B cells on the road to defeat cancer: A novel approach to generate tumorspecific human immunoconjugates from breast carcinomas and melanomas.

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Background: A great variety of solid tumors is characterized by lymphocytic infiltrates, especially T cells. The amount of B lymphocytes in tumors is variable. In some breast cancers heavy B cell infiltration (TIL-B) could be correlated with improved patient survival. Objectives: We aimed to investigate these B cells infiltrating breast carcinomas and melanomas. The question was, whether our way for tumor specific antibody fragment generation in medullary breast carcinoma would lead to similar results in melanomas? Methods: Breast carcinoma and melanoma tumor tissues were collected and processed. Heavy and light chain immunoglobulin variable region genes were amplified from B cells accumulated in the tumor tissues. Single chain Fv (scFv) antibody fragments were constructed. Phage libraries are to be generated for testing the specific tumor binding. Reaction pattern of tumor GD3 ganglioside specific antibody fragments on different breast cancer and melanoma cells were investigated in various systems: cell ELISA, immunofluorescence FACS analysis, confocal microscopy, and dot blot techniques. Results: With our previously developed novel technique we obtained human antibody fragmenst with unique GD3 ganglioside specificities on breast carcinomas and melanomas. The anti-GD3 antibody pattern was selective for the cancerous tissues and there was no or very faint reaction with normal cells. Heavy and light chain immunoglobulin variable region genes could be amplified from any of the selected breast cancer and melanoma tissue samples. This way single chain Fv antibody fragments of TIL-B origin could be successfully generated for further tumor specific binder selection process. Conclusion: Our results suggest that B cells accumulated in breast carcinomas have a potential capacity to reveal aberrant ganglioside structures characteristic for tumor cells. These data give evidence that GD3 ganglioside specific human antibody fragments may be generated from tumor tissue which reacts on breast carcinomas and melanoma. GD3 gangliosides are key molecules in tumorimmunology because of their special characteristics and functions. Our novel approach leads to the development of tumor specific human antibody fragments with diagnostic and immunotherapeutic potential. Acknowledgements: OTKA T048933, OTKA T030380, NATO CLG 978639, Rajko Medenica Research Foundation.