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Covalent Dimerization of Camelidae Anti-Human TNF-Alpha Single Domain Antibodies by the Constant Kappa Light Chain Domain Improves Neutralizing Activity
rgen Scheller2 Martin Giersberg,1 Doreen M. Floss,2 Sergey Kipriyanov,3 Udo Conrad,1 Ju Institute of Plant Genetics and Crop Plant Research (IPK), Phytoantibodies, D-06466 Gatersleben, Germany; telephone: 49-39482-5253; fax: 49-39482-5136; e-mail: conradu@ipk-gatersleben.de 2 t, D-24118 Kiel, Germany Department of Biochemistry, Christian-Albrechts-Universita 3 Aftech AS, Oslo Research Park, N-0349 Oslo, Norway
Received 26 August 2009; revision received 7 December 2009; accepted 10 December 2009 Published online 31 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22653
1

ABSTRACT: The tumor necrosis factor-alpha (TNFa) plays an important role in a number of chronic inammatory disorders. Monoclonal camelidae variable heavy chain domain-only antibodies (VHH) have been developed to antagonize the action of human TNFa (anti-TNF-VHH). Here we describe a strategy to obtain functional dimeric anti-TNF-VHH molecules, based on the C-terminal fusion of a k light chain domain to the anti-TNF-VHH. The resulting fusion protein was transiently expressed by use of viral vectors in Nicotiana benthamiana(Nb) leaves and puried. Competitive ELISA and cell cytotoxicity assays revealed that the dimerized anti-NbTNF-VHHCk proteins blocked TNFa-activity more effectively than either the monomeric Escherichia coli(Ec) produced anti-EcTNFVHH or the monomeric anti-NbTNF-VHHCk. We suggest that enhanced inhibition shown by dimeric anti-NbTNFVHHCk proteins is achieved by an increase in avidity. Biotechnol. Bioeng. 2010;106: 161166. 2009 Wiley Periodicals, Inc. KEYWORDS: TNFa; single domain antibody; VHH; nanobody; k light chain

Cytokines such as tumor necrosis factor-a (TNFa) control the proliferation, differentiation and viability of cells (Hehlgans and Pfeffer, 2005). Aberrant regulation of cytokine activity is associated with the onset and persistence of chronic diseases such as sepsis (Beutler et al., 1985), rheumatoid arthritis (Maini et al., 1995) and Crohns disease (Van Dullemen et al., 1995), while selective targeting of these proteins has proven to be clinically benecial in the treatment of a number of human diseases. Monoclonal
Correspondence to: U. Conrad Additional Supporting Information may be found in the online version of this article.

camelidae heavy-chain antibodies against both murine and human TNFa have been isolated (Coppieters et al., 2006), and their presence leads to an inhibition of TNFa binding to its cognate receptors (Plagmann et al., 2009). Camelidae heavy-chain antibodies recognize their antigen via a single variable heavy chain domain (VHH), since unlike conventional IgG antibodies, VHH molecules lack a light chain (Hamers-Casterman et al., 1993). As a result, they are about ten times smaller than a typical IgG antibody, their domains are highly temperature resistant, and their thermal unfolding is reversible (Arbabi Ghahroudi et al., 1997). Their small size is thought to confer improved tissue penetration, and they show less tendency than single-chain Fv (scFv) antibody fragments to aggregate and/or degrade proteolytically (Hamers-Casterman et al., 1993). Nonetheless, the specicity and antigen afnity of VHH antibodies have been shown to be generally comparable to those of IgG antibodies, with dissociation constants (KD) lying in the nanomolar range (Lauwereys et al., 1998). As a result, they have been proposed as an alternative drug format for the treatment of TNFa-mediated inammatory and autoimmune diseases. Dimers of antibody kappa (k) light chains, known as Bence Jones proteins, are secreted by myeloma cell lines (Bence-Jones, 1848). As a component of a tetrameric antibody, they contain the cysteines required for disulphide binding to the appropriate heavy chain. The same cysteine residues may also be involved in the formation of covalently linked k chain dimers (Stevenson and Straus, 1968). These antibodies lack the Fc component, which is advantageous where an interaction with Fc receptors following antibody-dependent cell-mediated cytotoxicity (ADCC) is undesirable (Liu et al., 1991). Their dimerization may result in a higher level of stability and an enhanced antigen binding capacity. In principle, it should be possible

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to engineer recombinant antibody fragments so that they assemble as stable multimeric oligomers with favorable binding behavior (avidity and specicity) to a wide range of target antigens (for a review, see Kortt et al., 2001). In the present work, we demonstrate the functional transient expression of an anti-TNF-VHH component fused to the constant region of the human k light chain constant domain (Ck) (anti-TNF-VHHCk) in Nicotiana benthamiana(Nb) leaves. The post-translational dimerization of Ck tagged anti-NbTNF-VHCk proteins generated a highly potent bivalent anti-NbTNF-VHHCk molecule, with a signicantly enhanced inhibition prole. To generate bivalent anti-TNF-VHH antibodies, the antiTNF-VHH cDNA fragment was concatenated with a cDNA coding for human k light chain constant domain (anti-TNFVHHCk) (Fig. 1A,B). Recombinant protein expression was achieved with the MagniconTM viral vector system, using N. benthamiana as the expression host. Recombinant anti-NbTNF-VHHCk proteins were puried by NTA-afnity chromatography (Fig. 1C). The nonoptimized expression of Nb TNF-VHHCk yielded in 80 mg recombinant protein per 150 g N. benthamiana leaves after NTA-afnity chromatography. This is in the range of expression to be expected for

the generally accepted MagniconTM system (Gleba et al., 2004). The recombinant anti-TNF-VHHCk derived from N. benthamiana leaves did not separate as a single size exclusion chromatography peak, indicating that 40% of the protein was present as monomers, 40% as dimers and 20% as higher polymers (Fig. 2A). The monomeric and dimeric anti-NbTNF-VHHCk proteins were separated from one another in a single step, using preparative size exclusion chromatography (Fig. 2A). When both the monomeric and

The expression and purication of anti-TNF-VHHCk. A: Schematic representation of the camelidae heavy-chain antibody with its reduced size, but full antigen-binding capacity VHH domain, and a novel dimerized fusion between the VHHdomain and the k light chain constant domain. B: Schematic illustration of the expression constructs encoding anti-TNF-VHHCk. C: Purication of anti-TNF-VHHCk by metalloafnity chromatography. Crude extracts (CE); ow-through (FT); imidazole washing steps (W1 5 mM imidazole; W2 20 mM); imidazole elution fractions (E1E3) separated by a SDSPAGE under reducing conditions and stained with Coomassie brilliant blue.

Figure 1.

Figure 2. The dimeric anti-TNF-VHHCk protein. A: Size exclusion chromatography of anti-NbTNF-VHHCk (2 mL) after purication of the recombinant protein via NTAagarose on a calibrated HiLoad 16/60 Superdex 75 prep grade column using a 10 sodium phosphate buffer, pH 7.5 as the mobile phase with a constant ow-rate of 1.0 mL/min. Fractions containing the monomeric and dimeric anti-NbTNF-VHHCk proteins were pooled separately and concentrated. B: SDSPAGE separation and staining with Coomassie brilliant blue of monomeric (M) and dimeric (D) anti-NbTNF-VHHCk under reducing (R) and non-reducing conditions (NR). C: Western blot analysis of monomeric (M) and dimeric (D) anti-NbTNF-VHHCk. Electrophoresis was performed under reducing (R) and non-reducing (NR) conditions.

