ICAM3-Fc Outperforms Receptor-Specific Antibodies Targeted Nanoparticles to Dendritic Cells for Cross-Presentation.

Optimal targeting of nanoparticles (NP) to dendritic cells (DCs) receptors to deliver cancer-specific antigens is key to the efficient induction of anti-tumour immune responses. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing tètanus toxoid and gp100 melanoma-associated antigen, toll-like receptor adjuvants were targeted to the DC-SIGN receptor in DCs by specific humanized antibodies or by ICAM3-Fc fusion proteins, which acts as the natural ligand. Despite higher binding and uptake efficacy of anti-DC-SIGN antibody-targeted NP vaccines than ICAM3-Fc ligand, no difference were observed in DC activation markers CD80, CD83, CD86 and CCR7 induced. DCs loaded with NP coated with ICAM3-Fc appeared more potent in activating T cells via cross-presentation than antibody-coated NP vaccines. This fact could be very crucial in the design of new cancer vaccines.

Targeting dendritic cells--why bother?

Vaccination is among the most efficient forms of immunotherapy. Although sometimes inducing lifelong protective B-cell responses, T-cell-mediated immunity remains challenging. Targeting antigen to dendritic cells (DCs) is an extensively explored concept aimed at improving cellular immunity. The identification of various DC subsets with distinct functional characteristics now allows for the fine-tuning of targeting strategies. Although some of these DC subsets are regarded as superior for (cross-) priming of naive T cells, controversies still remain about which subset represents the best target for immunotherapy. Because targeting the antigen alone may not be sufficient to obtain effective T-cell responses, delivery systems have been developed to target multiple vaccine components to DCs. In this Perspective, we discuss the pros and cons of targeting DCs: if targeting is beneficial at all and which vaccine vehicles and immunization routes represent promising strategies to reach and activate DCs.

Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines.

During the past decade, the immunotherapeutic potential of ex vivo generated professional antigen presenting dendritic cells (DCs) has been explored in the clinic. Albeit safe, clinical results have thus far been limited. A major disadvantage of current cell-based dendritic cell (DC) therapies, preventing universal implementation of this form of immunotherapy, is the requirement that vaccines need to be tailor made for each individual. Targeted delivery of antigens to DC surface receptors in vivo would circumvent this laborious and expensive ex vivo culturing steps involved with these cell-based therapies. In addition, the opportunity to target natural and often rare DC subsets in vivo might have advantages over loading more artificial ex vivo cultured DCs. Preclinical studies show targeting antigens to DCs effectively induces humoral responses, while cellular responses are induced provided a DC maturation or activation stimulus is co-administered. Here, we discuss strategies to target antigens to distinct DC subsets and to simultaneously employ adjuvants to activate these cells to induce immunity.

The C-type lectin receptor CLEC9A mediates antigen uptake and (cross-)presentation by human blood BDCA3+ myeloid dendritic cells.

CLEC9A is a recently discovered C-type lectin receptor involved in sensing necrotic cells. In humans, this receptor is selectively expressed by BDCA3(+) myeloid dendritic cells (mDCs), which have been proposed to be the main human cross-presenting mDCs and may represent the human homologue of murine CD8(+) DCs. In mice, it was demonstrated that antigens delivered with antibodies to CLEC9A are presented by CD8(+) DCs to both CD4(+) and CD8(+) T cells and induce antitumor immunity in a melanoma model. Here we assessed the ability of CLEC9A to mediate antigen presentation by human BDCA3(+) mDCs, which represent < 0.05% of peripheral blood leukocytes. We demonstrate that CLEC9A is only expressed on immature BDCA3(+) mDCs and that cell surface expression is lost after TLR-mediated maturation. CLEC9A triggering via antibody binding rapidly induces receptor internalization but does not affect TLR-induced cytokine production or expression of costimulatory molecules. More importantly, antigens delivered via CLEC9A antibodies to BDCA3(+) mDCs are presented by both MHC class I (cross-presentation) and MHC class II to antigen-specific T cells. We conclude that CLEC9A is a promising target for in vivo antigen delivery in humans to increase the efficiency of vaccines against infectious or malignant diseases.

Tracking targeted bimodal nanovaccines: immune responses and routing in cells, tissue, and whole organism.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), involved in the induction of immunity and currently exploited for antitumor immunotherapies. An optimized noninvasive imaging modality capable of determining and quantifying DC-targeted nanoparticle (NP) trajectories could provide valuable information regarding therapeutic vaccine outcome. Here, targeted poly(d,l-lactide-co-glycolide) nanoparticles (PLGA NPs) recognizing DC receptors were equipped with superparamagnetic iron oxide particles (SPIO) or gold nanoparticles with fluorescently labeled antigen. The fluorescent label allowed for rapid analysis and quantification of DC-specific uptake of targeted PLGA NPs in comparison to uptake by other cells. Transmission electron microscopy (TEM) showed that a fraction of the encapsulated antigen reached the lysosomal compartment of DCs, where SPIO and gold were already partially released. However, part of the PLGA NPs localized within the cytoplasm, as confirmed by confocal microscopy. DCs targeted with NPs carrying SPIO or fluorescent antigen were detected within lymph nodes as early as 1 h after injection by magnetic resonance imaging (MRI). Despite the fact that targeting did not markedly affect PLGA NP biodistribution on organism and tissue level, it increased delivery of NPs to DCs residing in peripheral lymph nodes and resulted in enhanced T cell proliferation. In conclusion, two imaging agents within a single carrier allows tracking of targeted PLGA NPs at the subcellular, cellular, and organismal levels, thereby facilitating the rational design of in vivo targeted vaccination strategies.

Antibodies and carbohydrate ligands binding to DC-SIGN differentially modulate receptor trafficking.

DCs are regarded as key APCs that initiate humoral and cellular immune responses. Consequently, targeted delivery of Ag toward DC-specific receptors enhances vaccine efficacy. DC-SIGN is a C-type lectin receptor that facilitates DC-specific delivery of Ag. This is accomplished by conjugating Ag to receptor-specific Ab or carbohydrate ligands that bind to its carbohydrate recognition domain. Here, we investigated the fate of DC-SIGN following receptor triggering with Ab. Both whole and single-chain Ab induced rapid internalization of about half of the surface receptor molecules. Biochemical studies showed that about half of the receptor molecules were still intracellular after 3 h, while minimal or no resurfacing of internalized or newly synthesized unbound DC-SIGN molecules was observed. Prolonged exposure of DCs to DC-SIGN Ab, but not carbohydrate ligands, resulted in reduced receptor expression levels, which lasted up to 2 days following removal of the Ab. In addition, exposure to DC-SIGN Ab reduced the ability of the receptor to internalize. Consequently, DC-SIGN showed a poor ability to accumulate targeting Abs within DCs. Vaccine efficacy may therefore be enhanced by strategies increasing the amount of Ag entering via a single receptor molecule, such as the use of targeting moieties allowing DC-SIGN recycling or Ab-coated vaccine carriers.

Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation.

Targeting antigens to dendritic cell (DC)-specific receptors, such as DC-SIGN, induces potent T cell-mediated immune responses. DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD). Thus far, only antibodies binding the CRD have been used to target antigens to DC-SIGN. We evaluated the endocytic pathway triggered by antineck antibodies as well as their intracellular routing and ability to induce CD8(+) T-cell activation. In contrast to anti-CRD antibodies, antineck antibodies induced a clathrin-independent mode of DC-SIGN internalization, as demonstrated by the lack of colocalization with clathrin and the observation that silencing clathrin did not affect antibody internalization in human DCs. Interestingly, we observed that anti-neck and anti-CRD antibodies were differentially routed within DCs. Whereas anti-CRD antibodies were mainly routed to late endosomal compartments, anti-neck antibodies remained associated with early endosomal compartments positive for EEA-1 and MHC class I for up to 2 hours after internalization. Finally, cross-presentation of protein antigen conjugated to antineck antibodies was approximately 1000-fold more effective than nonconjugated antigen. Our studies demonstrate that anti-neck antibodies trigger a distinct mode of DC-SIGN internalization that shows potential for targeted vaccination strategies.

Targeted delivery of TLR ligands to human and mouse dendritic cells strongly enhances adjuvanticity.

Effective vaccines consist of 2 components: immunodominant antigens and effective adjuvants. Whereas it has been demonstrated that targeted delivery of antigens to dendritic cells (DCs) improves vaccine efficacy, we report here that co-targeting of TLR ligands (TLRLs) to DCs strongly enhances adjuvanticity and immunity. We encapsulated ligands for intracellular TLRs within biodegradable nanoparticles coated with Abs recognizing DC-specific receptors. Targeted delivery of TLRLs to human DCs enhanced the maturation and production of immune stimulatory cytokines and the Ag-specific activation of naive CD8(+) T cells. In vivo studies demonstrated that nanoparticles carrying Ag induced cytotoxic T-lymphocyte responses at 100-fold lower adjuvant dose when TLRLs were co-encapsulated instead of administered in soluble form. Moreover, the efficacy of these targeted TLRLs reduced the serum cytokine storm and related toxicity that is associated with administration of soluble TLRLs. We conclude that the targeted delivery of adjuvants may improve the efficacy and safety of DC-based vaccines.

DEC-205 mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendritic cells.

DEC-205 is a type I C-type lectin receptor (CLR) that is expressed on various APC subsets and has been suggested to bind necrotic and apoptotic cells. Here we study DEC-205 characteristics in plasmacytoid DCs (pDCs) obtained from healthy individuals and assess its ability to mediate antigen presentation by isolating sufficient numbers of pDCs from apheresis material obtained from stage III/IV melanoma patients. The results demonstrate that DEC-205 is expressed on human pDCs. Internalization of DEC-205 after antibody ligation is clathrin- and dynamin-dependent as it is blocked by hypertonic shock or by inhibition of dynamin activity. Antibody targeting to DEC-205 does not affect TLR-induced expression levels of co-stimulatory and MHC molecules, but clearly impairs TLR-induced IFN-α secretion by 40%. We observed that TLR-mediated signaling increases DEC-205 expression levels without affecting receptor internalization. Moreover, human pDCs retained the capacity to present antigens via DEC-205 following TLR activation.

Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters.

PURPOSE: Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses. EXPERIMENTAL DESIGN: Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA. RESULTS: KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20 µg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9 %, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients. CONCLUSIONS: We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.

Human plasmacytoid dendritic cells phagocytose, process, and present exogenous particulate antigen.

Plasmacytoid dendritic cells (pDCs) play a major role in shaping both innate and adaptive immune responses, mainly via their production of large amounts of type I IFNs. pDCs are considered to primarily present endogenous Ags and are thought not to participate in the uptake and presentation of Ags from the extracellular environment, in contrast to their myeloid counterparts, which efficiently endocytose extracellular particulates. In this study, we show that human pDCs are able to phagocytose and process particulate forms of Ag entrapped in poly(lactic-coglycolic acid) microparticles. Furthermore, pDCs were also able to sense TLR ligands (TLR-Ls) incorporated in these particles, resulting in rapid pDC activation and high IFN-alpha secretion. Combining a tetanus toxoid peptide and TLR-Ls (CpG C and R848) in these microparticles resulted in efficient pDC activation and concomitant Ag-specific T cell stimulation. Moreover, particulate Ag was phagocytosed and presented more efficiently than soluble Ag, indicating that microparticles can be exploited to facilitate efficient delivery of antigenic cargo and immunostimulatory molecules to pDCs. Together, our results show that in addition to their potency to stimulate innate immunity, pDCs can polarize adaptive immune responses against exogenous particulate Ag. These results may have important consequences for the development of new immunotherapeutic strategies exploiting Ag and TLR-Ls encapsulated in microparticles to target APC subsets.

Multimodal imaging of nanovaccine carriers targeted to human dendritic cells.

Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and are currently exploited in immunotherapy against cancer and infectious diseases. The targeted delivery of nanovaccine particles (NPs) to DCs in vivo is a promising strategy to enhance immune responses. Here, targeted nanovaccine carriers were generated that allow multimodal imaging of nanocarrier-DC interactions from the subcellular to the organism level. These carriers were made of biodegradable poly(D,L-lactide-co-glycolide) harboring superparamagnetic iron oxide particles (SPIO) and fluorescently labeled antigen in a single particle. Targeted delivery was facilitated by coating the NPs with antibodies recognizing the DC-specific receptor DC-SIGN. The fluorescent label allowed for rapid analysis and quantification of specific versus nonspecific uptake of targeted NPs by DCs compared to other blood cells. In addition, it showed that part of the encapsulated antigen reached the lysosomal compartment of DCs within 24 h. Moreover, the presence of fluorescent label did not prevent the antigen from being presented to antigen-specific T cells. The incorporated SPIO was applied to track the NPs at subcellular cell organel level using transmission electron microscopy (TEM). NPs were found within endolysosomal compartments, where part of the SPIO was already released within 24 h. Furthermore, part of the NPs seemed to localize within the cytoplasm. Ex vivo loading of DCs with NPs resulted in efficient labeling and detection by MRI and did not abolish cell migration within collagen scaffolds. In conclusion, incorporation of two imaging agents within a single carrier allows tracking of targeted nanovaccines on a subcellular, cellular and possibly organism level, thereby facilitating rational design of in vivo targeted vaccination strategies.

A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

Activation of human plasmacytoid dendritic cells by TLR9 impairs Fc gammaRII-mediated uptake of immune complexes and presentation by MHC class II.

Human plasmacytoid dendritic cells (pDCs)(2) exploit Ag uptake receptors like CD32a for internalization of exogenous Ags. Activation of pDC by TLR9 ligand CpG-C induces strong maturation. Surprisingly, we observed that CpG-C-stimulated pDCs showed impaired Ag-specific T cell proliferation whereas the induction of allogeneic T cell proliferation was not affected. We demonstrated that signals from TLR9 caused a rapid down-regulation of the capacity of pDC to take-up Ab-Ag complexes without altering their CD32a expression, thus explaining the reduced Ag presentation. The recent contrasting biological responses that were observed upon TLR9 ligation in pDCs prompted us to study the effect of several TLR9 ligands. We observed that type I IFN-inducer CpG-A, localizing in the early endosomal compartment, did not affect CD32a function, whereas CpGs localizing in the late endosomes and inducing pDC maturation clearly inhibited CD32a-mediated Ag uptake and presentation. We conclude that TLR9 ligands not only determine the type of response, i.e., type I IFN production (innate immunity) or maturation (adaptive immunity), but also directly affect Ag presentation capacity of pDCs. We hypothesize that pDC, once activated via TLR9-ligands reaching the late endosomes, can only present initially sampled Ags and thus are protected from uptake and processing of additional potential self-Ags.

Relevance of DC-SIGN in DC-induced T cell proliferation.

The role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in DC-T cell communication was assessed by analyzing the effect of DC-SIGN-blocking mAb in MLR. The results show that the degree of inhibition by DC-SIGN and LFA-1 mAb depends on the magnitude of the MLR and the maturation status of the DC. Addition of DC-SIGN mAb at several time-points during MLR showed that DC-SIGN is involved early on in DC-T cell contacts. This initial role is masked by strong adhesive and costimulatory mechanisms, indicating a short-lived effect of DC-SIGN in DC-T cell interactions. To examine this concept in more detail, the percentage of PBL capable of binding DC-SIGN was determined. Analysis of several donors revealed that 1-20% PBL bind to beads coated with recombinant DC-SIGN, and the DC-SIGN-binding cells comprised all major cell subsets found in blood. PBL isolated from a donor with high DC-SIGN-binding capacity were more prone to blocking by DC-SIGN mAb in MLR than PBL from a donor with low DC-SIGN-binding capacity. This study indicates an initial and transient role for DC-SIGN in T cell proliferation, which becomes apparent when T cell proliferation is low and when the percentage of DC-SIGN binding PBL is high.

Controlled release of antigen and Toll-like receptor ligands from PLGA nanoparticles enhances immunogenicity.

AIM: Dendritic cells rapidly capture nanoparticles and induce a potent cellular immune response. It is yet unknown whether the immunological response induced by slow release of encapsulated versus soluble antigen and adjuvant is superior. MATERIALS & METHODS: The kinetics of poly(lactic-co-glycolic acid) PLGA nanoparticles antigen release was studied by the DQ-bovine serum albumin (BSA) self-quenching antigen model. The immunological response induced was evaluated by means of dendritic cell activation/maturation markers, cytokine production and their ability to drive antigen-specific T-cell proliferation. RESULTS & CONCLUSION: PLGA-encapsulated antigen and adjuvant showed an enhanced T-cell response when compared with soluble vaccine components by increasing antigenicity and adjuvanticity. Although the kinetic profile followed the same pattern, encapsulation increased strength and duration of the response.

Targeting antigens to dendritic cells in vivo.

Dendritic cells (DCs) play a key role in antigen-specific immune regulation. DCs take up and process antigens and present these as peptides through MHC molecules to T cells. Recent pre-clinical and clinical studies have exploited DCs as a means to improve vaccine efficiency. In these studies, monocyte-derived autologous DCs are loaded ex vivo with antigens and re-administered to the patient. These tailor-made vaccines are costly and labor intensive, and therefore less suitable for large-scale immunization programs. As a next step in the development of DC vaccines, it is proposed to load DCs with antigens in vivo. Drug delivery systems harboring antigens have been targeted to DCs via specific surface receptors preferentially expressed by DCs, resulting in priming of humoral and cellular immune responses. The present review focuses on the various antigen delivery systems that are currently in use and the DC surface receptors they target.

VLDL receptor deficiency enhances intimal thickening after vascular injury but does not affect atherosclerotic lesion area.

The very low density lipoprotein receptor (VLDLR) has been shown to modulate cell migration and foam cell formation in vitro. This suggests a role for the VLDLR in vascular pathology associated with intimal thickening and atherogenesis. In the present paper both intimal thickening and atherosclerosis were studied using VLDLR knockout and transgenic mouse models. The role of the VLDLR in intimal thickening was established in an in vivo model for vascular injury. A non-restrictive cuff was placed around the femoral artery of VLDLR deficient (VLDLR-/-), heterozygous deficient (VLDLR+/-) and wild type (WT) mice. Intimal thickening was assessed after 3 weeks by determining the intima to media (I/M) volume ratio. Both VLDLR-/- (I/M ratio 42%) and VLDLR+/- (I/M ratio 40%) mice showed a significant increase as compared with WT littermates (I/M ratio 25%). The effect of VLDLR deficiency on atherosclerosis was examined in VLDLR-/- mice on an LDLR deficient (LDLR-/-) background. In addition, we assessed whether increased endothelial VLDLR expression levels affect atherosclerotic lesion formation. Therefore, atherosclerosis was studied in LDLR deficient mice that over express the VLDLR in endothelial cells (PVL, LDLR-/-). Both VLDLR deficiency and endothelial VLDLR over expression did not affect the atherosclerotic lesion size. Interestingly, VLDLR-/-, LDLR-/- mice showed a high incidence of necrosis in both fatty streaks and atherosclerotic plaques as compared with LDLR-/- mice (75 vs. 0% and 76 vs. 45%, respectively). In conclusion, deficiency for the VLDLR profoundly increased intimal thickening after vascular injury.

Enhancing immunogenicity and cross-reactivity of HIV-1 antigens by in vivo targeting to dendritic cells.

Current retroviral treatments have reduced AIDS to a chronic disease for most patients. However, given drug-related side effects, the emergence of drug-resistant strains and the persistence of viral replication, the development of alternative treatments is a pressing need. This review focuses on recent developments in HIV immunotherapy treatments, with particular emphasis on current vaccination strategies for optimizing the induction of an effective immune response by the recruitment of dendritic cells. In addition to cell-based therapies, targeted strategies aiming to deliver synthetic HIV peptides to dendritic cell-specific receptors in vivo will be discussed.

Comparison of antibodies and carbohydrates to target vaccines to human dendritic cells via DC-SIGN.

Vaccine efficacy is improved upon specific delivery to professional antigen (Ag) presenting cells, such as dendritic cells (DCs). Antigenicity and adjuvanticity of vaccine components can be enhanced by encapsulation within nanoparticle (NP) vaccine carriers that are targeted to the human DC-specific C-type lectin receptor DC-SIGN. Here we used two strategies to target vaccines components to DC-SIGN: 1) carbohydrates as natural receptor ligands and 2) receptor-specific antibodies (Abs). To determine the optimal targeting strategy, we coated NP vaccines harboring MHC class I or II-restricted Ags and the TLR ligands (TLRLs) poly I:C and resiquimod with either the DC-SIGN ligands Lewis-X (Le(x)), mannosylated lipoarabinomannan (ManLAM), glycosylated HIV protein gp120, or three distinct DC-SIGN Abs. Although, because of their lower MW, surface coating of NP vaccines with carbohydrates resulted in a higher number of surface molecules per NP than coating with Abs, NP vaccines carrying Abs were more effectively bound and internalized by human DCs than carriers harboring Le(x), ManLAM or gp120. Furthermore, NP vaccines harboring TLRLs triggered significant induction of DC maturation markers when compared to those without TLRLs, irrespective of the targeting moiety. Ab- and gp120-mediated targeting induced equally high levels of proinflammatory cytokines and increased presentation of the MHC class I-restricted epitope. By contrast, presentation of the MHC class II-restricted epitope was more efficient upon Ab-mediated targeting than when using gp120, Le(x) or ManLAM. From these findings we conclude that receptor-specific Abs are more effective than carbohydrates for DC-targeted vaccination strategies.