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Background The ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging. Methods Using flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models. Results This longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model. Conclusions Taken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.

Vast improvements in tumor treatment have been achieved with immune checkpoint inhibitors. They have been approved for use in monotherapy or combinations for patients with different types of cancer. However, the treatments often yield limited benefits and strong responses are observed only in a minority of treated patients. For this reason, enormous efforts have been made to explore primary or acquired resistance mechanisms to improve the available current therapies. CNTN4 (contactin 4, alias BIG-2) is a neuronal cell adhesion molecule, which is mainly expressed in the human brain and functions in axonal growth and guidance. Some genetic evidence has indicated that CNTN4 may be associated with non-neurological disorders such as cancer and cardiovascular conditions as well as neurodevelopmental disorders. Recently we identified CNTN4 as a novel immunomodulatory protein, which is expressed on tumors. In this study, we investigate the potential of CNTN4 as a cancer immunotherapeutic target, using GENA-104A07 to target it. GENA-104A07 is a humanized IgG4 monoclonal antibody that specifically binds to human and mouse CNTN4 with sub-nanomolar binding affinity by ELISA assay, not to any other related proteins having similar sequences. This molecule competitively interferes with the binding of CNTN4 and its receptor protein. The in vitro functional effect showed that the treatment of GENA-104A07 alleviates the immune suppressive effect induced by CNTN4 on T cells. Consequently, the administration of GENA-104A07 induces significant suppression of the tumor growth as monotherapy in syngeneic mouse models with a high expression level of CNTN4. GENA-104A07 treatment increases the expression of activation markers and pro-inflammatory cytokines on T cells. Taken together with in vitro and in vivo studies, the efficacy of GENA-104A07 demonstrated that CNTN4 blockage with monoclonal antibody may provide an immunotherapeutic strategy for human cancer. Currently, the preliminary safety study is ongoing in mice, and IND enabling studies will be initiated early next year with the optimized antibody. Citation Format: Mi Young Cha, Youn Kyung Houh, Yun Yeon Kim, Hyunuk Kim, Joo-Yeon Chung, Kyung Mi Park, Hansoo Park. Anti-CNTN4 antibody, GENA-104A07 suppresses tumor growth in murine syngeneic tumor models by regulating T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 688.

Introduction Inducible T cell co-stimulator (ICOS) is a co-stimulatory receptor that is important for promoting immune activation and function. Despite reported clinical activity and a wide range of non-clinical studies supporting a role for ICOS in lymphocyte activation, proliferation and pro-inflammatory cytokine secretion, little is known regarding the potential of monoclonal agonist antibody-mediated ICOS signaling to drive cytotoxic T cell infiltration into tumors. Feladilimab (GSK3359609) is a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials. Here, we used PET/CT imaging to evaluate CD8+ T cell infiltration following treatment with a rodent surrogate of feladilimab (7E.17G9 mouse [m] IgG1) alone or in combination with anti-PD-1 mAb (RMP-14 rat IgG2a) in a syngeneic model of breast cancer (EMT6). Method Female BALB/c mice with established tumors (~150 mm3) received 10µg, IP of either IgG control mAbs, ICOS mAb, or ICOS + PD-1 mAbs on day 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 & 48 hrs post IV dose of 89Zr labeled CD8 minibody (IAB42M1-14, ImaginAb, CA) on day 0, 3, 5, 9, or 14. In addition to the CD8 minibody uptake in tumor & tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images. Top ranked features were used for hierarchical clustering to identify treatment effects. Results Tumor size regressed in all treated groups relative to IgG control, with a number of mice clearing tumors (ICOS: 4 mice, ICOS + PD-1: 9 mice). The in vivo uptake of CD8 minibody in TDLN was significantly higher in the ICOS + PD-1 group on day 4, 6, & 7 relative to IgG control P Conclusions Herein, we demonstrated for the first time that treatment of tumor-bearing mice with ICOS agonist mAb alone or in combination with PD-1 blockade can increase CD8+ T cell infiltration into tumors & TDLN, and is correlated with reduced tumor burden. Notably, radiomics features predicted an effect of treatment on CD8+ T cell infiltration earlier than the detection of absolute changes in the CD8 minibody uptake in tumor & TDLN. Overall, these data support the ongoing pivotal investigation of feladilimab. Moreover, this translational imaging method may be a useful tool to non-invasively monitor CD8+ T cell in response to immunotherapies and understand the temporal relationship between CD8+ T cell flux in tumor and in TDLN. Citation Format: Hasan Alsaid, Shih-Hsun Cheng, Meixia Bi, Mary V. Rambo, Tinamarie Skedzielewski, Bao Hoang, Sunish Mohanan, Andrew Gehman, Chih-Yang Hsu, Minh Doan, Fang Xie, M. Reid Groseclose, Christopher Hopson, Sara Brett, Ian A. Wilson, Andrew Nicholls, Marc Ballas, Jeremy D. Waight, Beat M. Jucker, Axel Hoos. Immuno-PET monitoring of CD8+ T cell infiltration post anti-ICOS agonist antibody treatment alone and in combination with PD-1 blocking antibody using a 89Zr anti-CD8+ mouse minibody in EMT 6 syngeneic tumor mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2816.

