Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or TRAIL-receptor agonistic monoclonal antibodies promote apoptosis in most cancer cells, and the differential ...expression of TRAIL-R2 between tumor and normal tissues allows its exploitation as a tumor-associated antigen. The use of these antibodies as anticancer agents has been extensively studied, but the results of clinical trials were disappointing. The observed lack of anticancer activity could be attributed to intrinsic or acquired resistance of tumor cells to this type of treatment. A possible strategy to circumvent drug resistance would be to strike tumor cells with a second modality based on a different mechanism of action. We therefore set out to generate and optimize a bispecific antibody targeting TRAIL-R2 and CD3. After the construction of different bispecific antibodies in tandem-scFv or single-chain diabody formats to reduce possible immunogenicity, we selected a humanized bispecific antibody with very low aggregates and long-term high stability and functionality. This antibody triggered TRAIL-R2 in an agonistic manner and its anticancer activity proved dramatically potentiated by the redirection of cytotoxic T cells against both sensitive and resistant melanoma cells. The results of our study show that combining the TRAIL-based antitumor strategy with an immunotherapeutic approach in a single molecule could be an effective addition to the anticancer armamentarium.
Myeloid-derived suppressor cells (MDSC) are well-known key negative regulators of the immune response during tumor growth, however scattered is the knowledge of their capacity to influence and adapt ...to the different tumor microenvironments and of the markers that identify those capacities. Here we show that the secreted protein acidic and rich in cysteine (SPARC) identifies in both human and mouse MDSC with immune suppressive capacity and pro-tumoral activities including the induction of epithelial-to-mesenchymal transition (EMT) and angiogenesis. In mice the genetic deletion of SPARC reduced MDSC immune suppression and reverted EMT.
c
MDSC were less suppressive overall and the granulocytic fraction was more prone to extrude neutrophil extracellular traps (NET). Surprisingly, arginase-I and NOS2, whose expression can be controlled by STAT3, were not down-regulated in
c
MDSC, although less suppressive than wild type (WT) counterpart. Flow cytometry analysis showed equal phosphorylation of STAT3 but reduced ROS production that was associated with reduced nuclear translocation of the NF-kB p50 subunit in
c
than WT MDSC. The limited p50 in nuclei reduce the formation of the immunosuppressive p50:p50 homodimers in favor of the p65:p50 inflammatory heterodimers. Supporting this hypothesis, the production of TNF by
c
MDSC was significantly higher than by WT MDSC. Although associated with tumor-induced chronic inflammation, TNF, if produced at high doses, becomes a key factor in mediating tumor rejection. Therefore, it is foreseeable that an unbalance in TNF production could skew MDSC toward an inflammatory, anti-tumor phenotype. Notably, TNF is also required for inflammation-driven NETosis. The high level of TNF in
c
MDSC might explain their increased spontaneous NET formation as that we detected both
and
, in association with signs of endothelial damage. We propose SPARC as a new potential marker of MDSC, in both human and mouse, with the additional feature of controlling MDSC suppressive activity while preventing an excessive inflammatory state through the control of NF-kB signaling pathway.
Lymphoma Immunotherapy: Current Status Zappasodi, Roberta; de Braud, Filippo; Di Nicola, Massimo
Frontiers in immunology,
09/2015, Letnik:
6
Journal Article
Recenzirano
Odprti dostop
The rationale to treat lymphomas with immunotherapy comes from long-standing evidence on their distinctive immune responsiveness. Indolent B-cell non-Hodgkin lymphomas, in particular, establish key ...interactions with the immune microenvironment to ensure prosurvival signals and prevent antitumor immune activation. However, reports of spontaneous regressions indicate that, under certain circumstances, patients develop therapeutic antitumor immunity. Several immunotherapeutic approaches have been thus developed to boost these effects in all patients. To date, targeting CD20 on malignant B cells with the antibody rituximab has been the most clinically effective strategy. However, relapse and resistance prevent to cure approximately half of B-NHL patients, underscoring the need of more effective therapies. The recognition of B-cell receptor variable regions as B-NHL unique antigens promoted the development of specific vaccines to immunize patients against their own tumor. Despite initial promising results, this strategy has not yet demonstrated a sufficient clinical benefit to reach the regulatory approval. Several novel agents are now available to stimulate immune effector functions or counteract immunosuppressive mechanisms, such as engineered antitumor T cells, co-stimulatory receptor agonist, and immune checkpoint-blocking antibodies. Thus, multiple elements can now be exploited in more effective combinations to break the barriers for the induction of anti-lymphoma immunity.
Background
The transcriptional repressor B-cell lymphoma 6 (BCL6) is dysregulated in several neoplasms, but its role in triple negative breast cancer (TNBC), a highly aggressive subtype which lacks ...effective treatment, is unclear. The presence of intratumoral cancer stem cells (CSCs) is a main cause of tumor relapse. The Notch signaling pathway is crucial for regulating CSC self-renewal and promoting breast cancer (BC) development and resistance to anticancer therapies. Here, we investigated signaling cascades of BCL6 in the CSC compartment of TNBCs, and the mechanisms that govern its activity, mainly through Notch signaling.
Methods
Gene expression, somatic copy number alterations and clinical data from the Cancer Genome Atlas and METABRIC were accessed through the Xena and cbioportal browsers. Public transcriptome profiles from TNBC datasets were retrieved from the Gene Expression Omnibus. Mammosphere formation efficiency was calculated after BCL6 knockdown via transient siRNA transfection, stable silencing or pharmacological inhibition. The effects exhibited via BCL6 inhibition in putative TNBC stem-like cells were evaluated by immunofluorescence and qRT-PCR analyses. Chromatin immunoprecipitation experiments were performed to validate a putative BCL6 responsive element located in the first intron of the
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gene and to define the circuit of corepressors engaged by BCL6 following its inhibition. Immunoprecipitation assays were carried out to investigate a novel interaction at the basis of BCL6 control of CSC activity in TNBC.
Results
In silico analyses of benchmarked public datasets revealed a significant enrichment of BCL6 in cancer stemness related pathways, particularly of Notch signaling in TNBC. In vitro stable inhibition of BCL6 significantly reduced tumor cell growth and, accordingly, we found that the mammosphere formation efficiency of BCL6 silenced cells was significantly impaired by pharmacological inhibition of Notch signaling. BCL6 was found to be expressed at significantly higher levels in TNBC mammospheres than in their adherent counterparts, and loss of BCL6 function significantly decreased mammosphere formation with preferential targeting of CD44-positive
versus
ALDH-positive stem-like cells. Functional interplay between BCL6 and the chromatin remodeling factor EZH2 triggered the BCL6/Notch stemness signaling axis via inhibition of
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transcription.
Conclusions
Our results may be instrumental for the prospective design of combination treatment strategies that selectively target novel TNBC-associated biomarker(s) whose activity is implicated in the regulation of cancer stemness (such as BCL6) and molecules in developmentally conserved signaling pathways (such as Notch) to achieve long-lasting tumor control and improve patient outcomes.
Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective ...clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4+CD25+FOXP3+ regulatory T cells, an increase in CD3−CD56dimCD16+ natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-γ–producing T-cell response to autologous tumor challenge. In one HLA-A*0201+ patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.
In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report ...on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3
and CD8
cell infiltrate, with few PD1
cells, rare CD4
cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B
NK and T cells, including CD4
T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4
T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.
The stabilization of G-quadruplex DNA structures by small molecules with affinity to oncogene promoters has emerged as a promising anticancer strategy, due to a potential role in gene expression ...regulation. We explored the ability of BMH-21 (
) and its analogue BA-41 (
) to bind the G-quadruplex structure present in the c-KIT promoter by biophysical methods and molecular modeling. We provide evidence that both compounds interact with the c-KIT 21-mer sequence. The stable monomeric intramolecular parallel G-quadruplex obtained by the mutation of positions 12 and 21 allowed the precise determination of the binding mode by NMR and molecular dynamics studies. Both compounds form a complex characterized by one ligand molecule positioned over the tetrad at the 3'-end, stabilized by an extensive network of π-π interactions. The binding constants (Kb) obtained with fluorescence are similar for both complexes (around 10
M
). Compound BA-41 (
) showed significant antiproliferative activity against a human lymphoma cell line, SU-DHL4, known to express substantial levels of c-KIT. However, the partial inhibition of c-KIT expression by Western blot analysis suggested that the interaction of compound
with the c-KIT promoter is not the primary event and that multiple effects provide a contribution as determinants of biological activity.
High-dose chemotherapy with peripheral blood progenitor cell (PBPC) autograft is effective in high-risk lymphoma, particularly with the addition of rituximab; however, it is associated with risk of ...secondary malignancy. These issues have been addressed in a series of 1,347 patients with lymphoma treated with a high-dose sequential (HDS) program.
A total of 1,024 patients with B-cell lymphoma, 234 patients with Hodgkin's lymphoma, and 89 patients with T-cell lymphoma were treated with HDS between 1985 and 2005 at 11 Gruppo Italiano Terapie Innovative Linfomi centers. HDS was given as salvage treatment to 707 patients (52%); 655 patients (49%) received a modified HDS, with high-dose cytarabine and two consecutive PBPC harvests. Rituximab-supplemented HDS was given to 523 patients (39%).
At a median follow-up of 7 years, the median overall survival (OS) was 16.2 years; in B-cell lymphoma the OS was significantly superior with rituximab HDS compared to HDS alone. The cumulative incidence at 5 and 10 years of secondary myelodysplasia/acute leukemia (sMDS/AL) were 3.09% and 4.52%, respectively, that of solid tumors were 2.54% and 6.79%, respectively. Factors associated with sMDS/AL were male sex and use of the second harvest PBPC for the graft; factors found to be associated with solid tumor were advanced age, post-HDS radiotherapy, and rituximab addition to HDS. Despite the increased risk of solid tumors, rituximab addition to HDS was still associated with survival advantages.
This analysis has relevant implications for the design and use of intensive chemoimmunotherapy with autograft. In addition, it offers useful insights toward the understanding and prevention of tumor development.
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