title: Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.
abstract: An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.
authors.affiliation: Lehrstuhl für Biologische Chemie, Technische Universität München, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Schlapschy
authors.forename: Marton
authors.initials: M
authors.lastname: Fogarasi
authors.forename: Helga
authors.initials: H
authors.lastname: Gruber
authors.forename: Oliver
authors.initials: O
authors.lastname: Gresch
authors.forename: Claudia
authors.initials: C
authors.lastname: Schäfer
authors.forename: Yasmine
authors.initials: Y
authors.lastname: Aguib
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007146:Immunoglobulin lambda-Chains / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017730:Ki-1 Antigen / Q000378:metabolism; D007694:Killer Cells, Natural; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009194:Myeloma Proteins / Q000378:metabolism; D017510:Protein Folding; D017452:Receptors, IgG / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012995:Solubility
chemical_list: D018033:Antibodies, Bispecific; C485700:Fcgr3 protein, mouse; D007140:Immunoglobulin Fab Fragments; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D017730:Ki-1 Antigen; D009194:Myeloma Proteins; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins