Blocking the citotoxic activity of ADP-ribosiltransferase ART2.2 on lymphocytes in vivo with single domain antibodies from llama

JAN REYELT; FRIEDRICH HAAG; VANINA ALZOGARAY; FERNANDO GOLDBAUM; FRIEDRICH KOCH-NOLTE

Hamburg

Congreso; NAD2008 Emerging roles of NAD and NAD-metabolites in cell signalling; 2008

University Medical Center Hamburg

Heavy-chain antibodies from llamas exhibit a remarkable propensity for binding to and blocking clefts on proteins (1). From a llama immunized with the toxin-related T cell ecto-ADP-ribosyltransferase, we have derived ART2-blocking single domain antibodies (VHHs) from a phage display library (2). Within 10 minutes after intravenous injection, the ART2.2-specific VHH domains (100μg/mouse) effectively shut-off the enzymatic and cytotoxic activities of ART2.2 on T cells in peripheral lymphatic organs. The blockade of ART2.2 in vivo was effective for 4-6 hours after injection, but was reversed within 24 hours, most likely as a result of rapid renal excretion of the small (15kd) proteins. With the goal of increasing the serum half life of ART2.2 inhibitors, we engineered a fusion protein of VHH and the Fc-portion of mouse IgG1 encompassing the hinge, CH2, and CH3 domains. Transfection into HEK cells confirmed resulted in high level secretion of a dimeric VHH-Fc fusion complete blockade of ART2.2 activity within 60 minutes after injection (even at doses as low as 1μg). The blockade was still essentially complete 24h after injection. Our results pose an interesting proof of principle demonstrating the utility of VHHs to block leucocyte ecto-enzymes in vivo. Considering that leucocyte ecto-enzymes play important roles in cell trafficking and inflammation. With their active sites exposed to the extracellular environment, these enzymes pose interesting targets for novel anti-inflammatory drugs(3).Similarily VHHs might pose a useful strategy to neutralize ADP-ribosylating toxins.