Route of Administration of the TLR9 Agonist CpG Critically Determines the Efficacy of Cancer Immunotherapy in Mice

Contains fulltext : 81648.pdf (publisher's version ) (Open Access)BACKGROUND: The TLR9 agonist CpG is increasingly applied in preclinical and clinical studies as a therapeutic modality to enhance tumor immunity. The clinical application of CpG appears, however, less successful than would be predicted from animal studies. One reason might be the different administration routes applied in most mouse studies and clinical trials. We studied whether the efficacy of CpG as an adjuvant in cancer immunotherapy is dependent on the route of CpG administration, in particular when the tumor is destructed in situ. METHODOLOGY/PRINCIPAL FINDINGS: In situ tumor destruction techniques are minimally invasive therapeutic alternatives for the treatment of (nonresectable) solid tumors. In contrast to surgical resection, tumor destruction leads to the induction of weak but tumor-specific immunity that can be enhanced by coapplication of CpG. As in situ tumor destruction by cryosurgery creates an instant local release of antigens, we applied this model to study the efficacy of CpG to enhance antitumor immunity when administrated via different routes: peritumoral, intravenous, and subcutaneous but distant from the tumor. We show that peritumoral administration is superior in the activation of dendritic cells, induction of tumor-specific CTL, and long-lasting tumor protection. Although the intravenous and subcutaneous (at distant site) exposures are commonly used in clinical trials, they only provided partial protection or even failed to enhance antitumor responses as induced by cryosurgery alone. CONCLUSIONS/SIGNIFICANCE: CpG administration greatly enhances the efficacy of in situ tumor destruction techniques, provided that CpG is administered in close proximity of the released antigens. Hence, this study helps to provide directions to fully benefit from CpG as immune stimulant in a clinical setting

Spontaneous Regression of Ovarian Carcinoma After Septic Peritonitis; A Unique Case Report

Despite advances in therapy, ovarian cancer remains the most lethal gynecological malignancy and prognosis has not substantially improved over the past 3 decades. Immunotherapy is a promising new treatment option. However, the immunosuppressive cancer microenvironment must be overcome for immunotherapy to be successful. Here, we present a unique case of spontaneous regression of ovarian carcinoma after septic peritonitis. A 79-year-old woman was diagnosed with stage IIIc ovarian cancer. The omental cake biopsy was complicated by sepsis. Although the patient recovered, her physical condition did not allow further treatment for her ovarian cancer. After 6 months, spontaneous regression of the tumor was observed during surgery. Analysis of the immune infiltrate in the tissues showed a shift from a pro-tumorigenic to an anti-tumorigenic immune response after sepsis. Strong activation of the immune system during sepsis overruled the immunosuppressive tumor microenvironment and allowed for a potent anti-tumor immune response. More understanding of immunological responses in cases with cancer and septic peritonitis might be crucial to identify potential new targets for immunotherapy

Neoadjuvant immune checkpoint blockade in women with mismatch repair deficient endometrial cancer: a phase I study

Neoadjuvant immune checkpoint blockade (ICB) has shown unprecedented activity in mismatch repair deficient (MMRd) colorectal cancers, but its effectiveness in MMRd endometrial cancer (EC) remains unknown. In this investigator-driven, phase I, feasibility study (NCT04262089), 10 women with MMRd EC of any grade, planned for primary surgery, received two cycles of neoadjuvant pembrolizumab (200 mg IV) every three weeks. A pathologic response (primary objective) was observed in 5/10 patients, with 2 patients showing a major pathologic response. No patient achieved a complete pathologic response. A partial radiologic response (secondary objective) was observed in 3/10 patients, 5/10 patients had stable disease and 2/10 patients were non-evaluable on magnetic resonance imaging. All patients completed treatment without severe toxicity (exploratory objective). At median duration of follow-up of 22.5 months, two non-responders experienced disease recurrence. In-depth analysis of the loco-regional and systemic immune response (predefined exploratory objective) showed that monoclonal T cell expansion significantly correlated with treatment response. Tumour-draining lymph nodes displayed clonal overlap with intra-tumoural T cell expansion. All pre-specified endpoints, efficacy in terms of pathologic response as primary endpoint, radiologic response as secondary outcome and safety and tolerability as exploratory endpoint, were reached. Neoadjuvant ICB with pembrolizumab proved safe and induced pathologic, radiologic, and immunologic responses in MMRd EC, warranting further exploration of extended neoadjuvant treatment

