which can be used in high throughput to determine the kinetic
binding constant of interacting proteins [3].
Here, we describe the expression and binding studies of the
Kallikrein-related protease 7 (KLK7) with improved inhibitory
anti-KLK7 antibodies. The functional integrity of the skin epidermis is highly dependent on cutaneous protease activity. The human
kallikrein-related peptidases (KLKs) are key players in skin homeostasis [4] and therefore important pharmacological therapeutic
targets [5].
In our approach, genetically engineered antibodies are investigated for the potential as KLK inhibitors [6]. Biologics like recombinant antibodies have high specificity. Inhibitory antibodies have
broad application and high acceptance in the community and
industry as a therapeutic tool. Efficient screening for appropriate
recombinant antibodies involves the small single-chain variable
fragment format (scFv), which can be isolated using the in vitro
phage display technology [7, 8].
For therapeutic use, however, the IgG molecule is still the most
widely accepted format. It allows for bivalent binding and immune
effector functions, along with a generally high stability, long serum
half-life, and low immunogenicity [9–11]. Accordingly, fragments
are routinely converted to IgG format after completed scFv selection and engineering. Nevertheless, there are other recombinant
formats that should be considered depending on the application.
Previous studies have shown that the conversion into the IgG
format can affect antigen binding both positively and negatively
[12, 13]. So, a structural compromise like the IgG-like scFv-Fc
format might better preserve the binding properties of the selected
scFv fragment. Due to their smaller size, recombinant antigenbinding fragments (like Fabs) of antibodies generally have better
tissue penetration, which may be particularly beneficial in applications for passing the skin barrier [14].
There are a variety of expression systems available today to
express the different antibody formats. Each has its own respective
advantages in terms of cost, ease of use, and the post-translational
modification profiles [15].
1.2 Expression
of Different
Recombinant Antibody
Formats
Below, the set of expression vectors to generate different antibody
formats and purification recombinant proteins expressed in
HEK293-6E cells is described. The HEK293-6E cell line is especially qualified for fast and simple protein production as this cell line
is compatible with the plasmid-based expression using polyethylenimine (PEI) [1, 2, 16]. The VH and VL coding fragments are
cloned in specific vector pairs to be able to generate scFv, homodimeric scFv-Fc, heterodimeric Fab, or tetrameric IgG antibodies
[9–11] (see Fig. 1)
60
Jens Ko ¨ nig et al.
binding constant of interacting proteins [3].
Here, we describe the expression and binding studies of the
Kallikrein-related protease 7 (KLK7) with improved inhibitory
anti-KLK7 antibodies. The functional integrity of the skin epidermis is highly dependent on cutaneous protease activity. The human
kallikrein-related peptidases (KLKs) are key players in skin homeostasis [4] and therefore important pharmacological therapeutic
targets [5].
In our approach, genetically engineered antibodies are investigated for the potential as KLK inhibitors [6]. Biologics like recombinant antibodies have high specificity. Inhibitory antibodies have
broad application and high acceptance in the community and
industry as a therapeutic tool. Efficient screening for appropriate
recombinant antibodies involves the small single-chain variable
fragment format (scFv), which can be isolated using the in vitro
phage display technology [7, 8].
For therapeutic use, however, the IgG molecule is still the most
widely accepted format. It allows for bivalent binding and immune
effector functions, along with a generally high stability, long serum
half-life, and low immunogenicity [9–11]. Accordingly, fragments
are routinely converted to IgG format after completed scFv selection and engineering. Nevertheless, there are other recombinant
formats that should be considered depending on the application.
Previous studies have shown that the conversion into the IgG
format can affect antigen binding both positively and negatively
[12, 13]. So, a structural compromise like the IgG-like scFv-Fc
format might better preserve the binding properties of the selected
scFv fragment. Due to their smaller size, recombinant antigenbinding fragments (like Fabs) of antibodies generally have better
tissue penetration, which may be particularly beneficial in applications for passing the skin barrier [14].
There are a variety of expression systems available today to
express the different antibody formats. Each has its own respective
advantages in terms of cost, ease of use, and the post-translational
modification profiles [15].
1.2 Expression
of Different
Recombinant Antibody
Formats
Below, the set of expression vectors to generate different antibody
formats and purification recombinant proteins expressed in
HEK293-6E cells is described. The HEK293-6E cell line is especially qualified for fast and simple protein production as this cell line
is compatible with the plasmid-based expression using polyethylenimine (PEI) [1, 2, 16]. The VH and VL coding fragments are
cloned in specific vector pairs to be able to generate scFv, homodimeric scFv-Fc, heterodimeric Fab, or tetrameric IgG antibodies
[9–11] (see Fig. 1)
60
Jens Ko ¨ nig et al.