Abstract
Production of recombinant antibody fragments in bacterial expression systems results in intentional or fortuitous differences compared to the original products prepared by hybridoma technology. These differences may have significant effects not only on antigen‐binding properties but also on pharmacokinetic and tumor‐seeking properties. Our major goal was to investigate some of these possible differences. We produced in Escherichia coli an rFab′ fragment containing only 1 cysteine residue in the hinge region; the fragment was derived from a mouse MAb (F6) specific for CEA. The rFab′ had a slightly lower m.w. and a higher isoelectric point relative to the corresponding nonrecombinant fragment (pFab′). This was explained by the absence of N‐glycosylation on the Vκ domain of rFab′. Vκ glycosylation had no significant effect on antibody‐binding affinity and kinetics. However, rFab′ was eliminated from the circulation much faster than pFab′, and the maximal dose accumulated in the tumor was reduced relative to pFab′. Thus, glycosylation appears to modify the targeting efficiency of antibody fragments. rF(ab′)~2~ fragments were obtained either spontaneously from the culture supernatant of E. coli or by chemical cross‐linking [rcF(ab′)~2~]. We observed improved tumor targeting with rcF(ab′)~2~ compared to rF(ab′)~2~, which could be explained by the greater stability of the thioether compared to the disulfide linkage. These results demonstrate that a single cysteine residue in the hinge region of rFab′ is particularly well suited to prepare stable, chemically coupled, bivalent or bispecific antibodies, avoiding intrahinge disulfide bonding and thus achieving higher production yields. © 2002 Wiley‐Liss, Inc.