TY - JOUR
T1 - Functional humanization of an anti-CD16 Fab fragment
T2 - Obstacles of switching from murine λ to human λ or κ light chains
AU - Schlapschy, Martin
AU - Fogarasi, Marton
AU - Gruber, Helga
AU - Gresch, Oliver
AU - Schäfer, Claudia
AU - Aguib, Yasmine
AU - Skerra, Arne
N1 - Funding Information:
The authors wish to thank Biotest Pharma GmbH, especially Dr. Matthias Germer and Dr. Michael Kloft, for advice and financial support in frame of a grant by the Bundesministerium für Bildung und Forschung (BMBF project no. 0311759).
PY - 2009/3
Y1 - 2009/3
N2 - An αCD30×αCD16 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 FcγRIII (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/λ MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine Vλ domain was converted to a humanized λ 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 κ 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 Vκ 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.
AB - An αCD30×αCD16 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 FcγRIII (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/λ MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine Vλ domain was converted to a humanized λ 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 κ 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 Vκ 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.
KW - Affinity maturation
KW - CD16
KW - Immunoglobulin subclass
KW - Light chain
KW - Natural killer cells
UR - http://www.scopus.com/inward/record.url?scp=60749137123&partnerID=8YFLogxK
U2 - 10.1093/protein/gzn066
DO - 10.1093/protein/gzn066
M3 - Article
C2 - 19022801
AN - SCOPUS:60749137123
SN - 1741-0126
VL - 22
SP - 175
EP - 188
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 3
ER -