Syngeneic Murine Tumor Models Research Articles

Abstract 1149: Humanized knock-in mouse models for evaluating in vivo efficacy of immune-oncology drugs targeting inhibitory immune checkpoint molecules

Abstract Recently, increasingly attention has been paid to the development of immuno-oncology (IO) drugs, due to the promising clinical results acquired by CTLA-4, PD-1 and PD-L1 antibodies. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step. Mouse surrogate antibodies were often used in immune-competent mice with murine syngeneic tumor models to evaluate in vivo efficacy of the IO drugs. However, the efficacy of a surrogate antibody cannot fully represent the human drug in the clinical scenario. In most cases, a human monoclonal antibody does not have mouse cross-reactivity. Therefore, we generated humanized knock-in mouse to evaluate the in vivo efficacy of human IO antibodies. For example, human PD-1 knock-in (B-hPD-1) mice were generated with a chimeric PD-1 receptor, which is recognizable by human PD-1 antibodies, and can be used to test human PD-1 antibody in vivo efficacy. Mouse cancer cell lines including colon cancer MC38, melanoma B16F10 and lung cancer LLC1 were genetically modified with over-expression of hPD-L1 and knock-out of mPD-L1, and used to evaluate the in vivo efficacy of human PD-L1 antibodies. Additionally, more knock-in mice targeting inhibitory immune checkpoint molecules were developed, such as B-hPD-L1, B-hCTLA-4, B-hLAG3, B-hTIM3, B-hTIGIT, B-hBTLA, B-hSIRPa et al,. Humanized knock-in mice, pairing with genetically modified mouse cancer cells, represent a promising in vivo efficacy model for the development of IO drugs, and contribute to the clinical translation of animal studies. Citation Format: Baozhu Zhang, Chaoshe Guo, Yuelei Shen, Yanan Guo. Humanized knock-in mouse models for evaluating in vivo efficacy of immune-oncology drugs targeting inhibitory immune checkpoint molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1149.

Abstract 4569: The CDK4/6 inhibitor abemaciclib synergizes with PD-L1 blockade to induce an immune inflamed tumor microenvironment through T cell and tumor cell intrinsic effects

Abstract The approval of abemaciclib and additional cyclin dependent kinases 4 & 6 (CDK4/6) inhibitors for the treatment of HR+ breast cancer has provided new therapeutic options to patients. As CDK4/6 inhibitors become part of the standard of care, combination strategies leveraging abemaciclib together with immunotherapy may represent an opportunity to extend benefit to more patients and additional cancers. Accordingly, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with PD-L1 blockade in immunocompetent murine syngeneic tumor models, and directly evaluated the tumor cell and immune cell intrinsic immunologic effect of abemaciclib in vitro. In vivo abemaciclib treatment of murine tumors (CT26, EMT6 and MC38) caused a dose-dependent delay in tumor growth and demonstrated the potential to induce complete tumor regression (CR ~10%). Combination with an anti-PD-L1 antibody after abemaciclib pretreatment greatly enhanced the anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy resulted in 50-60% CRs of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice maintaining CRs after cessation of combination therapy or abemaciclib monotherapy resisted later CT26 rechallenge, demonstrating the ability to generate immunological memory during abemaciclib therapy. Analysis of intra-tumor gene expression showed that abemaciclib monotherapy induced T cell activation and inflammation signatures. Combination therapy substantially enhanced this effect and was additionally associated with DC maturation, antigen presentation, cytokine signaling and helper T cell phenotype. Suppression of cell cycle genes indicative of inhibition of CDK4/6 was also more prominent during the combination therapy. In Jurkat and primary human T cells, treatment with abemaciclib in vitro resulted in a dose-dependent increase in NFAT activity upon TCR stimulation. This correlated with upregulation of both cell surface markers and genes associated with an enhanced T cell activation phenotype, while only modestly affecting T cell expansion. Abemaciclib also amplified expression of antigen presentation and other immune-related genes in human breast cancer cells. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results provide a strong rationale for combining abemaciclib with checkpoint immunotherapy to improve the anti-tumor efficacy. The T cell and tumor cell intrinsic effects, synergistic anti-tumor responses and intra-tumor immune activation, justify clinical investigation of this combination. Citation Format: David Schaer, Richard Beckmann, Jack Dempsey, Lysiane Huber, Amelie Forest, Nelusha Amaladas, Ying Cindy Wang, Erik Rasmussen, Darin Chin, Yanxia Li, Andrew Capen, Marianne Deroose, Carmine Carpenito, Xueqian Gong, Kirk Staschke, Linda Chung, Farhana Merzoug, Trent Stewart, Lacey Litchfield, Philip Iversen, Sean Buchanan, Alfonso de Dios, Ruslan Novosiadly, Michael Kalos. The CDK4/6 inhibitor abemaciclib synergizes with PD-L1 blockade to induce an immune inflamed tumor microenvironment through T cell and tumor cell intrinsic effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4569.

Abstract 2752: EGFR tyrosine kinase inhibitor limits tumor relapse through enhancing anti-tumor T cell responses

Abstract EGFR tyrosine kinase inhibitors (TKIs) have been clinically considered as the first line therapy for EGFR-driven tumor patients. Although the initial outcome of patients has significantly improved, almost all patients eventually relapse. Fully understanding the mechanisms of the anti-tumor effect of EGFR TKIs will help promote the clinical development of more efficient therapeutic strategies. The traditional view of the main mechanism is that the inhibitors can directly inhibit EGFR downstream signaling pathways to induce tumor cell death. Whether and how EGFR TKIs affect the host immune system is largely unknown. In current study, we have established syngeneic murine tumor models that are sensitive to EGFR TKIs treatment. To our surprise, we revealed that EGFR TKI treatment could control tumor growth and limit tumor relapse in T cells dependent manner. Mechanistically, EGFR TKI triggers type I IFN and its stimulation gene CXCL10 production through the Myd88 signaling pathway to enhance tumor specific T cells infiltration and activation. However, EGFR TKI cannot efficiently limit tumor relapse in advanced tumor models because of PD-L1 upregulation in the tumor microenvironment after treatment. We have observed that anti-PDL1 blockade can synergize with EGFR TKI to control advanced tumors. Our study will challenge the current regimen of EGFR TKI in clinic and provide new efficient therapeutic strategies for EGFR dependent cancer patients. Citation Format: Zhida Liu, Chuanhui Han, Aijun Shen, Chunbo Dong, Jian Qiao, Yang-Xin Fu. EGFR tyrosine kinase inhibitor limits tumor relapse through enhancing anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2752.

Abstract 4934: Immunostimulatory and oncolytic properties of rotavirus can overcome resistance to immune checkpoint blockade therapy

Abstract Immune checkpoint targeted therapies against PD-1, PD-L1 and CTLA-4 are currently revolutionizing cancer care. However, only a minority of patients generate objective tumor responses with these treatments. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors in order to overcome the resistance to immune checkpoint blockade therapy. Pattern recognition receptors (PRR) such as toll-like receptor agonists have been shown to overcome resistance to immune checkpoint targeted therapy in pre-clinical models. Besides their intrinsic ability to stimulate PRR, the oncolytic properties of common viruses can be exploited also for the priming of anti-tumor immune responses. Several oncolytic viruses are currently in clinical development for cancer immunotherapy. However the routine implementation of these therapies is limited by the ongoing regulations on GMOs. With the aim to analyze if anti-infectious vaccines can be used as a source or PRR agonists and/or oncolytic viruses, we infected different tumors cell lines with commercially available anti-infectious vaccines. We first confirmed that commercially available vaccines do have PRR agonist properties. More interestingly, we discovered that rotavirus vaccines also have oncolytic properties. These attenuated viruses can directly kill cancer cells with features of immunogenic cell death such as upregulation of calreticulin on dying cancer cells but doesn't have cytotoxicity on healthy primary fibroblasts. Moreover, they have pro-inflammatory properties and can activate the NF-KB pathway in a toll-like receptor and IRF3 independent manner. These in vitro biological properties translate into in vivo anti-tumor activity. Intra-tumoral (IT) rotavirus therapy has anti-tumor effects which are mainly immune mediated as demonstrated by their weaken activity in NSG mice. Interestingly, in immunocompetent syngeneic murine tumor models of neuroblastoma and lymphoma, IT rotavirus therapy can overcome resistance to immune checkpoint targeted therapy and in particular synergies with anti-CTLA4. This therapeutic effect relied on specific modifications of tumor immune infiltrates and immune activation pathways. IT rotavirus vaccines was associated to an increase of myeloid infiltrating cells expressing up-regulated level of CD86 in the tumor microenvironment, and upregulation of activation markers such as OX40/CD137 on T cells. Rotavirus vaccines are clinical grade products, including for children. Therefore, in situ immunization strategies with IT attenuated rotavirus can be implemented quickly in the clinic including in pediatric cancers. IT priming of the anti-tumor immunity with oncolytic and immunostimulatory rotavirus vaccines could be a feasible strategy to overcome resistance to anti-PD-1/anti-CTLA-4 therapy in patients with cancer.* co-last authors. Citation Format: Tala Shekarian, Stéphane Depil, Anne-Catherine Jallas, Christophe Bergeron, Christophe Caux, Aurélien Marabelle, Sandrine Valsesia-Wittmann. Immunostimulatory and oncolytic properties of rotavirus can overcome resistance to immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4934.

Abstract B46: Varying the inoculation site of mouse syngeneic tumors can result in aberrant inclusion of lymph node tissue at tumor harvest

Abstract In recent years, growing interest in immunotherapies for the treatment of cancer has led to increased reliance on murine syngeneic tumor models for preclinical study in immunocompetent animals. These studies typically examine changes in the quantities and phenotypes of tumor-infiltrating immune cells; thus, it is crucial to distinguish between these cells and extratumoral immune cells during tumor harvest. Here, we show that 4T1 breast tumors inoculated in mammary fat pads (MFP) numbers 4 or 9 (4/9) contained greater frequencies of B cells and naïve T cells than tumors inoculated in MFP numbers 5 or 10 (5/10). Immunohistochemical analysis for B-cell presence demonstrated that MFP 4/9 tumors encroached on, and often encapsulated, inguinal lymph nodes, such that lymph node tissue was unknowingly collected during tumor harvest. This prevented accurate enumeration and phenotyping of infiltrating immune cells by flow cytometry, as the majority of lymphocytes in the affected samples derived from lymph nodes, and had not infiltrated the tumor per se. We observed the same phenomenon in peritibial tumors using immunohistochemical analysis, which began to encapsulate popliteal lymph nodes at large tumor volumes (>1500 mm3). Our data highlight the importance of choosing an appropriate inoculation site for obtaining accurate and consistent results when evaluating immune cell infiltration in syngeneic tumor models. Citation Format: Renee Clift, Barbara Blouw, Susan Zimmerman, Sanna Rosengren, Curt Thompson, Benjamin Thompson. Varying the inoculation site of mouse syngeneic tumors can result in aberrant inclusion of lymph node tissue at tumor harvest [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B46.

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and invitro assays to investigate the impact of abemaciclib on Tcells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased Tcell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a Tcell inflamed phenotype, and enhanced cell cycle control. Invitro, treatment with abemaciclib resulted in increased activation of human Tcells andupregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate Tcell anti-tumor immunity.

The multi-receptor inhibitor axitinib reverses tumor-induced immunosuppression and potentiates treatment with immune-modulatory antibodies in preclinical murine models.

Cancer immunotherapies have significantly improved the prognosis of cancer patients. Despite the clinical success of targeting inhibitory checkpoint receptors, including PD-1 and/or CTLA-4 on T cells, only a minority of patients derive benefit from these therapies. New strategies to improve cancer immunotherapy are therefore needed. Combination therapy of checkpoint inhibitors with targeted agents has promisingly shown to increase the efficacy of immunotherapy. Here, we analyzed the immunomodulatory effects of the multi-receptor tyrosine kinase inhibitor axitinib and its efficacy in combination with immunotherapies. In different syngeneic murine tumor models, axitinib showed therapeutic efficacy that was not only mediated by VEGF-VEGFR inhibition, but also through the induction of anti-cancer immunity. Mechanistically, a significant reduction of immune-suppressive cells, including a decrease of tumor-promoting mast cells and tumor-associated macrophages was observed upon axitinib treatment. Inhibition of mast cells by axitinib as well as their experimental depletion led to reduced tumor growth. Of note, treatment with axitinib led to an improved T cell response, while the latter was pivotal for the therapeutic efficacy. Combination with immunecheckpoint inhibitors anti-PD-1 and anti-TIM-3 and/or agonistic engagement of the activating receptor CD137 resulted in a synergistic therapeutic efficacy. This demonstrates non-redundant immune activation induced by axitinib via modulation of myeloid and mast cells. These findings provide important mechanistic insights into axitinib-mediated anti-cancer immunity and provide rationale for clinical combinations of axitinib with different immunotherapeutic modalities.

Protoporphyrin IX (PpIX)-Coated Superparamagnetic Iron Oxide Nanoparticle (SPION) Nanoclusters for Magnetic Resonance Imaging and Photodynamic Therapy.

The ability to produce nanotherapeutics at large-scale with high drug loading efficiency, high drug loading capacity, high stability, and high potency is critical for clinical translation. However, many nanoparticle-based therapeutics under investigation suffer from complicated synthesis, poor reproducibility, low stability, and high cost. In this work, a simple method for preparing multifunctional nanoparticles is utilized that act as both a contrast agent for magnetic resonance imaging and a photosensitizer for photodynamic therapy for the treatment of cancer. In particular, the photosensitizer protoporphyrin IX (PpIX) is used to solubilize small nanoclusters of superparamagnetic iron oxide nanoparticles (SPIONs) without the use of any additional carrier materials. These nanoclusters are characterized with a high PpIX loading efficiency; a high loading capacity, stable behavior; high potency; and a synthetic approach that is amenable to large-scale production. In vivo studies of photodynamic therapy (PDT) efficacy show that the PpIX-coated SPION nanoclusters lead to a significant reduction in the growth rate of tumors in a syngeneic murine tumor model compared to both free PpIX and PpIX-loaded poly(ethylene glycol)-polycaprolactone micelles, even when injected at 1/8th the dose. These results suggest that the nanoclusters developed in this work can be a promising nanotherapeutic for clinical translation.

Abstract B013: Elevation of immune cell infiltration and interferon-stimulated gene expression is associated with niraparib treatment in murine syngeneic tumor models

Abstract Introduction: Niraparib is an orally available and selective poly (ADP-ribose) polymerase (PARP)-1/-2 inhibitor approved for maintenance treatment of patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer in complete or partial response to platinum-based chemotherapy. Here, we evaluated the effects of niraparib treatment on tumor-immune microenvironment and assessed the efficacy of niraparib in combination with anti-PD-1 therapy in mouse-derived syngeneic transplant models. Experimental Procedures: Mouse Apcmin/+ syngeneic skin squamous carcinoma model was established by subcutaneously transplanting tumor spontaneously developed from C57BL/6J-Apcmin mouse model into immunocompetent mice with similar genetic background. Niraparib was administered orally at 50mg/kg or 25mg/kg daily as single-agent or in combination with intraperitoneally administered anti-PD-1 antibody (RMP1-14) at 5mg/kg twice weekly after tumors reach 100-200mm3. The tumor growth was monitored twice per week post randomization. At the end of the study, tumors were collected for immunophenotyping and transcriptome profiling. Summary: Anti-PD-1 monotherapy demonstrated minimal effect in Apcmin/+ syngeneic tumors, which were sensitive to niraparib daily treatment at 50mg/kg. Upon niraparib treatment, numbers of infiltrating CD8+ and CD4+ cells were significantly elevated within intratumoral regions. Interferon response gene signatures were identified by gene set enrichment analysis (GSEA) as top differentially expressed gene sets upon niraparib treatment. Several immune-related transcripts were also found to be upregulated, including STING, PDL1, CXCL9, and CXCL10. The activation of STING pathway was further evaluated in BRCA-deficient MDA-MB-436 human triple-negative breast cancer cells, where levels of p-STING(Ser366) and p-TBK1(Ser172) were elevated following niraparib treatments. Consistent with the niraparib-induced changes in tumor microenvironments, combining niraparib treatment at suboptimal dose (25mg/kg) with anti-PD-1 antibody resulted in enhanced antitumor activity as compared to niraparib or anti-PD-1 antibody alone in Apcmin/+ syngeneic tumors. Conclusions: Niraparib treatment increased CD4+ and CD8+ immune cell infiltration and upregulated the expression of interferon-stimulated genes in Apcmin/+ syngeneic tumors. Elevated levels of STING transcription and phosphorylation were associated with niraparib treatment in niraparib-sensitive models. Niraparib and anti-PD-1 combination was well tolerated and demonstrated enhanced antitumor activities as compared to niraparib or anti-PD1 monotherapy. Citation Format: Sarah Wang, Kaiming Sun, Yonghong Xiao, Keith Mikule, Sridhar Ramaswamy, Jeffrey Hanke, Jing Yu Wang. Elevation of immune cell infiltration and interferon-stimulated gene expression is associated with niraparib treatment in murine syngeneic tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B013.

Abstract A168: The LSD1 specific inhibitor INCB059872 enhances the activity of mechanistically distinct immunotherapeutic agents in the syngeneic 4T1 mouse mammary tumor model

Abstract Despite the successes of immune checkpoint blockade for the treatment of a variety of cancers, effective combinatorial therapy strategies are needed to achieve more durable and complete clinical responses in patients. Pharmacologically inducing a more permissive tumor microenvironment to enhance patient responsiveness to immune modulatory therapies may offer a rational approach to address this medical need. In particular, targeting immune suppressive myeloid cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), and polymorphonuclear (PMN) cells in the tumor microenvironment may enhance the effectiveness of immune checkpoint blockade. Since MDSCs abundantly infiltrate syngeneic 4T1 mammary tumors compared with other commonly used syngeneic tumor models, this model was chosen for testing the hypothesis that the modulation of MDSC activity enhances antitumor activity driven by adaptive immunity. We have recently demonstrated (#4635, AACR 2017) that the selective Lysine Specific Demethylase 1 (LSD1) inhibitor, INCB059872, redirected myeloid differentiation toward monocyte/macrophages in vitro and in vivo and reduced the PMN-MDSC population in the syngeneic 4T1 murine mammary tumor model. The combination of INCB059872 and PD1/PDL1 axis blockade enhanced antitumor activity and was well tolerated in this model. In this study, we further tested if modulation of MDSCs with INCB059872 could enhance the effect of mechanistically distinct immunotherapeutic agents. The combination of INCB059872 with agonist anti-OX40 or anti-GITR T cell costimulatory monoclonal antibodies significantly augmented antitumor efficacy in the 4T1 model. These results consistently demonstrated that the inhibition of immune suppressive MDSCs increased antitumor activity of immune checkpoint modulatory monoclonal antibodies. Next, we tested the combination of INCB059872 with small-molecule inhibitors targeting the tumor microenvironment. The combination of INCB059872 with highly selective small-molecule immunotherapeutic inhibitors such as epacadostat (IDO1 inhibitor) and ruxolitinib (JAK1/JAK2 inhibitor) demonstrated similar marked increases in antitumor efficacy. The studies to understand mechanism of enhanced activity are under way. In summary, consistent with previous findings, the combination of INCB059872 with a variety of mechanistically distinct immunotherapeutic agents significantly enhanced antitumor efficacy in the syngeneic 4T1 murine mammary tumor model. These results strongly support the hypothesis that reshaping the tumor microenvironment by redirecting myeloid differentiation as a result of LSD1 inhibition enhances the responsiveness of the tumor microenvironment to immunotherapies, supporting the therapeutic rationale for the combination of an LSD1 inhibitor with various immunotherapeutic agents to improve clinical responses in cancer patients. Citation Format: Sang Hyun Lee, Melody Diamond, Antony Chadderton, Thomas Condamine, Huiqing Liu, Valerie Roman, Jin Lu, Yan Zhang, Maxim Soloviev, Chunhong He, Liangxing Wu, Holly Koblish, Timothy Burn, Andrew Combs, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. The LSD1 specific inhibitor INCB059872 enhances the activity of mechanistically distinct immunotherapeutic agents in the syngeneic 4T1 mouse mammary tumor model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A168.

Abstract B008: PD-1/PDL-1 blockade remarkably regulates abundance of gut commensal microbiota in a murine syngeneic tumor model

Abstract Microbiota has been linked to immune surveillance of tumor progression and tumor growth in cancer patients as well as in cancer mouse models. Recent research reveals that gut microbiota has the potential to act as a biomarker to predict responders or even as an alternative treatment approach. Therefore, several gut microbes discovered in this invention (e.g., Akkermansia, Adlercreutzia, Coprococcus) could possibly be used as a biomarker to predict PD-1 antibody efficacy or could combine with PD-1 antibody in preclinical and clinical settings. Considering the complexity of intestinal microorganisms, a larger panel or wider spectrum of microbiota species may be involved in this process. The current study was designed to explore the possibilities of identifying multiple strains of intestinal microbiota that may be involved in antitumor effects of immune-checkpoint inhibitors, using preclinical tumor models. Gut microbiota changes were determined before and after a series of anti-PD-1 mAb treatments had been administered. Sixteen-S (16S) rRNA-seq was performed using feces collected at 4 time points. The results indicate that significant antitumor activity was found to be associated with gut microbiota changes of a large panel of microbiota, including a few strains with robust upregulation or downregulation. Upon further characterization and confirmation, these findings could lead to identification of one or more microbiota strains that could have biomarker potentials for cancer immunotherapy. Citation Format: WenQing Yang, Jian Ding, Xiangchao Gu, Fei Chen, Juan Zhang, Qian Shi. PD-1/PDL-1 blockade remarkably regulates abundance of gut commensal microbiota in a murine syngeneic tumor model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B008.

Tumor Challenges in Immunotoxicity Testing.

Syngeneic murine tumor models have been widely used by researchers to assess changes in tumor susceptibility associated with exposure to toxicants. Two common tumor models used to define host resistance against transplanted tumors in vivo are EL4 mouse lymphoma cells (established from a lymphoma induced in a C57BL/6 mouse by 9,10-dimethyl-1,2-benzanthracene) and B16F10 mouse melanoma cells (derived through variant selection from a B16 melanoma arising spontaneously in C57BL/6 mice). While C57BL/6 mice are commonly used as the syngeneic host for these tumor models, other mouse strains such as B6C3F1 (C57BL/6×C3H) can also be used. Tumor challenge of the host can be done by subcutaneous (sc) or intravenous (iv) injection, depending upon whether the effects are to be examined on local tumor development or experimental/artificial metastasis. Materials and methodologies for injection of both tumor cell models are described in detail in the subsequent sections.

A nanoparticle-based approach to improve the outcome of cancer active immunotherapy with lipopolysaccharides

This study sought to develop a simple nanoparticle-based approach to enhance the efficiency and tolerability of lipopolysaccharide (LPS), a potent ligand of Toll-like Receptor 4 (TLR4), for immunotherapy in cancer. Despite holding promise within this context, the strong pro-inflammatory properties of LPS also account for its low tolerability given localized and systemic side effects, which restrict the administrable dosage. Herein, we investigated the effect of LPS decoration as a surface-active molecule on a polymeric matrix upon its efficiency and tolerability. The LPS-decorated nanoparticles (LPS-NP) were about 150 nm in size, with slightly negative zeta potential (about −15 mV) and acceptable LPS incorporation (about 70%). In vitro, the particles accounted for a higher induction of apoptosis in tumor cells cultured with murine splenocytes compared to LPS solution. When used for the treatment of a murine syngeneic colorectal tumor model, higher intratumoral deposition of the particle-bound LPS was observed. Furthermore, unlike LPS solution, which accounted for localized necrosis at high concentrations, treatment of tumor-bearing animals with equivalent doses of LPS-NP was well tolerated. We propose that the observed localized necrosis can be Shwartzman phenomenon, which, due to modulated 24-h post-injection systemic TNF-α and LPS concentrations, have been avoided in case of LPS-NP. This has in turn enhanced the therapeutic efficiency and enabled complete tumor regression at concentrations at which LPS solution was intolerable. The findings indicate that nanoparticles can serve as beyond carriers for the delivery of superficially decorated LPS molecules, but impact their overall efficiency and tolerability in cancer therapy.

Inhibition of PI4K IIIα radiosensitizes in human tumor xenograft and immune-competent syngeneic murine tumor model.

Phosphatidylinositol (PI) 4-kinase (PI4K) has emerged as a potential target for anti-cancer treatment. We recently reported that simeprevir, an anti-hepatitis C viral (HCV) agent, radiosensitized diverse human cancer cells by inhibiting PI4K IIIα in vitro. In this study, we investigated the radiosensitizing effect of simeprevir in an in vivo tumor xenograft model and the mechanism of its interaction. The immune modulatory effect of PI4K IIIα was evaluated in an immune-competent syngeneic murine tumor model.In in vivo xenograft models using BT474 breast cancer and U251 brain tumor cells, inhibition of PI4K IIIα induced by simeprevir combined with radiation significantly delayed tumor growth compared to either treatment alone. PI4K IIIα inhibition led to eversion of the epithelial-mesenchymal transition as suggested by decreased invasion/migration and vascular tube formation. Simeprevir down-regulated PI3Kδ expression and PI3Kδ inhibition using RNA interference radiosensitized breast cancer cells. PI4K IIIα inhibition enhanced the radiosensitizing effect of anti-programmed death-ligand 1 (PD-L1) and decreased the expression of PI3Kδ, phosphorylated-Akt, and PD-L1 in breast cancer cells co-cultured with human T-lymphocytes. The immune modulatory effect in vivo was evaluated in immune-competent syngeneic 4T1 murine tumor models. Simeprevir showed significant radiosensitizing effect and immune modulatory function by affecting the CD4(+)/CD8(+) ratio of tumor infiltrating lymphocytes.These findings suggest that targeting PI4K IIIα with an anti-HCV agent is a viable drug repositioning approach for enhancing the therapeutic efficacy of radiation therapy. The immune regulatory function of PI4K IIIα via modulation of PI3Kδ suggests a strategy for enhancing the radiosensitizing effect of immune checkpoint blockades.

Antitumor effect of oral cancer vaccine with Bifidobacterium delivering WT1 protein to gut immune system is superior to WT1 peptide vaccine

ABSTRACTDespite the revolutionary progress of immune checkpoint inhibitors (CPIs) for cancer immunotherapy, CPIs are effective only in a subset of patients. Combining CPIs and cancer vaccines to achieve better clinical outcomes is a reasonable approach since CPI enhances cancer vaccine-induced tumor-associated antigen (TAA) specific CTL. Among the various TAAs so far identified, WT1 protein is one of the most promising TAAs as a cancer vaccine target. Until now clinical trials of WT1 vaccine have demonstrated only modest clinical efficacy. These WT1 vaccines were based on peptides or dendritic cells (DCs), and there was no oral cancer vaccine. Recently, we developed a WT1 oral cancer vaccine using a recombinant Bifidobacterium displaying WT1 protein, which can efficiently deliver WT1 protein to the gut immune system, and we demonstrated that this oral cancer vaccine had a significant anti-tumor effect in a C1498-WT1 murine leukemia syngeneic tumor model. The WT1 protein displayed in this vaccine consists of about 70% of the WT1 amino acid sequence including multiple known CD4 and CD8 T-cell epitopes of WT1. In this commentary, we introduce our recent data indicating the superior anti-tumor effect of a WT1 oral cancer vaccine delivering WT1 protein to the gut immune system compared to a peptide vaccine.

Non-thermal plasma-treated solution demonstrates antitumor activity against pancreatic cancer cells in vitro and in vivo

Pancreatic cancer is associated with a high mortality rate. In advanced stage, patients often experience peritoneal carcinomatosis. Using a syngeneic murine pancreatic cancer cell tumor model, the effect of non-thermal plasma (NTP) on peritoneal metastatic lesions was studied. NTP generates reactive species of several kinds which have been proven to be of relevance in cancer. In vitro, exposure to both plasma and plasma-treated solution significantly decreased cell viability and proliferation of 6606PDA cancer cells, whereas mouse fibroblasts were less affected. Repeated intraperitoneal treatment of NTP-conditioned medium decreased tumor growth in vivo as determined by magnetic resonance imaging, leading to reduced tumor mass and improved median survival (61 vs 52 days; p < 0.024). Tumor nodes treated by NTP-conditioned medium demonstrated large areas of apoptosis with strongly inhibited cell proliferation. Contemporaneously, no systemic effects were found. Apoptosis was neither present in the liver nor in the gut. Also, the concentration of different cytokines in splenocytes or blood plasma as well as the distribution of various hematological parameters remained unchanged following treatment with NTP-conditioned medium. These results suggest an anticancer role of NTP-treated solutions with little to no systemic side effects being present, making NTP-treated solutions a potential complementary therapeutic option for advanced tumors.

Abstract 591: Efficacy of ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic tumor models alone and in combination with immune checkpoint inhibitors

Abstract ALKS 4230 is a novel immunotherapeutic agent being tested in an ongoing phase 1 study to evaluate safety and tolerability in the treatment of patients with refractory solid tumors. ALKS 4230 is a selective agonist of the intermediate-affinity interleukin 2 (IL-2) receptor that when administered to mice results in a significant increase in numbers of memory CD8+ T cells and NK cells with no expansion of CD4+ regulatory T cells (Tregs). The selectivity achieved by ALKS 4230 is due to the fact that the molecule is a fusion of circularly permuted IL-2 to the extracellular portion of the IL-2 receptor α, and the resulting fusion protein is sterically prevented from binding to the high-affinity IL-2 receptor expressed on Tregs. The efficacy of ALKS 4230 was compared to recombinant human IL-2 in a B16F10 lung tumor metastasis model. ALKS 4230 treatment resulted in dose-dependent reduction of lung tumor colonization, with 100% inhibition at the highest dose tested. In contrast, while IL-2 was able to reduce lung tumor colonization, the maximal level of inhibition achieved was 60-70% at multiple dose levels such that increasing doses did not result in greater inhibition. Thus, the activation and expansion of effector cells without expansion of Tregs in response to ALKS 4230 treatment correlates with its improved efficacy over IL-2, which non-selectively expands both effector cells and Tregs. The antitumor efficacy mediated by ALKS 4230 was further evaluated in several murine subcutaneous syngeneic tumor models, including B16F10, MC38 and EMT6. Treatment with ALKS 4230 or its murine ortholog resulted in inhibition of tumor growth and improved survival in multiple models. When dosed in combination with anti-CTLA-4 or anti-PD-1 antibodies, ALKS 4230 resulted in further improvement of tumor growth inhibition and survival. These results demonstrate the murine antitumor efficacy of ALKS 4230 alone and in combination with immune checkpoint inhibitors and support the ongoing clinical evaluation of ALKS 4230 as an immunotherapy for cancer. Citation Format: Heather C. Losey, Jared E. Lopes, Reginald L. Dean, Michael R. Huff, Rosemarie A. Moroso, Juan C. Alvarez. Efficacy of ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic tumor models alone and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 591. doi:10.1158/1538-7445.AM2017-591

Abstract 577: Antagonism of the co-inhibitory receptor BTLA enhances efficacy of anti-PD-1 treatment in murine syngeneic tumor models

Abstract Anti-PD1 therapies show a remarkable improvement in response over many standard of care regimens, but there is a significant need to further increase patient responses. Recent clinical studies show improved response rate when combining anti-PD1 and anti-CTLA4 therapies. This provides “proof of concept” that the combination of two immunotherapies can enhance efficacy. The goal of our work was to evaluate the anti-tumor efficacy of B and T lymphocyte attenuator (BTLA) antagonism with anti-PD1 therapy in preclinical mouse tumor models. BTLA is an inhibitory co-receptor that modulates T cell function and is a marker of “exhausted” T cells. The inhibitory signal mediated by BTLA is initiated following engagement with herpesvirus entry mediator (HVEM), a ubiquitous receptor that is highly expressed on malignant cells. One of the challenges to evaluating BTLA in mouse models is the presence of strain-specific allelic polymorphisms. These sequence differences occur adjacent to the HVEM binding sites and can interfere with the ability of certain antibodies to recognize BTLA in different strains of mice or block the HVEM-BTLA signaling. C57BL/6-specific and strain agnostic BTLA binding antibodies are commercially available, but none antagonize HVEM signaling through BTLA in BALB/c mice. To more comprehensively evaluate the anti-BTLA/anti-PD1 combination regimen we generated a first-in-class BALB/c strain polymorphism specific BTLA antagonist monoclonal antibody (40E4) and identified clone PJ196 as a C57BL/6 strain polymorphism specific antagonist. We show that 40E4 and PJ196 not only block BTLA:HVEM interaction as measured by cell ELISA, but also reverse HVEM-mediated suppression in primary T cells from the appropriate mouse strain. An effector function reduced mouse IgG1 (D265A) chimeric form of 40E4 was generated for in vivo use. In combination with anti-PD1 (mDX400), 40E4 mIgG1 (D265A) shows improved tumor growth inhibition (TGI) over mDX400 alone in preclinical subcutaneous mouse models of breast cancer (EMT6) and colon cancer (CT26). To show that anti-BTLA enhancement of the anti-tumor response to mDX400 is not unique to BALB/c mice, we tested the combination of a C57BL/6 BTLA strain-polymorphism specific monoclonal antibody (PJ196) with mDX400. Using the subcutaneous MB49 model of bladder cancer, we show that treatment with PJ196 and mDX400 significantly improved TGI over mDX400 alone. This is the first report evaluating a BTLA antagonist monoclonal antibody in preclinical mouse tumor models and the first report to show BTLA antagonism enhances the effect of anti-PD1 therapy. Citation Format: Nathan R. Miselis, Douglas Linn, Clifford Restaino, Toya Baral, Jane Xia, Roanna Ueda, Anandi Sawant, Jeanne Baker, Gopalan Raghunathan, Xinzhong Wang1, Edward Bowman, Selvakumar Sukumar. Antagonism of the co-inhibitory receptor BTLA enhances efficacy of anti-PD-1 treatment in murine syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 577. doi:10.1158/1538-7445.AM2017-577

