Personalized neoantigen viro-immunotherapy platform for triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) corresponds to approximately 20% of all breast tumors, with a high propensity for metastasis and a poor prognosis. Because TNBC displays a high mutational load compared with other breast cancer types, a neoantigen-based immunotherapy strategy could be effective. One major bottleneck in the development of a neoantigen-based vaccine for TNBC is the selection of the best targets, that is, tumor-specific neoantigens which are presented at the surface of tumor cells and capable of eliciting robust immune responses. In this study, we aimed to set up a platform for identification and delivery of immunogenic neoantigens in a vaccine regimen for TNBC using oncolytic vaccinia virus (VV). METHODS: We used bioinformatic tools and cell-based assays to identify immunogenic neoantigens in TNBC patients' samples, human and murine cell lines. Immunogenicity of the neoantigens was tested in vitro (human) and ex vivo (murine) in T-cell assays. To assess the efficacy of our regimen, we used a preclinical model of TNBC where we treated tumor-bearing mice with neoantigens together with oncolytic VV and evaluated the effect on induction of neoantigen-specific CD8+T cells, tumor growth and survival. RESULTS: We successfully identified immunogenic neoantigens and generated neoantigen-specific CD8+T cells capable of recognizing a human TNBC cell line expressing the mutated gene. Using a preclinical model of TNBC, we showed that our tumor-specific oncolytic VV was able to change the tumor microenvironment, attracting and maintaining mature cross-presenting CD8α+dendritic cells and effector T-cells. Moreover, when delivered in a prime/boost regimen together with oncolytic VV, long peptides encompassing neoantigens were able to induce neoantigen-specific CD8+T cells, slow tumor growth and increase survival. CONCLUSIONS: Our study provides a promising approach for the development of neoantigen-based immunotherapies for TNBC. By identifying immunogenic neoantigens and developing a delivery system through tumor-specific oncolytic VV, we have demonstrated that neoantigen-based vaccines could be effective in inducing neoantigen-specific CD8+T cells response with significant impact on tumor growth. Further studies are needed to determine the safety and efficacy of this approach in clinical trials.

HLA class I-restricted T cell epitopes isolated and identified from myeloid leukemia cells.

Leukemia-associated antigens (LAAs) and HLA-I epitopes published previously have shown promise in inducing leukemia-specific T cell responses. However, the clinical responses are limited, and clinical effectiveness is yet to be achieved. Limitations, among others, being the LAAs themselves, the indirect approach to HLA-I epitope identification by reverse immunology, and the use of single or few LAAs and HLA-I epitopes, which limits the spectrum of inducible tumor-specific T cells. Use of a direct approach to identify naturally processed and presented HLA-I epitopes from LAAs, and higher numbers of antigens for T cell-mediated immunotherapy for leukemia may enhance clinical responses and broaden clinical effectiveness. In a prior study we used immunoaffinity purification of HLA-I peptide complexes from the differentiated myeloid tumor cell lines MUTZ3 and THP1 coupled to high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). From this we identified in the current study seven new HLA-I epitopes and the corresponding LAAs for myeloid leukemia. In comparison, the myeloid HLA-I epitopes reported here were generally stronger HLA-binders that induce stronger T cell responses than those previously published, and their source LAAs had higher immunogenicity, higher expression levels in myeloid tumors cells compared to normal hemopoietin and other major normal tissues, and more protein interaction partners, and they are targeted by CD8 T cells in CML patients. This study analyses and compares the LAAs and HLA-I epitopes based on various immunotherapeutic targets selection criteria, and highlights new targets for T cell-mediated immunotherapy for leukemia.

Connecting signaling and metabolic pathways in EGF receptor-mediated oncogenesis of glioblastoma.

As malignant transformation requires synchronization of growth-driving signaling (S) and metabolic (M) pathways, defining cancer-specific S-M interconnected networks (SMINs) could lead to better understanding of oncogenic processes. In a systems-biology approach, we developed a mathematical model for SMINs in mutated EGF receptor (EGFRvIII) compared to wild-type EGF receptor (EGFRwt) expressing glioblastoma multiforme (GBM). Starting with experimentally validated human protein-protein interactome data for S-M pathways, and incorporating proteomic data for EGFRvIII and EGFRwt GBM cells and patient transcriptomic data, we designed a dynamic model for EGFR-driven GBM-specific information flow. Key nodes and paths identified by in silico perturbation were validated experimentally when inhibition of signaling pathway proteins altered expression of metabolic proteins as predicted by the model. This demonstrated capacity of the model to identify unknown connections between signaling and metabolic pathways, explain the robustness of oncogenic SMINs, predict drug escape, and assist identification of drug targets and the development of combination therapies.

Impact of Leishmania donovani infection on the HLA I self peptide repertoire of human macrophages.

Macrophages are specialized antigen-presenting cells that process and present self-antigens for induction of tolerance, and foreign antigens to initiate T cell-mediated immunity. Despite this, Leishmania donovani (LD) are able to parasitize the macrophages and persist. The impact of this parasitizing and persistence on antigen processing and presentation by macrophages remains poorly defined. To gain insight into this, we analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and compared the HLA-I self-peptidomes, proteasome compositions, HLA expression and activation states of non-infected and LD-infected THP1-derived macrophages. We found that, though both HLA-I peptidomes were dominated by nonapeptides, they were heterogeneous and individualized, with differences in HLA binding affinities and anchor residues. Non-infected and LD-infected THP1-derived macrophages were able to sample peptides from source proteins of almost all subcellular locations and involved in various cellular functions, but in different proportions. In the infected macrophages, there was increased sampling of plasma membrane and extracellular proteins, and those involved in immune responses, cell communication/signal transduction and metabolism/energy pathways, and decreased sampling of nuclear and cytoplasmic proteins and those involved in protein metabolism, RNA binding and cell growth and/or maintenance. Though the activation state of infected macrophages was unchanged, their proteasome composition was altered.

Combining R-DOTAP and a particulate antigen delivery platform to trigger dendritic cell activation: Formulation development and in-vitro interaction studies.

The utilization of the cationic lipid R-DOTAP as immune cell stimulant (e.g. its stimulating effects on immature dendritic cells) and correspondingly as possible adjuvant for vaccination is well known. Likewise, it is described in literature that solid polymer particles loaded with antigens can be size-tailored in a manner to be suitable for phagocytosis by antigen presenting cells. The effects of DOTAP-microparticle combinations, however, are not well understood. This study aimed therefore to explore the potential of R-DOTAP stabilized microparticles (MP) to act as a carrier platform for antigens e.g. for cancer vaccination. It was investigated whether or not a combination of R-DOTAP and PLGA leads to a boosted adjuvant effect in dendritic cell maturation. For proper comparison, neutral and negatively charged MPs of comparable sizes were developed. Toxicity, uptake, routing and maturation of the MP platform was assessed in-vitro on human immature dendritic cells (iDCs). Interestingly, none of the tested placebo formulations (without antigen) was capable to induce DC maturation when compared to LPS as positive control. This is in contrast to experiments previously reported in literature, where R-DOTAP (e.g. in liposomal form) triggered iDC maturation even without antigen. Possible reasons and further approaches are discussed in the paper.

Immature human DCs efficiently translocate endocytosed antigens into the cytosol for proteasomal processing.

Cross-presentation of endocytosed antigen is essential for induction of CD8 effector T cell responses and a hallmark of dendritic cells (DCs). The mode of antigen processing in this context is controversial and some models imply translocation of the antigen from the endosomes into the cytosol. To test this hypothesis we made use of the pro-apoptotic properties of cytochrome c when in the cytosol, and confirmed that it indeed triggered apoptosis of human immature DCs but only at high concentrations. Proteasome inhibitors reduced the required concentration of cytochrome c thousand-fold, indicating that protein translocated into the cytosol is rapidly degraded by proteasomes. Mature DCs were also susceptible to cytochrome c-triggered apoptosis at high concentrations but proteasome inhibitors did not increase their sensitivity. Other cross-presenting cells such as B cells and monocytes were not sensitive to cytochrome c at all, indicating that they do not shuttle internalized antigen into the cytosol. Thus, processing of internalized antigens seems to follow different pathways depending on cell type and, in case of DCs, maturation state. Immature DCs appear to have a unique capacity to shuttle external antigen into the cytosol for proteasomal processing, which could explain their efficiency in antigen cross-presentation.

