title: Improved binding activity of antibodies against major histocompatibility complex class I chain-related gene A by phage display technology for cancer-targeted therapy.
abstract: Major histocompatibility complex class I chain-related gene A (MICA) is an NKG2D ligand that is over-expressed under cellular stress including cancer transformation and viral infection. High expression of MICA in cancer tissues or patients' sera is useful for prognostic or follow-up markers in cancer patients. In this study, phage display technology was employed to improve antigen-binding activities of anti-MICA monoclonal antibodies (WW2G8, WW6B7, and WW9B8). The 12 amino acid residues in the complementarity determining regions (CDRs) on the V domain of the heavy chain CDR3 (HCDR3) of these anti-MICA antibodies were modified by PCR-random mutagenesis, and phages displaying mutated anti-MICA Fab were constructed. After seven rounds of panning, five clones of phages displaying mutant anti-MICA Fab which exhibited 3-7-folds higher antigen-binding activities were isolated. Two clones of the mutants (phage-displayed mutant Fab WW9B8.1 and phage-displayed mutant Fab WW9B8.21) were confirmed to have antigen-binding specificity for cell surface MICA proteins by flow cytometry. These phage clones are able to recognize MICA in a native form according to positive results obtained by indirect ELISA and flow cytometry. Thus, these phage particles could be potentially used for further development of nanomedicine specifically targeting cancer cells expressing MICA proteins.
authors.affiliation: Biomedical Sciences Program, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Achara
authors.initials: A
authors.lastname: Phumyen
authors.forename: Amonrat
authors.initials: A
authors.lastname: Jumnainsong
authors.forename: Chanvit
authors.initials: C
authors.lastname: Leelayuwat
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D001483:Base Sequence; D061505:Cell Surface Display Techniques / Q000379:methods*; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D008969:Molecular Sequence Data; D058990:Molecular Targeted Therapy / Q000379:methods*; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D009369:Neoplasms / Q000276:immunology* / Q000628:therapy*; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D022801:Complementarity Determining Regions; D015395:Histocompatibility Antigens Class I; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; C099187:MHC class I-related chain A; D050505:Mutant Proteins