, they go through a freezing process with several distinct phases (i.e., cooling until nucleation, ice nucleation, ice crystal growth and cooling to a final temperature). Conventional cell freezing approaches often employ a single cooling rate to describe and optimize the entire freezing process, which neglects its complexity and does not provide insight into the effects of the different freezing phases. The aim of this work was to elucidate the impact of each freezing phase by varying different process parameters per phase. Hereto, spin freezing was used to freeze Jurkat T cells in either a Me2SO-based or Me2SO-free formulation. The cooling rates before ice nucleation and after total ice crystallization impacted cell viability, resulting in viability ranging from 26.7% to 52.8% for the Me2SO-free formulation, and 22.5%-42.6% for the Me2SO-based formulation. Interestingly, the degree of supercooling upon nucleation did not exhibit a significant effect on cell viability in this work. However, the rate of ice crystal formation emerged as a crucial factor, with viability ranging from 2.4% to 53.2% for the Me2SO-free formulation, and 0.3%-53.2% for the Me2SO-based formulation, depending on the freezing rate. A morphological study of the cells post-cryopreservation was performed using confocal microscopy, and it was found that cytoskeleton integrity and cell volume were impacted, depending on the formulation-process parameter combination. These findings underscore the importance of scrutinizing all cooling and freezing phases, as each phase impacted post-thaw viability in a distinct way, depending of the specific formulation used.

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in quadrivalent influenza vaccine vaccinated mice.

There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses in vivo, which is important for vaccine safety and to prevent adverse effects upon viral exposure.

Thermally Triggered Triazolinedione-Tyrosine Bioconjugation with Improved Chemo- and Site-Selectivity.

A bioconjugation strategy is reported that allows the derivatization of tyrosine side chains through triazolinedione-based "Y-clicking". Blocked triazolinedione reagents were developed that, in contrast to classical triazolinedione reagents, can be purified before use, can be stored for a long time, and allow functionalization with a wider range of cargoes and labels. These reagents are bench-stable at room temperature but steadily release highly reactive triazolinediones upon heating to 40 °C in buffered media at physiological pH, showing a sharp temperature response over the 0 to 40 °C range. This conceptually interesting strategy, which is complementary to existing photo- or electrochemical bioorthogonal bond-forming methods, not only avoids the classical synthesis and handling difficulties of these highly reactive click-like reagents but also markedly improves the selectivity profile of the tyrosine conjugation reaction itself. It avoids oxidative damage and "off-target" tryptophan labeling, and it even improves site-selectivity in discriminating between different tyrosine side chains on the same protein or different polypeptide chains. In this research article, we describe the stepwise development of these reagents, from their short and modular synthesis to small-molecule model bioconjugation studies and proof-of-principle bioorthogonal chemistry on peptides and proteins.

Thermosensitive polymer prodrug nanoparticles prepared by an all-aqueous nanoprecipitation process and application to combination therapy.

Despite their great versatility and ease of functionalization, most polymer-based nanocarriers intended for use in drug delivery often face serious limitations that can prevent their clinical translation, such as uncontrolled drug release and off-target toxicity, which mainly originate from the burst release phenomenon. In addition, residual solvents from the formulation process can induce toxicity, alter the physico-chemical and biological properties and can strongly impair further pharmaceutical development. To address these issues, we report polymer prodrug nanoparticles, which are prepared without organic solvents via an all-aqueous formulation process, and provide sustained drug release. This was achieved by the "drug-initiated" synthesis of well-defined copolymer prodrugs exhibiting a lower critical solution temperature (LCST) and based on the anticancer drug gemcitabine (Gem). After screening for different structural parameters, prodrugs based on amphiphilic diblock copolymers were formulated into stable nanoparticles by all-aqueous nanoprecipitation, with rather narrow particle size distribution and average diameters in the 50-80 nm range. They exhibited sustained Gem release in human serum and acetate buffer, rapid cellular uptake and significant cytotoxicity on A549 and Mia PaCa-2 cancer cells. We also demonstrated the versatility of this approach by formulating Gem-based polymer prodrug nanoparticles loaded with doxorubicin (Dox) for combination therapy. The dual-drug nanoparticles exhibited sustained release of Gem in human serum and acidic release of Dox under accelerated pathophysiological conditions. Importantly, they also induced a synergistic effect on triple-negative breast cancer line MDA-MB-231, which is a relevant cell line to this combination.

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in QIV vaccinated mice.

Adjuvants can enhance vaccine effectiveness of currently licensed influenza vaccines. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus derived defective interfering (SDI) RNA, a RIG-I agonist, and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), TLR7/8 adjuvant. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating the direct delivery of a RIG-I agonist to the cytosol. We have previously tested SDI and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated in a licensed vaccine setting (quadrivalent influenza vaccine or QIV) against H1N1 influenza virus, showing robust induction of antibody titres and T cell responses. Depending on the adjuvant combination and LNP lipid composition (K-Ac7-Dsa or S-Ac7-Dog lipids), humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with protection during viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was examined against challenge with the vaccine-matching strain of H1N1 influenza A virus. Groups that received either LNP formulated with SDI, IMDQ-PEG-Chol or both showed very low levels of viral replication in their lungs at five days post virus infection. LNP ionizable lipid composition as well as loading (empty versus SDI) also skewed host responses to infection, as reflected in the cytokine and chemokine levels in lungs of vaccinated animals upon infection. These studies show the potential of LNPs as adjuvant delivery vehicles for licensed vaccines and illustrate the importance of LNP composition for subsequent host responses to infection, an important point of consideration for vaccine safety.

Lipid Nanoparticle Encapsulation Empowers Poly(I:C) to Activate Cytoplasmic RLRs and Thereby Increases Its Adjuvanticity.

Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.

A preclinical platform for assessing long-term drug efficacy exploiting mechanically tunable scaffolds colonized by a three-dimensional tumor microenvironment.

BACKGROUND: Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. METHODS: In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. RESULTS: The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. CONCLUSIONS: The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Smart hydrogels delivered by high pressure aerosolization can prevent peritoneal adhesions.

Postoperative peritoneal adhesions occur in the majority of patients undergoing intra-abdominal surgery and are one of the leading causes of hospital re-admission. There is an unmet clinical need for effective anti-adhesive biomaterials, which can be applied evenly across the damaged tissues. We examined three different responsive hydrogel types, i.e. a thermosensitive PLGA-PEG-PLGA, a pH responsive UPy-PEG and a shear-thinning hexapeptide for this purpose. More specifically, their potential to be homogeneously distributed in the peritoneal cavity by high pressure nebulization and prevent peritoneal adhesions was evaluated. Solutions of each polymer type could be successfully nebulized while retaining their responsive gelation behavior in vitro and in vivo. Furthermore, none of the polymers caused in vitro toxicity on SKOV3-IP2 cells. Following intraperitoneal administration, both the PLGA-PEG-PLGA and the hexapeptide hydrogels resulted in local inflammation and fibrosis and failed in preventing peritoneal adhesions 7 days after adhesion induction. In contrast, the pH sensitive UPy-PEG formulation was well tolerated and could significantly reduce the formation of peritoneal adhesions, even outperforming the commercially available Hyalobarrier® as positive control. To conclude, local nebulization of the bioresponsive UPy-PEG hydrogel can be considered as a promising approach to prevent postsurgical peritoneal adhesions.

Influenza breakthrough infection in vaccinated mice is characterized by non-pathological lung eosinophilia.

Eosinophils are important mediators of mucosal tissue homeostasis, anti-helminth responses, and allergy. Lung eosinophilia has previously been linked to aberrant Type 2-skewed T cell responses to respiratory viral infection and may also be a consequence of vaccine-associated enhanced respiratory disease (VAERD), particularly in the case of respiratory syncytial virus (RSV) and the formalin-inactivated RSV vaccine. We previously reported a dose-dependent recruitment of eosinophils to the lungs of mice vaccinated with alum-adjuvanted trivalent inactivated influenza vaccine (TIV) following a sublethal, vaccine-matched H1N1 (A/New Caledonia/20/1999; NC99) influenza challenge. Given the differential role of eosinophil subset on immune function, we conducted the investigations herein to phenotype the lung eosinophils observed in our model of influenza breakthrough infection. Here, we demonstrate that eosinophil influx into the lungs of vaccinated mice is adjuvant- and sex-independent, and only present after vaccine-matched sublethal influenza challenge but not in mock-challenged mice. Furthermore, vaccinated and challenged mice had a compositional shift towards more inflammatory eosinophils (iEos) compared to resident eosinophils (rEos), resembling the shift observed in ovalbumin (OVA)-sensitized allergic control mice, however without any evidence of enhanced morbidity or aberrant inflammation in lung cytokine/chemokine signatures. Furthermore, we saw a lung eosinophil influx in the context of a vaccine-mismatched challenge. Additional layers of heterogeneity in the eosinophil compartment were observed via unsupervised clustering analysis of flow cytometry data. Our collective findings are a starting point for more in-depth phenotypic and functional characterization of lung eosinophil subsets in the context of vaccine- and infection-induced immunity.

Hapten/Myristoyl Functionalized Poly(propyleneimine) Dendrimers as Potent Cell Surface Recruiters of Antibodies for Mediating Innate Immune Killing.

