Evaluation of tumour infiltrating immune cells into the orthotopic and metastatic tumour microenvironment using bioluminescent syngeneic cell line models in immune competent mice following treatment with checkpoint inhibitors

Poster Session – Animal Models, Thursday 1 December 2016 olaparib and carboplatin/paclitaxel. Tumor regression (TR) values were calculated, aligned to clinical RECIST criteria, and correlated with known literature-based response rates (RR). Results: A total of 51 ovarian PDX models were developed from 48 patients. Twenty-one (70%) models sequenced to date harbored deleterious mutations in BRCA 1/2. Of 9 BRCA negative models, 6 had mutations in molecules contributing to a BRCA-like phenotype such as PALB2 and FANCA, whilst 3 models had no identifiable mutations in these genes/pathways. We screened 14 ovarian models (3 BRCA 1/2 wild type, 8 BRCA1 mutated, and 3 BRCA2 mutated) against carboplatin/paclitaxel and olaparib. Based on tumor growth, 4 models were considered platinum resistant, 5 models platinum sensitive (definitive TR), and the remainder of intermediate sensitivity (TR equivalent to stable disease). TR values aligning with stable disease were observed in 4/14 (29%) ovarian PDX models treated with olaparib, which corresponds with reported clinical trial RR (~33%). There was no correlation between responses to carboplatin/paclitaxel and olaparib. Conclusion: In this pilot study, RR to olaparib in ovarian PDX models with BRCA mutations/BRCA-like phenotype was similar to clinical trial outcomes. In keeping with literature reports, platinum pretreatment did not affect model responsiveness to olaparib. Nevertheless, whilst the majority of models responding to olaparib harbored mutations in BRCA/BRCA-like genes, one was wild type with respect to these pathways. Further, other BRCA mutated/BRCA-like models failed to respond to PARP inhibition. This suggests a need for further interrogation of de novo resistance/sensitivity mechanisms, which the PDX platform is well suited to uncover. Conflict of interest: Ownership: Angela Davies, Daniel Ciznadija, Amanda Katz, David Sidransky. Board of Directors: David Sidransky. 336 Poster (Board P015) Characterizing the effect of immune checkpoint inhibitors on syngeneic tumor models through gut mircrobiome sequencing and immune-phenotyping Y. Kato Maves1 , H. Izadi1 , A. Calinisan1 , C. Talaoc1 , D. Yan1 , C. Echegaray1 , M. Blair1 , R. Mizukoshi1 , J. Thatte1 , T. Broudy1 . 1 Crown Bioscience Inc., San Diego, San Diego, USA Background: Syngeneic mouse models present an important tool to understand the local and systemic effects of immune modulating therapies. Immune checkpoint inhibitors have become a mainstay of oncology treatments, yet many patients experience partial responses. Thorough characterization of pre-clinical models is paramount in order to successfully predict response to treatment in the clinical setting. Material and Methods: We examined the efficacy of anti-PD-1, anti-PD-L1, and anti-CTLA-4 therapies on our syngeneic tumor model platform. Immune-phenotyping was conducted at various time points. Gut microbiome was examined by r16S sequencing to analyze statistically significant shifts longitudinally and between different arms and animals at cross-sectional time points. Results: Immune checkpoint inhibitors had variable efficacy across different tumor models. We also observed shifts in immune cell populations such as T regulatory cells and M1/M2 macrophages, as treatments were continued. Gut microbial community analysis enabled analysis of enriched microbes between groups and individual animals, as well as across time points. Conclusions: Here, we report on the establishment of syngeneic mouse models including efficacy, flow cytometry analysis of tumor-infiltrating lymphocytes, and gut microbial community analysis. No conflict of interest. 337 Poster (Board P016) Evaluation of tumour infiltrating immune cells into the orthotopic and metastatic tumour microenvironment using bioluminescent syngeneic cell line models in immune competent mice following treatment with checkpoint inhibitors M. Andrew1 , P. Nektaria1 , J. Simon1 , W. Jane2 , K. Jason3 , L. Vicky3 , J. Kelly3 , G. Russell4 , W. Neil5 , R. Kumari6 . 1 Crown Bioscience UK Ltd, Scientific Operations, Loughborough, United Kingdom; 2 Crown Bioscience UK Ltd, In Vitro Operations, Loughborough, United Kingdom; 3 Crown Bioscience UK Ltd, In Vivo Operations, Loughborough, United Kingdom; 4 KWS Biotest, Analytical Services, Bristol, United Kingdom; 5 KWS Biotest, KWS Biotest, Bristol, United Kingdom; 6 Crown Bioscience UK Ltd, Crown Bioscience UK Ltd, Loughborough, United Kingdom Background: Syngeneic models are widely used to model the impact of cancer immunotherapy on tumour growth and tumour invading leucocytes (TILs), and the majority of such work is typically carried out in the subcutaneous setting. However, orthotopic models are known to better model cancer in patients as they form a single focal disease area as

