Molecular structural and functional characterization of tumor suppressive anti-ErbB-2 monoclonal antibody by phage display system.
To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.
Itoh
Kunihiko
K
Department of Pharmaceutical Science, Akita University Hospital, 1-1-1 Hondo, Akita 010-8543, Japan. itohk@hos.akita-u.ac.jp
Inoue
Kazuyuki
K
Tezuka
Takehiko
T
Tada
Hitoshi
H
Hashimoto
Yoshiyuki
Y
Masuko
Takashi
T
Suzuki
Toshio
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D003429:Cross Reactions; D018604:Epitope Mapping; D000939:Epitopes; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases