Single domain antibodies from llama effectively and specifically block T cell ecto‐ADP‐ribosyltransferase ART2.2in 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.

NAD+ and ATP Released from Injured Cells Induce P2X7-Dependent Shedding of CD62L and Externalization of Phosphatidylserine by Murine T Cells

“…Moreover the small (15kd) sdAbs are rapidly eliminated via the kidney, with a serum half life of Ͻ5 min. Blockade of ART2.2 by sdAbs is reversible and ART2.2 activity on lymph node cells is largely restored 24 h after injection (41). However, systemic administration of etheno-NAD ϩ could have unwanted side effects as this would provide other members of the ARTfamily with substrate, leading to the etheno-ADP-ribosylation of other cell surface proteins.…”

“…32 P-NAD ϩ was obtained from Amersham Biosciences. mAbs Nika102 (anti-ART2.2) pAb K1G (anti-P2X 7 ) and single domain Abs sϩ16a and l-17 (anti-ART2.2) were prepared as described previously (12,40,41).…”

“…To distinguish between these possibilities, we sought conditions that would prevent these reactions either by blocking ART2.2 or by blocking the activation of P2X 7 . To this end, we first used two distinct, recently described single domain Abs (sdAbs) that block the enzymatic and cytotoxic activities of ART2.2 (41). As illustrated in Fig.…”

Development by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor

“…Camelid single-domain Ab fragments (V H Hs, nanobodies) specific for human ChemR23 were identified from Ig repertoires of genetically immunized llamas following a prime-boost regimen (30). The human ChemR23 coding sequence (GenBank accession number: Y14838.1 http:// www.ncbi.nlm.nih.gov/genbank) was cloned into the mammalian expression vector pVAX1 (Invitrogen), and endotoxin-free plasmid DNA was prepared using the EndoFree Giga kit (QIAGEN), according to the manufacturer's instructions.…”

Molecular Imprint of Enzyme Active Site by Camel Nanobodies

“…In several cases, the H3 loop was shown to protrude from the remaining paratope and insert into the active site cleft of enzymes (22)(23)(24). The unique property of camel antibody binding to the incompatible antigen, which behaved quite differently in comparison with the conventional antibody, has led several groups to efficiently develop the potent enzyme inhibitors (25)(26)(27)(28)(29).…”