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Appel, E.

Publications and source records attributed to Appel, E..

29 records · Page 2Linked to original sources

A regimen compression strategy for commercial vaccines leveraging an injectable hydrogel depot technology for sustained vaccine exposure

Equitable global access to vaccines requires we overcome challenges associated with complex immunization schedules and their associated economic burdens that hinder delivery in under resourced environments. The rabies vaccine, for example, requires multiple immunizations for effective protection and each dose is cost prohibitive, and therefore inaccessibility disproportionately impacts low- and middle-income countries. In this work we developed an injectable hydrogel depot technology for sustained delivery of commercial inactivated rabies virus vaccines. In a mouse model, we showed that a single immunization of a hydrogel-based rabies vaccine elicited comparable antibody titers to a standard prime-boost bolus regimen of a commercial rabies vaccine, despite these hydrogel vaccines comprising only half of the total dose delivered in the bolus control. Moreover, these hydrogel-based vaccines elicited similar antigen-specific T-cell responses and neutralizing antibody responses compared to the bolus vaccine. Notably, we demonstrated that while addition of a potent clinical TLR4 agonist adjuvant to the gels slightly improved binding antibody responses, inclusion of this adjuvant to the inactivated virion vaccine was detrimental to neutralizing responses. Taken together, these results suggest that these hydrogels can enable an effective regimen compression and dosesparing strategy for improving global access to vaccines.

bioengineering↗

Nanoparticle-Conjugated TLR9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines

Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and timescale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is critical need for new adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures, but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improve the magnitude and duration of antibody responses when compared to soluble CpG, and result in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhance the spatiotemporal control over co-delivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates comparable humoral responses as a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.

immunology↗

Broad and Durable Humoral Responses Following Single Hydrogel Immunization of SARS-CoV-2 Subunit Vaccine

Most vaccines require several immunizations to induce robust immunity, and indeed, most SARS-CoV-2 vaccines require an initial two-shot regimen followed by several boosters to maintain efficacy. Such a complex series of immunizations unfortunately increases the cost and complexity of populations-scale vaccination and reduces overall compliance and vaccination rate. In a rapidly evolving pandemic affected by the spread of immune-escaping variants, there is an urgent need to develop vaccines capable of providing robust and durable immunity. In this work, we developed a single immunization SARS-CoV-2 subunit vaccine that could rapidly generate potent, broad, and durable humoral immunity. We leveraged injectable polymer-nanoparticle (PNP) hydrogels as a depot technology for the sustained delivery of a nanoparticle COVID antigen displaying multiple copies of the SARS-CoV-2 receptor-binding-domain (RBD-NP), and potent adjuvants including CpG and 3M-052. Compared to a clinically relevant prime-boost regimen with soluble vaccines formulated with CpG/Alum or 3M-052/Alum adjuvants, PNP hydrogel vaccines more rapidly generated higher, broader, and more durable antibody responses. Additionally, these single-immunization hydrogel-based vaccines elicited potent and consistent neutralizing responses. Overall, we show that PNP hydrogels elicit improved anti-COVID immune responses with only a single administration, demonstrating their potential as critical technologies to enhance our overall pandemic readiness.

bioengineering↗

Injectable polymer-nanoparticle hydrogel for the sustained intravitreal delivery of bimatoprost

Vision impairment resulting from chronic eye diseases, such as macular degeneration and glaucoma, severely impacts patients quality of life and poses an immense global financial burden. Current standard of care for such diseases includes daily eye drops or frequent intravitreal (ITV) injections, which are burdensome treatment modalities resulting in low patient compliance. There remains a growing need for easily administered long-acting delivery technologies for prolonging exposure of ocular therapeutics with each administration. Here, we deploy a supramolecular polymer-nanoparticle (PNP) hydrogel for ITV delivery of the glaucoma drug bimatoprost. PNP hydrogels are shear-thinning and self-healing, key properties for injectability, and enable slow release of molecular cargo in vitreous humor (VH) mimics. An in vivo study in New Zealand white rabbits demonstrated intravitreally injected PNP hydrogels form depots that degrade slowly over time, maintaining detectable levels of bimatoprost in the VH up to eight weeks following injection. Ophthalmic examinations and histopathology identified a mild foreign body response (FBR) to the hydrogel, characterized by rare clusters of foamy macrophages and giant cells associated with minimal, patchy fibroplasia. This work shows that PNP hydrogels exhibit numerous desirable traits for sustained drug delivery and further work will be necessary to optimize tolerability in the eye.

bioengineering↗

Stable High-Concentration Monoclonal Antibody Formulations Enabled by an Amphiphilic Copolymer Excipient

Monoclonal antibodies are a staple in modern pharmacotherapy. Unfortunately, these biopharmaceuticals are limited by their tendency to aggregate in formulation, resulting in poor stability and often requiring low concentration drug formulations. Moreover, existing excipients designed to stabilize these formulations are often limited by their toxicity and tendency to form particles such as micelles. Here, we demonstrate the ability of a simple "drop-in", amphiphilic copolymer excipient to enhance the stability of high concentration formulations of clinically-relevant monoclonal antibodies without altering their pharmacokinetics or injectability. Through interfacial rheology and surface tension measurements, we demonstrate that the copolymer excipient competitively adsorbs to formulation interfaces. Further, through determination of monomeric composition and retained bioactivity through stressed aging, we show that this excipient confers a significant stability benefit to high concentration antibody formulations. Finally, we demonstrate that the excipient behaves as an inactive ingredient, having no significant impact on the pharmacokinetic profile of a clinically relevant antibody in mice. This amphiphilic copolymer excipient demonstrates promise as a simple formulation additive to create stable, high concentration antibody formulations, thereby enabling improved treatment options such as a route-of-administration switch from low concentration intravenous (IV) to high concentration subcutaneous (SC) delivery while reducing dependence on the cold chain.

bioengineering↗

Delivery of CAR-T Cells in a Transient Injectable Stimulatory Hydrogel Niche Improves Treatment of Solid Tumors

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers such as B cell malignancies, but traditional approaches to ACT are poorly effective in treating the multifarious solid tumors observed clinically. Locoregional cell delivery methods have shown promising results in treating solid tumors compared to standard intravenous delivery methods, but the approaches that have been described to date have several critical drawbacks ranging from complex manufacturing and poor modularity to challenging adminstration. In this work, we develop a simple-to-implement self-assembled and injectable hydrogel material for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improves treatment of solid tumors. We evaluate a range of hydrogel formulations to optimize the creation of a transient inflammatory niche that affords sustained exposure of CAR-T cells and cytokines. This facile approach yields increased CAR-T cell expansion, induces a more tumor-reactive CAR-T phenotype, and improves efficacy in treating solid tumors in mice.

bioengineering↗

Injectable Liposome Based Supramolecular Hydrogels for the Programmable Release of Multiple Protein Drugs

Directing and manipulating biological functions is at the heart of next-generation biomedical initiatives such as tissue and immuno-engineering. Yet, the ambitious goal of engineering complex biological networks requires the ability to precisely perturb specific signaling pathways at distinct times and places. Using lipid nanotechnology and the principles of supramolecular self-assembly, we have developed an injectable liposomal nanocomposite hydrogel platform to precisely control drug presentation through programing of the co-release of multiple protein drugs. These liposomal hydrogels exhibited robust shear-thinning and self-healing behaviors enabling facile injectability for local drug delivery applications. By integrating modular lipid nanotechnology into this hydrogel platform, we introduced multiple mechanisms of protein release based on liposome surface chemistry. When injected into immuno-competent mice, these liposomal hydrogels exhibited formulation-dependent rates of dissolution and excellent biocompatibility. To fully validate the utility of this system for multi-protein delivery, we demonstrated the synchronized, sustained, and localized release of IgG antibody and IL-12 cytokine in vivo, despite the significant size differences between these two proteins. Overall, these liposomal nanocomposite hydrogels are a highly modular platform technology with the ability the mediate orthogonal modes of protein release and the potential to precisely coordinate biological cues both in vitro and in vivo.

bioengineering↗

A cGAMP-containing hydrogel for prolonged SARS-CoV-2 RBD subunit vaccine exposure induces a broad and potent humoral response

The receptor binding domain (RBD) of the SARS-CoV-2 virus spike protein has emerged as a promising target for generation of neutralizing antibodies. Although the RBD subunit is more stable than its encoding mRNA, RBD-based subunit vaccines have been hampered by RBDs poor immunogenicity. We hypothesize that this limitation can be overcome by sustained co-administration with a more potent and optimized adjuvant than standard adjuvants. The endogenous innate immune second messenger, cGAMP, holds promise as potent activator of the anti-viral STING pathway. Unfortunately, delivery of cGAMP as a therapeutic exhibits poor performance due to poor pharmacokinetics and pharmacodynamics from rapid excretion and degradation by its hydrolase ENPP1. To overcome these limitations, we sought to create an artificial immunological niche enabling slow release of cGAMP and RBD to mimic natural infections in which immune activating molecules are co-localized with antigen. Specifically, we co-encapsulated cGAMP and RBD in an injectable polymer-nanoparticle (PNP) hydrogel. This cGAMP-adjuvanted hydrogel vaccine elicited more potent, durable, and broad antibody responses with improved neutralization as compared to dose-matched bolus controls and hydrogel-based vaccines lacking cGAMP. The cGAMP-adjuvanted hydrogel platform developed is suitable for delivery of other antigens and may provide enhanced immunity against a broad range of pathogens.

bioengineering↗

Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies

When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, we explore the ability of a slow-releasing injectable hydrogel depot to reduce the problematic dose-limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining their potent anti-cancer efficacy. We leverage a previously described polymer-nanoparticle (PNP) hydrogel system that exhibits shear-thinning and yield-stress properties that we hypothesized would improve locoregional delivery of the CD40a immunotherapy. Using PET imaging, we demonstrate that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.

bioengineering↗

Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, we found that clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum were unable to elicit neutralizing responses following a prime-boost immunization. Here we show that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

bioengineering↗

MRBLEs 2.0: High-throughput generation of chemically functionalized spectrally and magnetically-encoded hydrogel beads using a simple single-layer microfluidic device

Widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface. Here, we present a simple method (MRBLEs 2.0) for efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding (MRBLEs) bearing functional groups for downstream bioconjugation. Bead production in MRBLEs 2.0 relies on manual mixing of lanthanide/polymer mixtures (each of which comprises a unique spectral code) followed by droplet generation using single-layer, parallel flow-focusing devices and off-chip batch polymerization of droplets into beads. To streamline downstream analyte coupling, MRBLEs 2.0 crosslinks copolymers bearing functional groups on the bead surface simultaneously during bead generation. Using the MRBLEs 2.0 pipeline, we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes in high-throughput (>150,000/min and can be boosted to 450,000/min). We further demonstrate the efficient conjugation of oligonucleotides and entire proteins to carboxyl MRBLEs and biotin to amino MRBLEs. Finally, we show that MRBLEs can also be magnetized via simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space. We anticipate that MRBLEs 2.0 can be directly applied towards a wide variety of downstream assays from basic biology to diagnostics and other translational research.Competing Interest StatementStanford University and Chan Zuckerberg Biohub have filed a provisional patent application (U.S. Provisional Patent application No. 63/037,804) on the MRBLEs 2.0 methods described here and Y.F., A.K.W., J.B.H., and P.M.F. are named as inventors.View Full Text

bioengineering↗