Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles

We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-β inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles' cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8+ T cells relative to untargeted particles, and delivery of TGF-β inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases.Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)Melanoma Research AllianceNational Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. (Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Grant CA174795)National Institutes of Health (U.S.) (Grant CA172164)Horizon 2020 Framework Programme (European Commission). FutureNanoNeeds Projec

Vaccine delivery with microneedle skin patches in nonhuman primates

Transcutaneous drug delivery from planar skin patches is effective for small-molecule drugs and skin-permeable vaccine adjuvants. However, to achieve efficient delivery of vaccines and other macromolecular therapeutics into the skin, penetration of the stratum corneum is needed. Topically applied skin patches with micron-scale projections ('microneedles') pierce the upper layers of the skin and enable vaccines that are coated on or encapsulated within the microneedles to be dispersed into the skin. Although millimeter-scale syringes have shown promise for vaccine delivery in humans and technologies, such as the Dermaroller (Dermaroller, Wolfenbüttel, Germany), exist for creating microscale punctures in the skin for delivery of solutions of therapeutics, solid microprojection microneedles coated with dry vaccine formulations offer a number of valuable features for vaccination, including reduced risk of blood-borne pathogen transmission or needle-stick injury, the potential for vaccine administration by minimally trained personnel or even self administration and the use of solid-state vaccine formulations that may reduce or eliminate cold-chain requirements in vaccine distribution. Recent studies in mice have demonstrated the ability of microneedles to effectively deliver vaccines to the skin, eliciting protective immunity to influenza, hepatitis C and West Nile virus.Ragon Institute of MGH, MIT and HarvardMassachusetts Institute of TechnologyHarvard UniversityNational Institutes of Health (U.S.) (AI095109)National Institutes of Health (U.S.) (AI096040)National Institutes of Health (U.S.) (AI095985)National Institutes of Health (U.S.) (AI078526)National Institutes of Health (U.S.) (AI060354)United States. Dept. of Defense (Contract W911NF-07-D-0004

Antigen-Displaying Lipid-Enveloped PLGA Nanoparticles as Delivery Agents for a Plasmodium vivax Malaria Vaccine

The parasite Plasmodium vivax is the most frequent cause of malaria outside of sub-Saharan Africa, but efforts to develop viable vaccines against P. vivax so far have been inadequate. We recently developed pathogen-mimicking polymeric vaccine nanoparticles composed of the FDA-approved biodegradable polymer poly(lactide-co-glycolide) acid (PLGA) “enveloped” by a lipid membrane. In this study, we sought to determine whether this vaccine delivery platform could be applied to enhance the immune response against P. vivax sporozoites. A candidate malaria antigen, VMP001, was conjugated to the lipid membrane of the particles, and an immunostimulatory molecule, monophosphoryl lipid A (MPLA), was incorporated into the lipid membranes, creating pathogen-mimicking nanoparticle vaccines (VMP001-NPs). Vaccination with VMP001-NPs promoted germinal center formation and elicited durable antigen-specific antibodies with significantly higher titers and more balanced Th1/Th2 responses in vivo, compared with vaccines composed of soluble protein mixed with MPLA. Antibodies raised by NP vaccinations also exhibited enhanced avidity and affinity toward the domains within the circumsporozoite protein implicated in protection and were able to agglutinate live P. vivax sporozoites. These results demonstrate that these VMP001-NPs are promising vaccines candidates that may elicit protective immunity against P. vivax sporozoites.United States. Dept. of Defense (contract W911NF-07-D-0004)Ragon Institute of MGH, MIT and Harvar

Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

available in PMC 2011 September 1Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.Ragon Institute of MGH, MIT and HarvardBill & Melinda Gates FoundationUnited States. Dept. of Defense (contract W911NF-07-D-0004)National Institutes of Health (U.S.) (P41RR002250)National Institutes of Health (U.S.) (RC2GM092599

Redox-responsive interleukin-2 nanogel specifically and safely promotes the proliferation and memory precursor differentiation of tumor-reactive T-cells

Interleukin-2 (IL-2) is a potent T-cell mitogen that can adjuvant anti-cancer adoptive T-cell transfer (ACT) immunotherapy by promoting T-cell engraftment. However, the clinical applications of IL-2 in combination with ACT are greatly hindered by the severe adverse effects such as vascular leak syndrome (VLS). Here, we developed a synthetic delivery strategy for IL-2 via backpacking redox-responsive IL-2/Fc nanogels (NGs) to the plasma membrane of adoptively transferred T-cells. The NGs prepared by traceless chemical cross-linking of cytokine proteins selectively released the cargos in response to T-cell receptor activation upon antigen recognition in tumors. We found that IL-2/Fc delivered by T-cell surface-bound NGs expanded transferred tumor-reactive T-cells 80-fold more than the free IL-2/Fc of an equivalent dose administered systemically and showed no effects on tumor-infiltrating regulatory T-cell expansion. Intriguingly, IL-2/Fc NG backpacks that facilitated a sustained and slow release of IL-2/Fc also promoted the CD8 + memory precursor differentiation and induced less T-cell exhaustion in vitro compared to free IL-2/Fc. The controlled responsive delivery of IL-2/Fc enabled the safe administration of repeated doses of the stimulant cytokine with no overt toxicity and improved efficacy against melanoma metastases in a mice model. ©2019Swiss National Science Foundation (project grant: 315230_173243)NIH (award no. CA172164

Releasable Layer-by-Layer Assembly of Stabilized Lipid Nanocapsules on Microneedles for Enhanced Transcutaneous Vaccine Delivery

Here we introduce a new approach for transcutaneous drug delivery, using microneedles coated with stabilized lipid nanocapsules, for delivery of a model vaccine formulation. Poly(lactide-co-glycolide) microneedle arrays were coated with multilayer films via layer-by-layer assembly of a biodegradable cationic poly(β-amino ester) (PBAE) and negatively charged interbilayer-cross-linked multilamellar lipid vesicles (ICMVs). To test the potential of these nanocapsule-coated microneedles for vaccine delivery, we loaded ICMVs with a protein antigen and the molecular adjuvant monophosphoryl lipid A. Following application of microneedle arrays to the skin of mice for 5 min, (PBAE/ICMV) films were rapidly transferred from microneedle surfaces into the cutaneous tissue and remained in the skin following removal of the microneedle arrays. Multilayer films implanted in the skin dispersed ICMV cargos in the treated tissue over the course of 24 h in vivo, allowing for uptake of the lipid nanocapsules by antigen presenting cells in the local tissue and triggering their activation in situ. Microneedle-mediated transcutaneous vaccination with ICMV-carrying multilayers promoted robust antigen-specific humoral immune responses with a balanced generation of multiple IgG isotypes, whereas bolus delivery of soluble or vesicle-loaded antigen via intradermal injection or transcutaneous vaccination with microneedles encapsulating soluble protein elicited weak, IgG1-biased humoral immune responses. These results highlight the potential of lipid nanocapsules delivered by microneedles as a promising platform for noninvasive vaccine delivery applications.Howard Hughes Medical Institute (Investigator)United States. Dept. of Defense (W911NF-07-D-0004)Ragon Institute of MGH, MIT, and HarvardNational Institutes of Health (U.S.) (NIH AI095109)United States. Army Research Office. Institute for Soldier Nanotechnologie

Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming

Redox-responsive interleukin-2 nanogel specifically and safely promotes the proliferation and memory precursor differentiation of tumor-reactive T-cells

Interleukin-2 (IL-2) is a potent T-cell mitogen that can adjuvant anti-cancer adoptive T-cell transfer (ACT) immunotherapy by promoting T-cell engraftment. However, the clinical applications of IL-2 in combination with ACT are greatly hindered by the severe adverse effects such as vascular leak syndrome (VLS). Here, we developed a synthetic delivery strategy for IL-2 via backpacking redox-responsive IL-2/Fc nanogels (NGs) to the plasma membrane of adoptively transferred T-cells. The NGs prepared by traceless chemical cross-linking of cytokine proteins selectively released the cargos in response to T-cell receptor activation upon antigen recognition in tumors. We found that IL-2/Fc delivered by T-cell surface-bound NGs expanded transferred tumor-reactive T-cells 80-fold more than the free IL-2/Fc of an equivalent dose administered systemically and showed no effects on tumor-infiltrating regulatory T-cell expansion. Intriguingly, IL-2/Fc NG backpacks that facilitated a sustained and slow release of IL-2/Fc also promoted the CD8(+) memory precursor differentiation and induced less T-cell exhaustion in vitro compared to free IL-2/Fc. The controlled responsive delivery of IL-2/Fc enabled the safe administration of repeated doses of the stimulant cytokine with no overt toxicity and improved efficacy against melanoma metastases in a mice model