Immunostimulatory agents such as agonistic anti-CD137 and interleukin (IL)-2 generate effective anti-tumor immunity but also elicit serious toxicities, hampering their clinical application. Here we ...show that combination therapy with anti-CD137 and an IL-2-Fc fusion achieves significant initial anti-tumor activity, but also lethal immunotoxicity deriving from stimulation of circulating leukocytes. To overcome this toxicity, we demonstrate that anchoring IL-2 and anti-CD137 on the surface of liposomes allows these immune agonists to rapidly accumulate in tumors while lowering systemic exposure. In multiple tumor models, immunoliposome delivery achieves anti-tumor activity equivalent to free IL-2/anti-CD137 but with the complete absence of systemic toxicity. Immunoliposomes stimulated tumor infiltration by cytotoxic lymphocytes, cytokine production, and granzyme expression, demonstrating equivalent immunostimulatory effects to the free drugs in the local tumor microenvironment. Thus, surface-anchored particle delivery may provide a general approach to exploit the potent stimulatory activity of immune agonists without debilitating systemic toxicities.
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, IgG(1)-biased humoral immune responses. These results highlight the potential of lipid nanocapsules delivered by microneedles as a promising platform for noninvasive vaccine delivery applications.
Although cytokine therapy is an attractive strategy to build a more robust immune response in tumors, cytokines have faced clinical failures due to toxicity. In particular, interleukin-12 has shown ...great clinical promise but was limited in translation because of systemic toxicity. In this study, we demonstrate an enhanced ability to reduce toxicity without affecting the efficacy of IL-12 therapy. We engineer the material properties of a NP to meet the enhanced demands for optimal cytokine delivery by using the layer-by-layer (LbL) approach. Importantly, using LbL, we demonstrate cell-level trafficking of NPs to preferentially localize to the cell's outer surface and act as a drug depot, which is required for optimal payload activity on neighboring cytokine membrane receptors. LbL-NPs showed efficacy against a tumor challenge in both colorectal and ovarian tumors at doses that were not tolerated when administered carrier-free.
For subunit vaccines, adjuvants play a key role in shaping immunological memory. Nanoparticle (NP) delivery systems for antigens and/or molecular danger signals are promising adjuvants capable of ...promoting both cellular and humoral immune responses, but in most cases the mechanisms of action of these materials are poorly understood. Here, we studied the immune response elicited by NPs composed of multilamellar "stapled" lipid vesicles carrying a recombinant Plasmodium vivax circumsporozoite antigen, VMP001, both entrapped in the aqueous core and anchored to the lipid bilayer surfaces. Immunization with these particles and monophosphoryl lipid A (MPLA), a US Food and Drug Administration–approved immunostimulatory agonist for Toll-like receptor-4, promoted high-titer, high-avidity antibody responses against VMP001, lasting more than 1 y in mice at 10-fold lower doses than conventional adjuvants. Compared to soluble VMP001 mixed with MPLA, VMP001-NPs promoted broader humoral responses, targeting multiple epitopes of the protein and a more balanced Th1/Th2 cytokine profile from antigen-specific T cells. To begin to understand the underlying mechanisms, we examined components of the B-cell response and found that NPs promoted robust germinal center (GC) formation at low doses of antigen where no GC induction occurred with soluble protein immunization, and that GCs nucleated near depots of NPs accumulating in the draining lymph nodes over time. In parallel, NP vaccination enhanced the expansion of antigen-specific follicular helper T cells (Tfh), compared to vaccinations with soluble VMP001 or alum. Thus, NP vaccines may be a promising strategy to enhance the durability, breadth, and potency of humoral immunity by enhancing key elements of the B-cell response.
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.
Apoptosis is important in controlling hematopoietic stem cell (HSC) numbers. However, the specific BCL-2 family member(s) that regulate HSC homeostasis are not precisely defined. We tested myeloid ...leukemia-1 (MCL-1) as an attractive candidate that is highly expressed in HSCs and regulated by growth factor signals. Inducible deletion of Mcl-1 in mice resulted in ablation of bone marrow. This resulted in the loss of early bone marrow progenitor populations, including HSCs. Moreover, growth factors including stem cell factor increased transcription of the Mcl-1 gene and required MCL-1 to augment survival of purified bone marrow progenitors. Deletion of Mcl-1 in other tissues, including liver, did not impair survival. Thus, MCL-1 is a critical and specific regulator essential for ensuring the homeostasis of early hematopoietic progenitors.
Studies of chimeric mice demonstrated that the core Ig heavy chain (IgH) intronic enhancer (iEμ) functions in V(D)J and class switch recombination at the IgH locus. To more fully evaluate the role of ...this element in these and other processes, we generated mice homozygous for germ-line mutations in which the core sequences of iEμ (cEμ) were either deleted ( cEμΔ /Δmice) or replaced with a pgk-NeoRcassette ( cEμN/ Nmice). The cEμΔ /Δmice had reduced B cell numbers, in association with impaired D to JHand VHto DJHrearrangement, whereas cEμN/ Nmice had a complete block in IgH V( D) JHrecombination, confirming that additional cis elements cooperate with iEμ to enforce D to JHrecombination. In addition, developing cEμΔ /Δand cEμN/ NB lineage cells had correspondingly decreased levels of germ-line transcripts from the JHregion of the IgH locus (μ0 and Iμ transcripts); although both had normal levels of germ-line VHtranscripts, suggesting that cEμ may influence IgH locus V(D)J recombination by influencing accessibility of JHproximal regions of the locus. Consistent with chimera studies, peripheral cEμΔ /ΔB cells had normal surface Ig and relatively normal class switch recombination. However, cEμΔ /ΔB cells also had relatively normal somatic hypermutation of their IgH variable region genes, showing unexpectedly that the cEμ is not required for this process. The availability of mice with the iEμ mutation in their germ line will facilitate future studies to elucidate the roles of iEμ in VH( D) JHrecombination in the context of IgH chromatin structure and germ-line transcription.
B cell homeostasis is maintained by a balance between the continual generation of new cells and their elimination. Here we show proapoptotic BCL-2 family members BAX and BAK are essential for ...regulating the number of B cells at both immature and mature developmental stages. BAX and BAK are critical mediators of B cell death induced by multiple stimuli. In addition, BAX- and BAK- deficient B cells display defective cell cycle progression to B cell receptor crosslinking and lipopolysaccharide, but not to CpG-DNA. Furthermore, inducible deletion of Bax and Bak in adult mice results in the development of severe autoimmune disease.
Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in ...immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer‐by‐layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin‐12 (IL‐12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane‐binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL‐12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti‐tumor efficacy compared to carrier‐free IL‐12 or layer‐free liposomal NPs leading to a 30% complete survival rate.
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