Volume 23, Issue 8, Pages 1391-1400 (August 2015) mRNA-transfected Dendritic Cells Expressing Polypeptides That Link MHC-I Presentation to Constitutive TLR4 Activation Confer Tumor Immunity  Gal Cafri, Adi Sharbi-Yunger, Esther Tzehoval, Zoya Alteber, Tamar Gross, Ezra Vadai, Alon Margalit, Gideon Gross, Lea Eisenbach  Molecular Therapy  Volume 23, Issue 8, Pages 1391-1400 (August 2015) DOI: 10.1038/mt.2015.90 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Schemes of the β2m-based bi-functional constructs. (a) Genetic design. Major restriction sites are shown. br, bridge; lead, leader peptide; li, linker peptide; pr, promoter; p, antigenic peptide. (b) Anticipated configuration of the polypeptide products with a linked antigenic peptide in the context of an MHC-I heavy (α) chain. (c) Designations and expected molecular weights of the different constructs generated and explored in this study. Molecular Therapy 2015 23, 1391-1400DOI: (10.1038/mt.2015.90) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Expression and presentation of the peptide-β2m-TLR4 constructs in BMDCs. (a,b) Flow cytometry analysis for the expression of hβ2m. BMDCs transfected with gpTLR4, gpMix, and gpKb (a) or trTLR4, trMix, and trKb were stained with anti-hβ2m mAb. Peptide-loaded or unloaded BMDCs served as a positive and negative control, respectively. Mean fluorescence intensity (MFI) values for each analysis are shown. (c) Three million BMDCs were electroporated with 5 μg mRNA of gpTLR4, gpMix, gpKb, or loaded with 30 μg/ml hgp10025–33 (gpDCs and gpMDCs) or remained untreated (DCs). At 6–96 hours postelectroporation, BUSA14 hybridoma cells were added for 12 hours. Cells were then lysed and β-Gal enzymatic activity was detected with CPRG. Representative results of one of two experiments) are presented as ΔOD (sample OD-background OD) measured after 3 hours. Presented are results obtained in one of the two independent experiments. Molecular Therapy 2015 23, 1391-1400DOI: (10.1038/mt.2015.90) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 mRNA encoding the TLR4 anchor activates BMDCs. (a) TLR4-encoding mRNA promotes secretion of cytokines and chemokines. Five million BMDCs were electroporated with gpTLR4 or gpKb mRNA. Nonstimulated (DCs) or LPS-stimulated BMDCs (MDCs) served as a negative and positive control, respectively. Cells were cultured and analyzed 24 hours later. Secreted proteins were detected by the Mouse Cytokine Array Panel A protein profiler Kit (R&D Systems). Results are presented as normalized spot density analysis of secreted cytokines and chemokines as calculated by LABQUANT software. (b) IL-12 levels were measured by ELISA. Molecular Therapy 2015 23, 1391-1400DOI: (10.1038/mt.2015.90) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 BMDCs expressingTLR4 anchor are potent inducers of CTLs. (a) BMDCs expressing TLR4 anchor induce Pmel-1 T-cell proliferation. Two and a half million BMDCs were electroporated with 6 μg gpMix and 3 μg gpKb mRNAs. Immature (gpDCs) and mature (gpMDCs) BMDCs were loaded with 30 μg/ml hgp10025–33 and served as a positive control. For negative controls we used unloaded (DCs) or OVA257–264-loaded cells (OvDCs). Six hours postelectroporation, cells were transferred to 96-well plates in triplicates. Pmel-1-derived CD8 T cells were added in the indicated ratios and cocultured for 48 hours. 3H-thymidine uptake was measured using a β counter. (b,c) Active vaccination with gpTLR4-expressing BMDCs induces potent peptide-specific CTLs in-vivo. BMDCs (5 × 105) were electroporated with gpTLR4, gpKb, or gpMix mRNA and mice were vaccinated three times in 7 days interval. In vivo CTL killing assay was performed 10 days later following the administration of peptide-loaded cells differentially labeled with CFSE to evaluate vaccine efficiency. (b) Flow cytometry analysis for the presence of CFSE-labeled, peptide-loaded target cells in the splenocytes of immunized mice. Depicted are the results for one mouse in each group (n = 4) in one out of two independent experiments. (c) Summary of one out of these two independent in vivo killing assays, which yielded essentially the same results. Molecular Therapy 2015 23, 1391-1400DOI: (10.1038/mt.2015.90) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 mRNA-transfected DCs induce effector-memory peptide-specific CD8+ T cells. Mice (n = 4) were vaccinated three times using DCs transfected with 2pTLR4, 2pMix, and 2pKb. 2pMDCs were used as a positive control and 2pIMDCs or DCs were used as negative controls. Ten days later, spleens were harvested. (a) Percentage of CD8+ effector-memory cells (CD62L- CD44+). (b) Percentage of CD4+ T regulatory cells (CD25+ FOXP3+). (c,d). Percentage of CD8+ T cells secreting IFN-γ and TNF-α following restimulation with TRP2180–188 (c) and hgp10025–33 (d). *P < 0.05; **P < 0.001; ***P < 0.0001. Molecular Therapy 2015 23, 1391-1400DOI: (10.1038/mt.2015.90) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Vaccination with BMDCs transfected with 2pTLR4 confers tumor protection. (a) Experimental design (n = 8). DCs were transfected with BMDCs electroporated with 2pTLR4, 2pMix, and 2pKb. 2pMDCs were used as a positive control. (b) Survival rates were monitored until experimental endpoint. Significantly statistical differences between 2pMix and 2pTLR4 as compared to PBS (Mantel-cox test, ** p=0.0025) were evident. C. Lung weights were measured 36 days post-tumor inoculation. The vertical dashed lines represent the mean lungs weight of each group. Significantly statistical differences between pTLR4 and pMix as compared to pMDCs (student t-test, ***P = 0.002 and P = 0.0019 respectively) were evident. Line indicating normal lung weight (NLW) is presented. Molecular Therapy 2015 23, 1391-1400DOI: (10.1038/mt.2015.90) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Prolonged survival in mice vaccinated with pTLR4/pKb mRNA mix. (a) Mice (n = 8) were inoculated intrafootpad with 1.5 × 105 B16-F10 cells. When tumor reached 8–10 mm in size, footpads were amputated. Mice were vaccinated starting 3 days later. For vaccinations, DCs were transfected with 2pTLR4, 2pMix, and 2pKb. 2pMDCs were used as a positive control. (b) Survival was monitored until day 45; significantly statistical differences between 2pMix and 2pMDCs (Mantel-cox test, *P = 0.038) were evident. Presented are results obtained in one of two independent experiments. Molecular Therapy 2015 23, 1391-1400DOI: (10.1038/mt.2015.90) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions