title: Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines.
abstract: The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments.
authors.affiliation: Department I of Internal Medicine, Laboratory of Immunotherapy, University Hospital Cologne, D-50931 Cologne, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Rothe
authors.forename: A
authors.initials: A
authors.lastname: Klimka
authors.forename: M K
authors.initials: MK
authors.lastname: Tur
authors.forename: T
authors.initials: T
authors.lastname: Pfitzner
authors.forename: M
authors.initials: M
authors.lastname: Huhn
authors.forename: S
authors.initials: S
authors.lastname: Sasse
authors.forename: P
authors.initials: P
authors.lastname: Mallmann
authors.forename: A
authors.initials: A
authors.lastname: Engert
authors.forename: S
authors.initials: S
authors.lastname: Barth
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000276:immunology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008198:Lymph Nodes / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D007135:Immunoglobulin Variable Region; D019151:Peptide Library