Diverse HLA-I Peptide Repertoires of the APC Lines MUTZ3-Derived Immature and Mature Dendritic Cells and THP1-Derived Macrophages.

Dendritic cells (DCs) and macrophages are specialized APCs that process and present self-Ags for induction of tolerance and foreign Ags to initiate T cell-mediated immunity. Related to differentiation states they have specific phenotypes and functions. However, the impact of these differentiations on Ag processing and presentation remains poorly defined. To gain insight into this, we analyzed and compared the HLA-I peptidomes of MUTZ3-derived human immature and mature DC lines and THP1-derived macrophages by liquid chromatography tandem mass spectrometry. We found that the HLA-I peptidomes were heterogeneous and individualized and were dominated by nonapeptides with similar HLA-I binding affinities and anchor residues. MUTZ3-derived DCs and THP1-derived macrophages were able to sample peptides from source proteins of almost all subcellular locations and were involved in various cellular functions in similar proportion, with preference to proteins involved in cell communication, signal transduction, protein metabolism, and transcription factor/regulator activity.

Exposure to Melan-A/MART-126-35 tumor epitope specific CD8(+)T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS).

Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing.

Large molecular systems landscape uncovers T cell trapping in human skin cancer.

Immune surveillance of tumour cells is an important function of CD8 T lymphocytes, which has failed in cancer for reasons still unknown in many respect but mainly related to cellular processes in the tumour microenvironment. Applying imaging cycler microscopy to analyse the immune contexture in a human skin cancer we could identify and map 7,000 distinct cell surface-associated multi-protein assemblies. The resulting combinatorial geometry-based high-functional resolution led to discovery of a mechanism of T cell trapping in the epidermis, which involves SPIKE, a network of suprabasal keratinocyte projections piercing and interconnecting CD8 T cells. It appears initiated by clusters of infrabasal T and dendritic cells connected via cell projections across a fractured basal lamina to suprabasal keratinocytes and T lymphocytes.

Heterogeneity of Leishmania donovani parasites complicates diagnosis of visceral leishmaniasis: comparison of different serological tests in three endemic regions.

Diagnostic tests for visceral leishmaniasis that are based on antigens of a single Leishmania strain can have low diagnostic performance in regions where heterologous parasites predominate. The aim of this study was to investigate and compare the performance of five serological tests, based on different Leishmania antigens, in three endemic countries for visceral leishmaniasis. A total number of 231 sera of symptomatic and asymptomatic cases and controls from three endemic regions of visceral leishmaniasis in East Sudan, North India and South France were evaluated by following serological tests: rKLO8- and rK39 ELISA, DAT (ITMA-DAT) and two rapid tests of rK39 (IT LEISH) and rKE16 (Signal-KA). Overall, rKLO8- and rK39 ELISA were most sensitive in immunocompetent patients from all endemic regions (96-100%) and the sensitivity was reduced to 81.8% in HIV co-infected patients from France. Sera of patients from India demonstrated significantly higher antibody responses to rKLO8 and rK39 compared with sera from Sudan (p<0.0001) and France (p<0.0037). Further, some Indian and Sudanese patients reacted better with rKLO8 than rK39. Sensitivity of DAT (ITMA-DAT) was high in Sudan (94%) and India (92.3%) but low in France being 88.5% and 54.5% for VL and VL/HIV patients, respectively. In contrast, rapid tests displayed high sensitivity only in patients from India (96.2%) but not Sudan (64-88%) and France (73.1-88.5% and 63.6-81.8% in VL and VL/HIV patients, respectively). While the sensitivity varied, all tests showed high specificity in Sudan (96.7-100%) and India (96.6%).Heterogeneity of Leishmania parasites which is common in many endemic regions complicates the diagnosis of visceral leishmaniasis. Therefore, tests based on homologous Leishmania antigens are required for particular endemic regions to detect cases which are difficult to be diagnosed with currently available tests.

Role of IL-10 and TGF-ß in melanoma.

