Defining and targeting combination immunotherapies in mouse models of cancer

Checkpoint blockade has achieved long-lasting anti-tumour responses, unfortunately this is limited to a fraction of patients, highlighting the need for more effective therapies. This thesis focuses on the rational proposal and design of new cancer immunotherapies through: (1) proposing a novel immunomodulatory-target for cancer-immunotherapy, Inducible T-cell co-stimulator (ICOS), and studying its efficacy in murine models of cancer; and (2) the description of the immune tumour-microenvironment (TME) of mouse models of lung cancer, to propose strategies that promote increased immunogenicity and tumour rejection. In models of melanoma, the absence of ICOS/ICOSL pathway in ICOS-/- mice, impaired the efficacy of anti-CTLA-4 (Cytotoxic T-lymphocyte antigen-4) therapy. Additionally, patients that received ipilimumab (anti-CTLA-4) monoclonal antibody (mAb) had an increase in the frequency of ICOS+ T-cells. We hypothesised that an agonistic non-depleting anti-ICOS mAb will promote the function of activated T-cells in the TME. Here we show that an agonistic anti-ICOS mAb, with either mIgG1 (non-depleting) or mIgG2a (depleting) isotype, does not promote survival, either as a monotherapy or in combination with other antibody therapies. We also showed that both anti-ICOS isotypes eliminated T-cells in the TME and that anti-ICOS mIgG1 T-cell elimination was Fc-engagement independent. These results were replicated using mice expressing human Fcγ receptors (FcγRs) and anti-ICOS mAb with human (h)IgGs, demonstrating that anti-cancer therapy with anti-ICOS mAbs should be carefully evaluated before use in clinical trials. To design and test new combination therapies, we described the immune-TME of mouse models of lung cancer. Currently, lung cancer has the highest mortality among cancers, with immunotherapy-benefit limited to some patients. Here we described the TME of two mouse models of lung cancer: KPB6.F1 and CMT-167. We did not find significant differences in the TME of the KPB6.F1 model after radiotherapy and chemotherapy. To promote immunogenicity, combination therapy with anti-CD25 mAb and anti-4-1BB mAb was evaluated in both the KPB6.F1 and CMT-167 models. Anti-4-1BB promoted proliferation, granzyme B production and expression of activation markers on effector CD4+ and CD8+ T-cells. Whilst this combination reduced the tumour-burden of the CMT-167 model, no differences were observed in the KPB6.F1 model, suggesting intrinsic differences between them. Further work describing the differential response of both models to specific therapies could provide important information regarding resistant tumours in patients, together with strategies to overcome those resistances. The work presented in this thesis describes variations in the immune-TME following different therapies, suggesting that further investigation is crucial for understanding the biology of the mechanism of action of cancer immunotherapies and to improve their efficacy

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Dopaminergic Signalling Enhances IL-2 Production and Strengthens Anti-Tumour Response Exerted by Cytotoxic T Lymphocytes in a Melanoma Mouse Model

Dopamine has emerged as an important regulator of immunity. Recent evidence has shown that signalling through low-affinity dopamine receptors exerts anti-inflammatory effects, whilst stimulation of high-affinity dopamine receptors potentiates immunity in different models. However, the dopaminergic regulation of CD8+ T-cells in anti-tumour immunity remains poorly explored. Here, we studied the role of dopamine receptor D3 (DRD3), which displays the highest affinity for dopamine, in the function of CD8+ T-cells and its consequences in the anti-tumour immune response. We observed that the deficiency of Drd3 (the gene encoding DRD3) in CD8+ T-cells limits their in vivo expansion, leading to an impaired anti-tumour response in a mouse melanoma model. Mechanistic analyses suggest that DRD3 stimulation favours the production of interleukin 2 (IL-2) and the surface expression of CD25, the α-chain IL-2 receptor, which are required for expansion and effector differentiation of CD8+ T-cells. Thus, our results provide genetic and pharmacologic evidence indicating that DRD3 favours the production of IL-2 by CD8+ T-cells, which is associated with higher expansion and acquisition of effector function of these cells, promoting a more potent anti-tumour response in a melanoma mouse model. These findings contribute to understanding how dopaminergic signalling affects the cellular immune response and represent an opportunity to improve melanoma therapy

Dopamine Receptor D3 Signaling on CD4(+) T Cells Favors Th1-and Th17-Mediated Immunity

