EXPRESSION OF MONOVALENT FRAGMENTS DERIVED FROM A HUMAN-IGM AUTOANTIBODY IN ESCHERICHIA-COLI - THE INPUT OF THE SOMATICALLY MUTATED CDR1 CDR2 AND OF THE CDR3 INTO ANTIGEN-BINDING SPECIFICITY/

A hybridoma producing a polyspecific human monoclonal IgM antibody (na med CB03) has been derived from a fusion of mouse myeloma cells a with human spleen lymphocytes obtained from an autoimmune patient sufferin g from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study t he input of mutated complementarity regions (CDRs) into antibody speci ficity, the antigen binding features of the purified complete IgM anti body were compared with (i) a Fab fragment obtained by hot tryptic dig estion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and singl e chain Fv03 fragments containing the V-H and V-L genes connected by a linker sequence, (ii) scFc1.1, fragments containing the V-H germline equivalent and the CB03 mild-type CDR3 region, and (iii) scFv fragment s containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polysp ecific IgM antibody. The expression vectors contained at the 3' end ei ther a (His)(6) motif allowing purification on Ni2+-agarose or a c-myc tag for specifically detecting the expression products by a murine mo noclonal antibody. Western blotting and ELISA analyses of the expressi on products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 mu g from 1 litre b acterial culture), (ii) scFv fragments were obtained in suitable amoun ts from bacterial periplasm (800-1000 mu g/l), () the monovalent recom binant fragments as well as the Fab obtained by tryptic digestion refl ected the polyspecific antigen binding features of the complete IgM an tibody, but did bind to the antigens with much lower affinity, and (iv ) the CDR3 was found to be of critical importance for the antigen bind ing pattern of this particular IgM. We discuss the expression of recom binant. scFv fragments in E. coli as a suitable method in studying the role of somatic mutation in autoantibody generation.