FCS was then added to stop the enzymatic action, and tumor tissues were transferred to ice

FCS was then added to stop the enzymatic action, and tumor tissues were transferred to ice. T cell responses were observed against model-, neo-, and self-antigens and were highly potent in several murine tumor models. The safety and the immunogenicity of a human vaccine candidate designed for colorectal cancer treatment was demonstrated in a nonhuman primate model. This therapeutic vaccine approach, which we believe to be newly engineered, is promising for the treatment of poorly infiltrated tumors that do Indobufen not respond to currently marketed immunotherapies. 0.05; ** 0.01 (unpaired test). (C) THP1-XBlue-MD2-CD14 cells Indobufen were incubated with various concentrations of vaccine constructs, medium, or buffer. After 18 hours, supernatants were recovered, and SEAP activity was measured by QUANTI-Blue assay (InvivoGen). The EC50 of the Z13Mad5Anaxa and Mad5Anaxa was calculated from the obtained dose-response curves using Prism software. (D) The binding of ATP125 to TLR4, TLR2, and TLR3 was measured by surface plasmon resonance analysis for different concentrations of ATP125: 100, 200, 300 (in duplicate), 400, and Indobufen 500 nM and sensorgrams were obtained. All curves indicate the response after subtraction of nonspecific binding of molecules to a control channel. A self-adjuvanted cancer vaccine platform eliciting CD8 and CD4 T cell immune responses. The constructs were then compared in vivo in an EG7 mouse thymoma model. When compared with EDAZ13Mad5, Z13Mad5Anaxa showed the strongest antitumor effect (Figure 2A); therefore, Anaxa was selected as CREB3L4 the optimal TLRag for the next steps. Importantly, the antitumor activity of the vaccine was decreased when CPP Z13 was removed (Mad5Anaxa construct) (Figure 2B and Supplemental Figure 1B) or replaced by a different ZEBRA-derived CPP (Z14Mad5Anaxa construct) (Supplemental Figure 1B). Concomitant administration of a TLR4 agonist (Monophosphoryl lipid A [MPLA]) or a TLR2 agonist (synthetic tripalmitoylated lipopeptide Pam3CysSerLys4 [Pam3CSK4]) with vaccine Z13Mad5 displays less efficacious antitumor activity than vaccination with the whole construct Z13Mad5Anaxa (Figure 2B and Supplemental Figure 1C). Open in a separate window Figure 2 Z13Mad5Anaxa showed the strongest antitumor effect.(A) Tumor growth curve of C57BL/6 mice (= 7 mice/group) implanted s.c. with EG7-OVA cells and vaccinated twice (day 5 and day 13) with EDAZ13Mad5 or Z13Mad5Anaxa proteins. Values are represented as the mean SEM. One experiment shown is representative of 2. * 0.05; **** 0.0001 (2-way ANOVA). (B) Tumor growth curve of C57BL/6 mice (= 7 to 14 mice/group) implanted s.c. with EG7-OVA cells and vaccinated twice (day 5 and day 13) with Z13Mad5Anaxa, Mad5Anaxa, or Z13Mad5 with MPLA. Values are represented as the mean SEM. A pool of 2 independent experiments is shown. * 0.05; ** 0.01, **** 0.0001 (2-way ANOVA). (C) Mice were vaccinated twice (day 0 and day 14) with different constructs with or without adjuvants. One week after the last vaccination, multimer staining was performed on blood cells for detecting OVA257-264Cspecific CD8 T cells. A pool of 3 independent experiments is shown (mean SEM, = 4 to 6 mice/group). * 0.05 (Kruskal-Wallis test). (D) Mice were vaccinated 3 times (day 0, day 14, day 28) with 2 different constructs. One week after the last vaccination, multimer staining was performed on blood cells for detecting OVA257C264-specific CD8 T cells (mean SEM, = 2 to 4 mice/group). * 0.05 (Kruskal-Wallis test). The immunogenicity of Z13Mad5Anaxa was also compared to Z13Mad5 administered with or without the previously described adjuvants MPLA or Pam3CSK4 or to Mad5 fused to keyhole limpet hemocyanin (15). Indobufen Z13Mad5Anaxa was as immunogenic as MPLA or Pam3CSK4-adjuvanted Z13Mad5 and superior to nonadjuvanted (Figure 2C) or keyhole limpet hemocyaninCconjugated vaccines in terms of circulating antigen-specific CD8 T cells (Figure 2D). Optimization of the vaccination conditions established that a vaccine dose from 2 nmol was able to elicit a potent CD8 T cell immune response (Supplemental Figure 2A), with either synthetic peptide or recombinant.