title: Recombinant anti-human melanoma antibodies are versatile molecules.
abstract: The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.
authors: Cambridge Center for Protein Engineering, Medical Research Council Center, U.K.
authors: D
authors: D
authors: Neri
authors: P G
authors: PG
authors: Natali
authors: H
authors: H
authors: Petrul
authors: P
authors: P
authors: Soldani
authors: M R
authors: MR
authors: Nicotra
authors: R
authors: R
authors: Vola
authors: A
authors: A
authors: Rivella
authors: A M
authors: AM
authors: Creighton
authors: P
authors: P
authors: Neri
authors: M
authors: M
authors: Mariani
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008545:Melanoma / Q000276:immunology*; D051379:Mice; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D016324:Sequence Tagged Sites
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007074:Immunoglobulin G; D015335:Molecular Probes; D011994:Recombinant Proteins