Supplementary Figure S1: Summary of experiments Supplementary Figure S2: Gating strategy Supplementary Figure S3:Profiling by array CGH, whole-exome and targeted sequencing Supplementary Figure S4: Comparison of mutational profiles of murine syngeneic tumor cell lines and TCGA patient tumors Supplementary Figure S5: Differentially-expressed gene-sets in lymph node and spleen Supplementary Table S1: Cell line details Supplementary Table S2: Copy Number Variation Supplementary Table S3: List of 64 genes investigated by targeted sequencing Supplementary Table S4: Fluorescent antibodies used Supplementary Methods: Linear mixed-effect model

Supplementary dataset containing raw data from targeted sequencing, whole exome sequencing, array CGH and transcriptomic analysis

Abstract Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell–specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor “inflamed” and “non-inflamed” tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell–rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo. Cancer Immunol Res; 5(1); 29–41. ©2016 AACR.

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivoMEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti–PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti–CTLA-4, anti–PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR. Cancer Immunol Res; 3(9); 1052–62. 2015 AACR.

Cancerous cells emerge within the body following accumulation of deleterious genetic mutations. These mutations alter the phenotype of a cancer cell marking it as distinct from the surrounding host; an immunological state termed “altered self”. These cells, like other non-self entities such as viruses and bacteria, are recognised by the immune system and marked for destruction, a process known as “immune surveillance”. B7-H1 expression by tumour cells is believed to aid tumours in evading detection and elimination by the immune system. B7-H1 functions in this respect via several alternative mechanisms including driving exhaustion and anergy of tumour infiltrating T lymphocytes, stimulating secretion of immune repressive cytokines into the tumour micro-environment, stimulating repressive regulatory T cell function and protecting B7-H1 expressing tumour cells from lysis by tumour cell specific cytotoxic T cells. Using hybridoma technology and high throughput screening MedImmune has identified a series of fully human antibodies specific for human B7-H1. Further characterisation of these antibodies led to the identification of a single high affinity antibody, MEDI 4736, with the ability to relieve B7-H1 mediated suppression of T cell activation in vitro and to enhance sub-optimal T cell activation in a mixed lymphocyte reaction. In vitro testing shows that MEDI 4736 does not trigger non-specific cytokine release in whole blood, and is only able to activate T cells in the context of an active T cell receptor signal. A surrogate anti-mouse B7-H1 antibody shows significant anti-tumour activity in a syngeneic model when dosed in combination with chemotherapy. Similarly MEDI 4736 is able to inhibit tumour growth in a novel in vivo xenograft model, via a mechanism that is dependent on the presence of tumour specific human T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2011-LB-158