1 INTRODUCTION
Proteases are enzymes essential to the survival of organisms,
representing 2 to 4% of the whole proteome (Patel, 2017). Serine
proteases represent approximately one-third of all proteases identified
until 2009 (Cera & Di Cera, 2009); they are arranged in several clans
and families; human tissue kallikreins (KLKs) are serine proteases in
the PA clan (Patel, 2017). These enzymes are detected in many tissues
and biological fluids at the protein and mRNA levels (Diamandis,et al ., 2000; Turk, 2006). They are in the biggest
protease gene cluster in the human genome (Paliouras, Borgono, &
Diamandis, 2007; Prassas et al ., 2015) and show important
similarities in terms of their tertiary structures and protein and gene
levels (Shaw & Diamandis, 2007). This family is composed of 15 genesin tandem in the long arm of chromosome 19q13.3-13.4, where a
pseudogene can also be found (Avgeris, Mavridis, & Scorilas, 2012;
Kryza et al ., 2016). KLKs can be detected in a wide variety of
tissues, such as those of the skin, central nervous system, salivary
glands and biological fluids, including serum and seminal plasma. The
roles of many KLKs (KLK4 – KLK15) have not been completely elucidated,
but studies suggest that they can play roles in processing peptide
hormones within the pancreas (Komatsu et al., 2007) and the nervous
system (Blaber et al ., 2005) in dissolving the blood coagula
(Borgoño, Michael, & Diamandis, 2004), in maintaining epidermal
homeostasis (Ekholm & Egelrud, 1999) and in tooth development (Wanget al ., 2009).
KLKs 5 and 7 participate in the process of epidermal homeostasis,
particularly in skin desquamation (Wang et al ., 2009); these
enzymes are responsible for the cleavage of corneodesmosomes, structures
involved in cell cohesion (Lundwall & Brattsand, 2008). KLK7 is
secreted directly into the intracellular space, where it cleaves
desmocollin 1 and corneodesmosin (Ishida-Yamamoto et al ., 2004).
It has been shown that, in dermatologic pathologies such as atopic
dermatitis (Voegeli et al ., 2011), psoriasis (Komatsu et
al ., 2006) and Netherton syndrome (Komatsu et al ., 2008), KLK7
activity is higher than it is in normal skin. It is clear that
dysregulation of KLK activity in the stratum corneum leads to
pathological conditions such as atopic dermatitis (Igawa et al .,
2017; Komatsu et al ., 2006; Voegeli et al ., 2011),
psoriasis, lichen planus, lichen sclerosus and poroketarosis (Heet al ., 2017; Jonca et al ., 2011; Pampalakis &
Sotiropoulou, 2007). In addition, a mutation in the SPINK5 gene,
encoding the LEKTI inhibitor responsible for the inhibition of KLK5 and
KLK7 activities in epidermis and other stratified epithelia, leads to
Netherton syndrome (Chavanas et al ., 2000; Descargues et
al ., 2005; Ishida-Yamamoto & Igawa, 2015). In this way, it has been
noted that inhibition of KLK7 by exogenous molecules represents a new
potential strategy for therapeutic treatment of these skin diseases.
Phage display is a technique first described by Smith in 1985
(Smith, 1985), and antibody phage
display was developed in 1990–91 (Breitling et al ., 1991;
McCafferty et al ., 1990). This technology enables the generation
of human antibodies for research, diagnosis, therapy and in vitroselection processes independent of the restriction imposed by anin vivo immune response (Bradbury et al ., 2011; Schirrmannet al ., 2011). In 2016, six antibodies derived from phage display
were EMA/FDA approved (Frenzel, Schirrmann, & Hust, 2016), and three
years later, 12 antibodies generated by phage display were approved. In
this work, we used the human naïve scFv phage display libraries HAL9/10
(Kügler et al ., 2015) to generate soluble recombinant antibodies
capable of specifically inhibiting the proteolytic activity of KLK7.
