Plant Biotechnology Journal (2011) 9, pp.

2231

doi: 10.1111/j.1467-7652.2010.00523.x

ELPylated anti-human TNF therapeutic single-domain antibodies for prevention of lethal septic shock
Udo Conrad1, Ingo Plagmann2, Sven Malchow2, Markus Sack3, Doreen M. Floss2, Andrei A. Kruglov4, Sergei A. Nedospasov4,5, Stefan Rose-John2 and Ju rgen Scheller2,*
1 2 3 4 5

Institute of Plant Genetics and Crop Plant Research (IPK), Phytoantibodies, Gatersleben, Germany Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany Institute of Molecular Biotechnology, RWTH Aachen University, Aachen, Germany German Rheumatism Research Center (DRFZ), a Leibniz Institute, Inammation Biology, Berlin, Germany Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Received 18 November 2009; revised 5 February 2010; accepted 27 February 2010. *Correspondence (fax +49 431 880 5007; email jscheller@biochem.uni-kiel.de)

Summary
Tumour necrosis factor (TNF) is a major pro-inammatory cytokine involved in multiple inammatory diseases. The detrimental activity of TNF can be blocked by various antagonists, and commercial therapeutics based upon this principle have been approved for treatment of diseases including rheumatoid arthritis, Crohns disease and psoriasis. In a search for new, improved anti-inammatory therapeutics we have designed a single-domain monoclonal antibody (VHH), which recognizes TNF. The antibody component (TNF-VHH) is based upon an anti-human TNF Camelidae heavychain monoclonal antibody, which was linked to an elastin-like polypeptide (ELP). We demonstrate that ELP fusion to the TNF-VHH enhances accumulation of the fusion protein during biomanufacturing in transgenic tobacco plants. With this study, we show for the rst time that this plant-derived anti-human TNF-VHH antibody was biologically active in vivo. Therefore, therapeutic application of TNF-VHH-ELP fusion

Keywords: tumour necrosis factor, VHH, transgenic plants, elastin-like polypeptides, sepsis, nanobody.

protein was tested in humanized TNF mice and was shown to be effective in preventing death caused by septic shock. The in vivo persistence of the ELPylated antibody was 24 fold longer than that of non-ELPylated TNF-VHH.

Introduction
Cytokines control cellular proliferation, differentiation and viability. Disrupted regulation of cytokine activity is associated with the onset of chronic disease, and selective targeting of these proteins has proven to be clinically benecial for the treatment of a number of human diseases. Tumour necrosis factor (TNF) plays a pivotal role in several acute and chronic inammatory diseases, such as sepsis (Beutler et al., 1985), rheumatoid arthritis (Maini et al., 1995) and Crohns disease (Van Dullemen et al., 1995). TNF binds to the cell surface TNF receptors 1 (TNFR1, p55) and 2 (TNFR2, p75) (Smith et al., 1994), which undergo ligand-induced trimerization before becoming associated with signalling molecules such as the TNF receptorassociated death domain protein (TRADD) and various TNF receptorassociated factors (TRAFs),

which then initiate downstream intracellular cascades (Heyninck and Beyaert, 2001). Some commercially developed antibody-based anti-TNF drugs (Iniximab [RemicadeTM], adalimumab [HumiraTM], certolizumab pegol [CimziaTM, CDP870]) have been approved for the treatment of rheumatoid arthritis and Crohns disease (Sandborn et al., 2007; http://www.rxabbott.com), while the engineered soluble TNFR2 etanercept (EnbrelTM) is now licensed as a therapy for both rheumatoid arthritis and psoriasis (Ducharme and Weinberg, 2008). Camelidae heavy-chain antibodies have been suggested as an alternative drug format for the treatment of TNF-dependent diseases. These antibodies recognize the antigen through a single variable heavy-chain domain (VHH), a molecule which is about ten times smaller than the complete conventional IgG (Figure 1a). In contrast to IgG antibodies, single-domain antibodies lack a light chain

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2010 The Authors Plant Biotechnology Journal 2010 Society for Experimental Biology and Blackwell Publishing Ltd

