TY - JOUR
T1 - Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach
AU - Schlapschy, Martin
AU - Gruber, Helga
AU - Gresch, Oliver
AU - Schäfer, Claudia
AU - Renner, Christoph
AU - Pfreundschuh, Michael
AU - Skerra, Arne
N1 - Funding Information:
The authors thank Biotest Pharma GmbH, especially Dr Matthias Germer and Dr Michael Kloft, for a financial contribution and general advice. This work was supported by the Bundesministerium fur Bildung und Forschung (BMBF project No. 0311759).
PY - 2004/12
Y1 - 2004/12
N2 - CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the marine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an αCD30×αCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.
AB - CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the marine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an αCD30×αCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.
KW - Affinity maturation
KW - Antibody humanization
KW - CD16
KW - CD30
KW - Hodgkin's lymphoma
UR - http://www.scopus.com/inward/record.url?scp=15344341215&partnerID=8YFLogxK
U2 - 10.1093/protein/gzh098
DO - 10.1093/protein/gzh098
M3 - Article
C2 - 15708864
AN - SCOPUS:15344341215
SN - 1741-0126
VL - 17
SP - 847
EP - 860
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 12
ER -