Oncolytic virotherapy is an emerging treatment modality that may hold promise as a therapeutic option for cancer patients who do not respond to checkpoint inhibitors. Oncolytic viruses preferentially replicate in tumor cells, offer the ability to express transgenes and have been shown to modify the local and distant tumor microenvironments from immunosuppressive to immunostimulatory. We generated HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12, an oncolytic virus based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce murine FMS-like tyrosine kinase 3 ligand (mFLT3L) to enhance dendritic cell expansion and T cell priming, and murine interleukin-12 (mIL12) to potentiate type I helper and cytotoxic T cell responses. We confirmed the expression and bioactivity of mFLT3L and mIL12 in vitro and evaluated the efficacy of HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 alone and in combination with anti-PD-1 or anti-4-1BB treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used and only one tumor was treated via intratumoral injection with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12. Anti-tumor responses were evaluated in the injected tumor (local response), the uninjected tumor (abscopal) and via splenic immune profiling (systemic). HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 treatment alone led to tumor growth inhibition in both treated and contralateral tumors, and a significant increase in overall survival in the A20, Neuro2a and MC38 models. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with anti-PD-1 blocking antibody further improved tumor growth inhibition in both treated and contralateral tumors in the MC38 syngeneic model, and significantly increased median overall survival compared to either agent as a monotherapy. In addition, HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with an agonistic antibody targeting 4-1BB significantly increased overall survival compared to either agent as a monotherapy in the MC38 model. To understand antigen-specific anti-tumor T cell responses, MC38 tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell responses were measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 induced and/or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that treatment with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 can cause direct tumor lysis and induce an adaptive, systemic T cell-mediated anti-tumor immune response that can be further enhanced by concurrent PD-1 blockade or 4-1BB agonism in multiple syngeneic mouse tumor models. Citation Format: Keegan Cooke, Walter H. Meisen, Petia Mitchell, Juan Estrada, Jinghui Zhan, Jessica Orf, Peng Li, Christina de Zafra, Jing Qing, Jason DeVoss. Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1586.

