Innovative therapy, monoclonal antibodies, and beyond: Highlights from the eighth annual meeting.

The eighth annual conference of "Innovative therapy, monoclonal antibodies, and beyond" was held in Milan on Jan. 26, 2018, and hosted by Fondazione IRCCS-Istituto Nazionale dei Tumori (Fondazione IRCCS INT). The conference was divided into two main scientific sessions, of i) pre-clinical assays and novel biotargets, and ii) clinical translation, as well as a third session of presentations from young investigators, which focused on recent achievements within Fondazione IRCCS INT on immunotherapy and targeted therapies. Presentations in the first session addressed the issue of cancer immunotherapy activity with respect to tumor heterogeneity, with key topics addressing: 1) tumor heterogeneity and targeted therapy, with the definition of the evolutionary Index as an indicator of tumor heterogeneity in both space and time; 2) the analysis of cancer evolution, with the introduction of the TRACERx Consortium-a multi-million pound UK research project focused on non-small cell lung cancer (NSCLC); 3) the use of anti-estrogen agents to boost immune recognition of breast cancer cells; and 4) the high degree of functional plasticity within the NK cell repertoire, including the expansion of adaptive NK cells following viral challenges. The second session addressed: 1) the effectiveness of radiotherapy to enhance the proportion of patients responsive to immune-checkpoint blockers (ICBs); 2) the use of MDSC scores in selecting melanoma patients with high probability to be responsive to ICBs; and 3) the relevance of the gut microbiome as a predictive factor, and the potential of its perturbation in increasing the immune response rate to ICBs. Overall, a picture emerged of tumor heterogeneity as the main limitation that impairs the effectiveness of anti-cancer therapies. Thus, the choice of a specific therapy based on reproducible and selective predictive biomarkers is an urgent unmet clinical need that should be addressed in order to increase the proportion of long-term responding patients and to improve the sustainability of novel drugs.

Increased incidence of chronic GvHD and CMV disease in patients with vitamin D deficiency before allogeneic stem cell transplantation.

Vitamin D has emerged as a central player in the immune system, with its deficiency being implicated in the pathogenesis of several autoimmune diseases, including chronic GvHD. This is a retrospective cohort analysis of 166 patients, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) at the Karolinska University Hospital, evaluating GvHD, graft failure, infectious complications and survival after HSCT in relation to pre-transplantation vitamin D levels. Most of the patients were deficient in vitamin D before HSCT (median 42 nmol/L). In multivariate analysis, vitamin D level before HSCT was identified as a significant independent risk factor for development of cGvHD. The increased incidence of cGvHD was not coupled to better disease-free survival; instead there was a trend towards lower overall survival in the vitamin D-deficient patients. In addition, we found a significant correlation between vitamin D deficiency and incidence of CMV disease, with no case of CMV disease occurring in patients with sufficient levels of vitamin D before HSCT. Our results support a role of vitamin D in immune tolerance following HSCT. These findings could be highly relevant for the care of HSCT patients, and prospective, randomized studies on the effect of vitamin D supplementation are therefore needed.

Reduced DNAM-1 expression on bone marrow NK cells associated with impaired killing of CD34+ blasts in myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal stem-cell disorders characterized by ineffective hematopoiesis and risk of progression to acute myeloid leukemia. Increased apoptosis and suppressed functions of peripheral blood natural killer (NK) cells have been described in MDS patients, but only limited information is available on the phenotypic and functional integrity of NK cells in the bone marrow. In a cohort of 41 patients with distinct clinical subtypes of MDS, we here show that NK cells in the bone marrow show decreased surface expression of the activating receptors DNAM-1 and NKG2D. Notably, decreased receptor expression correlated with elevated bone marrow blast counts and was associated with impaired NK-cell responsiveness to stimulation with the K562 cell line, or co-activation by NKG2D or DNAM-1 in combination with the 2B4 receptor. Furthermore, antibody-masking experiments revealed a central role for DNAM-1 in NK cell-mediated killing of freshly isolated MDS blasts. Thus, given the emerging evidence for NK cell-mediated immune surveillance of neoplastic cells, we speculate that reduced expression of DNAM-1 on bone marrow NK cells may facilitate disease progression in patients with MDS.

Identification of a novel KIR3DL1*072 allele by sequence-based typing.

We report a novel KIR3DL1*072 allele that was found using a sequence-based typing approach.

The identification of a common pathogen-specific HLA class I A*0201-restricted cytotoxic T cell epitope encoded within the heat shock protein 65.

Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.

Inhibition of activated/memory (CD45RO(+)) T cells by oxidative stress associated with block of NF-kappaB activation.

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-gamma release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO(+)) T cells and correlated with a block in NF-kappaB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-gamma, TNF-alpha, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO(+), T cells found in the tumor milieu are functionally suppressed.

Identification of new HER2/neu-derived peptide epitopes that can elicit specific CTL against autologous and allogeneic carcinomas and melanomas.

Twenty-two new HLA-A2.1-binding peptides derived from the protooncogene HER2/neu were identified and analyzed for their capacity to elicit peptide and tumor-specific CTL responses. We used peptide-pulsed autologous DC from the ascites of patients with ovarian carcinomas to induce CTL. Of the 22 tested new HER2/neu-derived epitopes that could bind HLA-A2 with high (IC50 < 50 nM) or intermediate (50 nM < IC50 < 500 nM) affinity, we report the recognition by CTL of at least four novel epitopes, including HER2(9435), HER2(9665), HER2(9689), and HER2(10952), and confirm that of the known HER2 (9369) epitope. These epitopes were able to elicit CTL that specifically killed peptide-sensitized target cells and, most importantly, a HER2/neu-transfected cell line and the autologous tumor cells. We also confirm that HER2/neu is overexpressed in several melanoma lines, and as a new finding, report that some of these lines are sensitive to CTL induced by the HER2 (9369), HER2(9435), and HER2(9689) epitopes. Finally, CTL clones specific for HER2 (9369), HER2(9435), and HER2(9689) epitopes were isolated from tumor-specific CTL lines, further demonstrating the immunodominance of these epitopes. These findings broaden the potential application of HER2/neu-based immunotherapy.