PredictImmune

Abstract Background Biomarkers predictive of a patient’s future disease course are critical for personalised medicine. There are two Crohn’s disease (CD) assays with validated associations to a clinical outcome; seropositivity for anti-glycan antibodies (AGAs, Glycominds) and a transcriptional signature of T-cell exhaustion (PredictSURE-IBD™, PS-IBD). The optimal time to use these tests would be at diagnosis before bowel damage occurs. As AGAs have primarily been measured in patients after therapy it’s unclear if they provide prognostic information at diagnosis. To address this we performed a head-to-head comparison of both tests in newly diagnosed CD patients. Methods We recruited 74 CD patients with active disease; all patients gave written informed consent and were managed using an accelerated step-up strategy in accordance with international guidelines. Active disease was confirmed by one or more objective marker (raised CRP/calprotectin or endoscopic review) and active symptoms. AGA seropositivity was measured at Addenbrooke’s with the IBDX panel (Glycominds) according to manufacturer’s instructions. Patient stratification was also performed using the PS-IBDtest (PredictImmune Ltd) according to manufacturer’s instructions. Results Most (80%–59/74) patients were newly diagnosed; 58% (43/74) were seropositive for at least 1 AGA, 19% (14/74) were positive for ≥2 AGAs. Newly diagnosed patients had fewer AGAs (51%, 30/59) than previously diagnosed (87%, 13/15) patients (p = 0.018). We stratified patients into two groups based on seropositivity for 2 or more AGAs—associated with an increased risk of more severe disease but found no significant difference in time to, or frequency of, treatment escalation between the groups. Stratification of patients into IBDhi and IBDlo groups with PS-IBDrevealed a significant difference in the subsequent disease course with IBDhi patients experiencing more aggressive disease, characterised by a shorter time to treatment escalation compared with IBDlo patients (p = 0.001). Conclusion AGA seropositivity at diagnosis (as assessed using the IBDX antibody panel) did not predict the need to escalate treatment due to frequently-relapsing or chronically-active disease. Lower AGA seropositivity in newly diagnosed patients compared with those with previous diagnoses suggested that the extent of antibody positivity is a function of disease duration. Previously reported associations between AGA positivity and disease outcome may, therefore, reflect retrospective aggressive disease course and are unlikely to be useful for informing treatment decisions in newly diagnosed patients. The PS-IBD test result correlated with subsequent disease course indicating suitability for early prediction of clinical outcome.

Abstract Immuno-oncology (IO) therapies for cancer have recently delivered step-change improvements in patient outcomes, but the major proportion of patients remain unresponsive or become refractory to IO treatment. Because IO-based approaches necessarily rely on the interplay of multiple cell types, including immune cells, and other elements that constitute the tumor microenvironment (TME), innovative assays are critical to generate further translational insight. Indeed, results from in vivo mouse models and in vitro assays with human cell lines often do not correlate with clinical efficacy and as such, do not always represent relevant avatars. We have leveraged recent advances in tumor slice culture (TSC) techniques to enable in vitro mechanistic and novel biomarker testing/identification studies. TSC involves the in vitro culture of thick slices (200-300 μm) of fresh tissue prepared using a vibratome and is currently the only in vitro assay system which retains the 3-dimensional tissue architecture, complete with cell-cell and cell-matrix interactions. We are now regularly using the TSC together with tumor samples derived from patients and human cancer cell line derived xenograft or syngeneic mouse cancer models, to generate exciting pre-clinical data for several Oncology programs, including oncolytic viruses, modified RNA, bispecific antibodies and antibody-drug conjugates. For instance, the TSC platform has allowed us to analyse the strong type I IFN-driven inflammatory response initiated by infection of tumor nests within patient samples and demonstrate the consequent infiltration of immune cells from the surrounding stroma. Thus, using a tissue phenomics approach incorporating a range of endpoint assays, including multiplex whole-mount imaging, transcriptomics and cytokine expression, the data generated using the TSC platform will inform the underlying biology and translational strategies of multiple therapeutic candidates. Citation Format: Shannon Burke, Fabien Garcon, Kelli Ryan, James Harper, Robert W. Wilkinson. Tissue slice culture platform: A slice of reality in drug development [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 1190.

Immuno-oncology research relies heavily on murine syngeneic tumor models. However, whilst the median age for a cancer diagnosis is 65 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that aging has been shown to have a significant impact on the immune response. Using aged mice bearing CT26 tumors, we analysed how aging impacts the immune composition of the tumor, spleen and tumor-draining lymph nodes by flow cytometry. We found many age-related changes between aged (60-72 weeks old) and young (6-8 weeks old) mice, such as a reduction in the naïve T cell population and a decreased CD8/Treg ratio in aged animals. Profiling of co-inhibitory and co-stimulatory receptor expression levels on immune cells in aged versus young mice also identified altered expression profiles in both the periphery and tumor. We hypothesised that these differences may contribute to impaired anti-cancer immune responses in aged mice. To investigate this, we compared the anti-tumor efficacy of immune checkpoint blockade (PD-L1 and CTLA-4) and T-cell costimulation (OX-40) in aged versus young mice. Our data demonstrate that aged mice retained their capacity to generate effective anti-tumor immune responses, albeit often attenuated when compared to the responses observed in young mice. These differences highlight the potential importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.Citation Format: Suzanne I. Sitnikova, Michelle Morrow, Viia Valge-Archer, Robert W. Wilkinson, Simon J. Dovedi, Matthew J. Robinson. Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and reduce response to immuno-oncology treatments [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 4987.

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.

Diabetic Nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T‐cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T‐cell activation will ameliorate DN. Interaction of B7‐1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T‐cells, is essential for T‐cell activation. In this study we used a soluble CTLA4‐Fc fusion protein to block cell surface B7‐1, preventing the cellular interaction and inhibiting T‐cell activation.The CTLA4‐Fc Abatacept was dosed i.p. in the Streptozotocin‐induced Diabetic Nephropathy mouse model in both prevention and intervention modes. Renal damage was assessed by albuminuria and renal B7.1 positive cells and lymphocytes were measured by immunohistochemistry. Gene expression interrogated by real‐time PCR and protein expression by Western blotting. Differentiated human immortalized podocytes were used for in vitro studies.When CTLA4‐Fc was dosed in an animal model of DN, it reduced albuminuria in both prevention and intervention modes. The number of T‐cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria and treatment with CTLA4‐Fc reduced the number of renal T‐cells. As B7‐1 induction has been recently proposed to underlie podocyte damage in DN, CLTA4‐Fc could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7‐1 was not expressed in: 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients.We conclude that CTLA4‐Fc ameliorates DN by blocking T‐cell activation and not by protecting podocytes.

Abstract Immune-mediated therapy for cancer (IMT-C) biologics are an emerging class of cancer drugs which target cells of the immune system to enhance the immune response to cancer. Cross-reactivity of IMT-C mAbs and other biologics to rodents is rare, emphasizing the importance of human in vitro assays in IMT-C discovery and development. Typically there is a trade-off between the physiological relevance of an assay and its throughput, hence higher-throughput assays are used in the early stages of drug discovery, and more complex, lower-throughput assays are subsequently used to profile lead candidates. Here, we review a panel of in vitro cellular assays currently used for IMT-C drug programs at MedImmune including transgenic reporter cell systems, bead-based assays, and multi-cellular systems using cell-lines and primary human immune cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B300. Citation Format: Geoff Williams, Edmund Poon, Samantha Ireland, Janette Dillon, Ross Stewart, Robert Wilkinson. Discovering immune-mediated therapy for cancer biologics is informed by in vitro cellular assays. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B300.