IL-10 and TGF-ß are immunosuppressive cytokines expressed in tumors including melanoma and, therefore, deemed major cause for failing antitumor immune responses. Re-evaluating their role, we compared their expression by quantitative RT-PCR in melanoma and skin of healthy individuals, tested their induction in dendritic cells and T cells co-cultured with tumor cells, and their effects on the immune cells. Both cytokines as well as their receptors were expressed in melanoma at significantly lower levels than in healthy skin. Consequently, the expressions of IL-10-responsive SOCS-3 and TGF-ß-responsive Smad-7 were low in tumors but high in healthy skin. T cells co-cultured with tumor cells developed an anergic state without increased IL-10 or TGF-ß expression. In vitro tumor-induced immature dendritic cells produced high IL-10 levels and less efficiently induced T-cell proliferation. Nonetheless, they could be induced to mature, and blocking IL-10 did not alter the capacity of the resulting mature dendritic cells to stimulate T cells. Mature dendritic cells co-cultured with tumor cells produced increased IL-10 but decreased TGF-ß and more efficiently induced T-cell proliferation. The lack of correlation of IL-10 and TGF-ß with immune deficits in situ and in vitro suggests re-evaluating their roles in cancer.

Spatial separation of the processing and MHC class I loading compartments for cross-presentation of the tumor-associated antigen HER2/neu by human dendritic cells.

Cross-presentation is the process by which professional antigen presenting cells (APCs) (B cells, dendritic cells (DCs) and macrophages) present endocytosed antigens (Ags) via MHC-I to CD8+ T cells. This process is crucial for induction of adaptive immune responses against tumors and infected cells. The pathways and cellular compartments involved in cross-presentation are unresolved and controversial. Among the cells with cross-presenting capacity, DCs are the most efficient, which was proposed to depend on prevention of endosomal acidification to block degradation of the epitopes. Contrary to this view, we show in this report that some cargoes induce strong endosomal acidification following uptake by human DCs, while others not. Moreover, processing of the tumor-associated antigen HER2/neu delivered in nanoparticles (NP) for cross-presentation of the epitope HER2/neu369-377 on HLA-A2 depended on endosomal acidification and cathepsin activity as well as proteasomes, and newly synthesized HLA class I. However, the HLA-A*0201/HER2/neu369-377 complexes were not found in the endoplasmic reticulum (ER) nor in endolysosomes but in hitherto not described vesicles. The data thus indicate spatial separation of antigen processing and loading of MHC-I for cross-presentation: antigen processing occurs in the uptake compartment and the cytosol whereas MHC-I loading with peptide takes place in a distinct subcellular compartment. The findings further elucidate the cellular pathways involved in the cross-presentation of a full-length, clinically relevant tumor-associated antigen by human DCs, and the impact of the vaccine formulation on antigen processing and CD8+ T cell induction.

Modular multiantigen T cell epitope-enriched DNA vaccine against human leishmaniasis.

The leishmaniases are protozoal diseases that severely affect large populations in tropical and subtropical regions. There are only limited treatment options and preventative measures. Vaccines will be important for prevention, control and elimination of leishmaniasis, and could reduce the transmission and burden of disease in endemic populations. We report the development of a DNA vaccine against leishmaniasis that induced T cell-based immunity and is a candidate for clinical trials. The vaccine antigens were selected as conserved in various Leishmania species, different endemic regions, and over time. They were tested with T cells from individuals cured of leishmaniasis, and shown to be immunogenic and to induce CD4(+) and CD8(+) T cell responses in genetically diverse human populations of different endemic regions. The vaccine proved protective in a rodent model of infection. Thus, the immunogenicity of candidate vaccine antigens in human populations of endemic regions, as well as proof of principle for induction of specific immune responses and protection against Leishmania infection in mice, provides a viable strategy for T cell vaccine development.

Combinatorial chemistry by ant colony optimization.

BACKGROUND: Prioritizing building blocks for combinatorial medicinal chemistry represents an optimization task. We present the application of an artificial ant colony algorithm to combinatorial molecular design (Molecular Ant Algorithm [MAntA]). RESULTS: In a retrospective evaluation, the ant algorithm performed favorably compared with other stochastic optimization methods. Application of MAntA to peptide design resulted in new octapeptides exhibiting substantial binding to mouse MHC-I (H-2K(b)). In a second study, MAntA generated a new functional factor Xa inhibitor by Ugi-type three-component reaction. CONCLUSION: This proof-of-concept study validates artificial ant systems as innovative computational tools for efficient building block prioritization in combinatorial chemistry. Focused activity-enriched compound collections are obtained without the need for exhaustive product enumeration.

Exhaustive proteome mining for functional MHC-I ligands.

We present the development and application of a new machine-learning approach to exhaustively and reliably identify major histocompatibility complex class I (MHC-I) ligands among all 20(8) octapeptides and in genome-derived proteomes of Mus musculus , influenza A H3N8, and vesicular stomatitis virus (VSV). Focusing on murine H-2K(b), we identified potent octapeptides exhibiting direct MHC-I binding and stabilization on the surface of TAP-deficient RMA-S cells. Computationally identified VSV-derived peptides induced CD8(+) T-cell proliferation after VSV-infection of mice. The study demonstrates that high-level machine-learning models provide a unique access to rationally designed peptides and a promising approach toward "reverse vaccinology".

Direct activation of human dendritic cells by particle-bound but not soluble MHC class II ligand.

Dendritic cells (DCs) are key activators of cellular immune responses through their capacity to induce naïve T cells and sustained effector T cell responses. This capacity is a function of their superior efficiency of antigen presentation via MHC class I and class II molecules, and the expression of co-stimulatory cell surface molecules and cytokines. Maturation of DCs is induced by microbial factors via pattern recognition receptors such as Toll-like receptors, pro-inflammatory cytokines or cognate interaction with CD4(+) T cells. Here we show that, unexpectedly, the PanDR helper T cell epitope PADRE, a generic T helper cell antigen presented by a large fraction of HLA-DR alleles, when delivered in particle-bound form induced maturation of human DCs. The DCs that received the particle-bound PADRE displayed all features of fully mature DCs, such as high expression of the co-stimulatory molecules CD80, CD86, CD83, the MHC-II molecule HLA-DR, secretion of high levels of the biologically active IL-12 (IL-12p70) and induction of vigorous proliferation of naïve CD4(+) T cells. Furthermore, the maturation of DCs induced by particle-bound PADRE was shown to involve sphingosine kinase, calcium signaling from internal sources and downstream signaling through the MAP kinase and the p72syk pathways, and finally activation of the transcription factor NF-κB. Based on our findings, we propose that particle-bound PADRE may be used as a DC activator in DC-based vaccines.

Topical vaccination with functionalized particles targeting dendritic cells.

Needle-free vaccination, for reasons of safety, economy, and convenience, is a central goal in vaccine development, but it also needs to meet the immunological requirements for efficient induction of prophylactic and therapeutic immune responses. Combining the principles of noninvasive delivery to dendritic cells (DCs) through skin and the immunological principles of cell-mediated immunity, we developed microparticle-based topical vaccines. We show here that the microparticles are efficient carriers for coordinated delivery of the essential vaccine constituents to DCs for cross-presentation of the antigens and stimulation of T-cell responses. When applied to the skin, the microparticles penetrate into hair follicles and target the resident DCs, the immunologically most potent cells and site for induction of efficient immune responses. The microparticle vaccine principle can be applied to different antigen formats such as peptides and proteins, or nucleic acids coding for the antigens.

A polymorphic microsatellite repeat within the ECE-1c promoter is involved in transcriptional start site determination, human evolution, and Alzheimer's disease.

Genetic factors strongly contribute to the pathogenesis of sporadic Alzheimer's disease (AD). Nevertheless, genome-wide association studies only yielded single nucleotide polymorphism loci of moderate importance. In contrast, microsatellite repeats are functionally less characterized structures within our genomes. Previous work has shown that endothelin-converting enzyme-1 (ECE-1) is able to reduce amyloid ß content. Here we demonstrate that a CpG-CA repeat within the human ECE-1c promoter is highly polymorphic, harbors transcriptional start sites, is able to recruit the transcription factors poly(ADP-ribose) polymerase-1 and splicing factor proline and glutamine-rich, and is functional regarding haplotype-specific promoter activity. Furthermore, genotyping of 403 AD patients and 444 controls for CpG-CA repeat length indicated shifted allelic frequency distributions. Sequencing of 245 haplotype clones demonstrated that the overall CpG-CA repeat composition of AD patients and controls is distinct. Finally, we show that human and chimpanzee [CpG](m)-[CA](n) ECE-1c promoter repeats are genetically and functionally distinct. Our data indicate that a short genomic repeat structure constitutes a novel core promoter element, coincides with human evolution, and contributes to the pathogenesis of AD.