Abstract 2089: Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models

Abstract The development of therapies targeting tumor angiogenesis, tumor driver gene alterations and tumor immune evasion has made tremendous advancement in improving overall survival. However, efficacy may be limited and resistance often develops rapidly when targeting a single axis of tumorigenesis. Therefore, it is worthwhile to explore rational combination of therapies based on tumor-specific features. Fruquintinib is a potent and selective oral VEGFR inhibitor currently in Phase III clinical trials for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). We report here the evaluation of anti-tumor effect of fruquintinib in preclinical animal tumor models in combination with therapies targeting tumor driver gene alterations such as EGFR and c-MET or with immune checkpoints. In NSCLC xenograft models with EGFR activation such as activating mutations, gene amplification or protein overexpression, fruquintinib plus an EGFR tyrosine kinase inhibitor such as gefitinib or theliatinib (HMPL-309) was found to be more efficacious than either monotherapy. For instance, in PC-9 subcutaneous tumor model carrying EGFR exon 19 deletion, single agent treatment with fruquintinib at 2 mg/kg and gefitinib at 5 mg/kg produced the tumor growth inhibition (TGI) of 58% and 63%, respectively, while the combination treatment resulted in a TGI of 100% and tumor regression was observed in 11 of 16 mice treated with combinational therapy. In multiple xenograft models derived from lung cancer or renal cell cancer with c-MET activation (amplification or over-expression), addition of fruquintinib to a c-MET inhibitor savolitinib (AZD6094, HMPL-504) also improved the tumor growth inhibition substantially. At the end of the efficacy studies, CD31 and phosphorylation of EGFR, c-MET, AKT and ERK were analyzed with immunohistochemistry and western blotting method in tumor tissues. The results suggested that the enhanced anti-tumor effect in combination therapy could be attributed to the simultaneous blockade of cell signaling in tumor cells (EGFR or c-MET) and VEGFR suppression in the tumor microenvironment. Up-regulation of the immune inhibitory checkpoints induced by VEGF is one of the important mechanisms for tumor cells to escape immune surveillance. In a syngeneic murine tumor model, co-administration of fruquintinib and anti-PD-L1 antibody was found to provide improved anti-tumor effect compared to fruquintinib or anti-PD-L1 single agent alone. Studies to understand the mechanism responsible for the combination effect are under way. All combinations with fruquintinib described above were well tolerated. The efficacy observed in these models suggested that simultaneous blockade of tumor angiogenesis and tumor cell signaling or immune evasion may be a promising approach in improving treatment outcomes. Citation Format: Yongxin Ren, Qiaoling Sun, Jingwen Long, Shiming Fan, Renxiang Tang, Wei Zhang, Xuelei Ge, Jianxing Tang, Linfang Wang, Dongxia Shi, Hongbo Chen, Min Cheng, Weiguo Qing, Weiguo Su. Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2089. doi:10.1158/1538-7445.AM2017-2089

Abstract 1668: Comprehensive 10 color flow cytometry analysis of the neuroblastoma intratumoral immune response using the murine syngeneic neuro-2a tumor model

Abstract Neuroblastoma is the most prevalent solid tumor in childhood and the most common tumor in infants less than 1 year of age. In spite of aggressive multi-modal therapeutic approaches, the 5-year survival rate in patients with high-risk neuroblastoma remains poor at &amp;lt;50%. Recent advances have focused on harnessing the immune system to combat neuroblastoma. Antibodies that abrogate the immune checkpoint signaling pathway such as anti-CTLA-4 and anti-PD-L1 suppress neuroblastoma growth in certain mouse models of disease. Furthermore, the dinutuximab antibody that targets ganglioside GD2 expressing cells has been demonstrated to trigger neuroblastoma regression using a mechanism that involves several immune subsets. Although evidence suggest the host immune response can be exploited to fight neuroblastoma, the key immune subsets, their relative contribution, and mechanism of action to suppress neuroblastoma growth have yet to be fully characterized. To this end, we used flow cytometry to immunophenotype the intratumoral immune response in the neuro-2a model of neuroblastoma. This was accomplished by developing two 10-color panels to quantify the proportions of various myeloid and T cell subsets respectively. Using the myeloid panel, our results showed that total neuro-2a tumor immune cells were predominantly myeloid lineage-derived (CD11b+). A combination of Ly-6G and Ly-6C antibodies demonstrated that myeloid-derived suppressor cells (MDSCs) comprised the bulk of the myeloid cells and were more heavily skewed towards the granulocytic phenotype versus monocytic. An MDSC exclusion gate and a combination of F4/80, MHC class II, and CD206 antibodies enabled the analysis of tumor-associated macrophage subsets. Our results revealed that neuro-2a tumors contained relatively few activated M1 macrophages, which are typically associated with anti-tumor responses, and instead were dominated with a pro-tumor M2 macrophage response. The T cell antibody panel revealed that CD4+ T cells outnumbered CD8+ T cells by 2-fold. Regulatory T cells (Tregs) in the CD4+ T cell fraction were identified by a combination of FoxP3 and CD25 antibodies. The frequency of Tregs varied but represented &amp;lt;50% of total CD4+ T cells. Finally, the activation state of CD8+ T cells was examined using Ki-67, CTLA-4, and PD-1 antibodies. Analysis demonstrated that the majority of CD8+ T cells in neuro-2a tumors were actively proliferating based on the expression of Ki-67. However, most of the proliferating CD8+ T cells also co-expressed PD-1 suggesting a loss of anti-tumor functionality and an exhausted phenotype. This study provides a comprehensive immune profile of neuro-2a tumors and supports a platform with which to test new single agent and combination therapies designed to treat neuroblastoma. Citation Format: David D. Draper, Matt Thayer, Alden Wong, Dan Saims, Scott C. Wise. Comprehensive 10 color flow cytometry analysis of the neuroblastoma intratumoral immune response using the murine syngeneic neuro-2a tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1668. doi:10.1158/1538-7445.AM2017-1668