(word count: 239):Murine syngeneic tumor models are critical to novel immuno-based therapy development but the molecular and immunological 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 employed 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.4

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Studies have demonstrated that the clinical benefit of PD-1 blockade can be further improved by combination with an αCTLA-4 mAb in some indications. However, this increased activity is commensurate with significant immune related adverse events (irAE's). Therefore, novel approaches are required to uncouple toxicity from anti-tumour efficacy and realise the full potential of this combination. MEDI5752 is a monovalent bispecific human IgG1 monoclonal antibody (mAb) with an engineered fragment crystallisable (Fc) domain to reduce Fc effector function, that specifically binds two clinically validated negative T cell regulators; PD-1 (programmed cell death 1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). MEDI5752 has been designed to suppress the PD-1 pathway and provide modulated CTLA-4 inhibition to uncouple CTLA-4 dependent peripheral toxicity from tumour efficacy. PD-1 expression is a defining feature of tumour infiltrating lymphocytes (TILs). We show that MEDI5752 can saturate CTLA-4 on PD-1+ cells at orders of magnitude lower concentrations than required to saturate CTLA-4 on PD-1- cells. Moreover, our data demonstrate that monovalent targeting of CTLA-4 with MEDI5752 is significantly less potent (15 fold) than bivalent targeting with a parental αCTLA-4 mAb in reporter assays. In contrast, the switch to monovalent targeting of PD-1 has limited effect on potency (within 3-fold compared to a parental αPD-1 mAb) in a PD-1/L1 reporter assay. Together these data demonstrate the potential for MEDI5752 to inhibit CTLA-4 on TILs whilst sparing peripheral T cell populations and reducing toxicity. Furthermore, profiling of MEDI5752 in a range of primary T cell activation assays reveals equivalent activity to a combination of parental PD-1 and CTLA-4 antibodies. MEDI5752 is rapidly internalised upon target binding with kinetics similar to the parental αCTLA-4 mAb reflecting the rapid recycling of this receptor. However, in contrast to an αCTLA-4 mAb (or an αPD-1 mAb), MEDI5752, by tethering CTLA-4 to PD-1, leads to the internalisation and subsequent degradation of PD-1. This novel mechanism of action further differentiates MEDI5752 from a combination of mAb's targeting PD-1 and CTLA-4. MEDI5752 is a novel monovalent bispecific which may provide an improved therapeutic index when compared to a combination of bivalent αPD-1 and αCTLA-4 mAb's, and could provide benefit in cancer indications. Citation Format: Simon J. Dovedi, Yariv Mazor, Matthew Elder, Sumati Hasani, Bo Wang, Suzanne Mosely, Desmond Jones, Anna Hansen, Chuning Yang, Yanli Wu, Ikbel Achour, Nick Durham, Gareth Browne, Thomas Murray, James Hair, Michelle Morrow, Godfrey Rainey, Maria Jure Kunkel, John Gooya, Daniel Freeman, Ronald Herbst, Robert Wilkinson. MEDI5752: A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells [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 2776.

Objectives: Studies in murine syngeneic tumors are critical in the development of immune-based therapies, yet there still are knowledge gaps in the functional meaning (vs. response and resistance to treatments) of baseline molecular and immunological features in these tumors. We developed a quantitative systems model of immuno-oncology (IO), to (i) understand factors within the TME which may underlie anti PD-(L)1 and CTLA-4 efficacy in 6 syngeneic tumors (4T1, LLC, CT-26, MC-38, B16, RENCA); (ii) identify potential baseline factors which relate to treatment resistance. Methods: Our IO model [1] was used, firstly to incorporate rich datasets from the 6 syngeneic tumor types [2] and to characterize differences in baseline TME conditions. The model was then used to perform mechanistic population simulations of the initiation and development of anti-tumor T cell immune responses, linked to observed individual animal- and cohort-level tumor size dynamics (TSD) under anti PD-(L)1 and CTLA-4 treatments. Variability in individual tumor size dynamics was taken into account using a mixed-effects technique, implemented in the model at the level of tumor-infiltrating T cell influx. Results: The model adequately described individual- and cohort-level TSD patterns, for all treatment regimens in all 6 tumor types. The model incorporated in one quantitative framework immune cell count data measured in these tumors, by capturing empirical dependencies between TME properties and model parameters. Anti PD-L1 therapy was incorporated into the model via a direct increase in an immune activation rate (IAR) function in TME, validating our previous results [1]. Interestingly, an optimal model incorporating anti CTLA-4 mechanism of action was one considering an indirect effect on IAR through the decrease of immuno-suppressive cell (ISC) function, which supports the hypothesis that the driving force of anti CTLA-4 effects in syngeneic tumors would go through ISC deactivation, e.g., via regulatory T cell (Tregs) depletion. Also, higher counts of Tregs at baseline (e.g., CT26, RENCA) correlated well with responses to anti CTLA-4 treatment. Higher levels of macrophages and/or MDSC infiltration in lesser “immunologically hot” tumors (e.g., 4T1, MC38, LLC) were shown to be the main immuno-suppressive factors limiting tumor responsiveness to checkpoint inhibitor treatments. Conclusions: This quantitative model may be used as a platform to analyze immune-based treatment data from various tumor types, while providing mechanistic insights on the contributions of baseline TME conditions to response or resistance to treatment. The model may be further used to perform predictive tumor response simulations (monotherapies and combinations), of untested anti CTLA-4, PD-(L)1 dose schedules and of other novel IO agents beyond these two checkpoint inhibitors. Citation Format: Gabriel Helmlinger, Ivan Azarov, Yuri Kosinsky, Veronika Voronova, Lulu Chu, Suzanne Mosely, Simon Dovedi, Kirill Peskov. Linking tumor microenvironment properties in murine syngeneic tumors with resistance to immune checkpoint inhibitors: Insights from a quantitative systems approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1082.

<p>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</p>