Inadequate β-cell mass is essential for the pathogenesis of type 2 diabetes (T2D). Previous report showed that an immunomodulator FTY720, a sphingosine 1-phosphate (S1P) receptor modulator, sustainably normalized hyperglycemia by stimulating β-cell in vivo regeneration in db/db mice. We further examined the effects of FTY720 on glucose homeostasis and diabetic complications in a translational nonhuman primate (NHP) model of spontaneously developed diabetes. The male diabetic cynomolgus macaques of 18-19 year old were randomly divided into Vehicle (Purified water, n = 5) and FTY720 (5 mg/kg, n = 7) groups with oral gavage once daily for 10 weeks followed by 10 weeks drug free period. Compared with the Vehicle group, FTY720 effectively lowered HbA1c, blood concentrations of fasting glucose (FBG) and insulin, hence, decreased homeostatic model assessment of insulin resistance (HOMA-IR); ameliorated glucose intolerance and restored glucose-stimulated insulin release, indicating rejuvenation of β-cell function in diabetic NHPs. Importantly, after withdrawal of FTY720, FBG, and HbA1c remained at low level in the drug free period. Echocardiography revealed that FTY720 significantly reduced proteinuria and improved cardiac left ventricular systolic function measured by increased ejection fraction and fractional shortening in the diabetic NHPs. Finally, flow cytometry analysis (FACS) detected that FTY720 significantly reduced CD4 + and CD8 + T lymphocytes as well as increased DC cells in

BackgroundToll-like receptors (TLRs) serve critical roles in mediating innate immune responses against many pathogens. However, they may also bind to endogenous ligands and lead to the pathogenesis of autoimmunity. Although TLR8 belongs to the same TLR family as TLR7, its role in inflammation and tumor progression is not yet fully understood due to the lack of suitable animal models. In humans, both TLR7 and TLR8 recognize single-stranded self-RNA, viral RNA, and synthetic small molecule agonists.1, 2 However, mouse Tlr8 is non-functional due to the absence of 5 amino acids necessary for RNA recognition. In order to create a mouse model with functional TLR8, we replaced exon 3 of mouse Tlr8 with human TLR8, therefore developing a hTLR8 knock-in (KI) model. Both heterozygous and homozygous hTLR8 KI mice are viable with inflammatory phenotypes, i.e. enlarged spleens and livers, and significantly higher IL-12 p40 levels under TLR8 agonist treatment. In this study, we evaluated the potential use of hTLR8 mice for cancer immunotherapy studies.MethodshTLR8 mice, together with naïve C57BL/6 mice, were inoculated with MC38 syngeneic tumor cells. Tumor bearing mice were grouped at a mean tumor volume of approximately 100 mm3 for treatment with PBS or 10 mg/kg anti-PD-1 (RMP1-14) antibody. At the efficacy endpoint, spleens and tumors were collected for flow cytometry profiling.ResultsAnti-PD-1 treatment of MC38 tumors in naïve C57BL/6 led to moderate tumor growth inhibition (TGI = 54%). Interestingly, anti-PD-1 treatment showed improved efficacy in hTLR8 mice (TGI = 79%), including 2/10 tumors with complete tumor regression. In comparison, non-treated MC38 tumor growth rate was slower in hTLR8 mice than in naïve mice. Anti-PD-1 treated hTLR8 mice also had significantly increased IFN-γ and TNF-a positive CD4+ T cells in the spleen, along with higher numbers of differentiated effector T cells. In addition, hTLR8 mice have activated dendritic cells and macrophages, acting as critical steps in initiation of the inflammatory process, with higher levels of pro-inflammatory cytokines, such as IL-6, IFN-γ, TNF-a, and IL-1β, which may promote Th1 priming and differentiation of T cells into IFN-γ or TNF-a producing cells.ConclusionshTLR8 mice offer a great tool to model cancer immunotherapy in an inflammatory/autoimmunity prone background. Moreover, hTLR8 mice can be effectively used to shift a ‘cold’ tumor phenotype to ‘hot’ tumors in a syngeneic setting.Ethics ApprovalAnimal experiments were conducted in accordance with animal welfare law, approved by local authorities, and in accordance with the ethical guidelines of CrownBio (Taicang).ReferencesKugelberg E. Making mice more human the TLR8 way. Nat Rev Immunol 2014;14:6.Guiducci C, Gong M, Cepika A-M, et al. RNA recognition by human TLR8 can lead to autoimmune inflammation. J Exp Med 2013;210:2903–2919.

