Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: Application to brain imaging.

Normally, antibodies cannot bind to intracellular cerebral antigen because they cannot cross the blood-brain barrier (BBB). Here, we show for the first time that a novel class of antibodies can pass unimpeded through the BBB.
Native homodimeric heavy-chain antibodies made by camelids have a paratope with a single variable termed VHH. Recombinant VHH, a particular marker of astrocytes, was employed in this instance. It was directed towards human glial fibrillary acidic protein (GFAP). In vitro, the BBB could only be crossed by basic VHHs (such as those with pI=9.4) (7.8 vs. 0% for VHH with pI=7.7).
We demonstrated that these fundamental VHHs can cross the BBB in vivo, diffuse into the brain tissue, enter into astrocytes, and specifically label GFAP via intravenous and intracarotid injections into live mice. We then produced a recombinant fusion protein VHH-green fluorescent protein to assess their capacity to function as a particular transporter.
On mouse brain slices, these "fluobodies" preferentially identified GFAP, and an unmodified version of the fusion protein VHH-GFP (pI=9.3) was able to cross the BBB and mark astrocytes in vivo. It is now important to pay attention to VHHs' potential in the central nervous system as therapeutic or diagnostic tools.

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