HUMAN RECOMBINANT ANTIBODY FRAGMENTS SPECIFIC FOR A RYE-GRASS POLLEN ALLERGEN - CHARACTERIZATION AND POTENTIAL APPLICATIONS

One of the major allergens from the pollen of perennial rye grass (Lol ium 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 recombin ant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli p eriplasm. The DNA sequencing of the clones producing antibodies with t he highest binding activity showed three of them to be identical, whil e one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purif ied 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 K-d=2.63 x 10(-9) M, as demonstrated by the surface pla smon resonance technique, and was able to compete with a fraction of s erum IgE. Epitope mapping using synthetic peptides revealed that antig enic domains, located between amino acids 39 and 51 of Lol pII, are re cognized by Fab and polyclonal IgE from sera of allergic donors. The F ab fragments inhibited the binding of serum IgE to the allergen. in vi tro experiments on whole blood from allergic subjects showed that reco mbinant Fab fragments had a blocking activity on histamine release fro m 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 proces ses. Our molecular and functional findings altogether indicate that al lergen-specific human antibodies may be useful for the characterizatio n of the antigenic structure of allergens. We conclude that a phage li brary is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be poten tially innovative reagents for the treatment of atopic allergy. Copyri ght (C) 1996 Elsevier Science Ltd.