Production of functionally active murine and murine::human chimeric F(ab')2 fragments in COS-1 cells.
We report here the synthesis, in nonlymphoid cells, of two functionally active recombinant F(ab')2 fragments directed against the tumor marker, human placental alkaline phosphatase (hPLAP). The truncated heavy chain (HC) sequences, E6Hf2 and E6Hy3f2, of the murine F(ab')2 fragment, E6F2, and of the murine::human chimeric F(ab')2 fragment, E6(Hy3,kappa)F2, respectively, were engineered by introducing an in-phase stop codon within the second constant domain of the corresponding parental HC sequence. The antibody-encoding genes were placed under control of the simian virus 40 late promoter and each HC sequence, together with the light chain (LC) sequence, was transiently expressed in COS-1 cells. The truncated HCs were correctly synthesized, processed and assembled with the murine LC and subsequently secreted into the culture medium as functionally active entities with stable hinge region interactions. These results indicate that, under the conditions used, the hinge region was sufficient for the formation of divalent molecules. However, Western blotting revealed the presence of hPLAP-binding half-molecules of E6F2, which was not the case for E6(Hy3,kappa)F2. Since E6F2 and E6(Hy3,kappa)F2 mainly differ by the length of their hinge region (22 and 62 aa residues, respectively) and the number of inter-HC disulfide bridges (four and eleven, respectively), it may be concluded that F(ab')2 fragments with an extended hinge region and several inter-HC disulfide bridges are formed more efficiently.
De Sutter
K
K
Laboratory of Molecular Biology, State University, Gent, Belgium.
Feys
V
V
Van de Voorde
A
A
Fiers
W
W
D000469:Alkaline Phosphatase / Q000276:immunology / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002678:Chimera; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology; D010957:Plasmids; D011247:Pregnancy; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase