BACTERIALLY EXPRESSED FABS OF MONOCLONAL-ANTIBODIES NEUTRALIZING TUMOR-NECROSIS-FACTOR-ALPHA IN-VITRO RETAIN FULL BINDING AND BIOLOGICAL-ACTIVITY

Antibody fragments specific for the human tumour necrosis factor alpha (TNFalpha) have been cloned from lambda combinatorial expression libr aries using total RNA obtained from three different hybridoma cell lin es of therapeutic interest. The previously described bacteriophage lam bda vectors, lambdaHC2 and lambdaLC1, were modified to create unique a ntibody cloning sites in the combinatorial construct and a novel tag p eptide was inserted at the C-terminal end of the expressed Fd chain. S equence analysis of the cloned Fabs indicated that two of them were de rived from a single B cell. Expression in E. coli showed that the amou nt of recovered Fab in the bacterial culture medium was related to the sequences of the variable coding regions. Hybrid Fabs created by chai n exchange of similar antibodies were as active as the originally pair ed Fabs in binding assays. The relative affinities and the capacities of the bacterially expressed Fabs to neutralize TNFalpha cytotoxicity in vitro were identical to those of the parental antibodies. The resul ts demonstrate that, using an in vitro approach, it is possible to gen erate from existing hybridoma cell lines high affinity Fabs which reta in antigen specificity. The cloning sites incorporated into the C-term inal parts of these Fabs will now permit their further modification to include additional functional characteristics not possible with the o riginal hybridoma antibodies.