Overcoming transport barriers to immunotherapy.

Immunotherapies are designed to treat disease by modulating the activity of immune cells. Here, we consider how anatomy and microphysiology create transport barriers to immunotherapeutic delivery and retention at diseased sites, and summarize recent developments to overcome these barriers by exploiting immunobiology to engineer molecular and cellular engineering approaches. Creating impactful and practical solutions across these diseases requires the integration of the collective expertise of pathologists, clinicians, immunologists, biophysicists, immunoengineers, and more.

Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant.

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

Polymersomes Decorated with the SARS-CoV-2 Spike Protein Receptor-Binding Domain Elicit Robust Humoral and Cellular Immunity.

The COVID-19 pandemic underscores the need for rapid, safe, and effective vaccines. In contrast to some traditional vaccines, nanoparticle-based subunit vaccines are particularly efficient in trafficking antigens to lymph nodes, where they induce potent immune cell activation. Here, we developed a strategy to decorate the surface of oxidation-sensitive polymersomes with multiple copies of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to mimic the physical form of a virus particle. We evaluated the vaccination efficacy of these surface-decorated polymersomes (RBDsurf) in mice compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl-lipid-A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that a multivalent surface display of spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity.

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Lymphatic endothelial cells prime naïve CD8+ T cells into memory cells under steady-state conditions.

Lymphatic endothelial cells (LECs) chemoattract naïve T cells and promote their survival in the lymph nodes, and can cross-present antigens to naïve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However, the functional consequence of LEC priming of CD8+ T cells is unknown. Here, we show that while many proliferating LEC-educated T cells enter early apoptosis, the remainders comprise a long-lived memory subset, with transcriptional, metabolic, and phenotypic features of central memory and stem cell-like memory T cells. In vivo, these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall, and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.

Evaluating Nanoparticle Binding to Blood Compartment Immune Cells in High-Throughput with Flow Cytometry.

Nanoparticles are increasingly being utilized for in vivo applications, where they are implemented as carriers for drugs, contrast agents for noninvasive medical imaging, or delivery vehicles for macromolecular agents such as DNA or proteins. However, they possess many physical and chemical properties that cause them to become rapidly recognized by the immune system as a foreign body, leading to their clearance and elimination, even before they may accumulate to critical concentrations at anatomic and cellular sites of action. The techniques described in this chapter aim to identify potential interactions of test, fluorescently tagged nano-formulations with circulating immune cells, with the goal of predicting potentially problematic formulations that may be rapidly cleared following in vivo administration. The techniques make use of flow cytometry, a method commonly used in immunology to phenotype and identify immune cell subtypes based on their expression of signature surface marker profiles.

Emerging roles of lymphatic endothelium in regulating adaptive immunity.

Emerging research on the roles of stromal cells in modulating adaptive immune responses has included a new focus on lymphatic endothelial cells (LECs). LECs are presumably the first cells that come into direct contact with peripheral antigens, cytokines, danger signals, and immune cells travelling from peripheral tissues to lymph nodes. LECs can modulate dendritic cell function, present antigens to T cells on MHC class I and MHC class II molecules, and express immunomodulatory cytokines and receptors, which suggests that their roles in adaptive immunity are far more extensive than previously realized. This Review summarizes the emergent evidence that LECs are important in maintaining peripheral tolerance, limiting and resolving effector T cell responses, and modulating leukocyte function.

Ex vivo red blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Macrophage-specific RNA interference targeting via "click", mannosylated polymeric micelles.

Macrophages represent an important therapeutic target, because their activity has been implicated in the progression of debilitating diseases such as cancer and atherosclerosis. In this work, we designed and characterized pH-responsive polymeric micelles that were mannosylated using "click" chemistry to achieve CD206 (mannose receptor)-targeted siRNA delivery. CD206 is primarily expressed on macrophages and dendritic cells and upregulated in tumor-associated macrophages, a potentially useful target for cancer therapy. The mannosylated nanoparticles improved the delivery of siRNA into primary macrophages by 4-fold relative to the delivery of a nontargeted version of the same carrier (p < 0.01). Further, treatment for 24 h with the mannose-targeted siRNA carriers achieved 87 ± 10% knockdown of a model gene in primary macrophages, a cell type that is typically difficult to transfect. Finally, these nanoparticles were also avidly recognized and internalized by human macrophages and facilitated the delivery of 13-fold more siRNA into these cells than into model breast cancer cell lines. We anticipate that these mannose receptor-targeted, endosomolytic siRNA delivery nanoparticles will become an enabling technology for targeting macrophage activity in various diseases, especially those in which CD206 is upregulated in macrophages present within the pathologic site. This work also establishes a generalizable platform that could be applied for "click" functionalization with other targeting ligands to direct siRNA delivery.

Matrix Metalloproteinase Responsive, Proximity-activated Polymeric Nanoparticles for siRNA Delivery.

Small interfering RNA (siRNA) has significant potential to evolve into a new class of pharmaceutical inhibitors, but technologies that enable robust, tissue-specific intracellular delivery must be developed before effective clinical translation can be achieved. A pH-responsive, smart polymeric nanoparticle (SPN) with matrix metalloproteinase (MMP)-7-dependent proximity-activated targeting (PAT) is described here. The PAT-SPN was designed to trigger cellular uptake and cytosolic delivery of siRNA once activated by MMP-7, an enzyme whose overexpression is a hallmark of cancer initiation and progression. The PAT-SPN is composed of a corona-forming PEG block, an MMP-7-cleavable peptide, a cationic siRNA-condensing block, and a pH-responsive, endosomolytic terpolymer block that drives self-assembly and forms the PAT-SPN core. With this novel design, the PEG corona shields cellular interactions until it is cleaved in MMP-7-rich environments, shifting SPNζ-potential from +5.8 to +14.4 mV and triggering a 2.5 fold increase in carrier internalization. The PAT-SPN exhibited pH-dependent membrane disruptive behavior that enabled siRNA escape from endo-lysosomal pathways. Efficient intracellular siRNA delivery and knockdown of the model enzyme luciferase in R221A-Luc mammary tumor cellssignificantly depended on MMP-7 pre-activation. These combined data indicate that the PAT-SPN provides a promising new platform for tissue-specific, proximity-activated siRNA delivery to MMP-rich pathological environments.