Abstract
A human antibody library was displayed on the surface of filamentous bacteriophage and screened for binding to human interleukin-6 (IL-6). Two antibody-bearing phages were selected that bound IL-6. The complementary-determining region 3 loops of the variable heavy chains of these two antibodies differed in length and sequence and recognized two distinct epitopes. One of the single chain Fv fragments isolated (H1) was found to bind human (but not murine) IL-6 with an affinity comparable to that of the human IL-6 receptor. H1 also recognized newly synthesized human IL-6 intracellularly, as shown by indirect immunofluorescence. H1 did not neutralize human IL-6, and the H1 epitope was mapped to a region of IL-6 not involved in interactions with IL-6, IL-6 receptor, or the signal-transducing protein gp130. To target IL-6-secreting cells, we then constructed a bispecific antibody fragment (a diabody) comprising H1 and the antigen binding site of the T-cell activating monoclonal antibody OKT3. The diabody led to T-cell-mediated killing of cells secreting IL-6.
Highlights
A human antibody library was displayed on the surface of filamentous bacteriophage and screened for binding to human interleukin-6 (IL-6)
Selection of Phage Library against Human IL-6 —Recombinant phage with scFv expressed as fusion proteins on their tip were rescued from the culture supernatant of VCS-M13 helper phage (Stratagene, Heidelberg, Germany)-infected E. coli TG1 as described previously [20]. 1012–1013 phages were panned for three subsequent rounds of binding on immunotubes (Maxisorb, Nunc, Roskilde, Denmark) coated with recombinant human IL-6 at 90 g/ml
Screening for Human Recombinant Antibodies Binding to Human IL-6 —The library containing Ͼ108 recombinant phages was screened for adsorption to human recombinant IL-6 immobilized to immunotubes (Fig. 1A)
Summary
A human antibody library was displayed on the surface of filamentous bacteriophage and screened for binding to human interleukin-6 (IL-6). H1 did not neutralize human IL-6, and the H1 epitope was mapped to a region of IL-6 not involved in interactions with IL-6, IL-6 receptor, or the signal-transducing protein gp130. To target IL-6-secreting cells, we constructed a bispecific antibody fragment (a diabody) comprising H1 and the antigen binding site of the T-cell activating monoclonal antibody OKT3. The identification of the interaction sites between human IL-6, the IL-6 receptor (IL6R), and the signal-transducing protein gp130 has made it possible to construct IL-6 receptor antagonists, which have been shown to inhibit IL-6 responses in vitro (9 –16)
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