title: Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo.
abstract: The purpose of our study was to develop a tool for blocking the function of a specific leukocyte ecto-enzyme in vivo. ART2.2 is a toxin-related ecto-enzyme that transfers the ADP-ribose moiety from NAD onto other cell surface proteins. ART2.2 induces T cell death by activating the cytolytic P2x7 purinoceptor via ADP-ribosylation. Here, we report the generation of ART2.2-blocking single domain antibodies from an immunized llama. The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses. Their long CDR3 endows VHH domains with the extraordinary capacity to extend into and block molecular clefts. Following intravenous injection, the ART2.2-specific VHH domains effectively shut off the enzymatic and cytotoxic activities of ART2.2 in lymphatic organs. This blockade was highly specific (blocking ART2.2 but not the related enzymes ART1 or ART2.1), rapid (within 15 min after injection), and reversible (24 h after injection). Our findings constitute a proof of principle that opens up a new avenue for targeting leukocyte ecto-enzymes in vivo and that can serve as a model also for developing new antidotes against ADP-ribosylating toxins.
authors.affiliation: Department of Immunology, Diagnostic Center, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. nolte@uke.uni-hamburg.de
authors.forename: Friedrich
authors.initials: F
authors.lastname: Koch-Nolte
authors.forename: Jan
authors.initials: J
authors.lastname: Reyelt
authors.forename: Britta
authors.initials: B
authors.lastname: Schössow
authors.forename: Nicole
authors.initials: N
authors.lastname: Schwarz
authors.forename: Felix
authors.initials: F
authors.lastname: Scheuplein
authors.forename: Stefan
authors.initials: S
authors.lastname: Rothenburg
authors.forename: Friedrich
authors.initials: F
authors.lastname: Haag
authors.forename: Vanina
authors.initials: V
authors.lastname: Alzogaray
authors.forename: Ana
authors.initials: A
authors.lastname: Cauerhff
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Goldbaum
mesh_terms: D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D002161:Camelids, New World / Q000276:immunology*; D016923:Cell Death; D017931:DNA Primers; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000201:enzymology*
chemical_list: D000906:Antibodies; D017931:DNA Primers; D036002:ADP Ribose Transferases