David Vasquez-Dunddel

Myeloid-derived suppressor cells (MDSC) play a key immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). In this study, we characterized CD14+HLA-DR(-/lo) cells sorted from the tumors, draining lymph nodes, and peripheral blood of HNSCC patients. CD14+HLA-DR(-/lo) cells were phenotyped as CD11b+, CD33+, CD34+, arginase-I+, and ROS+. In all 3 compartments, they suppressed autologous, antigen-independent T cell proliferation in a differential manner. The abundance of MDSC correlated with stage, but did not correlate with previous treatment with radiation or subsites of HNSCC. Interestingly, MDSC from all 3 compartments showed high phosphorylated STAT3 levels that correlated with arginase-I expression levels and activity. Stattic, a STAT3-specific inhibitor, and STAT3-targeted siRNA abrogated MDSC’s suppressive function. Inhibition of STAT3 signaling also resulted in decreased arginase-I activity. Analysis of the human arginase-I promoter region showed multiple STAT3-binding elements, and ChIP demonstrated that phosphorylated STAT3 binds to multiple sites in the arginase-I promoter. Finally, rescue of arginase-I activity after STAT3 blockade restored MDSC’s suppressive function. Taken together, these results demonstrate that the suppressive function of arginase-I in both infiltrating and circulating MDSC is a downstream target of activated STAT3.

PURPOSE: Because toll-like receptor (TLR) agonists have been well characterized as dendritic cell (DC) activators, we hypothesized that the admixture of TLR4 agonist into a cellular vector could improve the antitumor response in vivo. EXPERIMENTAL DESIGN: Granulocyte macrophage colony stimulating factor secreting whole cell tumor cell vector (GVAX) was formulated with lipopolysaccharide (LPS), a TLR4 agonist, and its intratumoral therapeutic efficacy was tested in three different murine models. We utilized immunohistochemistry, fluorescence-activated cell sorting, enzyme-linked immunosorbent spot (ELISPOT), and in vivo CTL analysis to assess both local innate immune responses within the tumor tissue as well as the downstream generation of antitumor T-cell responses. RESULTS: Intratumoral treatment of LPS-absorbed GVAX showed efficacy in improving an antitumor response in vivo in comparison with GVAX alone. Improved antitumor efficacy of this novel admixture was not present in TLR4 signaling impaired mice. In the CT26 model, 40% to 60% of the mice showed regression of the transplanted tumor. When rechallenged with CT26 tumor cells, these mice proved to be immunized against the tumor. Tumors treated with TLR4 agonist-absorbed GVAX showed increased infiltrating CD4 and CD8 T cells as well as increased numbers of CD86(+) cells in the tumor tissue. Draining lymph nodes from the treated mice had enhanced number of activated CD86(+), MHCII(+), and CD80(+) DCs in comparison with GVAX alone and mock-treated groups. ELISPOT assay and in vivo CTL assay showed increased numbers of CTLs specific for the AH1 tumor antigen in mice treated with LPS-absorbed GVAX. CONCLUSION: TLR4 on antigen-presenting cells in the tumor microenvironment may be targeted by using cell-based vectors for improved antitumor response in vivo.

Abstract The blockade of immune checkpoints is a promising therapeutic avenue for cancer therapy, with durable objective responses observed in patients with a variety of solid tumors. Despite these successes, current animal models do not reliably identify targets with the greatest clinical potential, due in part to differences between human and murine immune systems. Hence, robust preclinical tools to test these drugs directly against human cancers in the context of a human immune system are required. Champions Oncology has now developed the ImmunoGraft, whereby two innovative technologies, the patient-derived xenograft and humanized mice (immunodeficient mice reconstituted with a human immune system), are combined in a single platform. Immune-compromised NOG (PrkdcscidIl2rgtm1Sug) mice were reconstituted (humanized) with human CD34+ cells and twelve weeks after humanization, mature human CD45+ cells comprised close to 50% of the leukocytes detected in the circulation and secondary lymphoid tissues of the now humanized animals. Growth of NSCLC tumors implanted into humanized NOG animals was comparable to those in non-humanized counterparts, and when humanized animals harboring NSCLC tumor implants were treated with ipilimumab, tumor growth was slowed, exhibiting marked regression. Systemic immune activation was observed in humanized mice following treatment with ipilimumab, characterized by robust proliferation of huCD3+ T cells and activated huCD4+ Th1 cells, as well as an increase in tumor-infiltrating huCD8+ cytotoxic T lymphocytes (all relative to vehicle controls). We have developed an innovative new preclinical cancer model, the ImmunoGraft, which will enable novel immunotherapeutic agents to be accurately evaluated for efficacy against virtually any cancer indication that can be engrafted. As a proof-of-concept of the utility of the ImmunoGraft, we demonstrated how effectively ipilimumab restricted the growth of NSCLC implants engrafted in humanized animals. This translational platform is, therefore, more reflective of the human tumor microenvironment (both immune and non-immune cell-based) and is the most clinically-relevant model for screening immune system-targeting therapies, as single agents or in combination with standard-of-care drugs and other immuno-modulators. The ImmunoGraft has strong potential to revolutionize the application of immunotherapy to both personalized oncology and translational drug discovery. Citation Format: Gilson Baia, David Vasquez-Dunddel, Daniel Ciznadija, David Sidransky, Amanda Katz, Keren Paz. A humanized mouse model for translational assessment of targeted immune checkpoint blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5031. doi:10.1158/1538-7445.AM2015-5031

Abstract Patient-derived xenograft (PDX) models maintain the complex intra-tumoral biology and heterogeneity of an intact malignancy, as well as the interplay with stromal components and other cells fluxing into the tumor environment. This intrinsic cross-talk between different elements of the tumor makes PDX models a superior tool for translational drug discovery research and personalized oncology studies. Champions PDX models were originally developed for personalizing cancer treatments through the different Champions clinical programs. These models accurately reflect the population of patients enrolling in clinical trials. We describe herein our extensive TumorBank of PDX models, a valuable resource for translational oncology research to predetermine target populations for intervention with novel therapeutics in specific cancer subtypes. Tumor tissue from over 950 patients with a variety of primary and metastatic solid malignancies, across all ages and ethnicities and encompassing both treatment-naïve and heavily-pretreated individuals, has been implanted into immunodeficient mice with successful engraftment observed in ∼72% of cases. Comprehensive and translational-relevant clinical annotations have been maintained for these PDX models, including patient demographics, disease stage, anatomic location, tumor grade and histology, and treatment history. Importantly, whole exome and RNA sequencing, tissue histopathology, and protein immunohistochemistry have all been applied to 297 of these models. Finally, 70 of the models were screened against the corresponding patient's treatment used in the clinic, demonstrating a sensitivity of 98%, specificity of 76%, positive predictive value of 89% and negative predictive value of 96%. This wealth of information can be accessed through the Champions TumorGraft Database. The combination of extensive molecular and clinical annotation, together with opportunities for unlimited prospective preclinical testing, makes Champions TumorBank a pioneering resource for pharmaceutical companies seeking to identify target populations for therapeutic intervention. Citation Format: Tin O. Khor, Ido Ben Zvi, Amanda Katz, David Vasquez-Dunddel, Ido Sloma, Daniel Ciznadija, David Sidransky, Keren Paz. A patient-centric repository of PDX models for translational oncology research. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3219. doi:10.1158/1538-7445.AM2015-3219