Identification and characterization of a human monoclonal antagonistic antibody AL-57 that preferentially binds the high-affinity form of lymphocyte function-associated antigen-1.

LFA-1 (alpha(L)beta(2)) mediates cell-cell and cell-extracellular matrix adhesions essential for immune and inflammatory responses. One critical mechanism regulating LFA-1 activity is the conformational change of the ligand-binding alpha(L) I domain from low-affinity (LA), closed form, to the high-affinity (HA), open form. Most known integrin antagonists bind both forms. Antagonists specific for the HA alpha(L) I domain have not been described. Here, we report the identification and characterization of a human antibody AL-57, which binds to the alpha(L) I domain in a HA but not LA conformation. AL-57 was discovered by selection from a human Fab-displaying library using a locked-open HA I domain as target. AL-57 Fab-phage bound HA I domain-expressing K562 cells (HA cells) in a Mg(2+)-dependent manner. AL-57 IgG also bound HA cells and PBMCs, activated by Mg(2+)/EGTA, PMA, or DTT. The binding profile of AL-57 IgG on PBMCs was the same as that of ICAM-1, the main ligand of LFA-1. In contrast, an anti-alpha(L) murine mAb MHM24 did not distinguish between the HA and LA forms. Moreover, AL-57 IgG blocked ICAM-1 binding to HA cells with a potency greater than MHM24. It also inhibited ICAM-1 binding to PBMCs, blocked adhesion of HA cells to keratinocytes, and inhibited PHA-induced lymphocyte proliferation with potencies comparable with MHM24. These results indicate that specifically targeting the HA I domain is sufficient to inhibit LFA-1-mediated, adhesive functions. AL-57 represents a therapeutic candidate for treatment of inflammatory and autoimmune diseases.

Efficient translocation and apoptosis induction by adenovirus encoded VP22-p53 fusion protein in human tumor cells in vitro.

BACKGROUND: p53 mutations are one of the most frequent genetic alterations in cancer. Various mechanisms of delivering p53 protein into tumor cells, such as plasmids, retroviruses and adenoviruses, have been widely used in experimental studies. Although these methods are relatively successful, the transduction rate into surrounding cells is still a limiting factor. Recent studies have shown that fusing VP22 (important for intercellular transport) to p53 and delivering this protein to cells in the form of an adenovirus is a very efficient method of getting p53 into cells. RESULTS: In the present study, the effect of adenovirus encoded VP22-p53 fusion protein on p53 negative human cancer cells (LNCaP, SK-OV-3, OVCAR-3, DU 145) was investigated. A functional VP22-p53 fusion protein was produced as verified by immunofluorescence and analysis of p21 expression. Induction of p21 expression (target gene for p53) confirmed p53 was functional and immunofluorescence staining using p53 antiserum demonstrated the intercellular trafficking ability of the VP22 portion of the fusion protein. CONCLUSION: In conclusion, VP22-p53 showed efficient translocation into tumor cells, inhibition of tumor cell proliferation and induction of apoptosis. These characteristics make the fusion protein an attractive method for introducing p53 into human cells as well as a potential candidate for gene therapy.