Oncodriver inhibition and CD4+ Th1 cytokines cooperate through Stat1 activation to induce tumor senescence and apoptosis in HER2+ and triple negative breast cancer: implications for combining immune and targeted therapies

In patients with HER2-expressing breast cancer many develop resistance to HER2 targeted therapies. We show that high and intermediate HER2-expressing cancer cell lines are driven toward apoptosis and tumor senescence when treated with either CD4+ Th1 cells, or Th1 cytokines TNF-α and IFN-γ, in a dose dependent manner. Depletion of HER2 activity by either siRNA or trastuzumab and pertuzumab, and subsequent treatment with either anti-HER2 Th1 cells or TNF-α and IFN-γ resulted in synergistic increased tumor senescence and apoptosis in cells both sensitive and cells resistant to trastuzumab which was inhibited by neutralizing anti-TNF-α and IFN-γ. Th1 cytokines induced minimal senescence or apoptosis in triple negative breast cancer cells (TNBC); however, inhibition of EGFR in combination with Th1 cytokines sensitized those cells causing both senescence and apoptosis. TNF-α and IFN-γ led to increased Stat1 phosphorylation through serine and tyrosine sites and a compensatory reduction in Stat3 activation. Single agent IFN-γ enhanced Stat1 phosphorylation on tyrosine 701 and similar effects were observed in combination with TNF-α and EGFR inhibition. These results demonstrate Th1 cytokines and antioncodriver blockade cooperate in causing tumor senescence and apoptosis in TNBC and HER2-expressing breast cancer, suggesting these combinations could be explored as non-cross-reactive therapy preventing recurrence in breast cancer.Fil: Rosemblit, Cinthia. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Datta, Jashodeep. University of Pennsylvania; Estados UnidosFil: Lowenfeld, Lea. University of Pennsylvania; Estados UnidosFil: Xu, Shuwen. University of Pennsylvania; Estados UnidosFil: Basu, Amrita. H. Lee Moffitt Cancer Center; Estados UnidosFil: Kodumudi, Krithika. H. Lee Moffitt Cancer Center; Estados UnidosFil: Wiener, Doris. H. Lee Moffitt Cancer Center; Estados UnidosFil: Czerniecki, Brian J.. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unido

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Aggregatibacter actinomycetemcomitans modulates toll-like receptors 2 and 4 in gingival epithelial cells in experimental periodontitis

Background: Periodontitis is a common bacterial infection precipitated by exaggerated host responses to the oral microorganisms, As the first cells to encounter the oral pathogens the gingival epithelial cells (GEC) respond via toll-like receptors (TLR) that recognize conserved microbial patterns. Here we investigated the expression of TLR-2 and TLR-4 in GEC of naοve mice in response to infection with the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). Methods: 7-9 weeks old mice were induced experimental periodontitis by inoculating the palatal gingival with 1x109 colony forming units of Aa. Mice were sacrificed 50 days later and intact maxilla harvested. The degree of alveolar bone loss was determined by micro-CT. Single suspensions of the epithelial cells isolated from palatal gingival tissues were assessed for the expressions of TLR-2 and TLR-4 proteins by flow cytometry and for TLR-2 and TLR-4 mRNA by reverse transcriptase PCR. Differences between the control and the disease groups were determined by students′t- test. Results: Both TLR-2 and TLR-4 proteins were significantly elevated in the GEC of mice infected with A. actinomycetemcomitans when compared to the control group. The ratio of TLR-4 to TLR-2 mRNA was upregulated in chornic periodontitis as compared with the control group. Conclusion: Our data suggest that TLR-2 and TLR-4 are regulated differentially in A. actinomycetemcomitans-induced periodontitis

Intralesional injection of rose bengal induces a systemic tumor-specific immune response in murine models of melanoma and breast cancer.

Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma. To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions. In a murine model of melanoma, B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases. Anti-tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10. Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models. In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed. Transfer of T cells isolated from tumor-bearing mice treated with IL PV-10 led to tumor regression in mice bearing B16 melanoma. These studies establish that IL PV-10 therapy induces tumor-specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies

Immune Checkpoint Blockade to Improve Tumor Infiltrating Lymphocytes for Adoptive Cell Therapy.

Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function

Expression of immune checkpoint receptors on MC-38 and B16 TIL.

<p>A&B) Flow cytometry analysis of PD-1, CTLA-4, BTLA, and LAG-3 expression on MC-38 (A) and B16 (B) TIL; C) PD-L1 expression on MC-38 tumor cells; D) fresh or cultured MC-38 TIL were co-cultured with MC-38 or irrelevant B16 tumor cells and incubated for 48 hours at 37°C. The number of IFN-γ producing cells in response to stimulation was evaluated in an ELISPOT assay. The number of spots was counted in triplicates and calculated using an automatic ELISPOT counter; E) TIL was cultured with ⁵¹Cr labeled MC-38 or B16 tumor cells at 50:1 and 25:1 effector to target ratios. After 5 hours, the percentage of specific ⁵¹Cr release was determined by the following equation: (experimental cpm − spontaneous cpm)/ (total cpm incorporated − spontaneous cpm) × 100. All determinations were done in triplicate, and the SE of all assays was calculated and was typically 5% of the mean or less.</p

Inhibitory immune checkpoint blockade delays tumor growth in MC-38 tumor bearing mice.

<p>A total of 1x10⁵ MC-38 colon cancer tumor cells were injected subcutaneously (s.c.) in C57BL/6 mice. Three days later, mice were injected with 300 ug of monoclonal antibodies (isotype, anti-PD-1, anti-PD-L1, anti-Lag-3, anti-CTLA-4 or anti-BTLA) intraperitoneally (i.p.). Mice continued to receive this treatment every 3–4 days until the tumor reached a size of 400mm². Tumor size was measured and recorded every two days. In another set of experiments, mice were euthanatized at day 21 after tumor injection. Tumors were harvested for <i>in vitro</i> assays A) Tumor growth B) Flow cytometry analysis of T cells infiltrating tumors after checkpoint blockade.</p