TNF single-domain antibodies 23

(a)
V

Temperature
CH2 CH3

Reversible aggregation

Serum half-life can be lengthened by ELPylation. Here, the single-domain antibody is fused to multiple repeats of an elastin-like pentapeptide (ELP), which typically has the sequence Val-Pro-Gly-Xaa-Gly (where Xaa represents any amino acid except proline) (Scheller et al., 2004, 2006). It is thought that this lengthening is brought about because the removal of the ELPylated antibody molecule via the reticuloendothelial system is hampered by its large molecular size. As ELPs are derived from mammalian tropoelastin, they are considered to be biocompatible (Urry et al., 1991; Rincon et al., 2006) making them suitable, in principle, for in vivo therapeutic applications (for reviews, see Chilkoti et al., 2006; Chow et al., 2008; Floss et al., 2010a,b). Expression of recombinant proteins in transgenic plants represents a versatile means of producing pharmaceutically active reagents, and a growing number of diagnostic and therapeutic antibodies have been produced in this way (Stoger et al., 2005; Twyman et al., 2005; Wycoff, 2005; Floss et al., 2007). The use of transgenic plants for the production of recombinant proteins in resource-poor areas with a low level of technological infrastructure is probably more realistic than the usage of bacterial, yeast, insect and mammalian cells (Twyman et al., 2005; Boehm, 2007; Sparrow et al., 2007). The combination of ELP fusion technology and plant-based expression systems results in increased accumulation of the protein of interest, simplied purication via temperature- and salt-dependent phase transition (inverse transition cycling, ITC, Figure 1b) and furthermore, plant-made ELPylated recombinant proteins retain their expected bioactivity and appropriate glycosylation patterns (for review, see Floss et al., 2010a). In this study, we have generated lines of transgenic tobacco plants (Nicotiana tabacum) expressing high quantities of ELPylated anti-human TNF (hTNF) monoclonal Camelidae heavy-chain antibody (NtTNF-VHHELP). We report the level of in vitro activity exhibited by this modied antibody and compare it with that shown by TNF-VHH produced in Escherichia coli (EcTNF-VHH). We show for the rst time that the in vivo half-life of plant-derived NtTNFVHHELP was signicantly extended over that of the non-ELPylated product, and that treatment with NtTNF-VHHELP was effective in preventing LPS-D-Gal-induced septic shock in humanized TNF mice.

Camelid heavy chain antibody (110 kDa)

VHH ( 15 kDa)

ELPylated VHHELP ( 57 kDa) (monomeric)

ELPylated VHHELP (multimeric)

(b) Nt

TNF-VHHELP NtTNF-VHH 3/19 10/25 150 ng TSP 7.5 g TSP 30 ng TSP 75 ng TSP 15 g TSP 3 g TSP

NtTNF-V H H ELP Control protein

0.5 ng

1.5 ng

15 ng

3 ng

6 ng

kDa 170 130 100 72 55 40 33 24 17

Figure 1 Expression of TNF-VHH and TNF-VHHELP. (a) Left panel: schematic representation of the Camelidae heavy-chain antibody and the size-minimized VHH-domain with full antigen-binding capacity. Right panel: purication procedure for NtTNF-VHHELP fusion proteins from tobacco leaves by temperature-dependent inverse transition cycling (ITC). (b) Western blot analysis of 30 ng, 75 ng and 150 ng total soluble protein, extracted from the leaves of transgenic tobacco line 10 25 expressing NtTNF-VHHELP, and 3, 7.5 and 15 lg of total soluble protein, extracted from the leaves of transgenic tobacco line 3 19 expressing NtTNF-VHH. Control lanes loaded with 0.5, 1.5, 3, 6 and 15 ng of ITC-puried NtTNF-VHHELP. The original western blot is shown as Figure S1d.

Nt

Nt

(Hamers-Casterman et al., 1993), instead comprising a single polypeptide (Arbabi Ghahroudi et al., 1997). VHH domains are highly temperature tolerant, and their thermal unfolding is reversible (Arbabi Ghahroudi et al., 1997). The antigen specicities and afnities of VHH and IgG antibodies are very similar, with dissociation constants (KD) lying in the nM range (Lauwereys et al., 1998). Monoclonal Camelidae heavy-chain antibodies against both murine and human TNFs have already been described (Coppieters et al., 2006), and in their presence, the binding of TNF to its cognate receptors is inhibited (Coppieters et al., 2006; Plagmann et al., 2009). Anti-murine TNF-VHH proteins can block the development of collagen-induced arthritis in mice (Coppieters et al., 2006). However, the serum half-life of anti-murine TNF-VHH antibodies is short (35 min), limiting their use as an in vivo therapy.

Results
The cDNAs representing the coding sequence of either TNF-VHH (NtTNF-VHH) or TNF-VHH fused to 100 ELP repeats

2010 The Authors Plant Biotechnology Journal 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 2231

24 Udo Conrad et al.

(NtTNF-VHHELP) were placed under control of the constitutive Cauliower mosaic virus (CaMV) 35S promoter (Figure S1a; for sequence information see Figure S1b,c) for expression in tobacco plants. The N-terminal LeB4 signal peptide promoted the translocation of NtTNF-VHH and TNF-VHHELP into the endoplasmic reticulum (ER), and the KDEL signal at the C-terminus resulted in their retention in the ER. A c-myc tag was included to screen for high producers of NtTNF-VHH and NtTNF-VHHELP by western blot analysis. Transgenic tobacco plants expressing TNF-VHH or NtTNF-VHHELP were generated and analysed by western blot for accumulation of the recombinant protein with an antibody against c-myc (data not shown). Transgenic plants with were were High a high accumulation of the recombinant proteins selected, self-fertilized and resulting progeny plants investigated by western blot (Figure 1b; Figure S1d). producers were chosen for further analysis (NtTNFNt Nt

(a) kDa
170 130 100 72 55 40 33 24

(b)
1 2 3 4 5 k Da 170 130 100 72 55 40 33 24 1 2 3 4 5

(c)

Conalbumin (76 kDa) Aldolase (158 kDa)

200

Ovalbumin (43 kDa)

NtTNFVHHELP

Absor rbance (a.u.)

150

100

VHH: 2 of 27 producers 41 tested, calculated molecular mass without signal peptide: 16.7 kDa; NtTNF-VHHELP: 2 of 13 producers 30 tested, calculated molecular mass without signal peptide: 57.1 kDa) (Fig. 1b,c). The molecular mass of monomeric NtTNF-VHHELP determined by SDSPAGE analysis is about 70 kDa. The difference to the calculated molecular mass of NtTNF-VHHELP (57.1 kDa) might be attributed to an aberrant behaviour of the ELP part on SDSPAA gels, which was previously observed in other experiments (Shimazu et al., 2003; Ge et al., 2006). The apparent increased size cannot be attributed to N-glycosylation, because there are no sites in the NtTNF-VHHELP. Fusion to 100ELP resulted in a substantial increase in the accumulation of VHH in tobacco leaves (Figure 1b). The different recombinant TNF-VHH variants (NtTNF-VHH and Nt TNF-VHHELP) accumulated to 0.003% total soluble protein (TSP), and 1.7% TSP, respectively. Relevant semi-quantitative western blot data are presented in Figure S1d. Proteins from transgenic plants expressing NtTNF-VHHELP were extracted from leaves with phosphate buffered saline (PBS). The expression of recombinant NtTNF-VHHELP could already be detected after SDS-PAA gel separation of raw leaf extracts followed by Coomassie staining (Figure 2a, lane 1). Recombinant NtTNF-VHHELP was puried by two rounds of inverse transition cycling as described in the material and methods section (Coomassie staining in Figure 2a, lane 15 and Western Blot in Figure 2b, lane 15). The second round of ITC was performed to concentrate the protein by a factor of 10. Purication of NtTNF-VHHELP was completed by size exclusion chromatography on a calibrated Superdex S-200 High Resolution column (Figure 2c). The purity of the recombinant NtTNF-VHHELP

50

30

40

50

60

70

80

90

100

Elution time (min)

(d) kDa
120 85 50 35 25

0.5

10

g protein

NtTNF-V H H ELP

Figure 2 Purication of NtTNF-VHHELP. (a) Coomassie-stained PAA gel documenting the purication of NtTNF-VHHELP from crude plant leaf extract. 1, crude leaf extract; 2, supernatant after rst round ITC; 3, soluble proteins after rst round ITC; 4, supernatant after second round ITC; 5, soluble proteins after second round ITC (concentrated by a factor of 10). (b) Western blot analysis documenting the purication of NtTNF-VHHELP. 1, crude leaf extract; 2, supernatant after rst round ITC; 3, soluble proteins after rst round ITC; 4, supernatant after second round ITC; 5, soluble proteins after second round ITC. (c) Size exclusion chromatography of NtTNF-VHHELP. The fractions eluting between 50 and 60 min were pooled and concentrated. Calibration molecular mass standards: aldolase 158 kDa; conalbumin 76 kDa; ovalbumin 43 kDa. (d) Dened amounts of NtTNF-VHHELP separated on 15% SDSPAA gel and stained with Coomassie.

was conrmed on a Coomassie-stained SDSPAA gel (Figure 2d). The majority of NtTNF-VHHELP behaved on the column like a 100 kDa protein as estimated by comparison with the calibration curve of molecular mass standards (large peak in Figure 2c). We assume that the presence of the ELP region resulted in an elongated protein that eluted in the position expected for a globular protein of higher molecular mass. A similar behaviour was previously

