Phage display cloning and characterization of monoclonal antibody genes and recombinant Fab fragment against the CD98 oncoprotein.
The Fab gene of anti-CD98 heavy chain (h.c.) monoclonal antibody (mAb) HBJ127 was cloned and expressed as a recombinant Fab (rFab) fragment by means of a phage display system. The variable heavy and light chain genes of HBJ127 were found to be derived from VOx-1 and IgVk8-30 germline, respectively. Extensive somatic mutation was found in the heavy chain complementarity determining region 2. rFab fragment was purified homogeneously from crude bacterial lysates by Ni-chelate chromatography in a yield of 71.4 mg from 100 ml of culture. rFab fragment was reactive with the cell surface of CD98-positive cells irrespective of tissues of origin, but not with CD98-negative cells. The recognition site of the rFab fragment was identical to that of mAb since the binding of rFab fragment to HeLaS(3) cells was completely inhibited by pretreatment with an excess of mAb. The relative affinity values of rFab fragment and mAb were found to be 0.11 x 10(8) and 0.35 x 10(8) M(-1), respectively. Three-fold lower affinity of rFab fragment may be due to the difference of valency of the antibody preparation. Cell growth inhibition in vitro by rFab fragment preincubated with anti-Fab suggests that the rFab fragment produced by cloned gene-bearing Escherichia coli was identical to the Fab part of HBJ127 mAb. These results show that a small fragment with antigen binding activity similar to that of the parent mAb can easily be prepared by using a phage display system. To our knowledge, this is a first report of the production of anti-CD98 h.c. rFab fragment.
Itoh
K
K
Department of Pharmaceutical Science, Akita University Hospital, Akita 010-8543, Japan. itohk@hos.akita-u.ac.jp
Inoue
K
K
Hirooka
K
K
Maruyama
K
K
Ohkawa
M
M
Matsui
K
K
Tada
H
H
Enomoto
T
T
Hashimoto
Y
Y
Suzuki
T
T
Masuko
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D001435:Bacteriophages / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular / Q000379:methods*; D005434:Flow Cytometry; D027261:Fusion Regulatory Protein-1 / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D027261:Fusion Regulatory Protein-1; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins