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Published as doi: 10.1096/fj.07-8661com.
 (The FASEB Journal. 2007;21:3490-3498.)
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Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo

Friedrich Koch-Nolte*,1, Jan Reyelt*, Britta Schößow*, Nicole Schwarz*, Felix Scheuplein*, Stefan Rothenburg*, Friedrich Haag*, Vanina Alzogaray{dagger}, Ana Cauerhff{dagger} and Fernando A. Goldbaum{dagger}

* Department of Immunology, Diagnostic Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and

{dagger} Fundación Instituto Leloir, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina

1Correspondence: Department of Clinical Pathology, Institute of Immunology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: nolte{at}uke.uni-hamburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.—Koch-Nolte, F., Reyelt, J., Schößow, B., Schwarz, N., Scheuplein, F., Rothenburg, S., Haag, F., Alzogaray, V., Cauerhff, A., and Goldbaum, F. A. Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo.


Key Words: recombinant antibodies • leukocyte ecto-enzymes • enzyme inhibitors • ADP-ribosylation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LEUKOCYTES EXPRESS A FLURRY OF ECTO-ENZYMES that have their active sites exposed in the extracellular environment. These enzymes play important roles in cell trafficking, inflammation, and apoptosis (1 , 2) . For example, NAD-dependent ADP-ribosyltransferases (ARTs, CD296) regulate the function of other cell surface proteins by posttranslational modification (3 4 5) ; other nucleotide-metabolizing ecto-enzymes (CD38, CD39, CD73, CD157, CD203) control the half-life of purinoceptor-ligands and/or generate second messengers, such as cyclic ADP-ribose, adenosine, and lysophosphatidic acid (6 7 8) ; and peptidases (CD10, CD13, CD26, CD156, CD249) control the shedding and processing of cytokines and cell adhesion molecules (9 , 10) . Given the important regulatory functions of these enzymes, specific inhibitors of their activities might pose new tools to modulate immune functions (2) .

Small molecule inhibitors and antibodies represent two possible approaches for inhibiting leukocyte ecto-enzymes (2 , 11) . Small-molecule enzyme inhibitors, however, often lack specificity and block more than one member of a protein family. As extracellular proteins, leukocyte cell surface ecto-enzymes are accessible to antibodies. Although conventional antibodies rarely block the activity of the enzyme against which they are raised, the unique heavy chain antibodies of llamas and other camelids exhibit a remarkable preference for binding into the active sites of enzymes (12 13 14) .

The variable domains of heavy-chain antibodies (VHH domains) represent the smallest known antigen-binding units generated by adaptive immune responses (15 16 17) . Camelid heavy-chain antibodies lack both the light chain and the first constant domain of the heavy chain (CH1) of conventional antibodies. Thus, the hinge region links the VHH directly to a conventional CH2-CH3 Fc-domain (see Fig. 1 A). VHH domains possess a number of potentially very useful features. Firstly, the heart of the antigen binding paratope, i.e., the complementarity determining region 3 (CDR3), is longer and more diverse in VHH domains than in the VH and VL domains of conventional antibodies (18 19 20) . Second, whereas conventional antibodies usually form planar interaction surfaces with their cognate antigen, the CDR3s of heavy chain antibodies can form fingerlike extensions, allowing the formation of convex paratopes. Consequently, the CDR3 of heavy-chain antibodies can reach into and fill out molecular crevices on proteins that are often inaccessible to conventional antibodies. Third, the region of the VHH surface that corresponds to the hydrophobic contact area of the VH with the VL domain in conventional antibodies contains hydrophilic amino acid residues that ensure good solubility of VHH domains. Consequently, when produced as recombinant proteins, VHH domains show much better solubility and stability than recombinant VH or VL domains of conventional antibodies. Fourth, owing to their small size VHH domains show excellent tissue penetration capacity in vivo yet elicit little if any anti-VHH antibody responses (21 22 23) .


Figure 1
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  		Figure 1. Immune serum and purified Ig subclasses from ART2.2-immunized llama Matahari recognize ART2.2-transfected but not untransfected lymphoma cells. A) Schematic diagram of the structures of llama heavy-chain antibodies (IgG2, IgG3) and conventional light- and heavy-chain (IgG1) antibodies. Note that heavy-chain antibodies lack light chains and the CH1 domain. The antigen-binding domain of heavy-chain antibodies is referred to as VHH domain, nanobody, or single-domain antibody. B) Untransfected and ART2.2-transfected DC27.10 lymphoma cells were incubated with serum or purified IgGs, and bound antibody was detected with FITC-conjugated goat anti-Llama IgG. pIS: preimmune serum, IS: immune serum.

Considering the high specificity, the high solubility, and the small size of camelid VHH domains, we hypothesized that these might be particularly well suited for the purpose of blocking leukocyte ecto-enzymes in vivo. To test this hypothesis, we chose the T cell ADP-ribosyltransferase ART2.2 as a model ecto-enzyme. ART2.2 is a glycosylphosphatidylinositol (GPI)-anchored, lipid raft-associated, T cell surface enzyme related in structure and function to ADP-ribosylating bacterial toxins (24 25 26) . ART2.2 transfers the ADP-ribose moiety from NAD onto arginine residues in other cell surface and secreted proteins (24 , 26) . Following the release of the ART substrate NAD, nicotinamide adenine dinucleotide, from cells during inflammation or tissue damage, ART2.2 ADP-ribosylates and activates the cytolytic P2x7 purinoceptor (27 28 29) . This induces within seconds the exposure of phosphatidyl-serine on the outer leaflet of the cell membrane and the shedding of CD62L. Chronic activation of P2x7 by NAD-dependent ADP-ribosylation culminates in cell death, and, hence, this mechanism has been dubbed NAD-induced cell death, NICD (27) . Given the important regulatory functions of ART2.2 and other leukocyte ecto-enzymes, inhibitors of these enzymes might pose new tools to modulate immune functions (2 , 30) .

The aims of this study were to raise single-domain antibodies that inhibit the enzymatic and cytotoxic activities of ART2.2 and to probe the utility of these antibodies for blocking ART2.2 on the T cell surface in vitro and in vivo. Our results show that ART2.2-specific VHH domains generated from an immunized llama effectively and specifically block the enzymatic and cytotoxic activities of both recombinant soluble ART2.2 and of native membrane-bound ART2.2, but not those of the related ecto-enzymes ART1 and ART2.1. Moreover, intravenous injection of ART2.2-specific VHH domains rapidly and effectively shut off the enzymatic and cytotoxic activities of ART2.2 on T cells in lymphatic organs. Our results provide an important proof of principle that could be useful for raising specific inhibitors of other leukocyte ecto-enzymes and of the ART2.2-related ADP-ribosylating bacterial toxins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Immunization of llamas and purification of immunoglobulin subclasses from immune sera
A llama (surname Matahari) was immunized with a modified DNA-prime protein-boost strategy, which we had previously used successfully to raise polyclonal and monoclonal antibodies against native ART2.2 in rats and rabbits (31 , 32) . Immunization was initiated with four intradermal gene gun immunizations with an expression vector encoding full-length ART2.2 (31) at 6- to 12-wk intervals on the shaved neck (12 shots of 1 µgDNA/mg gold at a pressure setting of 300 psi). To improve the penetration of DNA-conjugated gold particles into the rather tough llama skin, four subsequent boost DNA immunizations were performed at higher pressure settings (500–600 psi). To boost the titer of ART2.2-specific antibodies, two subsequent protein boosts were performed by subcutaneous injection of 50-µg epitope-tagged recombinant ART2.2 purified by immobilized metal affinity chromatography from E. coli periplasm (31) and emulsified in Specol adjuvant (Cedi Diagnostics, Lelystad, The Netherlands) (33) . Blood samples were collected 14 days after each immunization, and serum was assayed for reactivity against ART2.2 by indirect immunofluorescence analysis of ART2.2-transfected lymphoma cells. Conventional and the long-hinge and short-hinge classes of heavy-chain antibodies were purified from preimmune and immune sera of the ART2.2-immunized llama by consecutive affinity chromatography on protein A and protein G sepharose columns (15) .

PCR amplification, cloning, and sequence analyses of phage display expression libraries
The VHH-repertoire was PCR amplified from llama Matahari’s peripheral blood leukocytes using separate primer pairs for short-hinge and long-hinge VHH: shf: 5' GCT GGA TTG TTA TTA CTC GCG GCC CAG CCG GCC ATG GCC CAG GT(GC) (AC)A (AG) 3', shr: 5' GAT GGT GAT GAT GAT GTG CGG CCG CGC TGG GGT CTT CGC TGT GGT GCG 3', lhf: 5' GCT GGA TTG TTA TTA TCT GCG GCC CAG CCG GCC ATG GCC GAT GTG ACG CTG CAG GCG TCT GG(AG) GGA GG 3', lhr: 5' GAT GGT GAT GAT GAT GTG CGG CCG CTG GTT GTG GTT TTG GTG TCT TGG G 3'. PCR amplification products were purified, restriction digested, and cloned into the NcoI and NotI sites of the pHEN2 phagemid vector. This places the coding region of the VHH domain downstream of the PelB-leader peptide and upstream of the chimeric His6xMyc epitope tag. Ligation reactions were transformed into ultracompetent XL10-GOLD E. coli (Stratagene, Amsterdam, The Netherlands). Colonies were harvested by scraping in culture medium, and recombinant phages were produced by standard techniques following infection with helper phage M13KO7 (GE Healthcare, Uppsala, Sweden). Plasmid DNAs were prepared from individual clones, and inserts were sequenced by standard DNA-sequencing techniques using flanking primers LMB3: 5' CAG GCC ACA GCT ATG AC 3'and fdSeq1: 5' TGA ATT TTC TGT ATG AGG 3'. DNA and translated protein sequences were analyzed using DNAstar software.

Selection of binding phage from VHH phage display libraries
The VHH-short-hinge and VHH-long-hinge phage display libraries (containing 104–105 primary clones) were subjected to sequential negative and positive selection on untransfected and on ART2.2-transfected DC27.10 lymphoma cells (incubation of 109 phages and 106 cells in 1 ml RPMI medium containing 10% FCS for 1 h at 22°C). Cells were washed 10 times with 50 ml RPMI/10% FCS. ART2.2-bound phages were released by incubation of cells with phosphatidyl-inositol-specific phospholipase C (Invitrogen, Karlsruhe, Germany) (0.1 U/106 cells in 1 ml RPMI medium containing 10% FCS for 1 h at 37°), which cleaves ART2.2 from the cell surface at its GPI-anchor (26) . Cells were pelleted by centrifugation and supernatants were used to infect E. coli.

Production and purification of recombinant epitope-tagged VHH domains
For VHH production, plasmids were transformed into HB2151 E. coli cells (which recognize the amber stop codon between the His epitope tag and the gp3 phage capsid protein). Individual colonies were picked and cultivated in liquid cultures. Cells were harvested 3 h after induction of VHH-expression by IPTG (0.5 mM). Periplasma lysates were prepared and analyzed for the presence of recombinant fusion protein by SDS-PAGE. VHH domains were purified from periplasma lysates by immobilized metal affinity chromatography (IMAC) on HIS-Select matrix (Sigma-Aldrich, Seelze, Germany). The affinity of each VHH domain toward ART2.2 was measured by means of an IAsys biosensor, using cuvettes coated with recombinant VHH domains (34) .

Indirect immunofluorescent staining of ART-transfected cells with purified IgG and recombinant VHH domains
Untransfected and DC27.10 lymphoma cells stably transfected with ART1, ART2.1, or ART2.2 (32) were stained with Matahari preimmune or immune serum (1:400, 5x105 cells/100 µl) or purified IgG (1 µg/100 µl) for 30 min at 4°C followed by FITC-conjugated anti-llama IgG (1:100, Bethyl Laboratories, Montgomery, TX, USA) for 30 min at 4°C. For staining of cells with purified recombinant VHH domains, VHH domains were preincubated for 20 min at 4°C with FITC-conjugated anti-myc antibody (Santa Cruz Biotech, Heidelberg, Germany) at a molar ratio of 2:1 in order to allow formation of bivalent conjugates. DC27.10 cells (5x105/100 µl) were stained with these conjugates (150 ng VHH, 750 ng antimyc IgG) for 30 min at 4°C, washed and subjected to FACS analyses. Stained cells were analyzed on a FACS-Calibur using the Cellquest software (Becton Dickinson, Heidelberg, Germany). Gating was performed on living cells on the basis of propidium iodide exclusion.

Injections of VHH domains into mice and preparation of lymph node cells
BALBc/J and C57BL/6J mice were obtained from the Jackson Laboratories, Bar Harbor, ME, USA. CD38KOxBALBc mice were kindly provided by Dr. Frances Lund, NY (35) . Purified VHH domains were injected into the tail vein of mice in 300 µl PBS. Cells were harvested from lymph nodes of sacrificed mice by gentle passage through nytex membranes in RPMI medium, as described previously (27 , 36) .

Assays for etheno-ADP-ribosylation of cell surface proteins and NICD
Etheno-ADP-ribosylation of cell surface proteins was monitored as described previously (37) following incubation of freshly prepared mouse lymph node cells for 10–20 min with 5 µM etheno-NAD at 4°C and using Alexa 488-conjugated, etheno-adenosine specific mAb 1G4. Exposure of phosphatidyl-serine and uptake of propidium iodide induced by ADP-ribosylation of P2x7 was monitored as described previously (27) following incubation of lymph node cells in the absence or presence of exogenous NAD for 30–120 min at 37°C. For blocking experiments, cells were preincubated for 20 min in the presence of purified VHH domains (2 µg/106 cells/100 µl) before addition of NAD.

Assay for ADP-ribosylation of agmatine
ADP-ribosylation of the arginine analog agmatine (Sigma) was monitored by thin layer chromatography (TLC) after incubating 40 nM recombinant ART2.2 with 1 mM agmatine in 50 µl PBS containing 0.1 µM 32P-NAD for 30 min at 37°C, as described previously (24) . After 30 min, proteins were precipitated using StrataClean resin (Stratagene) and analyzed by SDS-PAGE. Reaction products in supernatants were separated on TLC plates (Merck, Darmstadt, Germany) and detected by autoradiography by exposing Kodak X Omat films for 1–20 h. For blocking experiments, ART2.2 was preincubated for 20 min in the presence of the indicated concentrations of purified VHH domains before addition of agmatine and 32P-NAD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of ART2.2-specific antibodies by DNA-prime, protein-boost immunization of llama Matahari and generation of VHH short-hinge and long-hinge phage display libraries
Using a DNA-prime protein-boost strategy to immunize llama Matahari with mouse ART2.2 yielded conventional light- and heavy-chain antibodies, as well as single-chain antibodies that specifically recognized native ART2.2 on the surface of transfected lymphoma cells, as assessed by flow cytometry (Fig. 1B ). Conventional antibodies (IgG1) and single chain antibodies harboring short (IgG2) and long (IgG3) hinges were readily purified from Matahari immune serum by sequential chromatography on protein G and protein A (Supplemental Fig. 1). The repertoires of the short-hinge and long-hinge variable VHH domains were PCR-amplified from peripheral blood leukocyte cDNA generated from Matahari 10 days after the last immunization. The PCR products were cloned into the pHEN2 phage display vector. Clones were picked randomly from the short-hinge and long-hinge libraries and analyzed by DNA-sequencing. Alignment of the deduced amino acid sequences revealed CDR3s ranging in length from 3 to 23 amino acids (Fig. 2 , Supplemental Fig. 2). With few exceptions, the second framework region (FR2) contained the characteristic hydrophilic motif in the region corresponding to the interface of VL and VH domains in conventional antibodies (residues 37, 44, 45, and 47). Approximately 40% of the clones contained an extra pair of cysteine residues that could form a disulfide bridge connecting the front end of the CDR2 to a variable position in the CDR3. A comparative analysis of CDR3 lengths revealed that clones containing an extra cysteine pair had, on average, longer CDR3s (Fig. 2A ), in accord with the notion that the extra disulfide bridge might stabilize long CDR3s (38) .


Figure 2
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  		Figure 2. ART2.2-specific VHH domains contain long CDR3s. A) The sequences of randomly picked and ART2-selected long-hinge (left) and short-hinge (right) VHH domains were analyzed with respect to the number of amino acid residues in their CDR3s. The height of each column indicates the number of VHH domains found to contain a CDR3 with the number of amino acid residues indicated below. VHH domains containing only the canonical disulfide are indicated by black columns. VHH domains containing an extra potential disulfide bridge are indicated by hatched columns. Asterisks mark the columns containing the four ART2-specific VHHs l-17, s-14, s+16a, and s+16b. B) Amino acid sequences of the ART2.2-binding VHH domains. Amino acids are color coded as follows: CDR1: red, CDR2: green, CDR3: blue, extra cysteine residues: yellow, the characteristic motif of hydrophilic amino acids in FR2: pink.

Selection and sequence analysis of ART2.2-specific single domain antibodies from the phage display libraries
Following selection on ART2.2-transfected lymphoma cells, 95% of the clones recovered bound to ART2.2-transfected but not to untransfected cells. The ART2.2-binding clones contained four distinct sequences (Fig. 2B ). They were designated s-14, s+16a, s+16b, and l-17 to reflect the presence of a short- or long-hinge (s, l), the presence or absence of an extra pair of cysteine residues (+/–), and the length of the CDR3 in the number of amino acid residues. Single-domain antibodies from these clones were produced as epitope-tagged recombinant proteins in E. coli and were readily purified from periplasma lysates by immobilized metal affinity chromatography (Fig. 3 A). Protein yields ranged from 1 to 5 mg of VHH per liter of E. coli culture.


Figure 3
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  		Figure 3. Three of four ART2.2-selected VHHs specifically recognize native ART2.2 but are not related ARTs on the cell surface of transfected lymphoma cells. A) Periplasma lysate was prepared from l-17 transformed E. coli and the hexa-histidine-tagged VHH domains were purified by immobilized metal affinity chromatography. Proteins were size fractionated by SDS-PAGE and silver stained. Lanes were loaded with total periplasma lysate (lane 1), flow through of the IMAC column (lane 2), final wash (10 mM imidazole) (lane 3), proteins eluted from the column by consecutive washes with 250 mM imidazole (lanes 4–7). B) Coomassie stain of purified ART2.2-specific VHHs l-17, s-14, s+16a, and s+16b. C) Untransfected and ART-transfected DC27.10 lymphoma cells were subjected to FACS analyses after staining with VHHs s-14, s+16a, s+16b, l-17, and FITC-conjugated secondary antibody directed against the C-terminal myc-tag. Panels 1–4: reactivity with ART2.2 vs. ART2.1 transfected cells. Panels 5–8: reactivity with ART2.2-transfected vs. ART1-transfected lymphoma cells. Dashed lines: reactivity with untransfected cells.

Specificity and affinity analyses of ART2.2-specific single-domain antibodies
To assess the specificities of the selected VHH domains, we performed comparative FACS analysis on lymphoma cells transfected with different ARTs (Fig. 3B ). VHHs s+16a, s+16b, and l-17 reacted only with ART2.2-transfected cells but not with cells transfected with ART2.1 or ART1, which share ~81% and 39% amino acid sequence identity with ART2.2, respectively (24) . VHH s-14 reacted weakly also with cells transfected with the more closely related ART2.1 (Fig. 3B , lane 1). The binding affinities of the VHH domains (36–55 nM kDa), as determined by IAsys biosensor analyses (Table 1 ), were in the ranges of those found for other enzyme-blocking, single-chain antibodies and for conventional antibodies (14) .


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  		Table 1. IAsys biosensor analyses reveal high binding affinities of ART2.2-specific VHH domains

Three of four selected VHH domains block the enzymatic activity of ART2.2 in vitro
To assess the capacity of the ART2.2-binding VHH domains to block the enzyme activity of ART2.2, we employed two established ART enzyme assays, that is, first, TLC-autoradiography analysis of the 32P-ADP-ribosylation of the low molecular weight arginine analog agmatine by soluble recombinant ART2.2 (24) , and second, FACS analysis of the etheno-ADP-ribosylation of cell surface proteins by native membrane-bound ART2.2 (24 , 37) . The results of the TLC analyses (Fig. 4 ) demonstrate that VHHs s+16a, s+16b, and l-17, but not VHH s-14 effectively blocked ADP-ribosylation of the small soluble arginine-analog agmatine. Dose-response analyses revealed that blocking of ART2.2 activity was effective at almost equimolar concentrations of VHH and ART2.2, with VHH s+16a, showing slightly stronger blocking activity than VHHs s+16b and l-17 (Fig. 4C ). The results of the etheno-ADP-ribosylation assay (Fig. 5 ) demonstrate that VHHs s+16a, s+16b, and l-17, but not VHH s-14 effectively blocked the ART2.2-catalyzed etheno-ADP-ribosylation of proteins on the cell surface of isolated murine T cells (Fig. 5 , panels 4–6 vs. panel 3).


Figure 4
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  		Figure 4. Three of four ART2.2-selected VHHs block 32P-ADP-ribosylation of the arginine analog agmatine by soluble recombinant ART2.2. Soluble recombinant ART2.2 (2 pmol/50 µl) was preincubated with increasing amounts of VHH domains for 10 min before addition of agmatine and 32P-labeled NAD. Reactions were stopped after 30 min by precipitation of proteins with StrataClean-resin. A) Precipitated proteins were analyzed by SDS-PAGE and silver staining. B) Reaction products in the supernatants were analyzed by thin-layer chromatography and autoradiography. Note that reactions contained serial 1:2 dilutions of VHH domains (i.e., from 64 pmol in lane 10 to 0.125 pmol in lane 1). C) Spots corresponding to ADP-ribosylagmatine were quantified by densitometry, and relative spot densities were plotted as a function of VHH-concentration. Results are representative of 3 independent experiments.


Figure 5
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  		Figure 5. Three of four ART2.2-selected VHHs block etheno-ADP-ribosylation of cell surface proteins by native membrane-bound ART2.2 in vitro. Purified C57BL/6J mouse lymph node T cells were incubated for 10 min with ART2.2-specific VHH domains and were then further incubated in the presence of 5 µM etheno-NAD for 20 min at 37°C (panels 3–6). Cells were washed and etheno-ADP-ribosylated cell surface proteins were detected with Alexa488-conjugated etheno-adenosine specific mAb 1G4. Counterstaining was performed with PE-conjugated anti-CD4 in order to distinguish ART-activity on helper (CD4+) and cytotoxic (CD4-) T cells. Control stainings were performed on T cells after incubation for 20 min in the absence (panel 1) or presence (panel 2) of etheno-NAD but without VHH domains. Results are representative of 3 independent experiments.

Three of four selected VHH domains block the cytotoxic activity of ART2.2 in vitro
ADP ribosylation of cell surface proteins on murine T cells activates the cytolytic P2x7 receptor, inducing the exposure of phosphatidyl serine on the outer leaflet of the plasma membrane and, ultimately, cell death (27) . The results presented in Fig. 6 show that purified murine T cells were protected from NAD-induced cell death (NICD) by the ART2.2-blocking single domain antibodies s+16a, s+16b, or l-17 but not by VHH s-14 (panels 4–6 vs. panel 3). Note, however, that treatment of freshly prepared T cells with ART2.2-blocking VHH domains had no effect on the background level of "spontaneous" apoptosis (e.g., 11–13% AnnexinV+ cells in the lower right quadrant in Fig. 6 , panels 1, 4–6). This background of spontaneously apoptotic cells is caused by ART2.2-catalyzed ADP-ribosylation of P2x7 in response to endogenous NAD released from cells before or during cell preparation (27 , 39) . The proportion of these spontaneously apoptotic cells is greatly elevated in mice carrying a genetic deficiency of the major NAD-degrading ecto-enzyme CD38 (36) . CD38KO mice, therefore, provide a convenient assay to test the efficacy of ART2.2-specific VHH domains in the living animal.


Figure 6
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  		Figure 6. Three of four ART2.2-selected VHHs block the cytotoxic activity of ART2.2 in vitro. Purified BALBc lymph node T cells were incubated with ART2.2-specific VHH domains for 10 min before the addition of the ART substrate NAD (25µM) (panels 3–6). Cells were incubated further for 30 min at 37°C, and then stained with FITC-conjugated AnnexinV and propidium iodide before FACS analyses. Numbers indicate the percentage of cells in each quadrant. Control stainings were performed on T cells after incubation for 30 min in the absence (panel 1) or presence (panel 2) of NAD but without VHH domains. Results are representative of 3 independent experiments.

Intravenous injection of VHH s+16a blocks the cytotoxic and enzymatic activities of ART2.2 on lymph node T cells
To test the hypothesis that VHH domains can be used as tools to block ecto-enzyme function in vivo, we injected purified VHH s+16a into the tail vein of CD38KO mice and used the high level of spontaneous PS-exposure by T cells from these mice to assess ART2.2-mediated cytotoxicity (Fig. 7 ). Mice were sacrificed 15 min and 90 min after VHH injection, and lymph node cells were assayed for spontaneous PS-exposure (Fig. 7A ) and for ART activity (Fig. 7B ). The results demonstrate that VHH s+16a, indeed, blocks ART2.2-mediated cytotoxicity for lymph node T cells in a dose-dependent manner in vivo. The cytotoxic activity of ART2.2 was effectively shut off within 15 min after intravenous injection of 300 µg VHH (Fig. 7A , panel 3). FACS-analyses using the 1G4 monoclonal antibody confirmed the effective shut-off of ART2.2-catalyzed etheno-ADP-ribosylation of cell surface proteins (Fig. 7B ). Time course analyses showed that the blockade of ART2.2 functions was still essentially complete 6 h after VHH injection but had largely subsided 24 h after injection (Supplemental Fig. 3).


Figure 7
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  		Figure 7. Intravenous injection of VHH s+16a blocks ART2.2 functions on lymph node T cells in vivo. BALBc_CD38KO mice were injected intravenous (i.v.) with 300 µl PBS or with PBS containing 50 µg or 300 µg of purified ART2-specific VHH s+16a and mice were sacrificed 15 min and 90 min after i.v. injection. A) Apoptosis of lymph node cells induced by endogenous NAD was assessed by staining of cells with AnnexinV and PI. Cells were costained with anti-CD3, and gates were set on CD3+ cells. B) Separate aliquots of cells were incubated with 5 µM etheno-NAD for 20 min at 4°C before costaining with etheno-adenosine-specific mAb 1G4 and anti-CD3 C). Results are representative of four independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study provides the first demonstration of an effective, reversible blockade of a leukocyte cell surface ecto-enzyme in vivo. Indeed, a single i.v. injection of VHH s+16a sufficed to effectively block the enzymatic and cytotoxic activities of ART2.2 on the cell surface of lymph node T cells (Fig. 7) . The blockade of ART2.2 by VHH s+16a was effective within 15 min after injection and lasted up to 6 h but subsided markedly 24 h after injection. These results are consistent with the notion that the small size (12–14 kDa) and high stability of single-domain antibodies ensure both, high tissue penetration capacity and rapid clearance via the kidney (17 , 21 , 22 , 38) . A rapid but reversible blockade of leukocyte cell surface enzymes would be desirable, for example, in settings of acute inflammation or tissue damage. In chronic conditions, longer durations of enzyme inhibition may be required and several available technologies could be applied to improve the avidity and/or serum half-life of VHH domains, including dimerization of VHH domains via a linker peptide, PEGylation, and/or fusion to long-lived serum proteins (17) .

Single-domain antibodies provide a number of important advantages vs. both, small molecule inhibitors and conventional antibodies. An advantage of single-domain antibodies over small-molecule inhibitors is the exquisite specificity of antibodies harnessed from in vivo immune responses. The three-dimensional structures of rat ART2 and its bacterial toxin cousins revealed very similar NAD-binding active site crevices with key conserved active site residues (25 , 40) , consistent with a common mechanism of NAD-binding and ADP-ribose transfer. Nicotinamide inhibits both, bacterial and eukaryotic ARTs, as do most other NAD analogues and small molecular weight ART antagonists (30 , 41) . Murine ART2.2 and its closest relatives ART2.1 and ART1 share 81% and 50% sequence identity, respectively (42) . These arginine-specific ARTs exhibit rather promiscuous target specificities, and, on transfected cells, they can modify the same membrane proteins, e.g., the integrin LFA-1 (37) . It has recently been shown that the association with lipid rafts restricts ART2.2 onto specific targets (26) . On release from the constraint of the plasma membrane, either by metalloprotease-mediated proteolytic cleavage of the juxtamembrane stalk or by PI-PLC-mediated cleavage of the GPI-anchor, soluble ART2.2 can modify many different soluble proteins, including nonphysiological targets such as arginine-rich histones and even the small arginine analog agmatine (43) . Similar activities are displayed by recombinant ART2.2 produced as a soluble protein by insect cells or E. coli (24 , 42) . It is likely that these differences in specificity of membrane bound vs. soluble ART2.2 are determined by target accessibility rather than by differences in the conformation of ART2.2, since all known ART2.2-specific monoclonal antibodies bind to both, soluble and membrane-bound ART2.2 (31 , 32) . Consistently, the three ART2.2-blocking VHH domains derived from llama Matahari inhibit the activity of both soluble and membrane-bound ART2.2 (Figs. 4 and 5) . The binding specificity of an antibody to its target antigen can be profoundly affected by subtle changes in the structure of the antigen. Indeed, in many cases, even a single conservative amino acid substitution in the antigenic paratope abrogates recognition by the cognate antibody. Consistently, the ART2.2-blocking VHH domains inhibit the activity of ART2.2 but do not affect the enzymatic activities of the close relatives ART2.1 and ART1. Indeed, these antibodies do not show any detectable binding to ART2.1 or ART1 (Fig. 3C ). Our results imply that VHH domains can be more efficient tools than substrate analogues for blocking specific members of ecto-enzyme-families. Moreover, the use of enzyme-specific VHH domains may preempt the unwanted side effects that can arise when small molecular weight inhibitors affect multiple members of an enzyme family.

Key advantages of single-domain antibodies over conventional antibodies include the lack of Fc domains, higher solubility and stability, and preference for binding to the active site of enzymes (Figs. 3 4 5 6 7) . Without Fc domains, recombinant VHH domains can not activate Fc-mediated effector mechanisms, thus circumventing unwanted side effects, such as the activation and/or depletion of the cell subset expressing the target antigen (44) . The preference of VHH domains for binding to and blocking the active site of enzymes is illustrated by our finding that three of four selected ART2.2-binding VHH domains blocked its enzymatic and cytotoxic activities, whereas none of 12 conventional ART2.2-specific rat monoclonal antibodies generated in our lab (31 , 32) blocked the enzymatic activity of ART2.2 (not shown). The observed high ratio of ART2.2 enzyme-blocking vs. binding VHH domains is in accord with a recent study on lysozyme-specific VHH domains obtained from an immunized dromedary (with 6/8 lysozyme-binding VHH domains exhibiting enzyme-blocking activity) (14) but stands in contrast to the low ratio of enzyme-blocking vs. binding VHH domains (1/8) obtained from a synthetic phage display library in case of potato starch branching enzyme (45) . Presumably, enzyme-blocking heavy-chain antibodies are preferentially selected by the immune systems of llamas and dromedaries during natural immune responses. VHH domains have previously been shown to display long CDR3 regions that tend to form convex paratopes rather than the flat interaction surfaces typical of conventional VH+VL domains (14) . These long convex paratopes display a propensity to fit into crevices on proteins such as the active sites of enzymes. It is conceivable that immunization of camelids with native enzymes leads to the display of these enzymes by follicular dendritic cells and, subsequently, to the selection of B cells for clonal expansion that express VHH domains with convex paratopes complementary to the active site crevice. In contrast, the (less frequent) B cells expressing VHH domains with flat paratopes would be selected by regions of the enzyme outside the catalytic crevice, similar to B cells displaying conventional antibodies. Consistent with this hypothesis, the three ART2.2-blocking single domain antibodies obtained from llama Matahari contain long CDR3s of 16–17 amino acids, whereas the single nonblocking VHH contains a shorter CDR3 of 14 amino acids (Fig. 2B ). It will be of great interest to determine whether a similar preferential selection of enzyme-blocking antibodies can be achieved in transgenic mice expressing camelid heavy chain antibodies (46) .

Our study provides confirmation for the underlying hypothesis that single domain antibodies can serve as tools to specifically block lymphocyte ecto-enzymes in vivo. This provides an important proof of principle. We propose that the ART2.2-blocking single-domain antibodies described here represent the prototype of a novel class of immunomodulating drugs that specifically target immunoregulatory ecto-enzymes (1 , 2) . Moreover, since a deep NAD-binding active site crevice is a common feature of ART2.2 and of its bacterial toxin cousins (25) , many of which have significant impact on human health (47 , 48) , we propose that the experimental strategy illustrated here also opens a new avenue for raising single-domain antibodies as toxin antidotes.


   ACKNOWLEDGMENTS
 
This work was supported by grants no. 310/4 and no. 310/5–1 from the Deutsche Forschungsgemeinschaft and the Bundesministerium für Wirtschaftliche Zusammenarbeit to F.K.N. and F.G. Parts of this work represent partial fulfillments of the requirements for the diploma thesis of BS and the graduate theses of J.R. and V.A. F.K.N. and F.G. designed and supervised this work with essential contributions by F. H. and S. R. to the study design. J. R. cloned the phage display library, selected the ART2.2-binding VHH domains, and performed the experiments illustrated in Figs. 2 3 4 5 and in Supplemental Fig. 2; B.S. performed the experiments illustrated in Fig. 1 and Supplemental Fig. 1; N.S. and F.S. performed those in Fig. 7 and Supplemental Figure 3; and V.A. and A.C. those in Table 1 . F.K.-N. wrote the paper. We thank Fenja Braasch, Marion Nissen, Fabienne Seyfried, Gudrun Dubberke, Michael Moeller, Hartmut Stein, and Dr. Irmgard Dobberstein for assistance with cloning, FACS-analyses, and immunizing llama Matahari. We thank Drs. Bernhard Fleischer and Peter Bannas, Hamburg, and Drs. Olivier Boyer, Michel Seman, and Sahil Adriouch, Rouen, for critical reading of the manuscript. The authors declare that they have no competing financial interests.

Received for publication March 30, 2007. Accepted for publication May 3, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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