Biomedical Engineering Reference
In-Depth Information
39
MODULAR ANTIBODY ENGINEERING: ANTIGEN
BINDING IMMUNOGLOBULIN Fc CH3 DOMAINS AS
BUILDING BLOCKS FOR BISPECIFIC ANTIBODIES (mAb 2 )
M AXIMILIAN W OISETSCHL ¨ GER , 1 F LORIAN R ¨ KER , 2 G EERT C. M UDDE , 1 G ORDANA W OZNIAK -K NOPP , 2
A NTON B AUER , 1 AND G OTTFRIED H IMMLER 1
1 f-star GmbH, Vienna, Austria
2 Department of Biotechnology, Christian Doppler Laboratory for Antibody Engineering, University of Natural Resources and Life
Sciences, Vienna, Austria
39.1 Introduction
39.2 Immunoglobulin Fc as a scaffold
39.3 Design of libraries based on the human IgG1 CH3 domain
39.4 TNF- a -binding Fcab: selection and characterization of Fcab
TNF353-2
39.5 Conclusions and future perspectives
Acknowledgments
References
25%, while their structural similarity is high, with a root-
mean-square deviation of C a atoms always below 3.9 A [1].
We therefore set out to explore whether this inherent
stability of the immunoglobulin fold allows loops of immu-
noglobulin domains other than the CDR loops to accommo-
date sequence variation without negatively impacting the
overall structure and stability of the protein. As shown in
Figure 39.1, the candidate loops of an immunoglobulin G1
(IgG1) for this kind of engineering are manifold, including
the N- and C-terminal loops of the constant domains as well
as C-terminal loops of the variable domains.
39.1
INTRODUCTION
39.2
IMMUNOGLOBULIN Fc AS A SCAFFOLD
The immune system creates binding sites of high specificity
and affinity in the variable domains of antibodies by gener-
ating sequence and consequently structural diversity in the
complementarity determining regions (CDR) loops, which
are located at the N-terminal ends of these domains.
Sequence variations in the CDR loops of an antibody
generally do not have a significant influence on the overall
structure of the variable domain that carries them. This
feature of variable domains is actually observed in a
more general sense in domains belonging to the immuno-
globulin-fold family, which are known to have a similar
general shape in the core ( b -barrel, and high structural
variability in the loops. Furthermore, overall sequence sim-
ilarity of immunoglobulin-fold domains is mainly below
Successful proof-of-concept of engineering non-CDR loops
has been obtained by randomizing the C-terminal loops of the
third constant domain (CH3) of human IgG1 [2]. CH3 domains
engineered with antigen-binding properties in the context of a
completeFc fragment (composedof thehinge, theCH2, and the
CH3 domains) were able to bind the tumor-associated antigen
HER2/neu with high affinity and specificity. Such antigen-
binding Fc proteins are called Fcabs TM (Fc molecules with
antigen-binding properties). In addition, such HER2/neu bind-
ing Fcabs, at a size of only
50 kDa, also possess all attractive
properties of complete antibodies such as their ability to trigger
effector functions via binding to Fc g receptors or C1q. Finally,
owing to the functional presence of the FcRn-binding site,
Fcabs also display the long in vivo half-life of antibodies.
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