Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.
We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.
Rheinnecker
M
M
MorphoSys GmbH, Munich, Germany.
Hardt
C
C
Ilag
L L
LL
Kufer
P
P
Gruber
R
R
Hoess
A
A
Lupas
A
A
Rottenberger
C
C
Plückthun
A
A
Pack
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D000951:Antigens, Neoplasm / Q000276:immunology*; D002240:Carbohydrate Sequence; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology*; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; C055966:immunoglobulin Fv