title: Human recombinant antibody fragments specific for a rye-grass pollen allergen: characterization and potential applications.
abstract: One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.
authors.affiliation: Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy.
authors.forename: C
authors.initials: C
authors.lastname: de Lalla
authors.forename: E
authors.initials: E
authors.lastname: Tamborini
authors.forename: R
authors.initials: R
authors.lastname: Longhi
authors.forename: E
authors.initials: E
authors.lastname: Tresoldi
authors.forename: M
authors.initials: M
authors.lastname: Manoni
authors.forename: A G
authors.initials: AG
authors.lastname: Siccardi
authors.forename: P
authors.initials: P
authors.lastname: Arosio
authors.forename: A
authors.initials: A
authors.lastname: Sidoli
mesh_terms: D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D052179:Antigens, Plant; D001667:Binding, Competitive; D018604:Epitope Mapping; D000939:Epitopes; D006632:Histamine / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D008129:Lolium / Q000276:immunology*; D008407:Mast Cells / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D010940:Plant Proteins / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; C056754:Lol p II protein, Lolium perenne; D019151:Peptide Library; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D006632:Histamine