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Dissecting humoral immune responses in melanoma and the design of antibody immunotherapy

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.

Identiferoai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:762267
Date January 2014
CreatorsKaragiannis, Panagiotis
ContributorsKaragiannis, Sophia ; Nestle, Frank Oliver ; Spicer, James Frederick
PublisherKing's College London (University of London)
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation
Sourcehttps://kclpure.kcl.ac.uk/portal/en/theses/dissecting-humoral-immune-responses-in-melanoma-and-the-design-of-antibody-immunotherapy(1b263e41-dedd-4d98-a045-97bfd6fa6f0b).html

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