Recruiting endogenous antibodies to the surface of cancer cells using antibody-recruiting molecules has the potential to unleash innate immune effector killing mechanisms against antibody-bound cancer cells. The affinity of endogenous antibodies is relatively low, and many currently explored antibody-recruiting strategies rely on targeting over-expressed receptors, which have not yet been identified in most solid tumors. Here, both challenges are addressed by functionalizing poly(propyleneimine) (PPI) dendrimers with both multiple dinitrophenyl (DNP) motifs, as anti-hapten antibody-recruiting motifs, and myristoyl motifs, as universal phospholipid cell membrane anchoring motifs, to recruit anti-hapten antibodies to cell surfaces. By exploiting the multivalency of the ligand exposure on the dendrimer scaffold, it is demonstrated that dendrimers featuring ten myristoyl and six DNP motifs exhibit the highest antibody-recruiting capacity in vitro. Furthermore, it is shown that treating cancer cells with these dendrimers in vitro marks them for phagocytosis by macrophages in the presence of anti-hapten antibodies. As a proof-of-concept, it is shown that intratumoral injection of these dendrimers in vivo in tumor-bearing mice results in the recruitment of anti-DNP antibodies to the cell surface in the tumor microenvironment. These findings highlight the potential of dendrimers as a promising class of novel antibody-recruiting molecules for use in cancer immunotherapy.

Sequence-defined antibody-recruiting macromolecules.

Antibody-recruiting molecules represent a novel class of therapeutic agents that mediate the recruitment of endogenous antibodies to target cells, leading to their elimination by the immune system. Compared to single-ligand copies, macromolecular scaffolds presenting multiple copies of an antibody-binding ligand offer advantages in terms of increased complex avidity. In this study, we describe the synthesis of sequence-defined macromolecules designed for antibody recruitment, utilising dinitrophenol (DNP) as a model antibody-recruiting motif. The use of discrete macromolecules gives access to varying the spacing between DNP motifs while maintaining the same chain length. This characteristic enables the investigation of structure-dependent binding interactions with anti-DNP antibodies. Through solid-phase thiolactone chemistry, we synthesised a series of oligomers with precisely localised DNP motifs along the backbone and a terminal biotin motif for surface immobilisation. Utilising biolayer interferometry analysis, we observed that oligomers with adjacent DNP motifs exhibited enhanced avidity for anti-DNP antibodies. Molecular modelling provided insights into the structures and dynamics of the various macromolecules, shedding light on the accessibility of the ligands to the antibodies. Overall, our findings highlight that the use of sequence-defined macromolecules can contribute to our understanding of structure-activity relationships and provide insights for the design of novel antibody-recruiting therapeutic agents.

LXR signaling controls homeostatic dendritic cell maturation.

Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell-mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

Successful batch and continuous lyophilization of mRNA LNP formulations depend on cryoprotectants and ionizable lipids.

The limited thermostability and need for ultracold storage conditions are the major drawbacks of the currently used nucleoside-modified lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccines, which hamper the distribution of these vaccines in low-resource regions. The LNP core contains, besides mRNA and lipids, a large fraction of water. Therefore, encapsulated mRNA, or at least a part of it, is subjected to hydrolysis mechanisms similar to unformulated mRNA in an aqueous solution. It is likely that the hydrolysis of mRNA and colloidal destabilization are critical factors that decrease the biological activity of mRNA LNPs upon storage under ambient conditions. Hence, lyophilization as a drying technique is a logical and appealing method to improve the thermostability of these vaccines. In this study, we demonstrate that mRNA LNP formulations comprising a reduction-sensitive ionizable lipid can be successfully lyophilized, in the presence of 20% w/v sucrose, both by conventional batch freeze-drying and by an innovative continuous spin lyophilization process. While the chemical structure of the ionizable lipid did not affect the colloidal stability of the LNP after lyophilization and redispersion in an aqueous medium, we found that the ability of LNPs to retain the mRNA payload stably encapsulated, and mediate in vivo and in vitro mRNA translation into protein, post lyophilization strongly depended on the ionizable lipid in the LNP formulation.

Towards a Rational Design of Antibody-Recruiting Molecules through a Computational Microscopy View of their Interactions with the Target Antibody.

Antibody recruiting molecules (ARMs) are an innovative class of chimeric molecules, consisting of an antibody-binding ligand (ABL) and a target-binding ligand (TBL). ARMs mediate ternary complex formation between a target cell of interest for elimination and endogenous antibodies that are present in human serum. Clustering of fragment crystallizable (Fc) domains on the surface of antibody-bound cells mediate destruction of the target cell by innate immune effector mechanisms. ARMs are typically designed by conjugating small molecule haptens to a (macro)molecular scaffold, without considering the structure of the respective anti-hapten antibody. Here we report on a computational molecular modeling methodology that allows for studying the close contacts between ARMs and the anti-hapten antibody, considering (1) the spacer length between ABL and TBL; (2) the number of ABL and TBL, and (3) the molecular scaffold onto which these are positioned. Our model predicts the difference in binding modes of the ternary complex and predicts which ARMs are optimal recruiters. Avidity measurements of the ARM-antibody complex and ARM-mediated antibody recruitment to cell surfaces in vitro confirmed these computational modeling predictions. This kind of multiscale molecular modelling holds potential for design of drug molecules that rely on antibody binding for their mechanism of action.

Visible Light Conjugation with Triazolinediones as a Route to Degradable Poly(ethylene glycol)-Lipids for mRNA Lipid Nanoparticle Formulation.

Polyethylene glycol (PEG) is considered as the gold standard for colloidal stabilization of nanomedicines, yet PEG is non-degradable and lacks functionality on the backbone. Herein, we introduce concomitantly PEG backbone functionality and degradability via a one-step modification with 1,2,4-triazoline-3,5-diones (TAD) under green light. The TAD-PEG conjugates are degradable in aqueous medium under physiological conditions, with the rate of hydrolysis depending on pH and temperature. Subsequently, a PEG-lipid is modified with TAD-derivatives and successfully used for messenger RNA (mRNA) lipid nanoparticle (LNP) delivery, thereby improving mRNA transfection efficiency on multiple cell cultures in vitro. In vivo, in mice, mRNA LNP formulation exhibited a similar tissue distribution as common LNPs, with a slight decrease in transfection efficiency. Our findings pave the road towards the design of degradable, backbone-functionalized PEG for applications in nanomedicine and beyond.

Impact of the polymer backbone chemistry on interactions of amino-acid-derived zwitterionic polymers with cells.

Zwitterionic polymers are known to interact with cells and have been shown to reveal cancer cell specificity. In this work, the importance of the chemistry of the polymer backbone for the cellular specificity of amino-acid-derived polyzwitterions is demonstrated. A series of glutamic acid (Glu)-based vinyl monomers (i.e., an acrylate, a methacrylate, an acrylamide, and a methacrylamide) were prepared and used for reversible addition-fragmentation chain-transfer (RAFT) polymerisation, yielding defined polymers with narrow size distribution (Р< 1.3). All Glu-functionalised, zwitterionic polymers revealed high cytocompatibility; however, differences in cellular association and specificity were observed. In particular, the methacrylamide-derived polymers showed high association with both, breast cancer cells and non-cancerous dendritic cells and, consequently, lack specificity. In contrast, high specificity to only breast cancer cells was observed for polyacrylates, -methacrylates, and -acrylamides. Detailed analysis of the polymers revealed differences in hydrophobicity, zeta potential, and potential side chain hydrolysis, which are impacted by the polymer backbone and might be responsible for the altered the cell association of these polymers. It is shown that a slightly negative net charge is preferred over a neutral charge to retain cell specificity. This was also confirmed by association experiments in the presence of competitive amino acid transporter substrates. The affinity of slightly negatively charged Glu-derived polymers to the xCT Glu/cystine cell membrane antiporter was found to be higher than that of neutrally charged polymers. Our results emphasise the importance of the polymer backbone for the design of cell-specific polymers. This study further highlights the potential to tailor amino-acid-derived zwitterionic materials beyond their side chain functionality.

Challenges in neoantigen-directed therapeutics.

A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against tumor-specific antigens, thereby eradicating cancer cells. Initial forays have been made in clinical environments utilizing vaccines and adoptive cell therapy; however, many challenges lie ahead. We provide an in-depth overview of the current state of the field with an emphasis on in silico neoantigen discovery and the clinical aspects that need to be addressed to unlock the full potential of this therapy.

Tumor-Selective Activation of Toll-Like Receptor 7/8 Agonist Nano-Immunomodulator Generates Safe Anti-Tumor Immune Responses upon Systemic Administration.

Agonists of innate pattern recognition receptors such as toll-like receptors (TLRs) prime adaptive anti-tumor immunity and hold promise for cancer immunotherapy. However, small-molecule TLR agonists cause immune-related adverse effects (irAEs) after systemic administration. Herein, we report a polymeric nano-immunomodulator (cN@SS-IMQ) that is inactive until it is selectively metabolized to an active immunostimulant within the tumor. cN@SS-IMQ was obtained via self-assembly of a cyclo(Arg-Gly-Asp-D-Phe-Lys)-modified amphiphilic copolymeric prodrug. Upon systemic administration, cN@SS-IMQ preferentially accumulated at tumor sites and responded to high intracellular glutathione levels to release native imidazoquinolines for dendritic cell maturation, thereby enhancing the infiltration of T lymphocytes. Collectively, cN@SS-IMQ tends to activate the immune system without irAEs, thus suggesting its promising potential for safe systemic targeting delivery.

RIG-I and TLR-7/8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine.

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)-PEG-Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant. Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centers during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.