In vitro modelling of local gene therapy with IL-15/IL-15Rα and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4+ T cells.

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with αPD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and αPD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-γ. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15Rα (IL-15/IL-15Rα) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4+ T cells or CD8+ T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8+ T cells. Gene-based delivery of IL-15/IL-15Rα to tumor cells, shows expansion of NK cells, activation of NK cells, CD4+ and CD8+ T cells, and killing of tumor spheroids. Both NK cells and CD8+ T cells are necessary for tumor cell killing and CD4+ T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4+ or CD8+ T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4+ T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15Rα to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with αPD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.

An mRNA mix redirects dendritic cells towards an antiviral program, inducing anticancer cytotoxic stem cell and central memory CD8+ T cells.

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

A synthetic DNA template for fast manufacturing of versatile single epitope mRNA.

A flexible, affordable, and rapid vaccine platform is necessary to unlock the potential of personalized cancer vaccines in order to achieve full clinical efficiency. mRNA cancer vaccine manufacture relies on the rigid sequence design of multiepitope constructs produced by laborious bacterial cloning and time-consuming plasmid preparation. Here, we introduce a synthetic DNA template (SDT) assembly process, which allows cost- and time-efficient manufacturing of single (neo)epitope mRNA. We benchmarked SDT-derived mRNA against mRNA derived from a plasmid DNA template (PDT), showing that monocyte-derived dendritic cells (moDCs) electroporated with SDT-mRNA or PDT-mRNA, encoding HLA-I- or HLA-II-restricted (neo)epitopes, equally activated T cells that were modified to express the cognate T cell receptors. Furthermore, we validated the SDT-mRNA platform for neoepitope immunogenicity screening using the characterized HLA-A2-restricted neoepitope DHX40B and four new candidate HLA-A2-restricted melanoma neoepitopes. Finally, we compared SDT-mRNA with PDT-mRNA for vaccine development purposes. moDCs electroporated with mRNA encoding the HLA-A2-restricted, mutated Melan-A/Mart-1 epitope together with TriMix mRNA-generated high levels of functional Melan-A/Mart-1-specific CD8+ T cells. In conclusion, SDT single epitope mRNA can be manufactured in a more flexible, cost-efficient, and time-efficient way compared with PDT-mRNA, allowing prompt neoepitope immunogenicity screening, and might be exploited for the development of personalized cancer vaccines.

Formatting and gene-based delivery of a human PD-L1 single domain antibody for immune checkpoint blockade.

Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (PD-1) and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single-domain antibody fragment, that can enhance T cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12 GS (glycine-serine) linker, were 313- and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy.

Infrared imaging in breast cancer: automated tissue component recognition and spectral characterization of breast cancer cells as well as the tumor microenvironment.

Current evaluation of histological sections of breast cancer samples remains unsatisfactory. The search for new predictive and prognostic factors is ongoing. Infrared spectroscopy and its potential to probe tissues and cells at the molecular level without requirement for contrast agents could be an attractive tool for clinical and diagnostic analysis of breast cancer. In this study, we report the successful application of FTIR (Fourier transform infrared) imaging for breast tissue component characterization. We show that specific FTIR spectral signatures can be assigned to the major tissue components of breast tumor samples. We demonstrate that a tissue component classifier can be built based on a spectral database of well-annotated tissues and successfully validated on independent breast samples. We also demonstrate that spectral features can reveal subtle differences within a tissue component, capturing for instance lymphocytic and stromal activation. By investigating in parallel lymph nodes, tonsils and wound healing tissues, we prove the uniqueness of the signature of both lymphocytic infiltrate and tumor microenvironment in the breast disease context. Finally, we demonstrate that the biochemical information reflected in the epithelial spectra might be clinically relevant for the grading purpose, suggesting potential to improve breast cancer management in the future.

The effect of anticancer drugs on seven cell lines monitored by FTIR spectroscopy.

Systemic approaches such as metabolomics are increasingly needed to improve the development of novel drugs. In this paper, we suggest a new strategy based on infrared spectroscopy which probes the global chemical composition of a sample. Seven cell lines from three tumour types were investigated and exposed to four classical anticancer drugs belonging to two classes characterized by a unique mechanism. First, each cell line was considered separately and a hierarchical clustering was built for the seven cell lines. Spectra clustered according to the drug mechanism of action for all the cell lines tested. Second, the similarities among drug mechanism spectral fingerprints were investigated for all the cell lines simultaneously. Difference spectra (the mean spectrum of the corresponding untreated cell line was subtracted) were computed so that the particular contribution of every cell line was eliminated and only the drug-induced differences could be compared. The hierarchical clustering shows a clear tendency to distinguish the two modes of action, revealing a very similar type of response to molecules with a similar mechanism. High throughput systems with 96-well plates are now available and a well established bioassay could be developed in order to provide an objective classifier for potential anticancer drugs.