For the comparative study of the transcutaneous penetration of whole antibody and scFv, ex vivo experiments were performed on pig ear skin. After shaving, ears were tape-stripped 25 times (Clinical&Derm, USA) to obtain the altered skin condition. Then, 8 mm diameter punches were treated with 5 µl phosphate buffered saline (PBS, Corning, USA), and 11 µM anti-CD44 scFv (Creative Biolabs, USA) or anti-CD44 Ab (Thermofisher Scientific, USA). Skin samples were placed at 37 °C for 6 h or 24 h and cryopreserved until Raman microspectroscopy analysis.
Three 14 µm thick sections were used per sample and three Raman acquisitions were read for each section totaling nine measures per sample. Briefly, Raman images were obtained using a confocal Raman microspectrometer (Horiba Jobin Yvon, France) operating with a 660 nm laser. Labspec 6 software (Horiba Jobin Yvon, France) was used for acquisition and data pre-processing. Raman maps were recorded in an area of X: 10-µm/Y: 150-µm with 5 µm step size in the XY directions. Raman spectra were acquired in the 400–2300 cm−1 spectral range. To prevent background noise, Raman spectra were smoothed using a 2nd order Savitzky-Golay type filter, baseline corrected using a 7th order polynomial function, and spectra with an intensity under 400 cts in the 1530–1730 cm−1 range were suppressed. Next, Raman spectra were cropped to keep only the 400–785 cm−1 range of interest, which was used for vector normalization. Finally, fitting (unmixing) by classical least squares with a non-negativity constraint (NCLS) was used to estimate the contribution of the various skin components leading to the images reflecting the distribution of the specific scFv or Ab through the skin cryo-sections.
To assess the neutralizing capacity of anti-hIL4 scFv in vitro, we used both a cell line model (HEK-Blue™ IL-4/IL-13 cells; InvivoGen, France) and NHKs as primary cells. HEK-Blue™ IL-4/IL-13 cells were cultured according to manufacturer’s instructions. Briefly, 50,000 cells were seeded in 96-well plates. Then, cells were stimulated with 10 ng/ml hIL-4 (Miltenyi Biotec, Germany) and treated with anti-hIL4 scFv (Creative Biolabs, USA) or Ab (Biotechne, USA) at different concentrations. The variable domains of the anti-hIL4 scFv and monoclonal Ab are distincts and the latter was used as a positive control. After 24 h, QUANTI-Blue™ substrate was added to supernatants. After 2h30 at 37 °C, SEAP levels were measured at 620 nm using a spectrophotometer.
NHKs were isolated from human skin samples from healthy donors (n = 4) undergoing medical surgery. The skin was collected after written informed consent from the donors and institutional approval. NHKs were stimulated with polyinosinic-polycytidylic acid (poly I:C) (2 µg/ml, Sigma, USA) ± hIL-4 (25 ng/ml, Peprotech, USA) and treated with anti-hIL4 scFv at different concentrations. After 24 h, supernatants were harvested to evaluate hIL-8 secretion by enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s instructions (R&D Systems, USA). Cell protein concentrations were determined using the BCA Protein Assay kit (Thermofisher Scientific, USA) according to manufacturer’s instructions. hIL-8 secretion was normalized to total protein amounts.
Statistical analysis was performed using Prism GraphPad software (GraphPad Software, USA). Significant differences between samples and control were evaluated by Student’s t-test. P values < 0.05 were considered significant.
Some detailed protocols are available in the Supporting Information.
