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

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation.

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes

Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated

Self-renewal of CSCs and mutation detection.

<p><i>A</i>. Flow cytometric analysis of the CD133+/EpCAM+ fraction in xenograft tumors generated by CSCs and differentiated progeny. Data are shown as the CSC frequency representing the percentage of CD133+/EpCAM+ cells in tumors of two distinct origins (mean ± SEM, n = 3; unpaired, two-tailed student t-test, P<0.05). <i>B</i>. Re-implantation of tumor fragments obtained from CSCs or differentiated cell-derived xenograft tumors in secondary SCID-bg mice. Tumor volumes are shown as the mean ± SEM (n = 5). The difference in the growth curves between the two groups is statistically significant (difference in slope by linear regression; P<0.05). <i>C</i>. Cell proliferation of the primary cells isolated from either CSCs or differentiated cell-derived xenograft tumors in the stem-cell culture condition. Viable cells were plated at 20,000 cells per well and cultured for 17 days. Cell numbers were manually counted, and total cell counts are shown as the mean ± SEM (n = 6; unpaired, two-tailed student t-test, P<0.01). <i>D</i>. Spheroid CSC cultures re-established under the stem cell conditions from xenograft tumors generated by the serial implantation of CSC-derived xenograft tumors. P₂, CSCs cultured from xenograft tumors derived from the original CSCs. P₃, CSCs cultured from xenograft tumors derived from the P₂ CSCs. <i>E</i>. Partial MALDI-TOF mass spectrum showing the H1047R mutation in <i>PIK3CA</i>. The red dotted lines indicate, from left to right, the unextended primer peak, the 3 potential peaks for the mutant G or T alleles and the wild type A allele. The high peak in the middle of the figure is a T allele of <i>PDGFRA</i> in the same MALDI-TOF mass spectrum.</p

Culture of spheroid CSCs and identification of CD133+/EpCAM+ cells.

<p><i>A</i>, Schema illustrating the work flow of spheroid CSC generation and differentiated cell populations as an experimental system, including the transplantation of a patient tumor into NOD/SCID mice, propagation of CSCs from P₁ xenograft tumors (spheroid; 10× objective magnification), and differentiation of CSCs into adherent cells with epithelial morphology (differentiated; 20×). <i>B</i>. Flow cytometric analysis of the primary cells isolated from the original patient tumor. PI staining excludes the dead cells (a). APC- and PE-conjugated isotype controls are shown in (b). A population of CD133+/EpCAM+ cells was detected (c). <i>C</i>. FACS of CD133+/EpCAM+ colon CSCs from the primary cell population derived from P₁ xenograft tumors. Dead cells and murine cells were first excluded by PI staining and using an anti-mouse specific monoclonal antibody H-2k[d], respectively (a & b). CD133+/EpCAM+ and CD133−/EpCAM+ populations were gated according to the baselines of isotype controls and sorted (c). Finally, enrichment of both populations in the sorted samples was confirmed by flow cytometry (d & e). <i>D</i>. Expression of the CSC markers in a fraction of cultured spheroid CSCs (right panel). Left panel shows isotype controls.</p

Enhanced tumorigenic potential of CSCs and histology of xenograft tumor tissues.

<p><i>A</i>. Tumor volumes in the <i>in vivo</i> limiting dilution assay for CSCs and their differentiated progeny in SCID-bg mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067258#s2" target="_blank">Materials and Methods</a> (mean ± SEM; n = 5). Differences in the growth rates were found between the CSC and differentiated cell groups inoculated with higher cell numbers (1×10⁶ and 1×10⁵; slope difference in lineage regression; P<0.01). <i>B</i>. Tumor take rates after 63 days post-implantation for CSCs and differentiated progeny. <i>C</i>. H&E stained sections of the primary patient tumor (<i>a</i>) and xenograft tumors derived from CSCs (<i>b</i>) and differentiated cells (<i>c</i>; upper panel, 40×; lower panel, 4×). Signet-ring cells were frequently identified in both patient and CSC-driven tumors and are indicated by arrows (a and b at 40×). A dot plot shows the frequency of signet-ring cells identified in the patient tumor, the CSC-derived xenograft and the differentiated cells-derived xenograft (d).</p

Antiproliferative and antitumor activities of PF-04691502 through the modulation of the PI3K/mTOR pathway.

<p><i>A</i>. Representative dose-dependent responses of CSCs and differentiated cells after treatment with PF-04691502. Cell viability, as measured by CellTiter Glo® assay, is shown as the mean ± SEM (n = 5). <i>B</i>. Tumor growth inhibition induced by the treatment with PF-04691502 in SCID-bg mice in comparison with the vehicle controls. *P≤0.05; **P≤0.001 (two-way ANOVA analysis). <i>C.</i> Relative change in body weight of mice treated in the experiment (%). <i>D.</i> Western blot analysis of treated xenograft tumors. The expression levels of pAKT (S473), pERK (T202/T204), and the loading control α-tubulin are shown in four individual xenograft tumors in each treatment group. <i>E.</i> Quantitative data of the expression levels of pAKT (S473) relative to the loading controls (mean ± SEM; n = 4). F. CSCs were treated with PF-04691502 (502) at 0.1, 0.5 and 1 µM in vitro for 3, 24, and 48 hours. The changes in pAKT (S473) and pERK (T202/T204) were evaluated by Western Blot. GAPDH served as a loading control.</p

PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models

SummaryWe report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases

Enhanced tumorigenic potential of CD133+/EpCAM+ CSCs, CSC differentiation and drug resistance.

<p><i>A</i>. Average tumor volumes in NOD/SCID mice inoculated with CD133+/EpCAM+ (3,400 cell/animal; putative CSCs) or CD133−/EpCAM+ (25,000 cell/animal; putative differentiated) cells after 12 weeks of implantation (mean±SEM; n = 5). <i>B</i>. Percentage of CD133+/EpCAM+ expressing cells in the CSC and differentiated cell populations (mean ± SEM, n = 3; unpaired, two-tailed student t-test, P<0.05). <i>C</i>. Representative results of cell proliferation rates and expression levels of cytokeratin in CSCs and differentiated cells. Cell numbers on Y axes are adjusted as a percentage of the maximum cell numbers analyzed. <i>D</i>. Representative data showing the <i>in vitro</i> drug sensitivity of CSCs and their differentiated progeny in response to oxaliplatin as assessed by CellTiter Glo® assay.</p