mAbs
`
`ISSN: 1942-0862 (Print) 1942-0870 (Online) Journal homepage: http://www.tandfonline.com/loi/kmab20
`
`“Inclonals”
`
`Rahely Hakim & Itai Benhar
`
`To cite this article: Rahely Hakim & Itai Benhar (2009) “Inclonals”, mAbs, 1:3, 281-287, DOI:
`10.4161/mabs.1.3.8492
`
`To link to this article: http://dx.doi.org/10.4161/mabs.1.3.8492
`
`Published online: 01 May 2009.
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`[mAbs 1:3, 281-287; May/June 2009]; ©2009 Landes Bioscience
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`Report
`“Inclonals”
`IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system
`
`Rahely Hakim and Itai Benhar*
`
`Department of Molecular Microbiology and Biotechnology; The George S. Wise Faculty of Life Sciences; Tel Aviv University; Ramat Aviv, Israel
`
`Abbreviations: PE38, truncated form of Pseudomonas exotoxin A; scFv, single-chain Fv composed of VH connected to VL through
`a short peptide linker; dsFv, disulfide-stabilized Fv fragment; IC50, concentration required to cause 50% inhibition of the measured
`phenotype
`
`Key words: IgG, IgG-toxin fusion protein, CD30, EGFR, PE38, inclusion bodies, refolding
`
`Full-length antibodies and antibodies that ferry a cargo to
`target cells are desired biopharmaceuticals. We describe the
`production of full-length IgGs and IgG-toxin fusion proteins
`in E. coli. In the presented examples of anti CD30 and anti
`EGF-receptor antibodies, the antibody heavy and light chains or
`toxin fusions thereof were expressed in separate bacterial cultures,
`where they accumulated as insoluble inclusion bodies. Following
`refolding and purification, high yields (up to 50 mg/L of shake
`flask culture) of highly purified (>90%) full-length antibodies and
`antibody-toxin fusions were obtained. The bacterially produced
`antibodies, named “Inclonals,” equaled the performance of the
`same IgGs that were produced using conventional mammalian
`cell culture in binding properties as well as in cell killing potency.
`The rapid and cost effective IgG production process and the high
`quality of the resultant product may make the bacterial produc-
`tion of full-length IgG and IgG-drug fusion proteins an attractive
`option for antibody production and a significant contribution to
`recombinant antibody technology.
`Introduction
`Antibodies are among the most powerful tools in biological and
`biomedical research and are presently the leading category of biop-
`harmaceuticals with annual sales exceeding $20 billion. Currently
`over 20 therapeutic antibodies are FDA-approved, and hundreds
`more are in late stages of clinical development.1 Although many
`formats of recombinant antibodies and antibody fragments popu-
`late the pipeline, the antibody market is dominated by full-length
`IgG antibodies both in research, diagnostic and clinical applications.
`
`*Correspondence to: Itai Benhar; Department of Molecular Microbiology and
`Biotechnology; The George S. Wise Faculty of Life Sciences; Tel Aviv University;
`Ramat Aviv 69978 Israel; Tel: +972.3.6407511; Fax: +972.3.6409407; Email:
`benhar@post.tau.ac.il
`
`Submitted: 02/25/09; Accepted: 03/19/09
`
`Previously published online as a mAbs E-publication:
`www.landesbioscience.com/journals/mabs/article/8492
`
`Unfortunately, many cancers are resistant to treatment with
`naked (unarmed) antibodies. Attaching a cytotoxic moiety to the
`antibody can provide several logs-fold improvement of potency
`in cell killing efficacy. Immunoconjugates are made by attaching
`chemotherapy drugs, radioisotopes or toxins to the antibody.
`Antibody-drug conjugates and antibody-toxin fusion proteins
`are also making headway in the clinical pipeline.2,3 However,
`conventional mammalian cell-based IgG production systems are
`not capable of expressing toxic proteins. Antibody-toxin conju-
`gates were originally made by chemical conjugation that, with a
`few exceptions, yielded heterogeneous products that contained a
`mixture of species with different molar ratios of drug to antibody,
`linked at different sites, each with distinct in vivo pharmacokinetic,
`efficacy and safety profiles. The unfavorable in vivo effects associ-
`ated with heterogeneity in the drug load and sites of attachment
`in antibody-drug conjugates could compromise their promise as
`cancer therapeutics.4
`Full-length monoclonal antibodies have traditionally been
`produced in mammalian cell culture. However, due to its simplicity
`and reduced production time and cost, Escherichia coli (E. coli) is
`the system of choice for the expression of recombinant proteins,
`including most recombinant antibody derivatives. Early, largely
`unsuccessful attempts to produce IgGs in bacteria were reported
`over 20 years ago.5,6 With advances in technology, full-length anti-
`bodies were recently obtained in E. coli by directing secretion of
`the antibody heavy and light chains to the bacterial periplasm.7-9
`With regard to E. coli-produced full length IgGs, two main
`obstacles remained unsolved: first is the purity of the final product
`that contains partially assembled species, and second is the limited
`yields, with approximately 1 mg (range of 0.2–1 mg/L9) antibody
`being produced from 1 liter of low density shake flask cultures.
`To overcome these obstacles, we have developed a highly effi-
`cient production method for full-length IgG and IgG-toxin fusion
`proteins in E. coli, named “Inclonals.”
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`E. coli produced IgGs and IgG-enzyme fusion proteins
`
`Figure 1. Expression and purification of T427 Inclonal in E. coli. (A)
`12% SDS/PAGE. Lane 1, un-induced E. coli culture. Lane 2, induced
`heavy-chain. Lane 3, induced light-chain Lane 4, unpurified refolded
`IgG. Lane 5, Protein-A purified IgG. M, MW marker, in kDa, Lane 6,
`cetuximab. Lane 7, protein-A purified T427 Inclonal. Lanes 1-5 were
`analyzed under reducing conditions while lanes 6-7 were not. Proteins
`were visualized by staining with GelCode Blue®. (B) Immunoblot using
`HRP-conjugated anti human antibody and ECL development. The lane
`arrangement is as in (A), except lane E = cetuximab.
`
`Results
`Production of chimeric IgGs in E. coli. The first model anti-
`body was an anti CD30 antibody, T427.10 T427 Inclonal IgG1
`was cloned into the pHAK expression vectors (Supplementary
`Fig. 1) and produced in E. coli as described in Materials and
`Methods. Fractions from the purification process are shown in Fig.
`1A and B. As shown, a high yield of highly purified preparation
`of chimeric T427 Inclonal was obtained. From 1 liter of shake
`flask culture we routinely obtain 100–200 mg of solubilized inclu-
`sion body protein. Refolding was initiated after mixing 50 mg of
`heavy chain and 50 mg of light chain inclusion bodies protein
`and reducing the mixture with 1,4-dithioerythritol (DTE). After
`refolding, dialysis and protein-A purification, up to 15 mg of
`pure (>90% according to densitometry of the SDS gel) IgG were
`obtained, which correspond to about 45 mg pure IgG per liter of
`heavy chain E. coli culture.
`Evaluation of the bacterially produced antibody. The
`bacterially produced Inclonal was compared to mammalian-cell
`produced IgG by gel-filtration chromatography, by measuring
`stability in serum, by antigen binding properties and by cell killing
`activity.
`An aliquot of the purified IgG was analyzed by gel-filtration
`chromatography on a TSK3000 column (Fig. 2). As shown, the
`T427 Inclonal (calculated MW 147,500) eluted from the column
`as a monomer (free of aggregates). The control mammalian-cell
`produced mAb cetuximab (MW 151,800) migrates as a slightly
`larger protein probably due to post-translational modifications
`
`Figure 2. Analysis of IgGs by gel filtration chromatography. IgG samples
`were separated on a TSK3000 column. The arrows mark the migration
`pattern of commercial size markers on the column.
`
`(glycosylation) that are absent in our E. coli produced IgG.
`Cetuximab and the mammalian cell produced chT427 IgG
`migrate similarly in gel filtration (not shown).
`To evaluate the stability of the Inclonal IgG T427 we compared
`its serum stability in 37ºC to that of mammalian-cell produced
`chimeric T427 IgG (T427 chIgG) that was prepared essentially
`as described.11 As shown (Fig. 3), the mammalian cell produced
`chT427 IgG and the T427 Inclonal were equally stable, losing no
`binding activity over the test period of four days at 37ºC.
`Antigen binding was studied by ELISA and by flow cytometry.
`As shown (Fig. 4A), the T427 Inclonal bound soluble antigen in
`ELISA with a similar avidity to the corresponding T427 chIgG
`that was produced in mammalian cell culture. Similarly, iden-
`tical binding properties could be observed in the flow cytometry
`analysis on CD30-expressing cells (Fig. 4B1). Binding specificity
`could be demonstrated by the competition of the T427 Inclonal
`binding signal by a T427(dsFv)-PE38 recombinant immunotoxin
`(prepared as described in Supplementary methods), as shown in
`(Fig. 4B3).
`The ability of the Inclonal antibodies to target tumor cells
`in vitro was evaluated by forming a complex with an antibody-
`binding toxin fusion protein (ZZ-PE38) (as described in reference
`11). The cytotoxicity evaluation also revealed that the T427
`Inclonal parallels the performance of the mammalian cell produced
`antibody (Fig. 4C).
`As an additional example, we produced an Inclonal derivative
`of the anti EGF receptor antibody 225. MAb 225 is the parental
`mouse monoclonal antibody from which the therapeutic anti-
`body cetuximab was derived.12 We compared the 225 Inclonal to
`cetuximab for antigen binding properties and by for cell killing
`activity as ZZ-PE38 immunocomplexes. As shown (Supplementary
`Fig. 5), the 225 Inclonal specifically bound EGFR expressing cells
`with about x10 lower avidity than that of cetuximab. Similarly, the
`225 Inclonal-ZZ-PE38 immunocomplex had cytotoxic activity
`on both high EGFR expressing A431 cell line and on low EGFR
`expressing 293 cell line, which was about x10 less potent than
`the cetuximab-ZZ-PE38 immunocomplex. This difference is in
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`E. coli produced IgGs and IgG-enzyme fusion proteins
`
`The cell killing potential of T427(di)-PE38 and T427(tetra)-
`PE38 Inclonal-toxin fusion proteins was tested on cultured
`CD30-expressing cells. As shown in Fig. 6B, both molecules inhib-
`ited the growth of the target cells with an IC50 of ~30 pM, while
`the monovalent immunotoxin T427(dsFv)-PE38 had an IC50 of
`~60 pM.
`Discussion
`This study demonstrates an expression and purification protocol
`we developed for producing full-length IgGs and IgG-toxin fusion
`proteins, by refolding E. coli-produced inclusion bodies of the
`antibody heavy and light chain. Our modified expression-refolding
`system enables an effective production of full length IgGs in
`E. coli. By applying this novel system we successfully obtained two
`antibodies: the anti-CD30 T427 antibody and the anti-EGFR
`225 antibody. The production process of the antibody chains from
`inclusion bodies revealed high quantity of over 200 mg of relatively
`pure protein. The entire refolding and purification process yielded
`up to 50 mg of IgG protein from 1 liter of shake flask culture,
`yields that were not reported before using bacterial expression
`systems for IgG production in low density culture. These produc-
`tion yields could benefit research laboratories that, in contrast
`to industrial laboratories, are generally not equipped with high
`density fermentors. The second important benefit of this system is
`the purity of the final product; following protein-A purification,
`the monomeric form of the antibody is notably the main form
`that was obtained. The purified protein is almost free of partially
`assembled species that were observed in previous studies.8,9 The
`advantage of the E. coli production system in time savings was
`considerable. The entire process in the mammalian system, (trans-
`fection, selection of a highly-expressing clone, expansion of the
`clone, IgG purification) required about eight weeks, while in the
`bacterial system, the production process was completed in about
`8–9 days. Inclonals equaled the performance of the same IgGs
`that were produced using conventional mammalian cell culture in
`binding properties, as well as in their potential to deliver toxins to
`cultured target cells. Moreover, the Inclonals method provided us
`the opportunity to generate full-length IgG that is genetically fused
`to a cytotoxic moiety, and consequently to explore IgG-enzyme
`fusion proteins.
`Our antibody production system thus provides several advan-
`tages over other systems. First, this system has the advantages of
`the bacterial expression systems (simple, cheaper, faster and easier
`to scale up compared to mammalian cell culture). Second, the
`bacterial produced antibodies are aglycosylated, and can be used
`where effector functions are either not required or are actually
`detrimental. Third, the separate expression of the antibody heavy
`and light chains enables mixing different heavy and light chains
`which can give rise to combinatorial shuffling in the protein level
`to obtain desired antigen specificities and affinity properties. The
`fourth and most significant advantage of production of targeting
`molecules in a non-mammalian host is the ability to express a
`cytotoxic moiety fused to the molecule as a single polypeptide.
`Refolding of therapeutic proteins is well established and in general
`refolded E. coli-produced proteins have a low endotoxin level
`
`Figure 3. Stability in serum. Analysis of the stability of mammalian-cells
`produced T427 (lower graph) and of the T427 Inclonal (upper graph)
`upon incubation in bovine serum. IgGs were diluted to a final concentra-
`tion of 30 μg/ml in 100% bovine serum and incubated at 37°C for the
`indicated time periods. Residual binding activity to MBP-CD30 of each
`fraction was evaluated by ELISA as described in materials and methods.
`
`accordance with the reported x10 affinity increase reported for
`cetuximab in comparison to the 225 mAb.12
`The Inclonal-PE38 fusion proteins production. By applying
`the Inclonals technology, we generate full-length IgGs that are
`genetically fused to a cytotoxic moiety. We prepared PE38 fusion
`proteins of the T427 Inclonal. Two derivatives were prepared;
`a T427(di)-PE38 derivative, with PE38 fused to the antibody
`heavy chain, and T427(tetra)-PE38, with PE38 fused to both the
`antibody heavy and light chains. The Inclonal-toxin fusion deriva-
`tives differ in their molecular weight (~225 kDa for the di-toxin
`and ~300 kDa for the tetra toxin) and in the number of toxin
`molecules payload delivered for each binding event (Fig. 5A). Both
`T427(di)-PE38 and T427(tetra)-PE38 were produced at a high
`purity (Fig. 5B and 5C), and at a high yield, similar to that we
`obtained for the IgG Inclonals.
`Evaluation of the Inclonal-PE38 fusion proteins. These
`novel T427(di)-PE38 and T427(tetra)-PE38 proteins were evalu-
`ated for their binding properties and for their cell-killing activity.
`As shown (Fig. 6A), the apparent binding affinity as evaluated from
`the ELISA signal for both T427(di)-PE38 and T427(tetra)-PE38
`is about 0.2 nM, which is similar to that of the T427 Inclonal
`and T427 chIgG (that are shown in Fig. 4A). Both IgGs bound
`with an apparent avidity, which was x10 higher than the affinity
`of the corresponding monovalent recombinant immunotoxin
`T427(dsFv)-PE38.
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`E. coli produced IgGs and IgG-enzyme fusion proteins
`
`Figure 4. Evaluation of T427 Inclonal. Binding properties: (A) Binding to MBP-CD30 in ELISA. Detection is with HRP-conjugated anti human IgG. (B)
`FACS analysis. (1) Stable A431/CD30 transfected cells were incubated with 10 nM of chT427-IgG made in mammalian cells or with T427 Inclonal.
`(2) FACS analysis of T427 Inclonal binding in the presence of X30 molar excess of T427(dsFv)-PE38 immunotoxin as competitor. Binding was detected
`using FITC-conjugated anti human antibody. (C) Specific cytotoxicity of T427-ZZPE38. A431/CD30 cells were incubated for 48 h with the indicated
`concentration of IgG-ZZPE38 immunoconjugates or the IgGs alone. The relative number of viable cells was determined using an enzymatic MTT assay.
`Each point represents the mean of a set of data determined in triplicate in three independent experiments. Error bars represent the standard deviation
`of the data.
`
`compared to proteins that are recovered from the bacterial
`periplasm.
`Our Inclonal-fusion technology resolves the issue
`of conjugate heterogeneity and should be applicable to
`production of a wide range of cytotoxic proteins. For
`research purposes, there is currently a great need to
`generate protein-specific affinity reagents to explore the
`human proteome. High-throughput methods to generate
`renewable antibodies are still immature.13 Antibody-
`enzyme or antibody-fluorophore fusion proteins that can
`be generated by the Inclonals technology may become
`very useful for such purposes. Cost-effective production
`of immunoconjugates, which are widely studied as anti-
`cancer treatments, is needed. We believe that our rapid
`and cost effective IgG and IgG-fusion protein production
`process and the high quality of the resultant product
`may make the bacterial production of full-length IgG
`and IgG-fusion proteins a viable and attractive option
`for antibody production for research and hopefully for
`clinical applications.
`Materials and Methods: Construction of Vectors for
`Expression of Inclonals
`Heavy chain vectors: the VH variable domain of anti
`CD30 antibody T427 with the C-region of human IgG1
`
`Figure 5. IgG-toxin fusion proteins. (A) Schematic representation of the Inclonals
`that were produced in this study. (B) Immunoblot of protein-A-purified T427 Inclonals
`under non-reducing conditions. Lane 1, IgG; Lane 2, IgG-(di)-PE38; Lane 3, IgG-
`(tetra)-PE38. (C) Immunoblot of protein-A-purified T427 Inclonal-PE38 fusion proteins
`under reducing conditions. Lane 1, IgG-(di)-PE38; Lane 2, IgG-(tetra)-PE38. M, MW
`marker, in kDa.
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`E. coli produced IgGs and IgG-enzyme fusion proteins
`
`Light chain vectors: the light chain of anti CD30
`antibody T427 with the human C-kappa region
`was subcloned from pMAZ-IgL-T427 (described in
`supplementary methods) into a T7-based, IPTG-
`inducible bacterial expression vector as follows: the
`entire light chain was amplified by PCR using plasmid
`pMAZ-IgL-T427 as template with primers CMV-Seq
`and CMV-antiseq-EcoRI-REV. The PCR product
`was digested with PstI and EcoRI and cloned into a
`pRB98Amp-T427VL(C105) plasmid vector15 that
`was linearized using the same enzymes. The resulting
`plasmid, pHAK-IgL-T427 can be used to express the
`light chain of T427 in a chimeric IgG1 format in E.
`coli. VL domains can be exchanged into this plasmid
`as NdeI-BsiWI fragments.
`A similar plasmid for the expression of the light
`chain of the anti EGFR antibody 225 in E. coli
`was constructed as follows: the V-Kappa variable
`domain was recovered by PCR using plasmid pCMV/
`H6myc/cyto-225(Fv)14 as template with primers
`225VK-NdeI-FOR and 225VK-BsiWI-REV. the PCR
`product was digested with NdeI and BsiWI and cloned
`into a pHAK-IgL-T427 vector (described above) that
`was linearized using the same enzymes. The resulting
`plasmid was named pHAK-IgH-225.
`Construction of vectors for expression of
`IgG-PE38 fusion. The heavy or light chain-PE38
`fusion protein expression vectors were constructed on
`the backbone of pHAK vectors that were modified by
`insertion of HindIII and EcoRI cloning site at the 3'
`end of the antibody constant regions as follows: For
`the heavy chain vector, the cloning site was inserted
`by PCR using plasmid pHAK-IgH as template with
`primers RGD/TAT-BsrGI-FOR and CH3-HindIII-
`EcoRI-REV. For the heavy chain vector, pHAK-IgL was used
`as template with primers BsiWI-Back-IgL and Cκ-HindIII-
`EcoRI-REV. The PCR products were digested with BsrGI and
`EcoRI for the heavy chain and with SacI-EcoRI for the light chain,
`respectively, and cloned into a pHAK-IgH vector and pHAK-IgL
`vector respectively that were linearized using the same enzymes.