Dopamine receptor D3 (DRD3) expressed on CD4(+) T cells is required to promote neuroinflammation in a murine model of Parkinson's disease. However, how DRD3 signaling affects T cell-mediated immunity remains unknown. In this study, we report that TCR stimulation on mouse CD4(+) T cells induces DRD3 expression, regardless of the lineage specification. Importantly, functional analyses performed in vivo using adoptive transfer of OVA-specific OT-II cells into wild-type recipients show that DRD3 deficiency in CD4(+) T cells results in attenuated differentiation of naive CD4(+) T cells toward the Th1 phenotype, exacerbated generation of Th2 cells, and unaltered Th17 differentiation. The reciprocal regulatory effect of DRD3 signaling in CD4(+) T cells favoring Th1 generation and impairing the acquisition of Th2 phenotype was also reproduced using in vitro approaches. Mechanistic analysis indicates that DRD3 signaling evokes suppressor of cytokine signaling 5 expression, a negative regulator of Th2 development, which indirectly favors acquisition of Th1 phenotype. Accordingly, DRD3 deficiency results in exacerbated eosinophil infiltration into the airways of mice undergoing house dust mite-induced allergic response. Interestingly, our results show that, upon chronic inflammatory colitis induced by transfer of naive CD4(+) T cells into lymphopenic recipients, DRD3 deficiency not only affects Th1 response, but also the frequency of Th17 cells, suggesting that DRD3 signaling also contributes to Th17 expansion under chronic inflammatory conditions. In conclusion, our findings indicate that DRD3-mediated signaling in CD4(+) T cells plays a crucial role in the balance of effector lineages, favoring the inflammatory potential of CD4(+) T cells.This work was supported by Fondo Nacional de Desarrollo Cientı´fico y Tecnolo´gico de Chile Grant 1130271, Comisio´n Nacional de Investigacio´n Cientı´fica y Tecnolo´gica de Chile Grant PFB-16, and Michael J. Fox Foundation Grant 10332 (to R.P.), as well as by the Programa de Formacio´n de Capital Humano Avanzado–Magı´ster Nacional, Comisio´n Nacional de Investigacio´n Cientı´fica y Tecnolo´gica de Chile Grants 22140120 (to F.O.-B.) and 22150983 (to E.L.), the Programa Atraccio´n e Insercio´n de Capital Humano Avanzado, Comisio´n Nacional de Investigacio´n Cientı´fica y Tecnolo´gica de Chile Grant 82130031 (to F.O.), and a Universidad Andre´s Bello Ph.D. fellowship (to D.E.)

Diabetes interactive diary: a new telemedicine system enabling flexible diet and insulin therapy while improving quality of life: an open-label, international, multicenter, randomized study

OBJECTIVE - Widespread use of carbohydrate counting is limited by its complex education. In this stud), we compared a Diabetes Interactive Diary (DID) with standard carbohydrate counting in terms of metabolic and Weight control, Lime required for education, quality of life, and treatment satisfaction. RESEARCH DESIGN AND METHODS - Adults with type 1 diabetes were randomly assigned to DID (group A, n = 67) or standard education (group 13, n = 63) and followed for 6 months. A subgroup also completed the SF-36 Health Survey (SF-36) and World Health Organization-Diabetes Treatment Satisfaction Questionnaire (WHO-DTSQ) at each visit. RESULTS - Of 130 patients (aged 35.7 +/- 9.4 years diabetes duration 16.5 +/- 10.5 years), 11 dropped out. Time for education was 6 h (range 2-15 h) in group A and 12 h (2.5-25 h) in group B (P = 0.07). A1C reduction was similar in both groups (group A from 8.2 +/- 0.8 to 7.8 +/- 0.8% and group B from 8.4 +/- 0.7 to 7.9 +/- 1.1%; P = 0.68). Nonsignificant differences in favor of group A were documented for fasting blood glucose and body weight. No severe hypoglycemic episode occurred. WHO-DTSQ scores increased significantly more in group A (from 26.7 +/- 4.4 to 30.3 +/- 4.5) than in group B (from 27.5 +/- 4.8 to 28.6 +/- 5.1) (P = 0.04). Role Physical, General Health, Vitality, and Role Emotional SF-36 scores improved significantly more in group A than in group B. CONCLUSIONS - DID is at least as effective as traditional carbohydrate counting education, allowing dietary freedom for a larger proportion of type 1 diabetic patients. DID is safe, requires less time for education, and is associated with lower weight gain. DID significantly improved treatment satisfaction and several quality-of-life dimensions