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dimeric forms of NbTNF-VHHCk were tested for stability by repassage through size exclusion chromatography, single elution peaks of the expected molecular weight were generated (data not shown). This result indicated that the monomeric and dimeric NbTNF-VHHCk were stable and not convertible into one another. The dimerization of anti-NbTNF-VHHCk was facilitated by the formation of intermolecular disulphide bridges within the k light chain constant domains. The polymerization of the puried anti-NbTNF-VHHCk proteins was assessed both by SDS PAGE under either reducing or non-reducing conditions, and by western blotting. Under reducing conditions, due to loss of intermolecular disulphide bridging, the monomeric and the dimeric anti-NbTNF-VHHCk protein fractions appeared as a single species of size 29 kDa, as expected for a monomeric anti-NbTNF-VHHCk. Under non-reducing conditions intermolecular disulphide bridges were conserved. As expected, the monomeric anti-NbTNF-VHHCk proteins were detected slightly below 30 kDa, and the dimeric anti-NbTNF-VHHCk proteins were detected at about 60 kDa, indicating that two anti-NbTNF-VHHCk proteins were connected by intermolecular disulphide bridges (Fig. 2B,C). Thus, in its puried form, both the recombinant monomeric anti-NbTNF-VHHCk and dimeric anti-NbTNFVHHCk proteins existed in a homogeneous and stable conformation. The binding afnities to human TNFa of monomeric and dimeric anti-NbTNF-VHHCk were determined by competitive ELISA. TNFa in solution prevents dependent on the concentration the binding of monomeric and dimeric anti-NbTNF-VHHCk to TNFa xed to the ELISA plate. The dissociation behavior of the two forms of anti-Nb TNFVHHCk is shown in Fig. 3. The calculated afnity value (KD) of the monomeric anti-Nb TNF-VHHCk was 2.2 nM, and that of the dimeric form 0.4 nM. As L929 cells are known to undergo apoptosis in the presence of human TNFa (Schmid et al. 1986), their behavior when exposed to TNFa was used as a cell-based assay of anti-Nb TNF-VHHCk protein inhibitory capacity. Monomers from both, anti-ECTNFVHH and anti-NbTNF-VHHCk inhibited the TNF-induced cell death of L929 in a dose-dependent manner, with rather similar IC50 values (respectively, 10.5 and 10.8 nM, see Fig. 4A). Importantly, the inhibitory proles of both proteins were almost identical to one another, thus indicating that the C-terminal Ck does not interfere with antigen binding. The monomeric anti-NbTNF-VHHCk or dimeric anti-NbTNF-VHHCk proteins were also compared with one another via the L929 cytotoxicity assay (Fig. 4B). At the molar level, the anti-Nb TNF-VHHCk dimer (IC50 1.1 nM) showed almost ten fold more inhibition of TNFa activity than the monomeric anti-NbTNF-VHHCk form (IC50 10.8 nM), indicating that dimerization produces a fully active bivalent antibody with enhanced biologic activity. We have described here the functional characterization of a dimerized anti-NbTNF-VHHCk antibody, achieved by its fusion to k light chain constant domains.

Figure 3. Competitive ELISA used to assess the binding properties of anti-NbTNF-VHHCk to human TNFa. Monomeric and dimeric anti-NbTNF-VHHCk have been puried by afnity chromatography and separated by size exclusion chromatography. Each data point consists of ve technical repeats. Standard deviation is given in bars.

Figure 4. Inhibition of TNFa-mediated cytotoxicity by dimeric anti-NbTNF-VHHCk. A: L929 cells incubated with 100 ng/mL TNFa and a variable quantity of anti-EcTNFVHH and monomeric anti-NbTNF-VHHCk. B: L929 cells incubated with 100 ng/mL TNFa and a variable quantity of monomeric anti-EcTNF-VHH or dimeric anti-NbTNF-VHHCk. Cellular proliferation was quantied by the CellTiter-Blue Cell Viability Assay.

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The IC50 of the monomeric protein, in terms of L929 cytotoxicity, was 10.8 nM, while that of the dimeric form was 1.1 nM. Here, molarity refers to the binding sites per molecule to allow comparison of the monomeric and the dimeric anti-NbTNF-VHHCk. The employed molarity of TNF refers to the trimeric molecule because we showed previously that only one anti-TNF-VHH binding site is accessible per trimeric TNFa and that the binding of one anti-TNF-VHH antibody is sufcient to neutralize one trimeric TNFa molecule (Plagmann et al. 2009). In the cytotoxicity test, TNFa is used at a concentration of 100 ng/ mL which is a molar concentration of about 1.66 nM. Thus, to inhibit the biologic activity of TNFa by 50% (IC50) an equal concentration of anti-TNF-VHH is sufcient. The improved neutralizing activity of the dimerized anti-NbTNFVHHCk was probably based on its greater avidity, a characteristic which combines the synergistic strength of multiple bond interactions. Until now, bispecic and bivalent VHH antibodies have been constructed by conventional genetic engineering, using linker peptides to join two VHH domains into a single protein (Conrath et al., 2001), followed by a post-translational dimerization effected by transglutaminases (Plagmann et al., 2009). This Ck mediated site-specic cross-linking may represent a generally applicable strategy for producing bivalent domain antibodies while avoiding the suppression of their afnity. Analysis of their in vivo TNFa neutralizing activity will depend on the availability of suitable animal models. In summary, we have shown that a combination of genetic engineering and a plant-based expression system has the potential to deliver the large scale production of dimerized VHH antibodies, which may be suitable as an in vivo therapeutic.

2009) and the c-myc tagged Ck gene from the plant expression vector encoding the light chain of the anti-HIV1 mAb 2G12 (Floss et al., 2009). Both fragments were fused and subcloned into the modied MagniconTM vector pICHM10990 (Marillonnet et al., 2004). The detailed amino acid sequence of anti-TNF-VHHCk is given in supplemental Figure 1. To prepare the vector pICHM10990 a DNA sequence encoding the poly-histidine tag was assembled from oligonucleotides and cloned into the BamHI and HindIII sites of pICH10990, thus resulting in the plasmid pICH-M10990. The plasmids pICH-M10990-aTNF-Ck, pICH14011 (integrase source) and pICH10570 (50 provector) were electroporated into Agrobacterium tumefaciens strain GV3101 (Marillonnet et al. 2004).

Expression and Purication of Anti-TNF-VHHCk From N. benthamiana MagniconTM vectors allow the accumulation of high levels of various recombinant proteins following transient expression in N. benthamiana (Gleba et al. 2004; Marillonnet et al. 2004). The method combines DNA delivery from Agrobacterium to plants with a virus-based expression of recombinant proteins. For agrobacteria-inltration of the N. benthamiana plants, 200 mL of overnight cultures of recombinant agrobacteria were sedimented by centrifugation (6,000g, 5 min) and resuspended in 1 L of 10 mM MES (pH 5.5), 10 mM MgSO4. The resulting bacterial suspension was mixed with equal amounts of two agrobacteria strains transformed with plasmids pICH14011 (encodes DNA integrase) and pICH10570 (50 provector). N. benthamiana plants were inltrated by immersing the whole plant into 3 L of the resulting agrobacteria suspension followed by vacuum application (0.8 bar) for 3 min. The gentle return to atmospheric pressure caused the agrobacterial suspension to spread inside the apoplastic system. Inltrated N. benthamiana plants were further grown in the greenhouse. Maximum product accumulation was observed after 912 days, as determined by western blot analysis of leaf discs. One hundred fty grams of leaf material was shock frozen in liquid Nitrogen and homogenized in extraction buffer (50 mM KH2PO4/K2HPO4) containing 100 mM ascorbic acid to prevent the typical browning effect of N. benthamiana extracts by the use of a Waring Blender. The homogenate cleared by ltration through Miracloth (Calbiochem, San Diego, CA) followed by centrifugation at 14,000 g and 48C for 20 min. The crude extract was further diluted with 3 volumes of binding buffer (20 mM TrisHCl, 0.5 M NaCl, 5 mM imidazole, pH 7.9). The anti-NbTNFVHHCk fragments were isolated from the cleared supernatant by batch incubation with Ni-NTA His-bind resin (Novagen-Merck, Nottingham, UK) for 2 h, at 48C under permanent stirring. Subsequent chromatography was performed under scaled up conditions, based on standard protocols from the manufacturer.

Materials and Methods

Cells and Reagents L929 cells (German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany) were grown in DMEM high glucose culture medium (PAA Laboratories, Marburg, Germany) supplemented with 10% (v/v) fetal calf serum, 60 mg/L penicillin and 100 mg/L streptomycin at 378C in a water saturated atmosphere containing 5% CO2. Recombinant human TNFa was kindly provided by Daniela N. Maennel, Dept. Immunology, University of Regensburg, Germany.

Cloning of Anti-TNF-VHH Into a Plant Expression Plasmid The gene encoding the anti-TNF-VHH was amplied from the pCRScript-anti-hTNF-VHH plasmid (Plagmann et al.,

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Expression and Purication of Anti-EcTNF-VHH From E. coli The plasmids pet23()-pelB-anti-TNF-VHH-c-myc-his (Plagmann et al. 2009) was transformed into E.coli BL21 pLysS and protein expression was achieved by growing bacteria in LB-Media for 18 h at 308C after induction with 1 mM Isopropyl-b-D-thiogalactopyranosid at an OD600 of 0.60.8. Cells were separated by centrifugation and the cleared anti-TNF-VHH-protein-containing supernatant was supplemented with an equal volume of phosphate buffer (500 mM NaCl, 50 mM Na2HPO4/NaH2PO4, pH 7.4). One milliliter Ni-NTA agarose was added per l supernatant and stirred at 48C for 2 h and then loaded on a column. Proteins were eluted with 500 mM imidazole in phosphate buffer.

