Abstract
Antibodies against melanoma antigens have been detected in patients but, despite known regulatory and activatory functions attributed to humoral immunity, the roles of B cells in solid tumours such as melanoma are inadequately understood. Insights into humoral responses and mechanisms of tumour-induced immune escape may in-form the design of more effective antibodies. The aims of this thesis are three-fold: a) to gain insights into regulatory mechanisms in tumour microenvironments that influence antibody expression; b) to examine whether humoral immune responses are associated with clinical outcomes with a view of defining biomarkers for melanoma; and c) to design antibody therapeutic strategies that may be less prone to tumour-induced immunomodulatory mechanisms. Th2-biased microenvironments favour production of IgG4 subclass antibodies, mainly through local expression of IL-10. Since IL-10 is expressed locally in melanoma tu-mours, B cell infiltration, IgG expression, cytokine production and IgG subclass distribution in melanoma tissues (n=57) were investigated and compared to samples from health volunteers (n=26). Consistent with Th2-biased inflammation, CD22+ and IgG4+ B cells infiltrated melanoma lesions. When cultured together ex vivo, B cells secreted increased VEGF and IgG4, while tumour cells enhanced IL-10 secre-tion. Two antibodies (IgG1, IgG4) against the tumour-associated antigen CSPG4 were engineered to examine the functional significance of IgG4 subclass. Despite ac-cumulation in tumours, anti-CSPG4 IgG4, in contrast to anti-CSPG4 IgG1, did not trigger effector cells to kill tumours in vitro and in vivo. IgG4 mediated IgG1 block-ade through the reduction of FcγRI activatory signalling, reducing immune effector cell capacity, and significantly impairing the potency of IgG1 in a humanised mouse model of cutaneous melanoma. Since IgG4 may impair anti-tumoural immunity, correlations between IgG4 serum levels and clinical outcomes were studied. Increased IgG4/IgGtotal ratios (G4-levels) in melanoma patient sera (n=173) were seen compared to those of healthy volun-teers (n=104). G4-levels were predictive of disease progression (ROC Curve analysis z=0.62; p=0.0065). Using 0.034 as a cut-off for G4-levels (Youden Index) higher ex-pression correlated with decreased progression-free survival (median 694 days; hazard ratio 2.559; 95% CI 1.555 to 4.211; p=0.0004) and overall survival (median 879 days; hazard ratio 1.871; 95% CI 1.0.45 to 3.349; P=0.035). These findings suggest that IgG4 may be further evaluated as a putative biomarker in sera of patients with melanoma. Tumour immune evasion may be overcome by employing antibodies less prone to Fcγ-mediated blockade, such as those of the IgE class. Two antibodies, anti-CSPG4 IgG1 and anti-CSPG4 IgE induced significant tumor cell death by differential mecha-nisms: antibody-dependent cell-mediated phagocytosis and antibody-dependent cell-mediated cytotoxicity, respectively, by human monocytes in vitro. Anti-CSPG4 IgE was however superior to IgG1 (p<0.05) in restricting subcutaneous human melanoma tumour growth in a humanized mouse model. IgE efficacy was confirmed in an or-thotropic patient tumour in mice populated with autologous patient PBMCs. In summary, this thesis reports a novel pathway of tumour evasion through melanoma favouring production of IgG4 subclass antibodies; provides evidence that IgG4 can be considered as a putative biomarker in melanoma and demonstrates a possible strategy to overcome Fcγ-mediated blockade by designing an IgE antibody against a melanoma-associated antigen and demonstrating its superiority to IgG1.| Date of Award | 2014 |
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| Original language | English |
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| Supervisor | Sophia Karagiannis (Supervisor), Frank Nestle (Supervisor) & James Spicer (Supervisor) |