Improving Multi-Epitope Long Peptide Vaccine Potency by Using a Strategy that Enhances CD4+ T Help in BALB/c Mice.

Peptide-based vaccines are attractive approaches for cancer immunotherapy; but the success of these vaccines in clinical trials have been limited. Our goal is to improve immune responses and anti-tumor effects against a synthetic, multi-epitope, long peptide from rat Her2/neu (rHer2/neu) using the help of CD4+ T cells and appropriate adjuvant in a mouse tumor model. Female BALB/c mice were vaccinated with P5+435 multi-epitope long peptide that presents epitopes for cytotoxic T lymphocytes (CTL) in combination with a universal Pan DR epitope (PADRE) or CpG-oligodeoxynucleotides (CpG-ODNs) as a Toll-like receptor agonist adjuvant. The results show that vaccination with the multi-epitope long peptide in combination with the PADRE peptide and CpG-ODN induced expansion of subpopulations of CD4+ and CD8+ cells producing IFN-γ, the average tumor size in the vaccinated mice was less than that of the other groups, and tumor growth was inhibited in 40% of the mice in the vaccinated group. The mean survival time was 82.6 ± 1.25 days in mice vaccinated with P5+435 + CpG+ PADRE. Our results demonstrate that inclusion of PADRE and CpG with the peptide vaccine enhanced significant tumor specific-immune responses in vaccinated mice

The percentages of IL-4-producing <i>CD4</i>+ T cells.

<p>Fourteen days after the last vaccination three mice per group were euthanized, and splenocytes were collected and characterized for CD4+ T cells using intracellular IL-4 staining followed by flowcytometry analysis. Data represent mean ± SEM (n = 3). ** denote significant differences from controls and all other groups, respectively.</p

Evaluation of the amount of IFN-γ produced in vaccinated mice.

<p>Nine BALB/c female mice per group were vaccinated three times subcutaneously with 100 μg/mouse of P₅₊₄₃₅ long peptide, P₅₊₄₃₅ in combination with CpG, or in combination with both 50 μg/mouse of PADRE and 25 μg/mouse of CpG. Two weeks after the last vaccination, splenocytes from three mice from each group were collected and activated with the long peptide. Immune responses were then determined using an IFN-γ ELISpot assay. The data indicate the mean ± SD. (n = 3). * denotes significant difference from all other groups (P < 0.001).</p

In vivo antitumor effects experiments.

<p>Six mice/group were immunized three times with P₅₊₄₃₅ long peptide alone, long peptide + CpG, or long peptide + PADRE + CpG. Control mice were immunized with PADRE, PADRE + CpG, or PBS. After 14 days the mice were challenged subcutaneously with 5× 10⁵ live TUBO cells. (A) Tumors were measured weekly and sizes were recorded. The values are means of tumor size and error bars indicate SD. (B) The survival times of the mice were analyzed by log-rank (P = 0.117) and Fleming-Harrington tests (P = 0.058) for 80 days. * denotes significant difference from PBS (P < 0.05).</p

The percentages of IFN-γ producing CD4+ and CD8+ T cells.

<p>Flow cytometry data were also analyzed according to the percentage of cytokine-producing cells and dot plots were drawn for each vaccinated group. Quadrants showing dot plot of CD8 and CD4 cells producing IFN-γ percentage. Spleen cells were analyzed using a gating strategy to exclude debris and identify CD4+ and CD8+ T cells. The subsequent analysis was on CD8+ or CD4+ gates to describe IFN-γ-producing T cells.</p

Catalase-gold nanoaggregates manipulate the tumor microenvironment and enhance the effect of low-dose radiation therapy by reducing hypoxia

Radiotherapy as a standard method for cancer treatment faces tumor recurrence and antitumoral unresponsiveness. Suppressive tumor microenvironment (TME) and hypoxia are significant challenges affecting efficacy of radiotherapy. Herein, a versatile method is introduced for the preparation of pH-sensitive catalase-gold cross-linked nanoaggregate (Au@CAT) having acceptable stability and selective activity in tumor microenvironment. Combining Au@CAT with low-dose radiotherapy enhanced radiotherapy effects via polarizing protumoral immune cells to the antitumoral landscape. This therapeutic approach also attenuated hypoxia, confirmed by downregulating hypoxia hallmarks, such as hypoxia-inducible factor α-subunits (HIF-α), vascular endothelial growth factor (VEGF), and EGF. Catalase stability against protease digestion was improved significantly in Au@CAT compared to the free catalase. Moreover, minimal toxicity of Au@CAT on normal cells and increased reactive oxygen species (ROS) were confirmed in vitro compared with radiotherapy. Using the nanoaggregates combined with radiotherapy led to a significant reduction of immunosuppressive infiltrating cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T-regs) compared to the other groups. While, this combined therapy could significantly increase the frequency of CD8+ cells as well as M1 to M2 macrophages (MQs) ratio. The combination therapy also reduced the tumor size and increased survival rate in mice models of colorectal cancer (CRC). Our results indicate that this innovative nanocomposite could be an excellent system for catalase delivery, manipulating the TME and providing a potential therapeutic strategy for treating CRC