Poster abstracts S111 in the patient situation, facilitate metastatic spread via intra- and extrathoracic lymph nodes and, in the case of syngeneic models, strain-specific tumour microenvironment interactions (immune and stromal components). Bioluminescent imaging (BLI) increases the usefulness of such models, as it allows for non-invasive longitudinal monitoring of tumour burden, allowing for optimal randomisation and reduction of false positives. It also allows continuous feedback allowing one to optimise treatment regimen mid-study. Herein we describe the generation of several bioluminescent variants of syngeneic cell lines commonly used for immunotherapy studies and assess the impact of orthotopic growth on response to immune checkpoint therapy. Materials and Methods: Bioluminescent cell line variants of syngeneic cell lines were established by lentiviral transduction for: 4T1 (breast), B16-F10 (melanoma), MBT-2 (bladder) H22 (Hepatoma), LL/2 (lung) and Pan02 (Pancreatic). Following establishment of stable cultures, DNA profiling and in vitro cytotoxicity assays was carried out to ensure there was no significant changes in DNA, cell doubling time or response to SoC agents following transduction. Subcutaneous growth of wild-type and bioluminescent variants was compared to assess any impact of luciferase expression on tumour growth, inflammation and TILs. Orthotopic models were established for most cell lines, and a metastatic model for B16-F10, BLI was carried out to assess real-time tumour growth and tumour burden at end stage (Spectrum CT; PerkinElmer). Immune checkpoint therapy was also assessed and TIL infiltration by FACs analysis and IHC. Results: Stable transduced bioluminescent cell lines were established; cell doubling time, morphology and growth in vitro was found to be consistent with their wild-type counterparts. Bioluminescent 4T1 cells exhibited growth consistent to that previously reported and readily metastasised to the lungs from both the orthotopic and subcutaneous sites; B16-F10 cells readily metastasise to bone following intracardiac administration. Subcutaneous response to immunotherapy did not appear to be affected. Modulation of immune cell infiltration will be reported and correlated to response. Conclusions: The growth and response to immunotherapy does not appear to be significantly impacted in the bioluminescent cell line models tested; as such they are a useful tool for further assessing the impact of complex orthotopic, spontaneous and experimental metastasis modelling in immune competent mice. Conflict of interest: Board of Directors: Dr Rajendra Kumari, Crown Bioscience UK Ltd; Prof Neil Williams, KWS Biotest Ltd. Corporatesponsored Research: Research has been funded by Crown Bioscience UK Ltd. 338 Poster (Board P017) Combination strategies with checkpoint immunotherapy and inducers of immunogenic cell death (ICD) in immune competent syngeneic models A. McKenzie1 , N. Papadopoulou1 , Y. Yin1 , S. Jiang1 , J. Wrigley2 , J. King3 , R. Garland4 , N. Williams5 , R. Kumari6 . 1 Crown Bioscience UK Ltd, Scientific Operations, Loughborough, United Kingdom; 2 Crown Bioscience UK Ltd, In vitro Operations, Loughborough, United Kingdom; 3 Crown Bioscience UK Ltd, In vivo Operations, Loughborough, United Kingdom; 4 KWS Biotest, Analytical Services, Bristol, United Kingdom; 5 KWS Biotest, KWS Biotest, Bristol, United Kingdom; 6 Crown Bioscience UK Ltd, CBUK, Loughborough, United Kingdom Background: A number of treatment strategies such as radiotherapy (RT), oncolytic viruses and chemotherapeutic agents such as oxaliplatin, doxorubicin, bortezomib and mitoxantrone have been highlighted as potential inducers of immunogenic cell death (ICD) through a well-defined mechanism resulting in the increased presentation of cell-associated antigens to CD4+ and CD8+ T lymphocytes by dendritic cells. Thus combination strategies of ICDs with immunotherapy (IT) could provide opportunities to harness the immune system to extend survival, even among metastatic and heavily pre-treated cancer patients and may increase the efficacy of immunotherapy in those cancer types with low immunogenic status. Here we report the application of RT and oxalipatin in combination with IT (anti-CTLA-4) to examine its impact on tumour growth and immunity. Materials and Methods: Bioluminescent 4T1 mammary carcinoma cells or CT26 mouse colon cells were implanted subcutaneously or orthotopically into BALB/c mice. Subcutaneous tumour growth was monitored by calliper measurement and bioluminescent imaging (BLI) was carried out to confirm orthotopic and/or metastatic growth. Established tumours were treated with IT in combination with chemotherapy, or RT; body weight and clinical condition of mice were monitored daily. At termination the tumours were collected and assessed for immune cell infiltration and/or ICD markers by FACS and IHC. Results: CT26 but not 4T1 tumours exhibited a moderate response to IT, whilst treatment with RT resulted in a statistically significant tumour growth inhibition (TGI) in both models. Combination of both regimens resulted in an additive TGI over monotherapy; additionally, for CT26, tumour response