Homodimeric VHHs offer new perspectives for in vivo immunodiagnosis. Because of their small size of about 12-14 kDa, VHHs rapidly pass the renal filter, which has a cutoff of about 60 kDa, resulting in rapid blood clearance. In addition, the small size results in a fast tissue penetration. The VHH short serum half-life of about 30 min, compared to 4h for scFv and 50h for IgG, is advantageous for in vivo diagnosis using imaging and for the targeting of VHHs coupled to a substance of interest for treating a disorder, as one can expect that unspecifically bound VHH will be quickly removed from the tissues. Moreover, VHH are able to cross the Blood brain barrier, to penetrate into cells and to bind an intracellular target.
We have develop a strategy to conjugate any VHH comprising a cystein residue at the C- or N- terminus, by thio-addition (conjugation step) with a maleimido compound bearing a substance of interest, such as a MRI contrast agent or a fluorescent tag. These labeled VHH are fully functional and recognize in vivo their targets.