Humoral immune response to melanoma: discovery and evaluation of anti-melanoma antibodies

Melanoma, a potentially lethal form of skin cancer, is widely thought to be immunogenic in nature. While numerous studies have examined T cell-mediated immune responses to melanoma and their therapeutic potential, there has been less focus on B cell-mediated immune responses and the tumor-reactive antibodies they produce. The aim of this work was three-fold: (1) to develop a methodology to detect antibodies secreted by human B cells that recognize melanoma cell surface proteins; (2) to evaluate the mature B cell repertoire of individuals with melanoma for antibody subclass composition and the presence and prevalence of anti-tumor antibodies; and (3) to study patient-derived antibodies and two engineered antibodies recognizing melanoma cells for their propensity to activate immune effector cells and their capacity to kill or restrict the growth of tumor cells. <br/>As part of this thesis, a novel tumor cell-based ELISA was developed for the detection of tumor-reactive antibodies. Utilizing this new assay, the presence and prevalence of melanoma-reactive IgG antibodies derived from ex vivo cultured peripheral blood B cells from a cohort of 21 patients with melanoma (Stage I, n=1; Stage II, n=8; Stage III, n=6; Stage IV, n=6) were compared to those from healthy volunteers (n=10). While B cells from melanoma patients secreted IgG antibody concentrations comparable to those from healthy volunteer B cell cultures, a significantly increased reactivity of antibodies derived from patients to primary and metastatic melanoma cells was measured compared to healthy volunteers (P&lt;0.001). Interestingly, there was a significant reduction in antibody responses to melanoma with advancing disease stage that was not found to be solely due to a -ii-reduction in the B cell memory compartment size. Comparing IgG antibody subclass distribution among cutaneous tumors, patient lymph nodes and peripheral blood B cells all isolated from individuals with metastatic disease, elevated proportions of IgG4 subclass antibodies were observed in cutaneous tumors which present a novel finding of this thesis. These findings point to differentially polarized humoral immune responses in cutaneous tumor microenvironments. <br/>Lastly, an antibody derived from a patient was then selected and preliminary evaluations of reactivity and specificity to a range of melanoma cell lines and primary human melanocytes were conducted. Using a live cell imaging cytotoxicity assay, this patient-derived melanoma-specific antibody was observed to kill melanoma cells via antibody-mediated cellular cytotoxicity. Additionally, two engineered monoclonal antibodies recognizing a melanoma associated antigen were found to partially restrict tumor cell migration and adhesion and to kill melanoma cells via antibody-dependent cellular cytotoxicity or phagocytosis. <br/>In summary, examining the humoral immune response to melanoma and the effector function of antibodies targeting melanoma cells provides insight into the discovery of new therapeutic strategies for the treatment of melanoma.<br/>

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2013

Author dc:creator

• Gilbert, Amy

Advisors dc:contributor.advisor

• Karagiannis, Sophia
• Nestle, Frank Oliver
• Spicer, James Frederick

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/34c0be73-e94a-4d3f-92b9-8888178c4cd2

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/34c0be73-e94a-4d3f-92b9-8888178c4cd2