Drug delivery systems are used to target cells, tissues and other
targets for therapy with different kinds of compounds (Tibbitt, Dahlman,
& Langer, 2016). Poloxamers (PLs) were first described in 1950 (Koffiet al ., 2006) as emulsifiers for pharmaceutical formulations. PLs
are composed of ethylene oxide and propylene oxide units arranged in a
triblock structure of the A-B-A type. They are found as registered
brands (Pluronic®, Synperonic®,
Tetronic®) and in many forms (Batrakova & Kabanov,
2008). Because of their amphipathic characteristics, they are able to
encapsulate both hydrophobic and hydrophilic compounds, suggesting their
value as drug delivery systems (Dumortier et al ., 2006); in
addition, studies have shown that poloxamers enhance the
pharmacodynamics and pharmacokinetics of the encapsulated agents (Akkariet al ., 2016; dos Santos et al ., 2015).
In this study, we report the use of a phage display to generate
recombinant human antibodies against KLK7, which can inhibit the action
of the target protease, and their incorporation into unique PL-based and
binary hydrogels composed of PL407 and PL403, where they are
characterized, in part, by considering the differences in their
hydrophilic-lipophilic balance. All of these systems were characterized
by their micelle interaction, rheological properties capacity for
release in vitro , inhibition effectivity and cytotoxicity.
2 MATERIALS AND METHODS
2.1 Antibody generation
Antibodies against recombinant KLK7 were selected in scFv format from
the human naïve antibody gene libraries HAL9/10 (Kügler et
al. , 2015). Recombinant KLK7 was produced as described previously (De
Souza et al ., 2013; Teixeira et al. , 2011). The selection
and screening were performed as described before (Russo et al .,
2018). Recombinant KLK7 was immobilized on Costar high-binding
microtiter plates (Sigma-Aldrich), and scFv phage from the HAL9/10
libraries was added to microtiter plates. Panning was performed at 37
°C. After three panning rounds, the soluble scFv antibodies were
produced and screened for KLK7 binding by antigen ELISAs. The DNA of
each binding candidate was isolated and sequenced. The unique scFv
sequences were recloned into pCSE2.6-hIgG1-Fc-XP (Beer et al .,
2018) using NcoI/NotI (New England Biolabs Inc.) for production as an
scFv-Fc antibody, which is an IgG-like antibody format. The production
in HEK293-6E cells and subsequent
protein A purification was performed as described (Jäger etal ., 2013). After the production and purification the
IC50 values for the scFv-Fc antibodies were determined
(Sebaugh, 2011).
scFv-Fc antibodies with lower IC50 values were selected
to undergo an affinity maturation protocol to enhance their specificity
(Hust, Jonas, & Tomszak, 2018). After the affinity maturation, the new
scFv antibodies were selected; these molecules were re-produced and
screened for KLK7 binding by antigen ELISA as described above. The new
candidates underwent the same process as the precursor antibodies, being
re-cloned into pCSE2.6-hIgG1-Fc-XP for production and purified with
protein A (Beer et al ., 2018; ,(Jäger et al.,
2013). The IC50 values were determined for the new
scFv-Fc antibodies.
2.1.1 Inhibitory assays of the scFv-Fc antibodies against KLK7
All enzymatic reactions were performed in 50 mM Tris-HCl buffer (pH =
7.5) in a Hitachi F2500 spectrofluorometer (wavelengths of excitation
and emission = 320 and 420 nm, respectively). The scFv-Fc molecules were
diluted to the appropriate concentration prior to the assays. These
molecules were screened against KLK7 at an initial concentration of 10
µM to verify the degree to which they inhibit the targeted enzyme. The
quartz cuvette was filled with 900 µL of the assay buffer, 0.2 µg of
recombinant KLK7 and the selected scFv-Fc antibody and incubated for 5
minutes at 37 ºC. The reactions were started by the addition of the FRET
substrate Abz-KLYSQ-EDDnp (GenOne Biotechnologies). Control assays were
performed without the inhibitors (negative control). The
IC50 values were independently determined by performing
rate measurements for at least five concentrations of inhibitor, ranging
from 0.1 nM to 1.0 µM. The values are presented as the mean ± SD of
three individual experiments. The IC50 values were
calculated by the GraFit 7 program.