Engineering Targeting Materials for Therapeutic Cancer Vaccines.

Therapeutic cancer vaccines constitute a valuable tool to educate the immune system to fight tumors and prevent cancer relapse. Nevertheless, the number of cancer vaccines in the clinic remains very limited to date, highlighting the need for further technology development. Recently, cancer vaccines have been improved by the use of materials, which can strongly enhance their intrinsic properties and biodistribution profile. Moreover, vaccine efficacy and safety can be substantially modulated through selection of the site at which they are delivered, which fosters the engineering of materials capable of targeting cancer vaccines to specific relevant sites, such as within the tumor or within lymphoid organs, to further optimize their immunotherapeutic effects. In this review, we aim to give the reader an overview of principles and current strategies to engineer therapeutic cancer vaccines, with a particular focus on the use of site-specific targeting materials. We will first recall the goal of therapeutic cancer vaccination and the type of immune responses sought upon vaccination, before detailing key components of cancer vaccines. We will then present how materials can be engineered to enhance the vaccine's pharmacokinetic and pharmacodynamic properties. Finally, we will discuss the rationale for site-specific targeting of cancer vaccines and provide examples of current targeting technologies.

Adjuvant-free immunization with infective filarial larvae as lymphatic homing antigen carriers.

Controlled infection with intestinal nematodes has therapeutic potential for preventing the symptoms of allergic and autoimmune diseases. Here, we engineered larvae of the filarial nematode Litomosoides sigmodontis as a vaccine strategy to induce adaptive immunity against a foreign, crosslinked protein, chicken egg ovalbumin (OVA), in the absence of an external adjuvant. The acylation of filarial proteins with fluorescent probes or biotin was not immediately detrimental to larval movement and survival, which died 3 to 5 days later. At least some of the labeled and skin-inoculated filariae migrated through lymphatic vessels to draining lymph nodes. The immunization potential of OVA-biotin-filariae was compared to that of an OVA-bound nanoparticulate carrier co-delivered with a CpG adjuvant in a typical vaccination scheme. Production of IFNγ and TNFα by restimulated CD4+ cells but not CD8+ confirmed the specific ability of filariae to stimulate CD4+ T cells. This alternative method of immunization exploits the intrinsic adjuvancy of the attenuated nematode carrier and has the potential to shift the vaccination immune response towards cellular immunity.

Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses.

BACKGROUND: Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization. OBJECTIVE: Small-molecule imidazoquinoline Toll-like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age- and species-specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs. METHODS: Chemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist-encapsulating polymersome formulations were then synthesized. Novel 96-well in vitro assays using neonatal human monocyte-derived DCs and humanized TLR8 mouse bone marrow-derived DCs enabled benchmarking of the TLR8 agonist-encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25-loaded BCG-mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice. RESULTS: Although alum-adjuvanted vaccines induced modest costimulatory molecule expression, limited TH-polarizing cytokine production, and significant cell death, BCG induced a robust adult-like maturation profile of neonatal DCs. Remarkably, TLR8 agonist polymersomes induced not only newborn DC maturation profiles similar to those induced by BCG but also stronger IL-12p70 production. On subcutaneous injection to neonatal mice, the TLR8 agonist-adjuvanted Ag85B peptide 25 formulation was comparable with BCG in inducing Ag85B-specific CD4+ T-cell numbers. CONCLUSION: TLR8 agonist-encapsulating polymersomes hold substantial potential for early-life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early-life vaccines.

Fibronectin EDA and CpG synergize to enhance antigen-specific Th1 and cytotoxic responses.

Subunit vaccines, employing purified protein antigens rather than intact pathogens, require the addition of adjuvants for enhanced immunogenicity with a correct balance between strong activation of the immune system and low toxicity. Here we show that the endogenous (i.e., autologous) non-toxic TLR4 agonist extra domain A type III repeat of fibronectin (FNIII EDA) can synergize with the exogenous (i.e., bacterial), toxic-at-high-dose, TLR9 agonist CpG to induce efficient cellular immune responses while keeping the dose of CpG low. The efficacy of the combined TLR agonists, even at half-doses, led to stronger dendritic cell activation, enhanced cytotoxic T lymphocyte activation as well as stronger humoral response, compared to the individual agonists given at full doses. Immune cells induced after vaccination with the co-adjuvanted formulation could mediate tumor regression in an E.G7-OVA tumor model, and eradicate circulating hepatitis B virus (HBV) in a transgenic HBV model. Together, these results show that endogenous TLR agonists, such as variants of FNIII EDA, can synergize with exogenous TLR ligands, such as CpG, and strongly enhance cellular immune responses, while improving their safety profile.

Nanoparticle conjugation enhances the immunomodulatory effects of intranasally delivered CpG in house dust mite-allergic mice.

