Malignant melanoma is an immunogenic tumour capable of inducing a humoral immune response, as shown by tumour-reactive serum antibody in patients. Lack of effective chemotherapy in association with the immunogenic nature of the malignancy, has stimulated interest in the immunological management of the malignancy by antibody. Many mouse monoclonal antibodies against melanoma antigens have been developed, and some have been shown to induce tumour regression. However, a limitation on the use of mouse monoclonal antibodies in patients is the induction of an immune response against the immunising xenogeneic protein. The employment of human monoclonal antibodies, may be expected to reduce the patient's immune response against the allogeneic protein. Although more difficult to produce than mouse monoclonal antibodies, several human monoclonal antibodies have been established which induce tumour regression. Here I describe the establishment of mouse/human heterohybridomas producing human monoclonal antibody, from tumour-draining lymph nodes. A series of novel assay systems, initially developed and characterised using melanoma reactive mouse monoclonal antibodies, were sequentially employed for the selection of human antibody exhibiting high tumour specificity. Several clones producing melanoma reactive human antibody were established. Clone MDT. 1 was selected for further characterisation, because of its highly selective reactivity against viable melanoma and other neuroectodermal cell lines, but lack of reactivity against other common malignant and non-malignant cell lines. Such restricted cell reactivity is characteristic of reactivity with class 2 tumour associated antigens. MDT. 1 was shown, in ELISA, to exhibit reactivity to ganglioside antigens GD3, GD2, GD1b, GM3 and GM2. These antigens are commonly associatedw ith the malignant transformation of melanocytes and other neuroectodermal cells. Enzymatic modification of GM3, with neuraminidase, identified the reactive minimal essential epitope as Neua2- 3Galß1-4GIc-. Reactivity with rat monoclonal antibody 9G4 and molecular analysis showed MDT. 1 is encoded by the highly conserved VH4 gene, VH4-21. Like other VH4-21 encoded autoantibodies MDT. 1 exhibits reactivity with the cold agglutinin T. Analysis of the structures of `i' and sialogangliosides has identified similar structural epitopes, which may confer MDT. 1 reactivity. VH4-21 encoded autoantibody 216 exhibits similar reactivity with tumour associated ganglioside antigens as MDT. 1. Sialo-ganglioside/`i' reactive VH4-21 encoded antibodies, could therefore represent an important aspect of autoantibodies in the overall host immune response to tumour.

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Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-cell Chronic Lymphocytic Leukaemia were generated by culture in Interleukin-4 and Granulocyte Macrophage-Colony Stimulating Factor. Lysate-pulsed autologous dendritic cells were used as antigen presenting cells in co-culture with autologous B-cell Chronic Lymphocytic Leukaemia T-cells. B-cell Chronic Lymphocytic Leukaemia T-cells stimulated with B-cell Chronic Lymphocytic Leukaemia lysate-pulsed autologous dendritic cells showed a significant increase in cell surface expression of Interleukin-2 Receptor (CD25), Interferongamma secretion and cytotoxicity against autologous B-cell Chronic Lymphocytic Leukaemia B-cell targets hut not against targets from healthy volunteers. Responses were only stimulated by the B-cell Chronic Lymphocytic Leukaemia B cell lysate. Cytotoxicity was Major Histocompatibility Complex Class II restricted. The addition of maturation agents such as Lipopolysaccharide, Tumour Necrosis Factor-alpha and Polyriboinosinic Polyribocytidylic Acid to monocyte derived dendritic cells was unsuccessful at increasing anti-tumour responses. Pre-treatment of T cells with Interleukin-15 before stimulation by lysate pulsed autologous dendritic cells increased numbers of activated cells, cytokine secretion and specific cytotoxicity to B-cell Chronic Lymphocytic Leukaemia 8-cells. Fusion of monocyte derived dendritic cells and B-cell Chronic Lymphocytic Leukaemia B-cells generated both Major Histocompatibility Complex Class I and Class II restricted cytotoxicity to B-cell Chronic Lymphocytic Leukaemia B-cell targets. When B-cell lysates were analysed using reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, a B-cell Chronic Lymphocytic Leukaemia specific hand at 42,000 Dalton and other patient specific bands were observed. Only the 65,000 Dalton and 42,000 Dalton hands were capable of stimulating comparable T cell responses as the whole lysate. The 65,000 Dalton band from normal healthy volunteers showed a dominant peptide that closely matched Human Serum Albumin. The 42,000 Dalton band from B-cell Chronic Lymphocytic Leukaemia patients showed a possible match with Human Actin.

Thesis (M.D.) -- Dept. of Obstetrics and Gynaecology, University of Adelaide, 1981. / Typescript (photocopy).

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Background: High-grade serous carcinoma (HGSC) can exhibit high intratumoral heterogeneity (ITH). Despite a strong association between tumor-infiltrating lymphocytes (TIL) and survival in HGSC, ITH may have profound impacts on the anti-tumor T cell response. Yet, it is unknown how anti-tumor T cell responses contend with ITH over time in HGSC. Previous studies in melanoma and HGSC both showed tumor-reactive T cell clones emerge over time with their cognate tumor-antigens. Therefore, I hypothesized patients would share a common mechanism of T cell evolution to respond to ITH in HGSC. If so, I expect to see similar patterns of tumor recognition between primary and recurrent disease. Methods: Tumor-associated lymphocytes (TAL) were expanded from primary and recurrent ascites samples using high-dose IL-2 and a rapid-expansion protocol (REP). Following expansion, TAL were assessed for recognition of autologous tumor by IFN-γ ELISPOT and flow cytometry for CD137. CD137+ tumor-reactive TAL were FACS-purified and the tumor-reactive T cell repertoire was profiled by deep sequencing of TCRβ chains (TCRseq). Tumor-reactive TCR clonotypes were compared between primary and recurrent disease to elucidate differences in tumor-reactive populations over time in HGSC. Results: Patient TAL recognized tumor in two out of three cases. In patient IROC 060, the tumor became more immunogenic between primary and recurrent disease, which may reflect expression of new antigens and/or loss of an immunosuppressive phenotype. In patient IROC 106, the tumor remained immunogenic between primary and recurrent disease, which may reflect maintenance of stable antigen expression and an immune-sensitive phenotype. Patient IROC 034 did not exhibit any tumor-reactivity, suggesting tumor-reactivity is not ubiquitous in HGSC. FACS-purification of CD137+ T cells followed by TCRseq was successfully performed on T cell populations of both high- and low-abundance, suggesting TCRseq can be performed on populations containing very few T cells. TCRseq results that profiled the clonal repertoire of tumor-reactive TAL from primary and recurrent disease in two patients, IROC 060 and IROC 106, showed both patients had evidence of T cell loss and T cell emergence between primary and recurrent disease. Further, IROC 106 had evidence of T cell clones that were maintained between primary and recurrent disease. Conclusions: Anti-tumor T cell responses from ascites are both diverse between patients and dynamic within a patient, suggesting various mechanisms of T cell evolution to contend with ITH in HGSC. I developed a pipeline for the identification of tumor-reactive TCR sequences without the need for a priori knowledge of specific antigens. Additionally, this pipeline is feasible for very low-abundance samples, such as tumor-reactive T cells. Significance: This study provides early insights into how TAL contend with ITH in HGSC. Ultimately, these results will inform the design of adoptive T cell therapy for recurrent HGSC. / Graduate

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