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.
Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.
Platform: Production de Protéines Recombinantes et d’Anticorps-Proteopole, Unité Genes, Synapses, et Cognition, Centre National de la Recherche Scientifique (CNRS), Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris cedex 15, France.
Tengfei
T
Li
Jean-Pierre
JP
Bourgeois
Susanna
S
Celli
Fabienne
F
Glacial
Anne-Marie
AM
Le Sourd
Salah
S
Mecheri
Babette
B
Weksler
Ignacio
I
Romero
Pierre-Olivier
PO
Couraud
François
F
Rougeon
Pierre
P
Lafaye
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001254:Astrocytoma / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D010962:Plasmodium berghei / Q000472:pathogenicity; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D005904:Glial Fibrillary Acidic Protein; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies