PEDS Advance Access published online on November 19, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn066
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Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine 
to human or 
light chains
Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany
1 To whom correspondence should be addressed. E-mail: skerra{at}wzw.tum.de
An 
CD30xCD16 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 Fc
RIII (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/
MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V domain was converted to a humanized 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 
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 VH and V 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.
Keywords: affinity maturation/CD16/immunoglobulin subclass/light chain/natural killer cells
Received September 30, 2008; revised September 30, 2008; accepted October 13, 2008.