Versuche zur Herstellung menschlicher monoklonaler Antikörper gegen Melanome unter Verwendung zervikaler Lymphknoten

Zusammenfassung

Zur Herstellung menschlicher monoklonaler Antikörper wurde ein System entwickelt, in dem B-Lymphozyten mit einer menschlichen B-Zellinie (Wi-42 729 NF) fusioniert werden können. Um auch humane monoklonale Antikörper gegen autologe Melanomzellen herzustellen, werden zervikale Lymphknoten von Melanom-patienten aufgearbeitet. Die so entstandenen Einzelzellpräparationen werden zur Gewinnung einer Melanomzellinie und als Quelle von Lymphozyten benutzt, die gegen diese Tumorzellen sensibilisiert sind.

Die Lymphozyten werden direkt oder nach Kryopräservation in vitro mit Poke-Weed-Mitogen stimuliert, mit Zellen einer B-Zellinie fusioniert und anschließend auf Antikörperproduktion gegen eigene Melanomzellen geprüft. Auf diese Weise wurden sowohl Melanomzellinien als auch Antikörper-produzierende Hybridome gewonnen.

Summary

Due to their specificity, constant properties and virtually unlimited supply monoclonal antibodies have given an important stimulus to almost every field of biomedical research within the last 10 years. The generation of mouse monoclonal antibodies includes immunisation of mice followed by fusion of mice spleen cells with a murine myeloma cell line. With this procedure hybridomas secreting monoclonal antibodies of a predefined specificity can be obtained.

For three major reasons we worked on the establishment of human hybridomas secreting specific antibodies:

• human antibodies are less immunogenic when used for diagnostic or therapeutic purposes,

• only human monoclonal antibodies allow the analyses of the human B cell repertoire,

• there is evidence that human monoclonal antibodies recognise epitopes different from those seen by murine monoclonal antibodies.

Therefore, we set out to generate human B cell hybridomas by cell fusion using the human lymphoblastoid B cell line Wi-L2-729 HF₂ and lymphocytes from melanoma patients. The lymphocytes were isolated from tumour-draining cervical lymph nodes, stimulated with pokeweed mitogens plus the autologous tumour cells in an enriched tissue culture medium and fused in the presence of polyethylene glycol. Supernatants of hybridomas were screened in a single cell immunosorbent assay with either autologous melanoma cells or established melanoma cell lines fixed to the bottom of Terasaki plates or on cytospin preparations of these cells using the immunoperoxydase staining procedure.

We could demonstrate that the tumour draining lymph nodes of these melanoma patients contained B lymphocytes capable of producing antibodies reacting with the tumour cells. Several monoclonal antibodies were established that showed reactivity with cytoplasmic or cell surface determinants.

Such reagents can be potentially useful as diagnostic or therapeutic tools in man.