Histone modification patterns associated with the human X chromosome

X inactivation is associated with chromosome-wide establishment of inactive chromatin. Although this is classically regarded as facultative heterochromatin that is uniform in nature, the exact distribution of associated epigenetic marks is not well defined. Here we have analysed histone modifications in human somatic cells within two selected regions of the X chromosome. Intergenic, coding and promoter regions are segregated into differentially marked chromatin. H3K27me3 is most prominent in intergenic and silenced coding regions, but is associated with some active coding regions as well. Histone H3/H4 acetylation and H3K4me3 are locally enriched at promoter regions but do not necessarily mark continuing transcription. Remarkably, H3K9me3 is predominant in coding regions of active genes, a phenomenon that is not restricted to the X chromosome. These results argue against the exclusiveness of individual marks to heterochromatin or euchromatin, but rather suggest that composite patterns of interdependent or mutually exclusive modifications together signal the gene expression status

Timing of i.v. CpG injections determines antitumor immunity.

<p>Established B16OVA tumors were treated with cryo ablation alone or in combination with i.v. CpG administration concurrent with ablation, or 1 day before or after ablation. Forty days later, naïve and treated tumor-free mice (7–10 mice per group) received a s.c. re-challenge with tumor cells (25.000–50.000 B16OVA cells) on the contra-lateral flank and survival was monitored. Representative of 2 experiments is shown.</p

Route of CpG administration affects T cell activation.

<p>(<b>A</b>) Percentages of IFN-γ⁺ T cells within the CD4⁺ or CD8⁺ populations after cryo ablation +/− CpG. B16-OVA tumor-bearing mice were treated with cryo ablation combined with CpG administration via the indicated routes. Seven days after ablation, spleen, tumor-draining lymph nodes and non-draining lymph nodes were isolated. Cells were stimulated with PMA/ionomycin and stained for CD8, CD4 and IFN-γ. * indicate significant differences (p<0.05) between the indicated CpG-treated group and the Naïve and Cryo group. (<b>B</b>) IFN-γ production after cryo ablation +/− CpG. Cells were stimulated with OVA for 96 hours and IFN-γ was measured in supernatant by ELISA. The mean levels of the Cryo+CpG i.v. groups in both organs are significantly different from all other groups (n = 6/group, 2 experiments).</p

Route of CpG administration determines induction of tumor-specific CTL.

<p>(<b>A</b>) Representative dot plots of CD8⁺OVA-Kb tetramer⁺ cells of individual mice. Mice bearing B16OVA tumors (6–9 mm) were subjected to cryo ablation alone or received additional CpG injections via the indicated routes. Ten days after tumor destruction, cells from spleen and tumor draining lymph nodes were isolated and re-stimulated with IFN-γ-treated γ-irradiated B16OVA tumor cells. Cultures were cleaned by a Ficoll step after 3–4 days of culture and at day 8–9 cells were analyzed for the presence of tumor-specific CTL using APC-labeled OVA-K_b tetramers. Cells were gated on CD8⁺ T cells. Data from one representative mouse per group is shown. (<b>B</b>) Quantitative analyses of collective data as shown in (A) (mean levels of 2 separate experiments (4–6 mice per group/experiment)). * indicates significant differences (p<0.05) of the indicated group compared to all other groups.</p

Quantity and quality of lymph node DC.

<p>(<b>A</b>) Absolute numbers of CD11c⁺ cells in the draining lymph nodes 2 days after ablation. Tumor-bearing mice were left untreated (NT) or were subjected to cryo ablation (cryo). In addition, some groups of mice received CpG via the indicated routes. Brackets indicate significant differences (p<0.05) between indicated groups (n = 4–6/group, 2 similar experiments). (<b>B</b>) Uptake of antigen after cryo ablation. OVA-Alexa488 was injected in B16OVA tumors (6–9 mm) just prior to cryo ablation to monitor the fate of antigen after in situ tumor destruction. Mice were additionally treated with CpG via the indicated routes. Two days after ablation, the uptake of antigen was analyzed in CD11c⁺ cells enriched from pools of tumor-draining lymph nodes. Numbers in the panels indicate the percentage of OVA⁺ of all CD11c⁺ cells. Data from one representative mouse is shown per group. (<b>C</b>) Quantitative analyses of replicates of experiments as shown in (B) (5 mice/group, representative of 2 experiments). (<b>D</b>) Uptake of antigen and CpG by CD11c⁺ cells monitored after p.t. and i.v. CpG-Cy5 administration and intra-tumoral injection of OVA-Alexa488. Data from one representative mouse is shown (n = 4/group). (<b>E</b>) The expression of CD80 on CD11c⁺ antigen-loaded cells. * indicates significantly different (p<0.05) from all other groups (n = 5/group, 2 similar experiments).</p