An emerging strategy in preventing and treating airway allergy consists of modulating the immune response induced against allergens in the lungs. CpG oligodeoxynucleotides have been investigated in airway allergy studies, but even if promising, efficacy requires further substantiation. We investigated the effect of pulmonary delivery of nanoparticle (NP)-conjugated CpG on lung immunity and found that NP-CpG led to enhanced recruitment of activated dendritic cells and to Th1 immunity compared to free CpG. We then evaluated if pulmonary delivery of NP-CpG could prevent and treat house dust mite-induced allergy by modulating immunity directly in lungs. When CpG was administered as immunomodulatory therapy prior to allergen sensitization, we found that NP-CpG significantly reduced eosinophilia, IgE levels, mucus production and Th2 cytokines, while free CpG had only a moderate effect on these parameters. In a therapeutic setting where CpG was administered after allergen sensitization, we found that although both free CpG and NP-CpG reduced eosinophilia and IgE levels to the same extent, NP conjugation of CpG significantly enhanced reduction of Th2 cytokines in lungs of allergic mice. Taken together, these data highlight benefits of NP conjugation and the relevance of NP-CpG as allergen-free therapy to modulate lung immunity and treat airway allergy.

The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine.

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8(+) T cell responses in two murine cancer models.

Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes.

The sentinel or tumor-draining lymph node (tdLN) serves as a metastatic niche for many solid tumors and is altered via tumor-derived factors that support tumor progression and metastasis. tdLNs are often removed surgically, and therapeutic vaccines against tumor antigens are typically administered systemically or in non-tumor-associated sites. Although the tdLN is immune-suppressed, it is also antigen experienced through drainage of tumor-associated antigens (TAA), so we asked whether therapeutic vaccines targeting the tdLN would be more or less effective than those targeting the non-tdLN. Using LN-targeting nanoparticle (NP)-conjugate vaccines consisting of TAA-NP and CpG-NP, we compared delivery to the tdLN versus non-tdLN in two different cancer models, E.G7-OVA lymphoma (expressing the nonendogenous TAA ovalbumin) and B16-F10 melanoma. Surprisingly, despite the immune-suppressed state of the tdLN, tdLN-targeting vaccination induced substantially stronger cytotoxic CD8+ T-cell responses, both locally and systemically, than non-tdLN-targeting vaccination, leading to enhanced tumor regression and host survival. This improved tumor regression correlated with a shift in the tumor-infiltrating leukocyte repertoire toward a less suppressive and more immunogenic balance. Nanoparticle coupling of adjuvant and antigen was required for effective tdLN targeting, as nanoparticle coupling dramatically increased the delivery of antigen and adjuvant to LN-resident antigen-presenting cells, thereby increasing therapeutic efficacy. This work highlights the tdLN as a target for cancer immunotherapy and shows how its antigen-experienced but immune-suppressed state can be reprogrammed with a targeted vaccine yielding antitumor immunity.

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose.

In subunit vaccines, strong CD8(+) T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector CD8(+) T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 µg CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity.

Peripherally administered nanoparticles target monocytic myeloid cells, secondary lymphoid organs and tumors in mice.

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to ∼50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.

Nanoparticle conjugation and pulmonary delivery enhance the protective efficacy of Ag85B and CpG against tuberculosis.

Vaccines that drive robust T-cell immunity against Mycobacterium tuberculosis (Mtb) are needed both for prophylactic and therapeutic purposes. We have recently developed a synthetic vaccine delivery platform with Pluronic-stabilized polypropylene sulfide nanoparticles (NPs), which target lymphoid tissues by their small size (∼ 30 nm) and which activate the complement cascade by their surface chemistry. Here we conjugated the tuberculosis antigen Ag85B to the NPs (NP-Ag85B) and compared their efficacy in eliciting relevant immune responses in mice after intradermal or pulmonary administration. Pulmonary administration of NP-Ag85B with the adjuvant CpG led to enhanced induction of antigen-specific polyfunctional Th1 responses in the spleen, the lung and lung-draining lymph nodes as compared to soluble Ag85B with CpG and to the intradermally-delivered formulations. Mucosal and systemic Th17 responses were also observed with this adjuvanted NP formulation and vaccination route, especially in the lung. We then evaluated protection induced by the adjuvanted NP formulation following a Mtb aerosol challenge and found that vaccination with NP-Ag85B and CpG via the pulmonary route displayed a substantial reduction of the lung bacterial burden, both compared to soluble Ag85B with CpG and to the corresponding intradermally delivered formulations. These findings highlight the potential of administrating NP-based formulations by the pulmonary route for TB vaccination.

Binding of silver nanoparticles to bacterial proteins depends on surface modifications and inhibits enzymatic activity.

Here we describe results from a proteomic study of protein-nanoparticle interactions to further the understanding of the ecotoxicological impact of silver nanoparticles (AgNPs) in the environment. We identified a number of proteins from Escherichia coli that bind specifically to bare or carbonate-coated AgNPs. Of these proteins, tryptophanase (TNase) was observed to have an especially high affinity for both surface modifications despite its low abundance in E. coli. Purified TNase loses enzymatic activity upon associating with AgNPs, suggesting that the active site may be in the vicinity of the binding site(s). TNase fragments with high affinities for both types of AgNPs were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Differences in peptide abundance/presence in mass spectra for the two types of AgNPs suggest preferential binding of some protein fragments based on surface coating. One high-binding protein fragment contained a residue (Arg103) that is part of the active site. Ag adducts were identified for some fragments and found to be characteristic of strong binding to AgNPs rather than association of the fragments with ionic silver. These results suggest a probable mechanism for adhesion of proteins to the most commonly used commercial nanoparticles and highlight the potential effect of nanoparticle surface coating on bioavailability.