2010 The Authors Plant Biotechnology Journal 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 2231

TNF single-domain antibodies 25

Fluorescence (560/590)

observed for the mini-sgp130-ELP fusion protein (Lin et al., 2006). A small amount of NtTNF-VHHELP dimers was also detected by Western blotting and could be separated by size exclusion chromatography (Figure 2a,b, small peak in 4250-mL elution volume in Figure 2c). NtTNF-VHHELP from the major peak was collected, concentrated and the nal retentate was quantied by UV spectroscopy. Therefore, the nal yield of puried NtTNF-VHHELP was 20 lg puried protein per gram leaf fresh weight. The recovery rate of this nonoptimized small-batch laboratory purication process was calculated to be approximately 11%. Puried Nt TNF-VHHELP protein was routinely stored at )20 C but was also stable at 4 C and room temperature without detectable loss of soluble recombinant protein integrity. Nt TNF-VHHELP, which was puried via ITC and size exclusion chromatography, was used as a standard for western blot analysis (Figure 1b; Figure S1d). The dissociation constants of NtTNF-VHHELP and recombinant TNF-VHH from E. coli (EcTNF-VHH) to hTNF were determined by competitive ELISA. The competitive binding of NtTNF-VHHELP and EcTNF-VHH to hTNF in solution was quantied by determination of the concentration of soluble hTNF that caused half-maximal binding of the antibody fragments to hTNF adsorbed to the plate. The dissociation constants of EcTNF-VHH and NtTNF-VHHELP revealed comparable performances at the nM level (EcTNF-VHH, 2.5 nM; TNF-VHHELP, 4 nM; Figure 3b). In addition, conventional ELISA experiments showed that EcTNF-VHH and Nt TNF-VHHELP bound to hTNF in a dose-dependent manner, both with a 50% binding at 3 nM of the antibodies (Figure 3a). The dissociation constants (KD) of NtTNF-VHHELP and EcTNF-VHH to hTNF measured by surface plasmon resonance (SPR) were, respectively, 1.3 0.098 and 0.59 0.032 nM (Figure S2ac). Again, both antibody variants bind in a comparable way. Biological activity can be estimated in several ways. While these ought to give a consistent ranking, they are all, to some extent, affected by the choice of experimental method and conditions (Weiergra ber et al., 1995; Kovaleva et al., 2006). As murine L929 cells undergo apoptosis in the presence of hTNF (Schmid et al., 1986), the ability of NtTNF-VHHELP to block the biological activity of hTNF was tested in a cell-based cytotoxicity assay. Both recombinant proteins, EcTNF-VHH and NtTNFVHHELP, inhibited hTNF-induced cell death in a dose-dependent manner, with IC50s of 6 and 9 nM (Figure 3c). At comparable antibody concentrations (3 nM) a half-maximal antibody binding in a conventional ELISA was measured (Figure 3a). These data support the view that EcTNF-VHH and NtTNF-VHHELP have comparable biological activity.
Nt

(a)
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

EcTNF-V NtTNF-V

HH

HHELP

10

20

30

40

50

60

VHH (nM)

(b)
Fluorescence (560/590)

3.0 2.5 2.0 1.5 1.0 0.5 0 0.1 1 10 hTNF (nM) 100 1000
EcTNF-V NtTNF-V HH HHELP

(c)
Cell viability fluorescence (560/590)

35 000 30 000 25 000 20 000 15 000 10 000 5000 0 0

EcTNF-V NtTNF-V

HH

HHELP

10

20

30

40

50

60

VHH (nM)
Figure 3 Binding properties of NtTNF-VHHELP. (a) Reaction of 0.1 1000 ng mL recombinant EcTNF-VHH from E. coli and 0.1 2500 ng mL plant-derived NtTNF-VHHELP in wells coated with hTNF. The binding of c-myc tagged EcTNF-VHH and NtTNF-VHHELP to hTNF was quantied on a molar basis. (b) Competitive ELISA, EcTNF-VHH or Nt TNF-VHHELP were mixed with various concentrations of hTNF. The binding of c-myc tagged EcTNF-VHH and NtTNF-VHHELP to hTNF was quantied on a molar basis. (c) Plant-derived NtTNF-VHHELP blocks hTNF-mediated cytotoxicity. Murine L929 cells were incubated with hTNF and increasing amounts of bacteria-derived EcTNF-VHH and plant-derived NtTNF-VHHELP. The inhibition of TNF-mediated cytotoxicity of c-myc tagged EcTNF-VHH and NtTNF-VHHELP to hTNF was quantied on a molar basis.

However, the serum half-life of NtTNF-VHHELP in C57 BL6 mice was 11.4 h and for EcTNF-VHH was about 28 min (Figure 4a), equivalent to a 24-fold increase. A comparable serum half-life of 35 min for the EcTNF-VHH was reported previously (Coppieters et al., 2006). The in vivo efcacy of NtTNF-VHHELP and as a control Ec TNF-VHH was tested using the LPS D-gal septic shock model applied to humanized TNF mice (Kuprash et al., 2002; Liepinsh et al., 2009), as treatment with TNF

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26 Udo Conrad et al.

Serum concentration (ng/mL)

(a)

60 000 50 000 40 000 30 000 20 000 10 000 10 20 30 40 50 60 70 80 t1/2 (NtTNFVHHELP) = 11.4 h t1/2 (EcTNFVHH) = 28 min

the accumulation of the transgene product from 0.003 to 1.7% of TSP and facilitated its purication via the saltand temperature-dependent phase transition from leaf material. ITC purication resulted in a nal yield of approximately 11% of pure recombinant NtTNF-VHHELP. TNF-VHHELP and EcTNF-VHH were tested side-by-side in ELISA, surface plasmon resonance and cytotoxicity assays. The plant-derived VHH-ELP fusion protein was not compromised with respect to its biological activity in comparison with non-ELPylated EcTNF-VHH from bacteria. The Nt TNF-VHHELP possessed a substantially longer serum half-life compared to the non-ELPylated VHH protein (24 fold, t1 2 = 11.4 h for NtTNF-VHHELP and 28 min for TNF-VHH). Recombinant VHH proteins can be produced in E.coli by periplasmic expression, leading to high-level accumulation of correctly folded single-domain antibodies in the cell culture supernatant. However, we were not able to produce TNF-VHHELP in E. coli (data not shown), which might be caused by the failure of the ELPs to be efciently secreted into the periplasmic space. Therefore, the plant-based expression system revealed to be a strategy way to produce ELPylated single-domain antibodies with prolonged serum half-life. In addition, the high expression levels achieved with ELP fusions in transgenic plants (Floss et al., 2009a,b) prompted us to use the plant system for single-domain antibody production. Plants potentially are considered to be an extremely cost-effective and efcient production system for pharmaceuticals, such as complete, single-chain or single-domain antibodies (Jobling et al., 2003; Peterson and Arntzen, 2004; Ismaili et al., 2007; Ramessar et al., 2008; Teh and Kavanagh, 2009; Winichayakul et al., 2009). Several ELP fusion proteins have been observed to be efciently and functionally accumulated in plants, as for instance spider silk proteins (Scheller et al., 2004), soluble gp130 (Lin et al., 2006), antigens (Floss et al., in press) single-chain Fv antibodies (Scheller et al., 2006) and complete antibodies (Floss et al., 2008, 2009a,b). The retention of recombinant proteins in the endoplasmic reticulum is one general strategy for the accumulation of high protein amounts in plants (Artsaenko et al., 1995; Scheller et al., 2001); however, this could be signicantly enhanced by ELPylation (Scheller et al., 2006). ELPylation has been exploited to modify a variety of therapeutically active proteins (for review, see Floss et al., 2010a,b), although little has been uncovered to date concerning either the effect this modication has on either in vivo biological activity or persistence in the serum. ELPs appear to be well suited to act as carriers for pharmaceutiEc Nt

Time (h)

(b)
100
Survival (%)

[0/5] [4/4]

[7/7]

[3/3]

[3/3]

[5/5] mice [survived/total]

80 60 40 20 0

Figure 4 Serum half-life of NtTNF-VHHELP and neutralization of LPS D-Gal toxicity by NtTNF-VHHELP. (a) Three mice were intravenously injected with 100 lg plant-derived NtTNF-VHHELP and for control with 100 lg EcTNF-VHHELP. Serum samples were prepared over a time range postinjection. Serum levels of NtTNF-VHHELP and EcTNF-VHHELP were quantied by ELISA. (b) LPS D-gal-induced septic shock was blocked by NtTNF-VHHELP and EcTNF-VHH. Survival was monitored for 24-h postinjection. Three to seven different mice per treatment were used.

inhibitory molecules improves survival in this animal model (Beutler et al., 1985). Mice treated with LPS D-Gal died during the rst 24 h, but mice decient for TNF were protected. All the mice treated with 1, 0.5 or 0.3 mg NtTNFVHHELP or 0.3 mg EcTNF-VHH survived (Figure 4b), demonstrating the in vivo ability of plant-derived block the activity of human TNF.
Nt

TNF-VHHELP to

Discussion
Fusion of elastin-like polypeptides provides many advantages in the large-scale production of biologicals including increased yields, simple downstream processing by inverse transition cycling and biocompatibility (for reviews, see Chilkoti et al., 2006; Chow et al., 2008; Floss et al., 2010a). In this study, we have shown that the ELPylation of a VHH single-domain antibody substantially enhanced

2010 The Authors Plant Biotechnology Journal 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 2231

TNF single-domain antibodies 27

cally active proteins. The biosafety of ELP proteins was demonstrated in terms of nonimmunogenicity, nonpyrogenicity and biocompatibility, and they have been classed as being overall highly biocompatible (Urry et al., 1991, 2002; Rincon et al., 2006). Of particular value is the observation that tumours subjected to 41.5 C by means of an external focused hyperthermia device encouraged the adherence of ELPs as micron-sized aggregates; but that when the temperature was returned to normal, these aggregates dissolved, leading to an increase in the localized concentration of ELPs. Because molecular ux into the tumour depends on concentration differences across the endothelium, the effect of this process was to drive ELPs into the tumour (Dreher et al., 2007). The same targeting property may also be relevant for the treatment of localized autoimmune diseases such as rheumatoid arthritis. Drugs are generally cleared in vivo through the reticuloendothelial system, and the lower their molecular weight, the more readily diffusible they are from the bloodstream into the target tissue (Graff and Wittrup, 2003). However, the clinical effectiveness of a small therapeutic protein is hampered by the fact that it has only a short serum halflife. In the case of the VHH antibodies, this disadvantage has been addressed either by increasing molecular size by the addition of polyethylene glycol (PEGylation; Harris and Chess, 2003; Kubetzko et al., 2006), or by the construction of fusion proteins (Osborn et al., 2002; Jazayeri et al., 2007), because they cannot be readily ltered into the kidney glomeruli (Behr et al., 1998). The binding of a short-lived protein to albumin has been shown to represent a further means of extending serum half-life (Dennis et al., 2002; Wunder et al., 2003; Coppieters et al., 2006). Here, we demonstrated that ELPylation increased the serum half-life of VHH by a factor of 24. Furthermore, TNF-VHHELP was able to reduce LPS D-Gal lethality in humanized TNF mice, verifying for the rst time its ability to block the biological activity of human TNF in vivo. Further experiments are now required to assess its efciency as a treatment against other chronic inammatory conditions, such as collagen-induced arthritis, as has been already shown for the anti-murine TNF-VHH fragment (Coppieters et al., 2006). The biological activity of the anti-human TNF-VHH fragment has not been tested in vivo so far. Recently, humanization of the VHH antibody format was described, which might allow its acceptance as human therapeutics (Vincke et al., 2008). Investigation of the therapeutic efcacy of Cimzia, a humanized anti-TNF PEGylated Fab fragment, has indicated that Fc-associated receptor functions and multivalency are not essential for
Nt

its in vivo activity (Sandborn et al., 2007; Emov et al., 2009). This drug shares several properties in common with Nt TNF-VHHELP. Both molecules are monovalent, and while Nt TNF-VHHELP has a C-terminal ELPylation of 100 ELP repeats, Cimzia is PEGylated at Cys221 of the heavy chain. ELPylation appears to represent a useful means of increasing the serum half-life of small protein molecules. Like Nt TNF-VHHELP, Cimzia lacks a heavy-chain Fc domain, and thus cannot trigger Fc-mediated functions, such as complement activation, antibody-dependent cellular cytotoxicity or apoptosis (Emov et al., 2009). Overall, we have demonstrated that ELPylated VHH antibodies represent promising molecules, which show major potential for therapeutic applications. There are still open questions about cGMP conditions for the production of plant-made proteins. However, transgenic plants might be specically grown in the green house or alternative systems for a fast and transient expression might be used (Gleba et al., 2007) to ensure an efcient, inexpensive and safe production of pharmaceutical proteins for developing countries, where the nancial resources to pay for medicine are limited, but where the demand is greatest.

Experimental procedures
Cells and reagents
Murine L929 cells, obtained from DSMZ (Braunschweig, Germany), were grown at 37 C under 5% CO2 in a water-saturated atmosphere 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. Ec TNF-VHH was produced in E. coli as previously described (Plagmann et al., 2009). Anti-c-myc antibodies were purchased from Cell Signalling, New England Biolabs (Schwalbach, Germany). All restriction enzymes were obtained from Fermentas (St. Leon-Rot, Germany).

Cloning of

Nt

TNF-VHH and

Nt

TNF-VHHELP

Standard cloning procedures were performed as described elsewhere (Sambrook and Russell, 2001). The pCRScript-TNF-VHH plasmid (Plagmann et al., 2009) was digested with NcoI and NotI, and the TNF-VHH was introduced into the plasmids pRTRA7 3-SO1-HOOK and pRTRA7 3-SO1-100xELP (Scheller et al., 2004) containing the constitutive Cauliower mosaic virus (CaMV) 35S promoter, the legumin B4 (LeB4) signal peptide (SP), a c-myc tag and the KDEL ER retention signal to form pRTRA-35S-NtTNF-VHH and pRTRA-35S-NtTNF-VHHELP. The resulting plasmids were digested with HindIII, and the fragments containing the plant expression cassettes (CaMV 35S promoter LeB4 SP TNF-VHH c-

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28 Udo Conrad et al.

myc tag KDEL CaMV 35S terminator or TNF-VHH c-myc tag 100xELP KDEL CaMV 35S terminator) were inserted into the binary vector pCB301-Kan (Gahrtz and Conrad, 2009), which is based on pCB301 (Xiang et al., 1999) to obtain the plasmids pCB301-Kan-35S-NtTNF-VHH and pCB301-Kan-35S-NtTNF-VHHELP, respectively.

Transformation of tobacco
pCB301-Kan-35S-NtTNF-VHH and pCB301-Kan-35S-NtTNF-VHHELP were transferred into Agrobacterium tumefaciens C58C1 (pGV2260) by electroporation. Leaf discs of Nicotiana tabacum cv. SNN were transformed as described elsewhere (Horsch et al., 1985). Regenerated transgenic plants were grown on Murashige and Skoog (MS) medium containing 50 mg L kanamycin and analysed by western blot, based on the monoclonal antibody antic-myc 9E10 to select for high producers of the transgene product (Conrad et al., 1997).

peroxidase diluted in PBS 1% BSA. Soluble peroxidase substrate (BM blue POD; Roche, Mannheim, Germany) was added to each well, and incubated at room temperature for 10 min, before stopping the reaction with 50 lL 1 M H2SO4. The absorbance of the reaction was measured at 560 590 nm. For competitive ELISAs, the wells were coated with 1 lg mL recombinant hTNF in PBS and incubated overnight at room temperature. After blocking with 3% w v BSA in PBS for 2 h, known amounts of either Ec TNF-VHH or NtTNF-VHHELP mixed with various concentrations of hTNF in 1% w v BSA in PBS-T were added to each well and the plates were incubated for 1.5 h at 25 C. VHH bound to the plate were visualized by treatment with anti-c-myc antibodies and antimouse IgG alkaline phosphatase diluted in PBS 1% BSA. The enzymatic substrate was pNP phosphate, and the absorbance (405 nm) was measured after 30 min incubation at 37 C.

L929 cytotoxicity assays with hTNF

L929 cells were seeded onto a 96-well plate at a density of 10 000 cells well and cultured for 24 h. Thereafter, the cells were incubated for additional 24 h either with or without 100 ng mL hTNF and either with or without known concentrations of NtTNFVHHELP or EcTNF-VHH. Quantication of cellular proliferation was effected using the CellTiter-Blue Cell Viability Assay (Promega, Mannheim, Germany), following the manufacturers instructions, and total uorescence was measured by a Lambda Fluoro 320 Fluorimeter (ex-lter 530 25, em-lter 590 35, sensitivity 75, Software KC4). All samples were measured in triplicate.

Purication of recombinant transition cycling

Nt

TNF-VHHELP by inverse

Ice-cold 50 mM TrisHCl, pH 9.0 (340 mL) was added to 100 g of frozen leaves and homogenized in a Waring blender for 4 min. The resulting extract was cleared by centrifugation. The supernatant was supplemented with 0.87% NaCl, incubated at 60 C for 15 min, subsequently cooled in an ice-water bath and centrifuged (5000 g, 20 min, 4 C). Sufcient NaCl was added to the supernatant to reach a nal concentration of 2 M, followed by incubation at 37 C for 15 min and centrifugation (14 000 g, 37 C, 1 h). The ELP-containing protein pellet was solubilized in 50 mM TrisHCl, pH 9.0 at 4 C. Remaining insoluble material was removed by centrifugation (16 000 g, 4 C, 1 h). The solubilized precipitate and the supernatant were analysed on a Coomassie-stained SDSPAA gel and by western blot.

Measurement of in mouse serum

Nt

TNF-VHHELP protein antibody levels

Into the wild-type C57 BL6 mice, 100 lg NtTNF-VHHELP and 100 lg EcTNF-VHH were injected intravenously. Serum samples were drawn at 0.5, 2, 4, 8, 12, 24, 48 and 72 h postinjection. Serum levels of NtTNF-VHHELP were quantied by ELISA at specic time points.

