PF-MC: a new human anti-inflammatory fusion protein engineered to target an inflammatory site

31. MAFFIA, PAULO; BOGADO, BETIANA.; TATEOSIAN, NANCY; GUERRIERI, DIEGO; AMIANO, NICOLAS; SANCHEZ, MERCEDES; CHULUYAN, EDUARDO

Buenos Aires

Congreso; First French-Argentine Immunology Congress; 2010

SAI

Secretory leukocyte protease inhibitor (SLPI) is a protein withanti-microbial, anti-inflammatory and wound healing activity. Invivo, SLPI is susceptible to proteolytic degradation. In order toimprove the therapeutic capacity of SLPI increasing the concentrationat the inflammatory site, we constructed a fusion proteincomprising an anchoring peptide domain named cementoinand human SLPI. Cementoin is a substrate of the enzyme transglutaminase(TG). TG is upregulated at the inflammatory site,crosslinking proteins on the cell membrane and the extrecellularmatrix. PF-MC was cloned and expressed in E. coli. The anti proteinaseactivity of PF-MC was 35±5 % higher than SLPI (p<0.001).The in vitro binding of PF-MC on TNFalpha treated-A549 cellsurface was analyzed by ELISA and fluorescence microscopy.The binding of PF-MC to A549 cells was higher compared toSLPI (p<0.05). In vivo, binding of the PF-MC was examined byusing an inflammatory air pouch model on mice. PF-MC or SLPI(52 μg) were inoculated into the air pouch; inflammatory recruitedcells were recovered after 2 h of treatment and PF-MCwas detected on the inflammatory cell surface by flow cytometry.We observed PF-MC on lymphocytes and monocytes, butnot on neutrophils. The binding was higher for PF-MC than SLPI(9±3 vs 4±1%; 31±7 vs 10±4% of lymphocytes and monocytes,respectively). A higher binding of PF-MC on lymphocytes wasconfirmed by analysing the cells of the popliteal lymph nodethat were injected with LPS (in the foot pad) and PF-MC (i.p.).The biological activities of PF-MC were similar or improved thanSLPI: the wound healing activity was augmented, since the diameterof the wound, measure at day 2 post-injury, was 35±8 %smaller in mice injected with PF-MC vs. SLPI. These results demonstratethat PF-MC binds to inflammatory cells both in vitroand in vivo, retains the biological activity of SLPI and, in somecases, improve it. Therefore, PF-MC may be a new tool to treatinflammatory diseases.