3.1 Antibody generation
After the panning procedures using the HAL9/10 libraries, the scFv antibodies were produced in XL1-Blue MRF’ E. coli, and ELISA assays were performed to select the soluble antibody fragments that bind to KLK7 (figure 1). Seventeen antibody clones were selected with interaction values higher than 0.1. These clones were sequenced, and it was found that many sequences were repeated or truncated, resulting in 9 unique functional scFv sequences against KLK7.
All nine selected scFv antibodies were produced in the scFv-Fc format and tested against recombinant KLK7 to determine the IC50 value. LUP-14G10 showed a powerful inhibitory response against recombinant KLK7, with a potency of 2.3 nM (figure 2).
To select the strong inhibitors, the clone with the lowest IC50 (LUP-14G10) was subjected to an affinity maturation process, which involves replacing its light chain fragment with a library of light chain fragments to generate a new LUP-14G10-based library. This new library was submitted to the panning procedures using KLK7 as the antigen. After the off-rate panning procedure was completed, four new scFv antibodies against KLK7 (LUP-37A10, LUP-37B10, LUP-37C11 and LUP-37D11) were selected with higher affinity for the target protease than that exhibited by LUP-14G10. These new scFv antibodies were produced in mammalian cells in the scFv-Fc format and purified with a protein A column. After production, they were also assessed for their inhibitory potential towards recombinant KLK7 (figure 3). Antibody LUP-37B10 displayed an IC50 value above that observed for the original antibody (6.2 nM); for that reason, we did not use this antibody in the other experiments.
Many molecules are capable of inhibiting KLK7, as described in the literature (Arama et al., 2015; Freitas et al ., 2012; Jendrny & Beck-Sickinger, 2016; Oliveira et al ., 2014). Molecules reported by Oliveira et al . and Freitas et al ., based on isomannide, showed the highest IC50 values, ranging from 10.2 to > 1000 µM (Oliveira et al ., 2014) and 13.5 to 205.2 µM (Freitas et al ., 2012), respectively. A more recent study by Jendrny and coworkers found compounds able to inhibit KLK7 at very low values (0.6 – 1.1 µM) based on a sunflower trypsin inhibitor (Jendrny & Beck-Sickinger, 2016). Although there are many other molecules described for inhibiting KLK7 with different inhibition constant values, ranging from medium nanomolar up to high micromolar, there are no reports about the use of the diverse universe of recombinant antibody fragments as inhibitors for KLK7. In this work, we present at least five new recombinant antibodies with the capacity to inhibit the target protease with an inhibition constant in the very low nanomolar range, which may be a new, powerful way to generate inhibitors for these family of proteases.
As the aim is the generation of inhibitors for human tissue kallikreins to be used as archetypes for the future development of new therapeutic agents for the pathologies in which KLKs appear to be related, we also decided to investigate the possibility of encapsulating these scFv-Fc antibodies against KLK7 in a poloxamer-based system of drug delivery.
3.2 Physicochemical characterization of the drug delivery system
3.2.1 Micellar hydrodynamic diameter
In table 1, the data for the hydrodynamic diameters (nm) and average distribution patterns (%) are available. These data refer to the DLS technique used to observe the effects of temperature, micelle composition and drug incorporation that is based on micellar hydrodynamic diameter and size distribution parameters.
Regarding the micelle population for both formulations, at 25 ºC, a population of micelles with diameters < 5 nm was found in all compounds (4.52 ± 0.55 nm with intensity = 5.2% and 4.73 ± 0.23 nm, with intensity = 3,1% for F1 and F2, respectively). At 32.5 ºC, no other populations were found.
For both formulations, the increase in temperature promoted a decrease in the micellar diameter, which was being more pronounced for LUP-37D11 in formulation F1 (25.61%) and for LUP-37A10 in formulation F2 (32.53%). Reductions in the micellar hydrodynamic diameter were directly related to the increase in temperature, which was promoted by the dehydration of the propylene oxide (PPO) units in the micellar core (Freitas Mariano et al ., 2019; Zhang, Lam, & Tan, 2005). The increase in temperature also influenced the hydrophobicity of the micellar core, favoring the formation of colloidal systems with low polydispersity (Freitas Mariano et al ., 2019;,(Su, Wang, & Liu, 2002). Similar results were reported on the influence of temperature on PL407 micelles only (Akkariet al ., 2016; dos Santos et al ., 2015; Freitas Marianoet al ., 2019; Nascimento et al ., 2018) There was one report in which PL407 was combined with PL403, and it confirmed the results shown in this work.(Freitas Mariano et al ., 2019)