Integrin-Blocking Antibodies Delay Keratinocyte Re-Epithelialization in a Human Three-Dimensional Wound Healing Model

The α6β4 integrin plays a significant role in tumor growth, angiogenesis and metastasis through modulation of growth factor signaling, and is a potentially important therapeutic target. However, α6β4-mediated cell-matrix adhesion is critical in normal keratinocyte attachment, signaling and anchorage to the basement membrane through its interaction with laminin-5, raising potential risks for targeted therapy. Bioengineered Human Skin Equivalent (HSE), which have been shown to mimic their normal and wounded counterparts, have been used here to investigate the consequences of targeting β4 to establish toxic effects on normal tissue homeostasis and epithelial wound repair. We tested two antibodies directed to different β4 epitopes, one adhesion-blocking (ASC-8) and one non-adhesion blocking (ASC-3), and determined that these antibodies were appropriately localized to the basal surface of keratinocytes at the basement membrane interface where β4 is expressed. While normal tissue architecture was not altered, ASC-8 induced a sub-basal split at the basement membrane in non-wounded tissue. In addition, wound closure was significantly inhibited by ASC-8, but not by ASC-3, as the epithelial tongue only covered 40 percent of the wound area at 120 hours post-wounding. These results demonstrate β4 adhesion-blocking antibodies may have adverse effects on normal tissue, whereas antibodies directed to other epitopes may provide safer alternatives for therapy. Taken together, we conclude that these three-dimensional tissue models provide a biologically relevant platform to identify toxic effects induced by candidate therapeutics, which will allow generation of findings that are more predictive of in vivo responses early in the drug development process

Immunoblocking of integrins decreases the rate of cell motility.

<p>The initial scratch wound was carried out on uncoated culture dishes with confluent keratinocyte cultures (A), and on keratinocyte cultures treated with ASC-3 (B) and ASC-8 (C). Panels D–F illustrate repopulation of the wound surface after 24 hours by keratinocytes without the presence of antibodies (D), or in the presence of ASC-3 (E), or ASC-8 (F). Cells that migrated into the wound gap were calculated for each condition and the differences were expressed as a percentage of wound closure of NHK cell culture (G). Each bar represents the mean and the standard deviation of triplicate determinations in two separate experiments.</p

Characterization of β4-specific antibody effects on human keratinocytes.

<p>The binding of control or β4-specific antibodies ASC-3 and ASC-8 to normal human keratinocytes (A) and HaCaT cells (B) was measured by flow cytometry. To determine the effects on adhesion, serum starved cells were either left untreated (NT), or incubated with control or β4-specific antibodies prior to plating on rat laminin-5 (C) and a mixture of human laminins (D) Relative adhesion was measured using a luciferase based luminescent viability assay. Experiments were performed twice in triplicate and results shown are from one representative experiment.</p

Immunofluorescent detection of the Basement Membrane Proteins.

<p>The immunodetection of alpha6 integrin shows no difference between the control (A) and the presence of ASC-3 and ASC-8 antibodies in the medium (B and C). The same observation can be made for laminin-5 (D, E and F). Note that in all cases (C),(F) ASC-8 is associated with large disruption of the tissue between dermis and epidermis (white arrow).</p

Immunodetection of the blocking antibodies in the Human Skin Equivalents.

<p>H and E staining of tissues revealed no differences in tissue architecture or numbers of cell layers between the control (A) and the ASC-3 (B) or ASC-8 (C) treated HSEs. No antibody was detected in the control tissues not exposed to antibodies. (D). While both ASC-3 (E) and ASC-8 (F) antibodies detected inside the tissue at the junction between dermis and epidermis, indicating specific localization by target along the basement membrane interface (C and F) ASC-8 is associated with a large disruption of the tissue integrity between the dermis and epidermis (F, white arrow).</p