recombinant human TNFa in PBS and incubated overnight at room temperature. After blocking with 3% (w/v) bovine serum albumin (BSA), 0.05% (v/v) Tween-20 in PBS (PBST) for 2 h, pre-determined quantities of either monomeric or dimeric anti-NbTNF-VHHCk mixed with various concentrations of TNFa in 1% (w/v) BSA in PBST were added to each well, and the plates were incubated for 1.5 h at 258C. Bound anti-NbTNF-VHHCk was visualized by treatment with anti-cmyc antibody and rabbit anti-mouse IgG alkaline phosphatase conjugate (SigmaAldrich, St. Louis, MO) diluted in 1% (w/v) BSA in PBST. The enzymatic substrate was pNP phosphate, and the absorbance was measured after a 30 min incubation at 378C. The KD values were calculated from the competition curves at 50% inhibition from the molarities of free soluble antigen (Friguet et al. 1985).

Size Exclusion Chromatography Recombinant anti-NbTNF-VHHCk and anti-EcTNF-VHH proteins were further puried by passing through a calibrated HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) using 10 mM sodium phosphate buffer (10 mM Na2HPO4/NaH2PO4, pH 7.5) or 10 mM sodium phosphate buffer with 150 mM NaCl as eluent, at a constant ow rate of 1 mL/min. The column was calibrated with a low molecular mass standard, containing Blue Dextran 2000 (2000 kDa), bovine serum albumin (67.6 kDa), ovalbumin (42.7 kDa), chymotrypsinogen A (25.1 kDa), and ribonuclease A (13.8 kDa) (Amersham Biosciences, Freiburg, Germany). Consecutive 2.5 mL fractions were collected, analyzed by SDSPAGE, pooled where appropriate, and concentrated. L929-Cytotoxicity Assays With Human TNFa L929 cells were seeded into a 96-well plate at 10,000 cells/ well and cultured for 24 h. The cells were then incubated for additional 24 h in either the presence or absence of 100 ng/mL TNFa, plus a given concentration of anti-EcTNFVHH, monomeric and dimeric anti-NbTNF-VHHCk. The CellTiter-Blue Cell Viability Assay (Promega, Mannheim, Germany) was used to quantify cellular proliferation, and measured on a Lambda Fluoro 320 Fluorimeter (ex-lter 530/25, em-lter 590/35, sensitivity 75, Software KC4). The peak uorescence (560/590) values were dependent on the uorescence development time. All samples were measured in triplicate.

Immunoblotting and Enhanced Chemiluminescence (ECL) Detection A 10 ng aliquot of puried anti- TNF-VHHCk was denatured by heat treatment (5 min at 958C), separated by SDSPAGE, and transferred by a semi-dry electroblotting procedure to a polyvinylidene diuoride membrane (Hybond-P, Amersham Biosciences). The membrane was blocked in TBS (10 mM TrisHCl, pH 8, 150 mM NaCl) supplemented with 0.02% (v/v) Tween-20 and 6% (w/v) skimmed milk powder, and exposed overnight at 48C to a solution of anti-c-myc antibody (Cell Signaling, New England Biolabs, Schwalbach, Germany). Post hybridization, the membranes were incubated with horseradish peroxidase-conjugated sheep anti-mouse antibody (GE Healthcare UK Ltd., Little Chalfont, UK), and reacting proteins were detected via chemiluminescence (ECL plus western Blotting Detection System, Amersham Biosciences).
Nb

Nomenclature
TNFa VHH human tumor necrosis factor-alpha heavy-chain only antibody variable domain

We thank Stefanie Schnell and Isolde Tillack for their excellent technical assistance. We acknowledge the gift of recombinant human nnel. This research was supported by TNFa from of Dr. Daniela Ma the Deutsche Forschungsgemeinschaft to J.S. (SFB415, Project B5) and the German cluster of excellence Inammation at Interfaces (J.S.). We also thank colleagues involved in COST action Molecular Farming: Plants as a Production Platform for High Value Proteins FA0804 for helpful discussions.

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