Background: The tyrosine kinase inhibitor cabozantinib showed clinical activity in 1L RCC as well as RCC and HCC in 2L after treatment with anti-angiogenic therapy where it prolonged survival. Immune checkpoint inhibition (ICI) is rapidly becoming a 1L standard of care across tumor types. We evaluated the effectiveness of cabozantinib in a 2L post-ICI setting. Experiments focused on anti-tumor efficacy of cabozantinib ± ICI after ICI in one of several syngeneic mouse tumor models partly refractory to ICI. Cabozantinib monotherapy was also tested. Methods: Colon38 tumors were established in C57Bl6 mice and dosed with anti-PDL1 (5 mg/kg ip q2d 3x/wk) as 1L ICI treatment for 2 weeks. Animals were defined as initial responders (R) or non-responders (NR) based on, respectively, a >10% decrease or increase in tumor volume (TV) during week 2 of ICI therapy. 2L treatment was initiated in week 3 with cabozantinib (10 mg/kg po qd) or ICI for 3 weeks alone or combined in R and NR arms (n=10/cohort). Cabozantinib was also dosed as monotherapy from week 1 (cohort Cwk1) or week 3 (cohort Cwk3). Time (days) to reach a TV of 1000mm³ (TTRV1000) was used as efficacy measure. Results: Cabozantinib monotherapy was effective with 1-3 complete regressions (CR) seen for cohorts Cwk3 with a TTRV1000 of 38.5d (21.5-98) and Cwk1 with a TTV1000 of 47.5d (36.5-98). Vehicle-dosed tumors had a median TTRV1000 of 18.5d (range 14-34.5) after randomization. R tumors reacted well to 1L ICI with a TTRV1000 of 45.5d (29-98) and 4-6 CR seen in 2L R cohorts regardless of 2L treatment. The remaining non-CR tumors had for 2L ICI treatment a TTRV1000 of 36.5d (30.5-43; n=4) as compared to 40d (29-47; n=6) for 2L vehicle-dosed controls, suggesting they had become refractory to ICI. 2L treatment with cabozantinib had a TTRV1000 of 58d (45.5-80.5; n=6), while cabozantinib+ICI had a TTRV1000 of 56d (42-98; n=5). 2L cabozantinib ± ICI thus led to longest TTRV1000 values for refractory non-CR tumors after 1L ICI across R cohorts. NR tumors receiving ICI had a TTRV1000 of 27d (13.5-32) and thus behaved similar to vehicle alone. 2L cabozantinib ± ICI effects were comparable in magnitude to the efficacy of cabozantinib monotherapy in cohort Cwk3; 2L cabozantinib after 1L ICI in NR tumors had a TTRV1000 of 40.5d (26-47.5), while 2L cabozantinib+ICI after 1L ICI yielded a TTRV1000 of 43.5d (20-59.5). Conclusion: Cabozantinib showed robust anti-tumor efficacy in refractory post-ICI settings. Further investigation of the mechanisms of action of sequential combination is warranted. Citation Format: Sylvie Rolland, Stephan Klinz, Sophie Chaumeron, Florence Meyer-Losic, Marc Hillairet de Boisferon. Efficacy of cabozantinib after immune checkpoint inhibition in a syngeneic tumor model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO053.

Inhibition of the immune-checkpoint axis that involves PD-L1/PD-1 has improved responses and outcomes for certain cancer patients, but there remains unmet need as only a fraction of patients respond to these therapies. Talimogene laherparepvec (T-VEC) is an FDA-approved, first-in-class oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce granulocyte-macrophage stimulating factor (GM-CSF) to enhance a tumor-antigen specific adaptive immune response. Intratumoral treatment with T-VEC has been shown to promote an inflammatory tumor microenvironment both preclinically and clinically. Combining T-VEC with immune checkpoint inhibitors is of high interest to potentially broaden and deepen the clinical responses in checkpoint-resistant tumors. In the current study, we evaluated the efficacy of OncoVEXmGM-CSF (an HSV modified like T-VEC, except that it produces murine GM-CSF) in combination with anti-PD-1/PD-L1 treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used, where only one tumor was treated with OncoVEXmGM-CSF (representing the local oncolytic effect), allowing the evaluation of systemic abscopal effect in the uninjected contralateral tumors. OncoVEXmGM-CSF in combination with anti-PD-L1/PD-1 enhanced tumor growth inhibition in both treated and contralateral tumors in the A20 and MC38 syngeneic models and overcame anti-PD-L1 resistance in the CT26 model. The combination treatment also led to a significant increase in median overall survival compared to either agent as a monotherapy. In addition, tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell response was measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. OncoVEXmGM-CSF induced and /or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that OncoVEXmGM-CSF treatment can cause direct tumor lysis along with the potentiation of an adaptive, systemic T cell-mediated anti-tumor immune response that can be enhanced by the addition of PD-L1/PD-1. Our study provides a strong rationale for the ongoing clinical evaluation of the combination of T-VEC and PD1-/PD-L1-targeted therapy in cancer patients. Citation Format: Keegan Cooke, Juan Estrada, Petia Mitchell, Jinghiu Zhan, Pedro J. Beltran, Jing Qing, Jude Canon. OnxoVEXmGM-CSF promotes systemic response to PD-L1 /PD-1 blockade in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2219.

