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

Mey, Wout de; Schrijver, Phaedra De; Autaers, Dorien; Pfitzer, Lena; Fant, Bruno; Locy, Hanne; Esprit, Arthur; Lybaert, Lien; Bogaert, Cedric; Verdonck, Magali; Thielemans, Kris; Breckpot, Karine; Franceschini, Lorenzo

doi:10.1016/j.omtn.2022.08.021

Cited by 11 publications

(6 citation statements)

References 78 publications

(123 reference statements)

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“…1C). Subsequently, we investigated the expression of PD-1, CD134, CD137 and CD16 on NK cells, CD4 + and CD8 + T cells, as these markers correlate with early immune cell activation 21,[25][26][27] . IL-15 significantly increased the expression of CD134 and CD137 on NK cells and the expression of PD-1 on T cells (Supplementary Fig.…”

Section: Results Il-15 Stimulates Tumor Cell Killing and Modulates Im...mentioning

confidence: 99%

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

Awad,

De Vlaeminck,

Meeus

et al. 2023

Sci Rep

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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.

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Section: Results Il-15 Stimulates Tumor Cell Killing and Modulates Im...mentioning

confidence: 99%

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

Awad,

De Vlaeminck,

Meeus

et al. 2023

Sci Rep

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“…Plasmid DNA was generated using the in-house developed plasmid pLMCT ( 61 ). gBlocks for the different inserts were purchased from IDT and cloned into pLMCT using the Gibson assembly kit™ (NEB) and XL2-Blue Ultracompetent Cells (Agilent).…”

Section: Methodsmentioning

confidence: 99%

“…Transfection of mRNA to moDCs was performed by electroporation as described in de Mey et al ( 61 ). In short cells were extensively washed in serum-free OptiMEM (Life Technologies, Belgium).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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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.

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“…Supernatants from the co-cultured cells were collected to quantify IFNγ in ELISA (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. An STD p53 mRNA was used as a positive control [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

“…Cells w sively washed in serum-free OptiMEM (Gibco-Thermo Fischer Scientific, Wal USA). The electroporation was performed in 200 µL of OptiMEM medium in a troporation cuvette (Cell Projects, Harrietsham, UK) using the following p square wave pulse, 500 V, 2 ms, 1 pulse for K562-A2 + cells; and square wave pu 5 ms, 1 pulse for T-cells, using the Gene Pulser Xcell device (Bio-Rad, Hercules, p53 TCRα and TCRβ-chain mRNA (5 µg each, 4/10 6 cells) was electroporated in cells [19]. T-mRNA with TAG (5 µg each, 2/10 6 cells) was electroporated in K56…”

Section: Transfection Of Mrna To K562-a2 + Cells and Cd8 T-cells By E...mentioning

confidence: 99%

Assessment of Human SARS CoV-2-Specific T-Cell Responses Elicited In Vitro by New Computationally Designed mRNA Immunogens (COVARNA)

Esteban,

Pastor-Quiñones,

Usero

et al. 2023

Vaccines

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The COVID-19 pandemic has brought significant changes and advances in the field of vaccination, including the implementation and widespread use of encapsidated mRNA vaccines in general healthcare practice. Here, we present two new mRNAs expressing antigenic parts of the SARS-CoV-2 spike protein and provide data supporting their functionality. The first mRNA, called RBD-mRNA, encodes a trimeric form of the virus spike protein receptor binding domain (RBD). The other mRNA, termed T-mRNA, codes for the relevant HLA I and II spike epitopes. The two mRNAs (COVARNA mRNAs) were designed to be used for delivery to cells in combination, with the RBD-mRNA being the primary source of antigen and the T-mRNA working as an enhancer of immunogenicity by supporting CD4 and CD8 T-cell activation. This innovative approach substantially differs from other available mRNA vaccines, which are largely directed to antibody production by the entire spike protein. In this study, we first show that both mRNAs are functionally transfected into human antigen-presenting cells (APCs). We obtained peripheral blood mononuclear cell (PBMC) samples from three groups of voluntary donors differing in their immunity against SARS-CoV-2: non-infected (naïve), infected-recovered (convalescent), and vaccinated. Using an established method of co-culturing autologous human dendritic cells (hDCs) with T-cells, we detected proliferation and cytokine secretion, thus demonstrating the ability of the COVARNA mRNAs to activate T-cells in an antigen-specific way. Interestingly, important differences in the intensity of the response between the infected-recovered (convalescent) and vaccinated donors were observed, with the levels of T-cell proliferation and cytokine secretion (IFNγ, IL-2R, and IL-13) being higher in the vaccinated group. In summary, our data support the further study of these mRNAs as a combined approach for future use as a vaccine.

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

Cited by 11 publications

References 78 publications

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

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

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

Assessment of Human SARS CoV-2-Specific T-Cell Responses Elicited In Vitro by New Computationally Designed mRNA Immunogens (COVARNA)

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