mRNA-transfected Dendritic Cells Expressing Polypeptides That Link MHC-I Presentation to Constitutive TLR4 Activation Confer Tumor Immunity

Abstract

Recently, we have developed a novel genetic platform for improving dendritic cell (DC) induction of peptide-specific CD8 T cells, based on membrane-anchored β2-microglobulin (β2m) linked to a selected antigenic peptide at its N-terminus and to the cytosolic domain of toll-like receptor (TLR)4 C-terminally. In vitro transcribed mRNA transfection of antigen presenting cells resulted in polypeptides that efficiently coupled peptide presentation to cellular activation. In the present study, we evaluated the immunogenicity of such constructs in mRNA-transfected immature murine bone marrow-derived DCs. We show that the encoded peptide β2m-TLR4 products were expressed at the cell surface up to 72 hours and stimulated the maturation of DCs. In vivo, these DCs prompted efficient peptide-specific T-cell activation and target cell killing which were superior to those induced by peptide-loaded, LPS-stimulated DCs. This superiority was also evident in the ability to protect mice from tumor progression following the administration of B16F10.9 melanoma cells and to inhibit the development of pre-established B16F10.9 tumors. Our results provide evidence that the products of two recombinant genes encoding for peptide-hβ2m-TLR4 and peptide-hβ2m-K(b) expressed from exogenous mRNA can cooperate to couple Major Histocompatibility Complex (MHC-I) peptide presentation to TLR-mediated signaling, offering a safe, economical and highly versatile genetic platform for a novel category of CTL-inducing vaccines.

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

“…20 Moreover, we observed an increased antitumor immune reactivity indicated by an elevated cytokine production by effector CD8 C T cells and their enhanced cytotoxic activity in in vivo killing assay as compared to peptide-loaded mature BMDCs. 20 This system allows continuous peptide presentation and improves the prospects of peptide-bearing DCs to enter the LNs and encounter peptide-specific naive T cells. Moreover, by using mRNA rather than cDNA as gene carrier, we can prevent the development of long-term toxic effects arising from continuous TLR4 activation.…”

“…The use of both anchors, and not only one, was due to our previous data showing that although the K b anchored peptides are more stable on the cell surface, the TLR4 anchor is important for transmitting DC maturation signals. 20 We utilize the recombinant bi-functional b2m-based polypeptides system for the immunization of melanoma-bearing mice. The vaccination by a single peptide (derived from gp100 or TRP-2) with both anchors (K b and TLR4) is designated as a double construct.…”

“…Moreover, by using mRNA rather than cDNA as gene carrier, we can prevent the development of long-term toxic effects arising from continuous TLR4 activation. 19,20 However, the ability of a transplantable model system to adequately predict therapeutic responses in human patients is limited. In this study, we attempt to utilize our mRNA constructs system in ret transgenic mice that develop spontaneously skin tumors and metastases in the BM, lungs, liver and brain.…”

“…Procedure was performed as previously described by Cafri et al 20 Shortly, BMDCs cultured for 8 d, were washed twice with OptiMEM medium (GibcoBRL) and resuspended in 150 mL OptiMEM medium containing 20 mg transcribed mRNA and electroporated using BTX ECM 830 electroporator (BTX, Holliston, MA) 400 V, 0.9 ms, one pulse, using a 2 mm cuvette. Cells were resuspended in 5 mL growth medium and transferred into tissue culture plates for further incubation.…”

“…19 We also showed that these constructs can mediate an effective antitumor activity in the transplantable B16 melanoma model. 20 In this study, we utilize this chimeric mRNA construct system to examine whether DCs immunization will affect melanoma progression in a spontaneous ret transgenic mouse melanoma model that closely resembles human melanoma regarding histopathology and clinical development. 21,22 In addition, primary skin melanomas and metastases in lymph nodes (LNs) and distant organs of transgenic mice express similar MAAs to those expressed in human melanoma and B16 tumors (such as gp100, Tyr, TRP-1 and TRP-2).…”