Transfer of Cellular Content from the Allogeneic Cell-Based Cancer Vaccine DCP-001 to Host Dendritic Cells Hinges on Phosphatidylserine and Is Enhanced by CD47 Blockade.

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell-cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

A novel allogeneic off-the-shelf dendritic cell vaccine for post-remission treatment of elderly patients with acute myeloid leukemia.

In elderly acute myeloid leukemia (AML) patients post-remission treatment options are associated with high comorbidity rates and poor survival. Dendritic cell (DC)-based immunotherapy is a promising alternative treatment strategy. A novel allogeneic DC vaccine, DCP-001, was developed from an AML-derived cell line that uniquely combines the positive features of allogeneic DC vaccines and expression of multi-leukemia-associated antigens. Here, we present data from a phase I study conducted with DCP-001 in 12 advanced-stage elderly AML patients. Patients enrolled were in complete remission (CR1/CR2) (n = 5) or had smoldering disease (n = 7). All patients were at high risk of relapse and ineligible for post-remission intensification therapies. A standard 3 + 3 dose escalation design with extension to six patients in the highest dose was performed. Patients received four biweekly intradermal DCP-001 injections at different dose levels (10, 25, and 50 million cells DCP-001) and were monitored for clinical and immunological responses. Primary objectives of the study (feasibility and safety) were achieved with 10/12 patients completing the vaccination program. Treatment was well tolerated. A clear-cut distinction between patients with and without detectable circulating leukemic blasts during the vaccination period was noted. Patients with no circulating blasts showed an unusually prolonged survival [median overall survival 36 months (range 7-63) from the start of vaccination] whereas patients with circulating blasts, died within 6 months. Long-term survival was correlated with maintained T cell levels and induction of multi-functional immune responses. It is concluded that DCP-001 in elderly AML patients is safe, feasible and generates both cellular and humoral immune responses.

The development of standard samples with a defined number of antigen-specific T cells to harmonize T cell assays: a proof-of-principle study.

The validation of assays that quantify antigen-specific T cell responses is critically dependent on cell samples that contain clearly defined measurable numbers of antigen-specific T cells. An important requirement is that such cell samples are handled and analyzed in a comparable fashion to peripheral blood mononuclear cells (PBMC). We performed a proof-of-principle study to show that retrovirally TCR-transduced T cells spiked at defined numbers in autologous PBMC can be used as standard samples for HLA/peptide multimer staining. NY-ESO-1157-165-specific, TCR-transduced CD8+ T cell batches were successfully generated from PBMC of several HLA-A*0201 healthy donors, purified by magnetic cell sorting on the basis of HLA tetramer (TM) staining and expanded with specific antigen in vitro. When subsequently spiked into autologous PBMC, the detection of these CD3+CD8+TM+ T cells was highly accurate with a mean accuracy of 91.6 %. The standard cells can be preserved for a substantial period of time in liquid nitrogen. Furthermore, TM staining of fresh and cryopreserved standard samples diluted at decreasing concentrations into autologous cryopreserved unspiked PBMC revealed that the spiked CD3+CD8+TM+ T cells could be accurately detected at all dilutions in a linear fashion with a goodness-of-fit of over 0.99 at a frequency of at least 0.02 % among the CD3+CD8+ T cell population. Notably, the CD3+CD8+TM+ cells of the standard samples were located exactly within the gates used to analyze patient samples and displayed a similar scatter pattern. The performance of the cryopreserved standard samples in the hands of 5 external investigators was good with an inter-laboratory variation of 32.9 % and the doubtless identification of one outlier.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Design of neo-glycoconjugates that target the mannose receptor and enhance TLR-independent cross-presentation and Th1 polarization.

Cross-presentation is an important mechanism by which DCs present exogenous antigens on MHC-I molecules, and activate CD8(+) T cells, cells that are crucial for the elimination of tumors. We investigated the feasibility of exploiting the capacity of the mannose receptor (MR) to improve both cross-presentation of tumor antigens and Th polarization, processes that are pivotal for the anti-tumor potency of cytotoxic T cells. To this end, we selected two glycan ligands of the MR, 3-sulfo-Lewis(A) and tri-GlcNAc (N-acetylglucosamine), to conjugate to the model antigen OVA and assessed in vitro the effect on antigen presentation and Th differentiation. Our results demonstrate that conjugation of either 3-sulfo-Lewis(A) or tri-GlcNAc specifically directs antigen to the MR. Both neo-glycoconjugates showed, even at low doses, improved uptake as compared with native OVA, resulting in enhanced cross-presentation. Using MR(-/-) and MyD88-TRIFF(-/-) bone marrow-derived DCs (BMDCs), we show that the cross-presentation of the neo-glycoconjugates is dependent on MR and independent of TLR-mediated signaling. Whereas proliferation of antigen-specific CD4(+) T cells was unchanged, stimulation with neo-glycoconjugate-loaded DCs enhanced the generation of IFN-γ-producing T cells. We conclude that modification of antigen with either 3-sulfo-Lewis(A) or tri-GlcNAc enhances cross-presentation and permits Th1 skewing, through specific targeting of the MR, which may be beneficial for DC-based vaccination strategies to treat cancer.

Tumour-associated glycan modifications of antigen enhance MGL2 dependent uptake and MHC class I restricted CD8 T cell responses.

We recently showed that MGL2 specifically binds tumour-associated glycan N-acetylgalactosamine (GalNAc). We here demonstrate that modification of an antigen with tumour-associated glycan GalNAc, targets antigen specifically to the MGL2 on bone marrow derived (BM)-DCs and splenic DCs. Glycan-modification of antigen with GalNAc that mimics tumour-associated glycosylation, promoted antigen internalisation in DCs and presentation to CD4 T cells, as well as differentiation of IFN-γ producing CD4 T cells. Furthermore, GalNAc modified antigen enhanced cross-presentation of both BM-DCs and primary splenic DCs resulting in enhanced antigen specific CD8 T cell responses. Using MyD88-TRIFF(-/-) BM-DCs we demonstrate that the enhanced cross-presentation of the GalNAc modified antigen is TLR independent. Our data strongly suggest that tumour-associated GalNAc modification of antigen targets MGL on DCs and greatly enhances both MHC class II and class I presentation in a TLR independent manner.

Targeting glycan modified OVA to murine DC-SIGN transgenic dendritic cells enhances MHC class I and II presentation.

Dendritic cells have gained much interest in the field of anti-cancer vaccine development because of their central function in immune regulation. One of the receptors that facilitate DC-specific targeting of antigens is the DC-specific C-type lectin DC-SIGN. Although DC-SIGN is specifically expressed on human DCs, its murine homologue is not present on any murine DC subsets, which makes in vivo evaluation of potential DC-SIGN targeting vaccines very difficult. Here we describe the use of DC-SIGN transgenic mice, as a good model system to evaluate DC-SIGN targeting vaccines. We demonstrate that glycan modification of OVA with DC-SIGN targeting glycans, targets antigen specifically to bone marrow (BM)** derived DCs and splenic DCs. Glycan modification of OVA with Lewis X or Lewis B oligosaccharides, that target DC-SIGN transgenic DCs, resulted in efficient 10-fold induction of OT-II compared to unmodified OVA. Interestingly, glycan modified OVA proteins were significantly cross-presented to OT-I T cells by wild type DC, 10-fold more than native OVA, and the expression of DC-SIGN further enhanced this cross-presentation. Targeting of glycosylated OVA was neither accompanied with any DC maturation, nor the production of inflammatory or anti-inflammatory cytokines. Thus, we conclude that glycan modification of antigens and targeting to DC-SIGN enhance both CD4 and CD8 T cell responses. Furthermore, our data demonstrate that DC-SIGN transgenic mice are valuable tool for optimisation and efficiency testing of DC vaccination strategies that are designed to target in particular the human DC-SIGN receptor.

Characterization of murine MGL1 and MGL2 C-type lectins: distinct glycan specificities and tumor binding properties.

Antigen presenting cells (APC) express a variety of pattern recognition receptors, including the C-type lectin receptors (CLR) that specifically recognize carbohydrate structures expressed on self-tissue and pathogens. The CLR play an important role in antigen uptake and presentation and have been shown to mediate cellular interactions. The ligand specificity of the human macrophage galactose-type lectin (MGL) has been characterized extensively. Here, we set out to determine the glycan specificity of the murine homologues, MGL1 and MGL2, using a glycan array. Murine MGL1 was found to be highly specific for Lewis X and Lewis A structures, whereas mMGL2, more similar to the human MGL, recognized N-acetylgalactosamine (GalNAc) and galactose, including the O-linked Tn-antigen, TF-antigen and core 2. The generation of MGL1 and MGL2-Fc proteins allowed us to identify ligands in lymph nodes, and MGL1-Fc additionally recognized high endothelial venules. Strikingly, MGL2 interacted strongly to adenocarcinoma cells, suggesting a potential role in tumor immunity.

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions.

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFalpha, and IL-1beta were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA-DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.