Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity.
Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule death receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family receptors, avidity-mediated oligomerization of DR4 augments the receptor-mediated apoptotic cell death by promoting intracellular cell death signaling.
Dept. of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea.
Seung-Hyun
SH
Lee
Dong-Woon
DW
Park
Eun-Sil
ES
Sung
Hye-Ran
HR
Park
Jin-Kyoo
JK
Kim
Yong-Sung
YS
Kim
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D045325:HCT116 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000819:agonists* / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment
D000906:Antibodies; D055767:Immobilized Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011993:Recombinant Fusion Proteins