title: Generation of a highly stable, internalizing anti-CD22 single-chain Fv fragment for targeting non-Hodgkin's lymphoma.
abstract: The generation of a single chain Fv (scFv) fragment derived from the anti-CD22 monoclonal antibody LL2 resulted in a molecule with good antigen binding but very poor stability properties, thus hampering its clinical applicability. Here we report on the construction of an engineered LL2 scFv fragment by rational mutagenesis. The contribution of uncommon wild-type sequence residues for providing stability to the conserved common core structure of immunoglobulins was examined. Aided by computer homology modeling, 3 destabilizing residues within the core of the wild-type VH domain were identified. Owing to the conserved nature of the buried core structure, mutagenesis of these sites to respective consensus residues markedly stabilized the molecule but did not influence its antigen binding properties: the engineered scFv MJ-7 exhibited exceptional biophysical stability with a half-life not reached after 6 days of incubation in human serum at 37 degrees C, while fully retaining the epitope specificity of the monoclonal antibody, and antigen binding affinity of the wild-type scFv. Furthermore, both the monoclonal antibody LL2 and the engineered scFv fragment became fully internalized after only 30 min of incubation at 37 degrees C with CD22+ tumor cells. These properties predict scFv MJ-7 could become a novel powerful tool to selectively deliver cytotoxic agents to malignant CD22+ cells.
authors.affiliation: National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA.
authors.forename: Michaela A E
authors.initials: MA
authors.lastname: Arndt
authors.forename: Jürgen
authors.initials: J
authors.lastname: Krauss
authors.forename: Robert
authors.initials: R
authors.lastname: Schwarzenbacher
authors.forename: Bang K
authors.initials: BK
authors.lastname: Vu
authors.forename: Shailen
authors.initials: S
authors.lastname: Greene
authors.forename: Susanna M
authors.initials: SM
authors.lastname: Rybak
mesh_terms: D015703:Antigens, CD / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology*; D001666:Binding Sites, Antibody; D015815:Cell Adhesion Molecules; D003198:Computer Simulation; D006801:Humans; D006860:Hydrogen Bonding; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D007167:Immunotherapy / Q000379:methods; D007700:Kinetics; D037102:Lectins / Q000276:immunology*; D008228:Lymphoma, Non-Hodgkin / Q000628:therapy*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D051918:Sialic Acid Binding Ig-like Lectin 2; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; D015815:Cell Adhesion Molecules; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D037102:Lectins; D051918:Sialic Acid Binding Ig-like Lectin 2