To examine how baseline tumor immunophenotyes can influence therapeutic outcome, we used the CT26 colorectal and 4T1-Luc (4T1) breast cancer models, which are immunologically warm and cold tumors, respectively. We treated tumor-bearing mice with control or anti-mCTLA-4 antibodies (anti-CTLA-4), or left them untreated, and excised tumors to measure immune cell infiltrates, TIL cytokine/granzyme B production, and activation marker expression. Baseline analysis in untreated mice revealed that CD8+ T-cell infiltrates were similar between models (5-6% of total CD45+ cells). NKT cells were reduced in CT26 (0.5% vs. 1.2%). The myeloid compartment in CT26 was mostly M2 macrophages (43% of CD45+ cells), whereas G-MDSCs dominated in 4T1 (40% of CD45+ cells). CD69 and PD-1 activation markers were expressed on >90% of CD8+ T cells from CT26 tumors compared to 4T1, which were Citation Format: David Draper, Philip Lapinski, Stacey Roys, Scott Wise, Maryland Rosenfeld Franklin. Comparative immunophenotypic analysis of immunogenically warm and cold syngeneic tumor models at baseline and after anti-mCTLA-4 treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A86.

GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the tumor necrosis factor (TNF) ligand superfamily. GITRL binds and activates the co-stimulatory surface receptor GITR, which promotes proliferation and activation of effector T cells (Teff) and inhibits suppressive activity of regulatory T cells (Treg). It is thus hypothesized that co-stimulation of GITR by agonist agents will promote anti-tumor immunity. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc) that shows robust single agent antitumor efficacy and immune effects in multiple syngeneic mouse models, suggesting its potential benefit in cancer immunotherapy To investigate the prevalence of GITR expression in human tumors, RNA-Seq data analyses of 33 tumor types in TCGA showed GITR is highly expressed in a subset of solid tumors, including head & neck, lung, breast, esophageal, and bladder cancers. In most solid tumors, GITR expression correlated poorly with T cell markers, implying that GITR may not be exclusive to immune cells and may be expressed in tumor cells as well. Similar findings emerged from RNA-Seq data analysis of patient-derived xenograft (PDX) samples from 24 tumor types. The gene expression data was corroborated by immunohistochemistry (IHC) analysis of GITR expression in 17 tumor types which showed that in addition to immune cells, GITR was expressed on tumor cell membranes. A multi-platform approach was taken to investigate GITRL-Fc pharmacodynamic (PD) biomarkers in tumors and in matched whole blood samples from mice bearing CT26 colon, 4T1 breast, and B16F10 melanoma carcinoma models. Global gene expression levels were profiled by microarray on treated and control tissues. We also monitored the changes of immune cell populations and cytokine secretions by flow cytometry, Luminex and IHC. Immune gene changes were more robust in tumors than in blood samples. In tumor samples, GITRL-Fc increased the gene expression associated with T cells, CD8 T cells, cytotoxicity, Th1 cells, interferon gamma (IFN-γ), natural killer cells, Teff cells, and T cell activation markers. These gene changes were validated by quantitative real-time PCR. Similarly, flow cytometry analysis showed that GITRL-Fc promoted activation of CD4+ effector cells, decreased Treg frequency, and increased the ratio of CD8+ T cell/Treg in the tumor. GITRL-Fc also modulated secretion of cytokines in splenocytes, including an increase in IFN-γ. Taken together, the PD biomarker changes in immune-related gene expression, immune cell populations, and cytokine secretions observed in these preclinical tumor models are consistent with GITRL-Fc mechanism of action and demonstrated target engagement of GITRL-Fc. Additional approaches, including in-silico sorting, to monitor rare immune cell populations in tumor samples will be discussed. Citation Format: Min Wang, Fiore Cattaruzza, Pete Yeung, Alayne Brunner, Erwan LeScolan, Yuwang Liu, Jennifer Cain, Gilbert OYoung, Earth Light Lowe, Belinda Cancilla, Rose Harris, Tim Hoey, Austin Gurney, John Lewicki, Gretchen Argast, Ann M. Kapoun. Prevalence of GITR expression and pharmacodynamic (PD) biomarkers in syngeneic tumor models treated by a GITR agonist (GITRL-Fc) [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 5621. doi:10.1158/1538-7445.AM2017-5621

