Local Gene Expression Changes after UV-Irradiation of Human Skin

UV-irradiation is a well-known translational pain model inducing local inflammation and primary hyperalgesia. The mediators and receptor proteins specifically contributing to mechanical or heat hyperalgesia are still unclear. Therefore, we irradiated buttock skin of humans (n = 16) with 5-fold MED of UV-C and assessed the time course of hyperalgesia and axon reflex erythema. In parallel, we took skin biopsies at 3, 6 and 24 h after UVC irradiation and assessed gene expression levels (RT-PCR ) of neurotrophins (e.g. NGF, BDNF, GDNF), ion channels (e.g. NaV1.7, TRPV1), inflammatory mediators (e.g. CCL-2, CCL-3) and enzymes (e.g. PGES, COX2). Hyperalgesia to mechanical impact (12 m/s) and heat (48°C) stimuli was significant at 6 h (p<0.05 and p<0.01) and 24 h (p<0.005 and p<0.01) after irradiation. Axon reflex erythema upon mechanical and thermal stimuli was significantly increased 3 h after irradiation and particularly strong at 6 h. A significant modulation of 9 genes was found post UV-C irradiation, including NGF (3, 6, 24 h), TrkA (6, 24 h), artemin, bradykinin-1 receptor, COX-2, CCL-2 and CCL-3 (3 and 6 h each). A significant down-regulation was observed for TRPV1 and iNOS (6, 24 h). Individual one-to-one correlation analysis of hyperalgesia and gene expression revealed that changes of Nav1.7 (SCN9A) mRNA levels at 6 and 24 h correlated to the intensity of mechanical hyperalgesia recorded at 24 h post UV-irradiation (Pearson r: 0.57, p<0.04 and r: 0.82, p<0.001). Expression of COX-2 and mPGES at 6 h correlated to the intensity of heat-induced erythema 24 h post UV (r: 0.57, p<0.05 for COX-2 and r: 0.83, p<0.001 for PGES). The individual correlation analyses of functional readouts (erythema and pain response) with local expression changes provided evidence for a potential role of Nav1.7 in mechanical hyperalgesia

Pain ratings (VAS, 0–100) upon (A) mechanical and (B) heat stimuli.

<p>Mechanical impact stimuli were delivered at 12 m/s and continuous heat applied for 5 sec at 48°C. Responses recorded at 3, 6 and 24 h post irradiation are depicted in open columns from untreated skin and those recorded from UV-C treated skin (5-fold MED) in black columns. Asterisks indicate significant differences between control and UV-C skin (p<0.01, Fishers LSD test).</p

Correlation analysis of mRNA expression (n = 14) and 24 h hypersensitivity upon (A) heat and (B) mechanical impact.

<p> Individual one-to-one correlation analyses of functional hypersensitivity 24 h post UV-C irradiation and mRNA expression changes at 6 h (left panel) and 24 h (right panel). All values were normalized to control skin. (<b>A</b>): Correlation of relative increase of cyclooxygenase 2 (COX-2, upper panels) and prostaglandin synthase (mPGES, middle panels) expression with relative increase of heat-evoked erythema (48°C, 5 sec) recorded at 24 h after irradiation. (<b>B</b>): Correlation of NaV1.7 (gene SCN9A) expression changes at 6 h (left) and 24 h (right) after UV-C irradiation with relative increase of mechanical impact pain (12 m/s) recorded 24 h post irradiation. Significant correlations (p<0.05, Bonferroni corrected) are marked by asterisks.</p

Group analysis of mRNA expression increase (RT-PCR, n = 14).

<p>Changes upon UV-C irradiation are depicted as fold increase above baseline (dashed horizontal line represents no change) assessed at 3, 6 and 24 h post irradiation. Error bars indicate the 95% confidence interval.</p

Compilation of relative gene expression 3, 6 and 24 h after UV-C irradiation (n = 14).

<p>Values depict ratios to baseline condition (geometric mean and 95% confidence interval, bold face indicates means outside the 95% confidence interval). (*, **, ***: p<0.05, 0.01, 0.001; repeated measures ANOVA for 3, 6 and 24 h expression level for each gene).</p

An Adaptable Antibody-Based Platform for Flexible Synthetic Peptide Delivery Built on Agonistic CD40 Antibodies

The agonistic potentials of therapeutic anti-CD40 antibodies have been profiled in relation to antibody isotype and epitope specificity. Still, clinical impact relies on a well-balanced clinical efficacy versus target-mediated toxicity. As CD40-mediated immune activation must rely on a combination of stimulation of antigen-presenting cells (APCs) alongside antigen presentation, for efficient T cell priming, alternative approaches to improve the therapeutic outcome of CD40-targeting strategies should focus on providing optimal antigen presentation together with CD40 stimulation. Herein, a bispecific antibody targeting CD40 as a means to deliver cargo (i.e., synthetic peptides) into APCs through a non-covalent, high-affinity interaction between the antibody and the cargo peptide, further referred to as the Adaptable Drug Affinity Conjugate (ADAC) technology, has been developed. The ADAC platform demonstrated a target-specific CD4(+) and CD8(+) T cell expansion in vitro and significantly improved peptide-specific CD8(+) T cell proliferation in vivo. In addition, the strategy dramatically improved the in vitro and in vivo half-life of the synthetic peptides. Future applications of ADAC involve pandemic preparedness to viral genetic drift as well as neoepitope vaccination strategies where the bispecific antibody is an off-the-shelf product, and the peptide antigen is synthesized based on next-generation sequencing data mining

Fed-batch production assessment of a tetravalent bispecific antibody : A case study on piggyBac stably transfected HEK293 cells

The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 x 106 viable cells/mL with over 90 % viability 12 days postinoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 s, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody