1 Department of Molecular Biotechnology, 52074 Aachen, Germany, 2 Talwar Research Foundation, New Delhi 110068, 3 Immunoendocrinology Laboratory, National Institute of Immunology, New Delhi 110067, India and 4 Fraunhofer IME, Department of Molecular Biology, 52074 Aachen, Germany
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Abstract |
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Key words: antibody engineering/HCG/plant expression system/recombinant antibodies
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Introduction |
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HCG is a heterodimeric glycoprotein of 40 kDa composed of two non-identical - and ß-subunits (Epstein and Levin, 1987
). HCG and other related pituitary hormones, such as human LH (hLH), human FSH (hFSH), and human thyroid stimulating hormone (hTSH), share nearly identical
-chains. However, their ß-chains show a variable degree of amino acid sequence heterogeneity, which can be specifically distinguished by monoclonal antibodies (mAbs). The mAb PIPP used for the studies presented in this publication recognizes the ß-chain of HCG and does not react with other pituitary hormones (Gupta and Talwar, 1980
).
HCG circulates from the trophoblast to the ovaries, and is amenable to inactivation by antibodies. Since immune response to vaccines varies greatly between individuals, passive immunization using antibodies neutralizing HCG has been proposed as a contraceptive measure.
The therapeutic use of murine mAbs is limited because they can have serious disadvantages: (i) short half-life in serum; (ii) mouse antibodies only weakly recruit the human effector functions; and (iii) the mAbs may elicit a human anti-mouse antibody response (hAMA) when administered to patients (Hasholzner et al., 1997). For in-vivo diagnostic and/or therapeutic approaches chimeric, humanized or human antibodies or antibody fragments are desirable in order to minimise an adverse immune response to the diagnostic and/or therapeutic agent. Small antibody fragments, such as single chain variable fragment (scFv) consist of the variable heavy and light chain antibody domains linked by a flexible peptide linker (Huston et al., 1988
). These antibody fragments have been shown to penetrate tissue better and to be cleared faster from the circulation than full-size antibodies (Milenic et al., 1991
), which are desirable traits in tumour imaging and therapy. However, this fragment is monovalent and due to the small size, 29 kDa, it has a fast clearance rate and this might result in a low total dose accumulation. The efficacy can be improved by using multimeric formats with increased avidity and a molecular weight slightly above the renal filtration threshold. One strategy to produce a multimeric antibody fragment is to shorten the flexible peptide linker of scFv antibody fragments to make it impossible to form monomers (Holliger et al., 1993
). These so-called diabodies have a molecular weight of 60 kDa and they have been shown to be stable under in-vivo conditions (Adams et al., 1993
; Nielsen et al., 2000
).
Antibodies are needed in large quantities for many medical and biotechnological applications. They can be produced in heterologous expression systems such as microbes, animal cells, transgenic animals, plant tissue culture, transient plant expression and transgenic plants. While bacteria are an inexpensive, convenient production system, they are incapable of many of the post-translational modifications necessary for the activity of many mammalian proteins. These limitations, the cost of expression of proteins in mammalian cells and potential safety issues prompted us to explore plants as a cheap, safe and efficient alternative. Antibodies have already been successfully expressed in plants (Hiatt et al., 1989; Ma and Hein, 1995
; Voss et al., 1995
). They are functionally equivalent to those produced by hybridoma (Hiatt et al., 1989
; Voss et al., 1995
) and further refinements have made it possible to produce chimeric mousehuman therapeutic antibodies transiently in plants in sufficient quantities for pre-clinical trials (Zeitlin et al., 1998
; Vaquero et al., 1999
).
In order to investigate the efficacy of different antibody formats specific for HCG, we engineered the scFv (Huston et al., 1993), diabody (Hudson and Kortt, 1999
) and full-size chimeric (Morrison et al., 1984
) mAb PIPP and expressed all three recombinant antibodies transiently (Vaquero et al., 1999
; Fischer and Emans, 2000
) in tobacco leaves to levels of 2040 mg of pure antibody per kg fresh weight of leaves. ELISA and EMSA confirmed antibody specificity to ß-HCG and HCG. Antibody efficacy was confirmed in-vitro by inhibiting HCG induced production of testosterone by Leydig cells and blocking HCG induced rise in mouse uterine weight in vivo. These HCG specific recombinant antibodies may have clinical utility as (i) contraceptive measures and (ii) diagnostic and therapeutic tools of HCG expressing cancers.
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Materials and methods |
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Chimeric full-size antibody PIPP
Chimeric mAb PIPP heavy and light chain genes were generated by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts (kappa, IgG1) using splice overlap extension (SOE)-PCR and cloned into two independent plant expression vectors as previously described (Vaquero et al., 1999). In-vivo assembly of full-size chimeric mAb PIPP was achieved by co-expression of the chimeric light and heavy chains after vacuum infiltration of tobacco leaves with the respective clones of recombinant Agrobacterium. The chimeric constructs contained the endoplasmatic reticulum (ER) retention signal KDEL (Denecke et al., 1992
) that facilitated targeting of the protein to the ER.
Plant cultivation
Nicotiana tabacum, cultivar Petit Havana SR1, was cultivated in the greenhouse in DE73 standard soil. Leaves from 36 week old plants were used for vacuum infiltration.
Agrobacterium mediated transient expression of scFv, diabody and chimeric mAb PIPP
Agrobacterium tumefaciens strain GV3101 were transformed by electroporation with the plant expression vectors pSSH1 containing scFv PIPP, diabody PIPP or chimeric heavy and light chain constructs of mAb PIPP. Transformed cells were incubated for 3 days at 28°C on YEB plates [0.5% (w/v) nutrient broth, 0.1% (w/v) yeast extract, 0.5% (w/v) bactotryptone, 0.5% (w/v) sucrose, 2 mmol/l MgSO4, pH 7.4, 1.5% agar] containing 100 µg/ml carbenicillin, 25 µg/ml kanamycin and 100 µg/ml rifampicin. Recombinant clones were analysed by PCR and cultured in liquid YEB media containing antibiotics for 23 days on a gyratory shaker at 28°C. Cells were harvested by centrifugation and resuspended in 1/20 of the initial volume of medium. Stocks were frozen at 80°C in 50% (v/v) glycerol containing 100 mmol/l MgSO4, 25 mmol/l Tris, pH 7.4. For transient expression, YEB medium containing antibiotics was inoculated with recombinant Agrobacterium and incubated overnight at 28°C. Cells were harvested and resuspended in induction medium [YEB medium, pH = 5.6, 20 µmol/l acetosyringone, 10 mmol/l 2-N-morpholino-ethanesulphonic acid (MES)] and incubated overnight at 28°C. The bacteria were centrifuged and resuspended in MMA medium [4.43 g/l Murashige and Skoog (MS) basal medium, 20 g/l sucrose, 10 mmol/l MES, pH 5.6] containing 200 µmol/l acetosyringone. The density of the suspension was adjusted to OD600 = 1.0 and the bacteria were incubated for 2 h at room temperature.
Vacuum infiltration of Agrobacterium was carried out as previously described (Kapila et al., 1996). For co-infiltration experiments equal volumes of Agrobacterium cultures carrying the chimeric mAb PIPP heavy and light chain constructs were mixed and used for vacuum infiltration.
Extraction of recombinant protein from infiltrated leaves
Proteins were extracted from infiltrated leaves (Vaquero et al., 1999). Briefly, 1 kg of infiltrated tobacco leaves was ground in liquid nitrogen to a fine powder. Soluble protein was extracted using 2 ml of extraction buffer (200 mmol/l Tris-HCl, 5 mmol/l EDTA, 0.1 mmol/l DTT, 0.1% Tween20, pH 7.5) per gram of leaf material. Cell debris was removed by centrifugation (20 000 g for 30 min at 4°C). The supernatant was used for purification and characterization of the recombinant proteins.
Protein purification
For purification via metal ion affinity chromatography (Hochuli et al., 1988) (scFv and diabody) or Protein A (chimeric mAb) plant extracts were centrifuged at 15 000 g for 30 min at 4°C and the supernatant used for further processing. The pH of the supernatant was adjusted to 8.0, and 500 mmol/l NaCl was added to prevent any non-specific interaction with the purification matrix. The extract was incubated at 4°C for 12 h and centrifuged at 7500 g for 30 min. Supernatant was filtered through Whatman paper and applied to purification matrices. Proteins were purified following manufacturers instructions.
Protein quantification
Absorbance at 280 nm was measured with a UVIKON photometer. The concentration of chimeric mAb PIPP was theoretically calculated to be OD278nm = 1 = 1.35 mg/ml and 0.7 mg/ml for the scFv and diabody.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
Purified proteins (110 µg/ml) or 4 µl plant extract were separated on a 12% SDS-PAGE under reducing conditions or on an 8% SDS-PAGE under non-reducing conditions. Proteins were visualized by staining with 0.05% (w/v) Coomassie brilliant blue. For estimation of molecular weights the M12 marker (BioRad, München, Germany) was used.
For Western blot analysis proteins were electro-transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Freiburg, Germany). Membranes were blocked for 1 h at room temperature with 4% (w/v) skimmed milk dissolved in PBS. Bound scFv and diabody were detected with a mouse-anti-KDEL (StressGen Biotechnologies, York, UK) or anti-HIS6 (Qiagen, Hilden, Germany) antibody followed by a goat-anti-mouse alkaline phosphatase (AP) conjugated antibody. Chimeric full-size antibody was detected by AP conjugated polyclonal goat anti-human antibodies (heavy and light chain specific) (1 µg/ml). Proteins were visualized by incubation with Nitro Blue Tetrazolium chloride/5-bromo 4-chloroindol-3-yl phosphate (NBT-BCIP, Pierce, St. Augustin, Germany). Between incubation steps membranes were washed three times with PBS containing 0.05% Tween 20.
ELISA
Microtitre plates (M129B; Greiner Bio-One GmbH, Solingen, Germany) were coated with 100 ng HCG (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) per well in 50 mmol/l bicarbonate buffer pH 9.6 overnight at 4°C and after washing blocked with 300 µl/well 1% (w/v) BSA in PBS for 2 h at room temperature. Plant extracts or purified proteins were serially diluted and transferred to coated and blocked plates. Bound scFv and diabody were detected with a mouse-anti-KDEL (StressGen Biotechnologies) or anti-HIS6 (Qiagen) antibody followed by a goat-anti-mouse AP conjugated antibody. Chimeric full-size antibody was detected by AP conjugated polyclonal goat anti-human antibodies (heavy and light chain specific) (1 µg/ml). The ELISA was developed with 1 mg/ml p-nitrophenyl phosphate in substrate buffer [1.5% (v/v) ethanolamine, 0.15 mol/l NaCl, 1 mmol/l MgCl2, pH 9.8]. A405nm was measured using a microplate reader (Molecular Devices, Munich, Germany) and evaluated using Microcal Origin 5.0. Between all incubation steps plates were washed three times with PBS containing 0.05% Tween 20.
For competition ELISA 50 ng/ml hybridoma derived murine mAb PIPP was added as competitor.
Electrophoretic mobility shift assay (EMSA)
The electrophoretic mobility of varying amounts of HCG added to 2 µg of purified scFv, diabody and chimeric mAb PIPP was compared with the electrophoretic mobility of purified scFv, diabody and chimeric mAb PIPP and HCG alone. Samples were loaded on a native 9% non-reducing PAGE pH 9 and run at 100V for 5 h. Proteins were visualized by Coomassie blue staining.
Gel filtration
A Sephadex 200 column HR10/30 (Amersham Pharmacia Biotech) was equilibrated with PBS. Affinity purified scFv, diabody and chimeric mAb PIPP were loaded and run at 1 ml/min. Cytochrome C (12.4 kDa), carbonic anhydrase (29 kDa), BSA (66 kDa), ß-amylase (200 kDa), and Dextran Blue (2000 kDa) were run as molecular weight standards (Sigma-Aldrich Chemie GmbH). The eluates were monitored by UV 260 nm and 280 nm, with Dextran Blue additionally monitored by 610 nm.
Stability of scFv, diabody and chimeric mAb PIPP
Storage stability was monitored by running purified protein after storage for 68 months at 4°C on an SDS-PAGE and visualizing the proteins by Coomassie blue staining.
In-vitro HCG neutralization assay of scFv, diabody and chimeric mAb PIPP (Leydig cell bioassay)
The Leydig cell bioassay was carried out according to an established method (Van Damme et al., 1974) as modified by Rao et al. (Rao et al., 1988
). NMRI adult male mice were killed in the morning, to negate any diurnal changes in hormone levels, and the testis removed and rinsed in DMEM medium supplemented with 3% fetal calf serum (FCS). The tissues were minced and filtered through nylon membrane followed by culturing at 34°C with 5% CO2 for 1 h to liberate the endogenous testosterone. The cells were pelleted for 10 min at 1000 g at 4°C and resuspended in 10 ml DMEM supplemented with 3% FCS. A total of 200 µl of cell suspension was incubated with: (i) 200 µl steroid buffer (2.35 g/l NaH2PO4, 11.2 g/l Na2HPO4, 8.8 g/l NaCl, 0.1 g/l thiomersal, 1.0 g/l gelatine); (ii) 100 µl buffer plus 100 µl HCG (concentration of HCG 250 pg/ml); or (iii) 100 µl HCG and 100 µl of recombinant antibody fragment. The reaction was mixed gently by hand and incubated for 3 h at 34°C with 5% CO2. The reaction was stopped by addition of steroid buffer and assayed for testosterone by the WHO Matched Assay Reagents as described in the WHO Methods Manual (WHO, 1993).
In-vivo HCG neutralization assay of chimeric mAb PIPP
The HCG neutralization assay was carried out as previously described (Pal et al., 1990). Briefly, three groups each comprising three immature female Balb/c mice (1618 days), were used for the assay. One group was injected with (i) 200 µl/dose saline, (ii) 200 µl/dose HCG (1.5 IU/ml) in PBS and (iii) 200 µl HCG followed by a 50 µg/ 100 µl/dose of chimeric mAb PIPP after 15 min at a different site. All injections were i.p. The procedure was repeated on three consecutive days and on day 4 the mice were killed. The uterus of each mouse was removed and weighed.
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Results |
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Discussion |
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It was possible to purify 2040 mg of recombinant antibodies from 1 kg of vacuum infiltrated tobacco leaves in only 3 days. In addition, we could show that co-infiltration of tobacco leaves with two populations of Agrobacteria containing either the heavy or light chain cDNA was efficient in producing functional full-size chimeric mAb. Moreover, the transient expression system is a timesaving and cost-effective alternative to other eukaryotic systems in expressing proteins, especially complex (full-size IgG) recombinant proteins. The chimeric mAb PIPP had a high affinity for binding HCG (KD = 3x1010 mol/l). The large quantities in which this antibody can be readily produced by transient plant expression technologies provides high quality recombinant proteins suitable for diagnostic purposes such as pregnancy testing, detection of HCG synthesizing cancer cells, Downs syndrome pregnancies (Knight et al., 1998) or pre-eclampsia (Bahado-Singh et al., 1998
).
Work is in progress to fully humanize this antibody (R.Sriraman, M.Sack, H.Speigel, G.P.Talwar, R.Fisher and R.Finnern, unpublished data) so that it can be used for detection and regulation of fertility and as a tool for the construction of improved cancer diagnostics and therapeutics.
The possibility that plant-derived therapeutic proteins could elicit an immune or sensitization response in patients is of concern, although clinical application might still be possible depending on the dose requirements and blood clearance rate (Miele, 1997). In addition, glycosylation differences might not be an issue for non-glycosylated proteins and peptides, such as scFv or diabodies, and may only be a problem for therapeutic glycoproteins administered to patients by i.v. injection. Plant-derived antibodies given orally have been shown in human trials not to elicit titres of serum IgG, IgA or IgM antibodies capable of binding to the foreign protein (Ma et al., 1998
). The HCG specific recombinant antibodies are currently being tested in marmosets. It might be feasible to use these plant-expressed proteins in immunoassays and for in-vivo contraceptive measures. On radio-tagging they can also be used for imaging of HCG-synthesizing tumours and their metastasis, a purpose for which the use of a mouse mAb bears potential risks. Other useful applications for these antibodies would include the immunotherapy of cancers. Antibody fragments and chimeric full-size antibodies have already been used with benefit for immunotherapy of cancers (Baselga et al., 1996
; McLaughlin et al., 1998
; van Zanten-Przybysz et al., 2001
). Their safety and benefits are evident from such studies.
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Acknowledgements |
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Notes |
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5 Present address: Experimental Hepatology, University Düsseldorf, 40225 Düsseldorf, Germany
6 To whom correspondence should be addressed at: Pharmaceutical Product Development, Fraunhofer IME, Worringerweg 1,52074 Aachen, Germany. E-mail: finnern{at}molbiotech.rwth-aachen.de
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Submitted on September 10, 2002; resubmitted on January 7, 2002; accepted on April 25, 2002.