The kinetics of skin cancer induction by UV radiation has been extensively studied in hairless mice and described by Weibull statistics in which the time till 63% of the mice bear tumors is a primary parameter. However, the kinetics of the associated immunosuppression remained to be determined. To this end, we implanted a syngeneic UV-induced skin carcinoma cell line (T51/6.53) in the ventral skin of HRA/SKH hairless mice after various periods of daily dorsal UV exposure, either 150 or 75 mJ/cm2 per day UV from F40 sunlamps (regimens that when continued yield 63% of the mice with 1 mm tumors in 11.5 or 16.2 weeks, respectively). Both exposure regimens achieved a 100% acceptance (after 7 and 16 weeks, respectively). The implants failed to grow in all unirradiated control mice, but the percentage of mice in which the implants grew increased with the UV treatment time and dose. The estimated times to 63% implant acceptance were 4.3 +/- 0.8 and 8.2 +/- 0.8 weeks for the high and low daily doses, respectively. As reported earlier for shaved haired mice, there appears to be a straight reciprocity between daily UV dose and the time to tumor acceptance, i.e. the latter fully depends on the cumulative UV dose, whereas the tumor induction does not. The latter probably also depends on a pure elapse of time, i.e. UV-independent processes. A further analysis of the Weibull description indicates that immunosuppression toward the tumor requires fewer UV-driven steps that tumor induction.

Background: Syngeneic tumor models have proven to be one of the most robust model systems for cancer immunotherapy. However, in vitro screens that are frequently used in oncology to quickly identify cells, models, and PD effects for further study often fail in immuno-oncology, due to immunotherapeutics targeting the complex host immune system. Alternatively, an in vivo screen with a panel of models addresses many of these questions, e.g. PD and efficacy, but may be cost prohibitive. Material and methods: Leveraging in-house detailed profiling data on our syngeneic models, including efficacy benchmarking with aPD-1, aPD-L1 and aCTLA-4 antibodies, RNAseq data on tumor samples, and FACS analysis on both baseline and treatment response tumor samples, CrownBio recently launched a new service platform: MuScreen. MuScreen includes up to 20 well-characterized syngeneic models in a 3 month screening run. Both PD and efficacy may be determined in the run, allowing researchers to make decisions based on results observed from a large dataset. To address the cost issue, test agents from multiple clients are pooled together for each run (sharing vehicle and other common groups) providing a significant reduction in the number of animals used and the associated costs. Results: CrownBio has established the largest collection of syngeneic models with well-characterized immunotherapy data. In the first MuScreen run, we have generated new data on common IO agents, e.g. aPD-1 antibodies, with FACS analysis, IHC, and efficacy data. The first complete set of MuScreen data will be shown. Design parameters, such as starting tumor size, group size, of the MuScreen studies will be discussed. Conclusions: MuScreen is the first in vivo screening tool for cancer immunotherapeutics. It provides detailed response data on a panel of syngeneic models, therefore enabling decision making in a cost and time efficient manner. Citation Format: Ying Jin, Lan Zhang, Juan Zhang, Eric Murphy, Zhongliang Li, Meng Qiao, Qian Shi. A panel of well characterized syngeneic models for in vivo screening analysis: MuScreenTM [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B098.

Background: The recent successes of cancer immunotherapies have stimulated interest in the potential application of these approaches in many solid and hematologic tumors. Syngeneic tumor models have been widely used in cancer immunotherapy assessment. However, most of these experimental models were established as subcutaneous models, while there may be significant difference in immune microenvironment comparing orthotopic vs. subcutaneous tumors. In addition, drug delivery and metabolism are different in orthotopic tumors vs. subcutaneous tumors. To address this unmet need, we set out to validate a panel of orthotopic syngeneic models for anti-cancer therapeutics (mainly immunotherapeutics) evaluation. Results: A large collection of orthotopic syngeneic models covering breast cancer, liver cancer and various hematological cancers were established in immunocompetent mice. Survival curves were established and the responsiveness to anti-PD1, anti-PDL1 and anti-CTLA4 antibodies were evaluated in the orthotopic models. Orthotopic liver tumors were monitored with ultrasound imaging to obtain longitudinal growth curves. The correlation in drug efficacy between the orthotopic model and its subcutaneous counterpart was analyzed. The role of NK cells in the development of a panel of systemic hematological tumor models was investigated with NK depleting antibodies. Conclusion: Despite the obvious difficulties, orthotopic syngeneic models represent closer model systems to human diseases and may be a valuable tool to the evaluation of cancer immunotherapies and their combination strategies. Citation Format: Juan Zhang, Sheng Guo, Zhensheng Wang, zhongliang Li, Meng Qiao, Qian Shi. Orthotopic syngeneic tumor models for preclinical evaluation of cancer immunotherapy strategies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5176.

The pre-clinical assessment of immuno-oncology (IO) therapies can be enabled by the use of murine syngeneic tumors established in immuno-competent mice. With the aims of selecting relevant models and of minimizing animal experimentation by reducing the number of models tested, the full characterisation of syngeneic models at the transcriptomic and genomic level is a key objective for pre-clinical scientists. Model characterisation includes global aCGH, exon array analysis and FACS profiling alongside exome sequencing. The model data is undergoing hypothesis free and driven analyses which are already generating valuable insights. Comparison of in vivo tumor samples with their in vitro equivalents has highlighted enrichment for a number of immune pathways; as has the comparison of different tumor lines. The genomic, transcriptomic and ‘proteomic’ model data are being integrated to give a functional output which will act as a ‘Syngenomic Fingerprint’ for each model. The resulting Syngenomic fingerprints will help pre-clinical scientists to refine their in vivo plans through an improved understanding of the limits and advantages as well as the clinical relevance of some of our preclinical models. It is also supporting the targeted modification of models to better match specific human cancer types. Citation Format: John E. Prime, Suzanne Mosely, Jens-Oliver Koopmann, Dennis YQ Wang, Danielle Greenawalt, James Harper, Miika J. Ahdesmaki, Rebecca Leyland, Olivia Harris, Ross Stewart, Philip Brohawn, Brandon Higgs, Bryony Langford, Athula Herath, Robert Kozarski, Jane Coates-Ulrichsen, Judith Anderton, Michelle Morrow, Richard C. A Sainson, Robert W. Wilkinson. Syngenomic fingerprint: the biomic characterization of the mouse syngeneic tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4186.

Contains fulltext : 80074.pdf (Publisher’s version ) (Closed access) Liver metastases of colorectal carcinoma occur in about 50-60% of patients. To improve survival of these patients, there is an urgent need for new treatment strategies. For this purpose, the availability of a preclinical model to develop and test such treatments is mandatory. An ideal animal model for studying liver metastases of colorectal origin should mimic all aspects of the metastatic development in humans and be practical, predictable, and optimal in terms of ethical considerations. Thus far, no model has been developed which satisfies all these conditions. As a consequence, choosing an animal model for the study of liver metastases requires compromises and choices about the necessary characteristics that depend on the purpose of the intended experiments. This overview addresses the advantages and disadvantages of different animal models used for research on experimental liver metastases of colorectal origin. Based on data available in literature, we conclude that heterotopic injection of undifferentiated syngeneic tumor cells in immunocompetent rodents covers most of the desired characteristics. Both subcapsular as well as intraportal injection will yield suitable models and the eventual choice will depend on the aim of the study.

A study to evaluate the mechanisms of tumoricidal activity resulting from orally administered extract of Agaricus blazei Murill (A. blazei) was performed in mice bearing syngeneic and xenogeneic tumors. Tumor regression was comparably seen in both syngeneic and xenogeneic tumor-bearing mice when administered oral extract preparations. In addition, in a murine syngeneic tumor model, oral administration of water-soluble extracts of A. blazei resulted in significant production of cytokines such as IFN-gamma, and TNF-alpha in peritoneal exudate cells, in parallel with the marked regression of tumor development. The water-soluble extracts also induced pronounced antioxidant activity in in vitro and in vivo assays using two different methods. These results indicate the A. blazei extract may enhance not only the immnunomodulatory effects that promote activity of peritoneal exudate cells for tumor regression but also potentially result in the direct destruction of tumor cells through its antioxidant activity.

The growth of MethA tumor was significantly inhibited by oral administration of the alpha-glucan SPR-901 in BALB/c (+/+) mice but not in nude mice. Mice treated orally with SPR-901 exhibited an augmentation of antigen-specific resistance against rechallenge with the tumor cells. The tumor-neutralizing activity of regional lymph node cells from MethA-bearing mice against the tumor was augmented by oral administration of SPR-901. The tumor-neutralizing activity of lymph node cells from SPR-901-treated mice mainly appeared in Lyt2+ cells. Furthermore, lymphokine-activated killer activity of these cells was enhanced by administration of SPR-901. The antitumor effect of SPR-901 was abrogated in mice depleted of either L3T4+ or Lyt2+ cells, and in cyclosporin-A-treated mice. These results suggest that Lyt2+ cells are important effector cells in MethA-bearing mice orally administered SPR-901 and that functional exertion of both Lyt2+ and L3T4+ T cells is necessary for the antitumor effect of orally administered SPR-901 in vivo.

Antibodies against cancer-relevant targets can recruit Fc receptor-bearing myeloid and natural killer cells to mediate antibody dependent cellular cytotoxicity (ADCC). Toll-like receptors are potent activators of the innate immune system and generate signals leading to the initiation of an adaptive immune response that can be utilized for therapeutic purposes. We tested the efficacy of E6020, a TLR4 agonist, administered in combination with the clinically approved anti-HER2 antibody trastuzumab. A four-week course of therapy with trastuzumab and E6020 protected against the outgrowth of syngeneic D5 melanoma cells transfected with the human tumor antigen HER2 in immunodeficient C57BL/6 SCID mice, in immunocompetent wild-type C57BL/6 mice as well as in human HER2-tolerant C57BL/6 hmHER2 transgenic mice. In each tested strain of mice, the addition of E6020 to trastuzumab significantly increased the proportion of animals that survived initial tumor challenge compared to trastuzumab treatment alone (83% vs. 33% in C57BL/6 hmHER2 transgenic mice; p < 0.05). To determine if apparently cured mice had been effectively immunized against human HER2, the transgenic mice were inoculated subcutaneously with 103 D5-HER2 cells and then received a 4-week course of trastuzumab+E6020, or trastuzumab alone. Animals that survived the primary tumor challenge were followed for at least 120 days and then rechallenged with 5 × 103 D5-HER2 or unmodified D5 cells and followed for a minimum of 60 additional days. Mice that had been initially treated with the combination of trastuzumab and E6020 demonstrated superior protection (22/30; 73%) to those treated with trastuzumab alone (16/36; 44%; p < 0.05) against rechallenge with D5-HER2, but no protection was seen against D5 challenge. Both CD4 and CD8 T-cells were required for full protection against rechallenge. These findings suggest that trastuzumab therapy can induce a host-protective adaptive immune response. Moreover, combined treatment with trastuzumab and a TLR4 agonist enhances both the direct anti-tumor effects of trastuzumab and a host-protective human HER2-directed adaptive immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-324.