Eosinophil accumulation predicts response to melanoma treatment with immune checkpoint inhibitors.

Eosinophils have been identified as a prognostic marker in immunotherapy of melanoma and suggested to contribute to anti-tumor host defense. However, the influence of immune checkpoint inhibitors (ICI) on the eosinophil population is poorly studied. Here, we applied routine laboratory tests, multicolor flow cytometry, RNA microarray analysis, and bio-plex assay to analyze circulating eosinophils and related serum inflammatory factors in 32 patients treated with pembrolizumab or the combination of nivolumab and ipilimumab. We demonstrated that clinical responses to ICI treatment were associated with an eosinophil accumulation in the peripheral blood. Moreover, immunotherapy led to the alteration of the eosinophil genetic and activation profile. Elevated serum concentrations of IL-16 during ICI treatment were found to be associated with increased frequencies of eosinophils in the peripheral blood. Using immunohistochemistry, we observed an enhanced eosinophil degranulation and a positive correlation between eosinophil and CD8+ T cell infiltration of tumor tissues from melanoma patients treated with ICI. Our findings highlight additional mechanisms of ICI effects and suggest the level of eosinophils as a novel predictive marker for melanoma patients who may benefit from this immunotherapy.

A universal anti-cancer vaccine: Chimeric invariant chain potentiates the inhibition of melanoma progression and the improvement of survival.

For many years, clinicians and scientists attempt to develop methods to stimulate the immune system to target malignant cells. Recent data suggest that effective cancer vaccination requires combination immunotherapies to overcome tumor immune evasion. Through presentation of both MHC-I and II molecules, DCs-based vaccine platforms are effective in generating detectable CD4 and CD8 T cell responses against tumor-associated antigens. Several platforms include DC transfection with mRNA of the desired tumor antigen. These DCs are then delivered to the host and elicit an immune response against the antigen of interest. We have recently established an mRNA genetic platform which induced specific CD8+ cytotoxic T cell response by DC vaccination against melanoma. In our study, an MHC-II mRNA DCs vaccine platform was developed to activate CD4+ T cells and to enhance the anti-tumor response. The invariant chain (Ii) was modified and the semi-peptide CLIP was replaced with an MHC-II binding peptide sequences of melanoma antigens. These chimeric MHC-II constructs are presented by DCs and induce proliferation of tumor specific CD4+ T cells. When administered in combination with the MHC-I platform into tumor bearing mice, these constructs were able to inhibit tumor growth, and improve mouse survival. Deciphering the immunological mechanism of action, we observed an efficient CTLs killing in addition to higher levels of Th1 and Th2 subsets in the groups immunized with a combination of the MHC-I and MHC-II constructs. These universal constructs can be applied in multiple combinations and offer an attractive opportunity to improve cancer treatment.

Optimized dendritic cell vaccination induces potent CD8 T cell responses and anti-tumor effects in transgenic mouse melanoma models.

Despite melanoma immunogenicity and remarkable therapeutic effects of negative immune checkpoint inhibitors, a significant fraction of patients does not respond to current treatments. This could be due to limitations in tumor immunogenicity and profound immunosuppression in the melanoma microenvironment. Moreover, insufficient tumor antigen processing and presentation by dendritic cells (DC) may hamper the development of tumor-specific T cells. Using two genetically engineered mouse melanoma models (RET and BRAFV600E transgenic mice), in which checkpoint inhibitor therapy alone is not efficacious, we performed proof-of-concept studies with an improved, multivalent DC vaccination strategy based on our recently developed genetic mRNA cancer vaccines. The in vivo expression of multiple chimeric MHC class I receptors allows a simultaneous presentation of several melanoma-associated shared antigens tyrosinase related protein (TRP)-1, tyrosinase, human glycoprotein 100 and TRP-2. The DC vaccine induced a significantly improved survival in both transgenic mouse models. Vaccinated melanoma-bearing mice displayed an increased CD8 T cell reactivity indicated by a higher IFN-γ production and an upregulation of activation marker expression along with an attenuated immunosuppressive pattern of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). The combination of DC vaccination with ultra-low doses of paclitaxel or anti-PD-1 antibodies resulted in further prolongation of mouse survival associated with a stronger reduction of MDSC and Treg immunosuppressive phenotype. Our data suggest that an improved multivalent DC vaccine based on shared tumor antigens induces potent anti-tumor effects and could be combined with checkpoint inhibitors or targeting immunosuppressive cells to further improve their therapeutic efficiency.

mRNA-based dendritic cell immunization improves survival in ret transgenic mouse melanoma model.

Malignant melanoma is characterized by a rapid progression, metastasis to distant organs and resistance to chemo and radiotherapy. Although melanoma is capable of eliciting an immune response, the disease progresses and the overall results of immunotherapeutic clinical studies are not satisfactory. Recently, we have developed a novel genetic platform for improving an induction of peptide-specific CD8(+) T cells by dendritic cell (DC) based on membrane-anchored ß2-microglobulin (ß2m) linked to a selected antigenic peptide at the N-terminus and to the cytosolic domain of TLR4 at the C-terminus. In vitro transcribed mRNA transfection of antigen-presenting cells (APCs) resulted in an efficient coupling of peptide presentation and cell activation. In this research, we utilize the chimeric platform to induce an immune response in ret transgenic mice that spontaneously develop malignant skin melanoma and to examine its effect on the overall survival of tumor-bearing mice. Following immunization with chimeric construct system, we observe a significantly prolonged survival of tumor-bearing mice as compared to the control group. Moreover, we see elevations in the frequency of CD62L(hi)CD44(hi) central and CD62L(lo)CD44(hi) effector memory CD8(+) T-cell subsets. Importantly, we do not observe any changes in frequencies of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the vaccinated groups. Our data suggest that this novel vaccination approach could be efficiently applied for the immunotherapy of malignant melanoma.