SPECIFIC DETECTION OF MYELOMA PLASMA-CELLS USING ANTIIDIOTYPIC SINGLE-CHAIN ANTIBODY FRAGMENTS SELECTED FROM A PHAGE DISPLAY LIBRARY

Bone marrow plasma cells constitute the bulk of malignant cells in mul tiple myeloma patients. B-lymphocytes having immunoglobulin heavy chai n gene rearrangements identical to those of the malignant clone (clona lly related B-lymphocytes) may function as malignant plasma cell precu rsors. We and others proposed the use of anti-idiotypic antibodies to isolate and study clonally related B-lymphocytes. This strategy failed until now because anti-idiotypic antibodies raised by conventional hy bridoma techniques proved to react frequently with epitopes shared by different idiotypes. Recently, we succeeded in selecting specific sing le chain Fv antibodies from phage libraries. To select single chain Fv bearing phages specifically directed against the immunoglobulin idiot ype expressed by myeloma tumor cells we panned a semisynthetic phage l ibrary against purified myeloma paraprotein Fab fragments. The selecti on was performed in the presence of soluble polyclonal immunoglobulin as a competitor. Three independent selections for three myeloma patien ts yielded 10-26 clones. Between two and seven of the selected clones were reactive with patient Fab and not with polyclonal immunoglobulin in enzyme-linked immunosorbent assays. Five out of six anti-idiotypic single chain Fvs were able to specifically stain fixed monoclonal plas ma cells in myeloma bone marrow. Idiotype specificity of these single chain Fvs was confirmed by flow cytometry since they did not react wit h monoclonal plasma cells of other patients, a panel of nine myeloma c ell lines, isolated polyclonal bone marrow plasma cells and cultured B -lymphocytes. Using these anti-idiotypic reagents we were able to dete ct 25 myeloma plasma cells in a background of 50 000 immunoglobulin is otype-matched cells of the myeloma cell line UM-1 or 50 000 donor bone marrow cells (sensitivity 0.05%). This paper shows that highly specif ic anti-idiotypic single chain Fv antibody fragments selected from a p hage display library can be used to detect rare idiotypic cells in pat ient samples.