Background: The rapid development of immuno-oncology (I/O) therapies for various cancer types has transformed the paradigm of cancer treatment from targeting the tumor to targeting the immune system, and now to combination strategies. Numerous Phase I-III trials with immunotherapeutics and combinations are being conducted; however, a lot of these are failing. More preclinical models are required to help investigate efficacy, uncover mechanisms of action, and to design more rational combination strategies. The most widely used models for immunotherapy evaluation are models with functional murine immunity such as syngeneic allograft models and genetically engineered mouse models (GEMMs). Syngeneic models are used in nearly all I/O therapy programs as these are well-characterized and straightforward to set-up as subcutaneous models. However, one of the major disadvantages of subcutaneous models is the lack of a clinically relevant tumor microenvironment (TME) including stromal cells, immune cells, extracellular matrix etc. The dynamic interplay of these cells drive alterations in cellular functions and impact study outcomes. Using bioluminescent imaging (BLI), we have developed and characterized a panel of orthotopic models where tumor cells are inoculated in a relevant organ-specific location to recapitulate the immune and stromal component interactions with the tumor, which can also facilitate metastatic spread. These models can be used to evaluate various therapies in both the orthotopic and metastatic settings. Method: A panel of syngeneic cell lines were labeled with bioluminescence by transducing the cell lines with a lentiviral vector carrying the firefly luciferase gene. This panel represents a diverse range of cancer types including liver (Hepa 1-6 and HT22), breast (4T1), colon (CT26.WT), brain (GL261), prostate (RM-1), and pancreas (Pan02). These bioluminescent cells were orthotopically implanted into immune competent mice. In-life tumor growth and metastasis were assessed using the IVIS® Spectrum In Vivo Imaging System. Tumor-infiltrating lymphocytes were assessed by flow cytometry (BD LSRFortessa™) and multiplex IHC. Results: Orthotopic implantation of syngeneic models was successfully demonstrated for all models as confirmed by in-life imaging and ex vivo imaging at termination. Baseline response towards various checkpoint inhibitors and chemotherapies were established. For the Hepa 1-6 liver model, response to an anti-PD-1 antibody was significant in both the subcutaneous and orthotopic setting. In contrast, the H22 liver model responded to both sorafenib and anti-mCTLA-4 antibody in the subcutaneous model but not in the orthotopic setting. In addition, anti-mCTLA-4 treatment led to more significant inhibition of pancreatic Pan02 tumor growth in the orthotopic model in comparison with its corresponding subcutaneous setting. Conclusion: Bioluminescent syngeneic models in the orthotopic setting provide a valuable tool for testing immunotherapies, with a more clinically relevant TME in comparison to the corresponding subcutaneous model. The establishment of a range of models covering diverse cancer types allows for a thorough interrogation of immunotherapeutic potential. Citation Format: Diandong Jiang, Bryan Miller, Yanrui Song, Rajendra Kumari, Annie An, Jie Cai, Davy Xuesong Ouyang, Henry Qixiang Li, Yinfei Yin. Characterization of a panel of orthotopic syngeneic models using bioluminescent imaging for the evaluation of immuno oncology therapeutics [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 2788.

BackgroundCD137 (4-1BB) is a powerful T cell co-stimulatory molecule belonging to the TNF receptor superfamily, which promotes cytotoxic T cell survival and memory formation upon CD137L ligation. CD137 has become an attractive immuno-oncology therapeutic target with multiple agonistic antibodies in clinical trials, including urelumab and utomilumab, with promising response in combination with anti-PD1 immunotherapies such as nivolumab. Clinical applications of CD137 agonistic antibodies are hampered, however, by dose-limiting off-tumor liver toxicity (urelumab) or lower efficacy (utomilumab). The cause of liver toxicity is reported primarily to be due to Fcγ receptor mediated cross linking;1–3 CD137 agonistic antibodies may also trigger hepatotoxicity through activation of IL-27 secreting liver Kupffer cells and monocytes.4 The remaining challenge in decoupling efficacy from liver toxicity is the lack of preclinical mouse models which can be used to assess both efficacy and the immune-related adverse events (irAE) of human CD137 agonistic antibodies.MethodsTo mimic the clinical outcomes of urelumab, we utilized humanized CD137 knock-in mice in Balb/c background (Balb/c CD137 HuGEMM) to evaluate its efficacy with CT26.WT syngeneic tumors. Liver toxicity was analyzed by monitoring fasting serum ALT/AST levels at different time points.ResultsUrelumab showed moderate anti-tumor response at the dose level of 5 mg/kg, while serum ALT/AST levels showed no difference compared to isotype control suggesting that, due to the different binding capacity of the human IgG4 Fc domain to mouse FcγR, the human version of the agonistic antibody cannot fully recapitulate its effect on HuGEMM mice. Therefore, a chimeric antibody with mouse IgG1 Fc domain (urelumab-mIgG1) was created to dissect the potential role of FcγR mediated cross linking on both efficacy and liver toxicity; an urelumab-mIgG1-DANA variant with D265A/N297A mutation to abolish Fc effector function was also included as a dominant negative control. We found that urelumab-mIgG1 showed further enhanced efficacy compared to urelumab alone through FcγR mediated cross linking, while urelumab-mIgG-DANA showed compromised anti-tumor response. With regards to liver toxicity, urelumab-mIgG1 caused chronic liver inflammation and hepatocyte damage indicated by immune cell infiltration in the liver and significantly elevated serum ALT levels, which was abolished by the urelumab-mIgG1-DANA variant. The study also compared urelumab treatment in CD137 HuGEMM head-to-head with the mouse surrogate agonistic antibody (3H3) in wild-type BALB/c mice. 3H3 showed robust tumor growth inhibition as well as dramatic ALT elevation.ConclusionsWe faithfully recapitulated the clinically observed tumor growth inhibition and liver toxicity of urelumab by using a chimeric version of urelumab in CD137 HuGEMM, indicating the importance of both the mouse model and antibody version in evaluation of efficacy and irAE.Ethics ApprovalAnimal experiments were conducted in accordance with animal welfare law, approved by local authorities, and in accordance with the ethical guidelines of CrownBio (Taicang).ReferencesClaus C, Ferrara C1, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 2019; 11:eaav5989.Qi X, Li F, Wu Y, et al. Optimization of 4-1BB antibody for cancer immunotherapy by balancing agonistic strength with FcγR affinity. Nat Commun 2019;10:2141–2151.Compte M, Harwood SL, Muñoz IG, et al. A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity. Nat Commun. 2018;9:4809–4821.Bartkowiak T, Jaiswal AR, Ager CR, et al. Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27-dependent pathway. Clin Cancer Res 2018;24:1138–1151.

Mouse tumors of homografts in the corresponding syngeneic mouse strains have become the key experimental system in immune-oncology (I/O) research and evaluating mouse surrogate I/O therapies. There are several types of mouse tumor homografts, including syngeneic cell line-derived, spontaneous or carcinogen-induced tumor-derived, or tumorigeneic GEMM (genetic engineered mouse model) -derived. We have created an increasingly larger panel of such diverse murine tumor homografts (now > 40 syngeneic cell line-derived and > 60 GEMM primary tumor-derived, or MuPrime®, usually driven by defined activated oncogenes frequently seen in human cancers) to support global I/O researches, including discovery of new drugs or drug targets, discovery/validation of biomarkers of both predictive and pharmacodynamic (PD) natures, as well as mechanism of actions (MOA). We are also systematically characterizing these heterogeneous library of murine tumors, in terms of genomic (Transcriptome-sequencing, or RNAseq), immunological (TILs and cytokine profiles), histopathological (lung, PDAC, breast, prostate, liver, CRC, bladder, kidney carcinoma, lymphoma, melanoma, sarcoma, leukemia, etc), growth and pharmacological (SOC, I/O) profiles. With the broad applications of these models at different laboratories and different times, it is prudent to track and QC the tumor to ensure their absolute correct identification (ID) by a readily routine assay. Unlike human-originated tumors (e.g. xenografts), either cancer cell line-derived or patient tumor-derived (PDX) whose genetic ID can readily be tracked by STR (short tandem repeat) genotyping or HLA tissue typing, murine tumors come from limited strains of inbred experimental mice, thus lack of sufficient genetic and/or tissue diversity to be used, in a readily performed assay, for individual murine model ID. To this end, we have established a novel efficient model quality control and tumor ID method in murine tumor model systems, in which we sought out to identify unique gene features in each individual murine tumor among a cohort of > 100. We have validated many such unique gene features in the original cell lines or primary tumors and/or their subsequent passages. Our results demonstrated that these unique gene features can served as genetic fingerprints for murine model ID. Citation Format: Jie Cai, Wubin Qian, Bin Fan, Xiaobo Chen, Sheng Guo, Davy Ouyang, Wenqing Yang, Annie An, Jinglei Bi, Dongliang Mo, Henry Li. Development of murine tumor homograft panels and their genetic fingerprints for their identification to ensure the quality controlled I/O studies using these models [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 5116.