Size exclusion chromatography

TNF-VHHELP was further puried by passing through a calibrated SuperdexTM 200 column (Amersham Biosciences, Freiburg, Germany) using 1 PBS as the mobile phase with a constant owrate of 1.0 mL min. Fractions (2.5 mL) containing the ELP fusion protein were pooled and concentrated by ultraltration. Puried Nt TNF-VHHELP was stored at )20 C. Protein concentration was estimated following Waxman et al. (1993).
Nt

LPS D-gal sepsis model

LPS (10 lg mouse) and D-gal (20 mg mouse) were injected intraperitoneally into hTNF LT Tg mTNF LT) ) mice. NtTNF-VHHELP was injected intraperitoneally 30 min later. Survival was monitored during the following 24 h. The hTNF LT Tg mTNF LT) ) mice were generated by integrating a cosmid containing genomic human TNF LT locus into the mouse genome. The resultant hTNF LT Tg mice were back-crossed into a triple-decient TNF LT) ) background resulting in hTNF LT Tg TNF LT) ) mice, which were physiologically normal but expressed exclusively human TNF (Kuprash et al., 2002; Liepinsh et al., 2009).

Enzyme-linked immunosorbent assay (ELISA)

Microtitre plates (Greiner Microlon, Solingen, Germany) were coated with 10 lg mL recombinant hTNF in PBS and held overnight at room temperature. After blocking with 3% w v bovine serum albumin (BSA) in PBS for 2 h, at room temperature, NtTNFVHHELP in 1% w v BSA PBS-T (PBS with 0.05% v v Tween-20) was added and incubated for 1 h at room temperature. The binding of antigen to the plate was visualized by treatment with anti-c-myc-antibody followed by anti-mouse IgG horseradish

Acknowledgements
We thank Stefanie Schnell, Isolde Tillack and Christine Helmold for their excellent technical assistance and

2010 The Authors Plant Biotechnology Journal 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 2231

TNF single-domain antibodies 29

nnel (Dept. Immunology, University of RegensDaniela Ma burg, Germany) for her gift of hTNF protein. We thank Charles J. Arntzen (Arizona State University, Tempe, Arizona, USA) for critical reading of the manuscript. We also thank colleagues involved in COST action Molecular Farming: Plants as a production platform or high value proteins FA0804 or helpful discussions. This research was supported by the Deutsche Forschungsgemeinschaft (SFB415, Project B5) and the German cluster of excellence project Inammation at Interfaces.

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Data S1 Supporting experimental procedures. Figure S1 (a) Schematic representation of the plant expression cassettes for NtTNF-VHH and NtTNF-VHHELP. (b) Protein-sequence of NtTNF-VHH. Black, LeB4 signal peptide; red, TNF-VHH; green, linker sequences; blue, c-myc tag; pink, ER retention signal. (c) Protein sequence of NtTNFVHHELP. Black, LeB4 signal peptide; red, TNF-VHH; green, linker sequences; blue, c-myc tag; orange, ELP; pink, ER retention signal. (d) Western blot analysis of transgenic TNF-VHH plant lines and transgenic NtTNF-VHHELP plant lines. Offspring of high expressors were selected and analyzed by western blot. Different amounts of TSP were loaded on the gel. Recombinant proteins were visualized using anti-c-myc antibodies. Lanes from L1 to L23L1, 30 ng TSP NtTNF-VHHELP 10 25; L2, 75 ng TSP NtTNFVHHELP 10 25; L3, 150 ng TSP NtTNF-VHHELP 10 25; L4, 30 ng TSP NtTNF-VHHELP 17 26; L5, 75 ng TSP NtTNFVHHELP 17 26; L6, 150 ng TSP NtTNF-VHHELP 17 26; L7, 30 ng TSP NtTNF-VHHELP 28 32; L8, 75 ng TSP NtTNFVHHELP 28 32; L9, 150 ng TSP NtTNF-VHHELP 28 32; L10, 3 lg TSP NtTNF-VHHELP 3 19; L11, 7.5 lg TSP NtTNF-VHH 3 19; L12, 15 lg TSP NtTNF-VHH 3 19; L13, 3 lg TSP TNF-VHH 12 17; L14, 7.5 lg TSP NtTNF-VHH 12 17; L15: 15 lg TSP NtTNF-VHH 12 17; L16: 3 lg TSP NtTNF-VHH 13 11; L17, 7.5 lg TSP NtTNF-VHH 13 11; L18, 15 lg TSP Nt TNF-VHH 3 19; L19, 0.5 ng NtTNF-VHHELP; L20, 1.5 ng TNF-VHHELP; L21, 3 ng NtTNF-VHHELP; L22, 6 ng NtTNFVHHELP; L23, 15 ng NtTNF-VHHELP. ITC puried NtTNFVHHELP was used as control. Figure S2 Surface plasmon resonance analysis of the
Nt Nt Nt Nt

antigen binding TNF-VHHELP.

properties

of

the

Ec

TNF-VHH

and

Supporting information
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2010 The Authors Plant Biotechnology Journal 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 2231