Membrane-attached Cytokines Expressed by mRNA Electroporation Act as Potent T-Cell Adjuvants

Abstract

Proinflammatory cytokines are widely explored in different adoptive cell therapy protocols for enhancing survival and function of the transferred T cells, but their systemic administration is often associated with severe toxicity which limits their clinical use. To confine cytokine availability to the therapeutic T cells, we expressed 3 key cytokines, IL-2, IL-12, and IL-15, as integral T-cell membrane proteins. To prevent permanent activation of growth signaling pathways, we delivered these genes to T cells through mRNA electroporation. The engineered cytokines could be detected on the surface of mRNA-transfected cells and binding to their cell-surface receptors mainly occurred in cis. The 3 human cytokines supported the ex vivo growth of activated human CD8 and CD4 T cells for at least 6 days posttransfection, comparably to high-dose soluble IL-2. Similarly, membrane IL-2, membrane IL-12, and, to a lesser extent, membrane IL-15, were comparable with their soluble counterparts in supporting proliferation of splenic mouse CD8 T cells. Following electroporation of human CD8 T cells and antimelanoma tumor-infiltrating lymphocytes, membrane cytokines synergized with constitutively active toll-like receptor 4 in inducing interferon-γ secretion. Efficient cooperation with TLR4 was also evident in the upregulation of the activation molecules CD25, CD69, CD137 (4-1BB), and CD134 (OX40). Taken together, membrane cytokines expressed through mRNA transfection emerge as effective tools for enhancing T-cell proliferation and function and may have potential use in adoptive T-cell therapy.

Electrotransfer of Cytokine Genes for Cancer Treatment

“…For cancer treatment, studies have been done on various therapeutic cytokines such as interferons (IFN), interleukins (IL), growth factors and tumor necrosis factor family members. In vitro T cell function and proliferation is enhanced through the delivery of cytokine mRNA by electroporation (Weinstein-Marom et al, 2016). For adoptive cell cancer therapy, these findings may have potential.…”

The Emerging Role of In Vitro-Transcribed mRNA in Adoptive T Cell Immunotherapy

“…Recently, improvement in TIL degranulation through transient expression of membranebound cytokines was reported. 26 The IVT mRNA encoding membrane-bound interleukin (IL)-2, IL-12, and IL-15 were electroporated into human CD8 + T cells and melanoma TILs. Membrane-linked cytokines bound to the corresponding receptors on the T cells in cis, thus providing a localized stimulatory effect to those T cells only and not to nearby untransfected T cells.…”

Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes

“…Although transient, mRNA transfection can drive functional expression of the introduced genes up to 5-7 days and more. [32][33][34][35][36] The use of mRNA entirely obviates the risk of cellular transformation and allows the co-introduction of several genes as pre-defined mixtures, which is often limited with other gene delivery vehicles.…”