`The resulting vectors were linearized with HindIII and EcoRI
`and ligated with the PE38 DNA fragment that was recovered
`form plasmid pRB98Amp-T427VH(C44)-PE38 using the same
`enzymes. The resulting vectors were named pHAK-IgH-PE38 and
`pHAK-IgL-PE38.
`Expression of Inclonals in E. coli. The Inclonals and Inclonal-
`PE38 fusion proteins were expressed in E. coli BL21(DE3)
`pUBS500 cells16 that were transformed with the expression
`vectors. For the production of IgGs, cells were transformed with
`pHAK-IgH and pHAK-IgL. For the production of IgG-(di)
`PE38, cells were transformed with pHAK-IgH-PE38 and
`pHAK-IgL. For the production of IgG-(tetra)PE38, cells were
`transformed with pHAK-IgH-PE38 and pHAK-IgL-PE38. Cells
`were grown in SB medium (35 gr/L tryptone (Difco, USA), 20
`gr/L yeast extract (Difco, USA), 5 gr/L NaCl, 6.3 gr/L glycerol
`
`Figure 6. Evaluation of the T427 Inclonal-toxin fusion proteins. (A) Binding to MBP-CD30
`in ELISA. Detection is with HRP-conjugated anti human IgG. (B) Specific cytotoxicity:
`A431/CD30 cells were incubated for 48 h with the indicated concentration of recom-
`binant PE38 fusion proteins. The relative number of viable cells was determined using
`an enzymatic MTT assay. Each point represents the mean of a set of data determined in
`triplicate in three independent experiments. Error bars represent the standard deviation
`of the data.
`
`was subcloned from pMAZ-IgH-T427 (described in supple-
`mentary methods) into a T7-based, IPTG-inducible bacterial
`expression vector as follows: the entire heavy chain was amplified
`by PCR using plasmid pMAZ-IgH-T427 as template with primers
`CMV-Seq and CMV-antiseq-EcoRI-REV (All the PCR primers
`are described in Supplementary Table 1). The PCR product was
`digested with PstI and EcoRI and cloned into a pRB98Amp-
`T427VH(C44)-PE38 vector that was linearized using the same
`enzymes. The resulting plasmid, pHAK-IgH-T427 can be used to
`express the heavy chain of T427 in a chimeric IgG1 format in E.
`coli. VH domains can be exchanged into this plasmid as NdeI-NheI
`fragments.
`A similar plasmid for the expression of the heavy chain
`of the anti EGFR antibody 225 in E. coli was constructed
`as follows: the VH variable domain was recovered by PCR
`using plasmid pCMV/H6myc/cyto-225(Fv)14 as template with
`primers 225VH-NdeI-FOR and 225VH-NheI-REV. The PCR
`product was digested with NdeI and NheI and cloned into a
`pHAK-IgH-T427 vector (described above) that was linearized
`using the same enzymes. The resulting plasmid was named pHAK-
`IgH-225.
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`E. coli produced IgGs and IgG-enzyme fusion proteins
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`(Frutarom, Israel), 12.5 gr/L K2HPO4, 3.8 gr/L KH2PO4, 0.48
`gr/L MgSO4, 0.4% (w/v) glucose) supplemented with 100 μg/
`ml ampicillin and 50 μg/ml kanamycin at 37ºC shaking at 250
`RPM. The bacterial cultures were induced for protein expression
`in the late exponential growth phase (OD600 of 2.5) with 1 mM
`isopropyl-1-thio-β-D-galactopyranoside for 3 h at 37ºC. The
`recombinant proteins accumulated as insoluble inclusion bodies
`and were isolated from lysed bacteria cells by centrifugation.
`From 500 ml of culture about 3 gr of wet cell paste was collected.
`The cells were suspended in 50 mM Tris (HCl) pH 8.0, 20 mM
`EDTA, using a tissuemizer and further processed as described.17
`The inclusion bodies were completely solubilized in 6 M guanidine
`hydrochloride, 50 mM Tris (HCl) pH 8.0, 20 mM EDTA, mixed,
`reduced and refolded essentially as described.15 After refolding,
`the protein was dialyzed against phosphate buffer pH 7.4 (20 mM
`containing 77% Na2HPO4 and 23% NaH2PO4). The refolded
`active protein was then filter sterilized using a 0.45 μm filter and
`separated from contaminating bacterial proteins, excess light chains
`and from improperly folded protein by protein-A chromatography.
`Purified IgG was stored at 4ºC. Typically, from a refolding initiated
`by mixing 50 mg of heavy chain with 50 mg of light chain protein,
`we obtain ~12.5 mg of pure Inclonal.
`The anti EGFR 225 Inclonal was produced in the same way
`using cultures of cells carrying pHAK-IgH-225 and pHAK-
`IgL-225.
`Preparation of IgG-ZZ-PE38 immunocomplexes. The
`immunocomplex of T427 or 225 IgGs with ZZ-PE38 fusion
`protein was carried out by mixing IgGs with ZZ-PE38 fusion
`protein and purifying the immunocomplex by Superdex 200
`(Amersham Pharmacia Biotech, now GE healthcare, USA) gel
`filtration chromatograph essentially as described.11
`Gel filtration chromatography. Analytical separation of
`chimeric IgGs was carried out by gel-filtration chromatography
`using a 30 ml TSK3000 column (TosoHaas, Japan) on a fast protein
`liquid chromatography (FPLC), (Pharmacia LKB-Pump-P500)
`according to supplier’s recommendations. About 200 micrograms
`of sample were loaded in 500 μl with PBS as buffer at a flow rate
`of 0.5 ml/min.
`Evaluation of IgG stability in serum. To compare the stabili-
`ties of an Inclonal IgG T427 to that of the corresponding chT427
`IgG that was produced in mammalian cell culture, a serum
`stability assay was carried out as follows: The IgGs were diluted
`to a final concentration of 30 μg/ml in 100% bovine serum (Beit
`Haemek, Israel) and incubated at 37ºC for the indicated time
`periods. Residual binding activity to MBP-CD30 of each fraction
`was evaluated by ELISA as describes below.
`Evaluation of antigen binding by ELISA and whole-cell
`ELISA. Antigen binding by chimeric IgGs was tested in ELISA
`as follows: ELISA plates were coated with a solution of 5 μg/ml
`MBP-CD30 in PBS at 4ºC for 20 h and blocked with 3% (v/v)
`non-fat milk in PBS for 1-2 h at 37ºC. All subsequent steps were
`done at room temperature (25ºC). Protein-A purified IgGs were
`applied onto the plates in a five-fold dilution series and tested for
`their affinity to MBP-CD30. Following incubation the plates were
`washed thee times with PBST. HRP-conjugated goat anti human
`
`antibodies were used as secondary antibodies diluted x5,000 dilu-
`tion in PBST. The ELISA was developed using the chromogenic
`HRP substrate TMB and color development was terminated with
`50 μl/well of 1 M H2SO4. The results were plotted as absorbance
`at 450 nm and the binding-avidity was roughly estimated as the IgG
`concentration that generates 50% of the maximal signal.
`Cellular EGFR binding by 225 Inclonal and cetuximab, was
`tested by whole-cell ELISA as follows; the human epidermoid
`carcinoma A431 cells were seeded in 96-well plate at a density
`of 2x104 cells/well in DMEM supplemented with 10% FBS for
`16 h. The medium was aspirated and the cells were fixed with 3%
`glutaraldehyde for 15 minutes at 25ºC. The wells were blocked
`with 3% (v/v) non-fat milk in PBS for 1-2 h at 37ºC. Next, IgGs
`were added to the wells at a 5 fold dilution series in PBS +3%
`BSA and incubated for 1.5 h at 25ºC. After cells were washed
`three time with PBS +3% BSA, 100 μl of HRP-conjugated goat
`anti human antibodies (x5000 dilution in PBS + 3% BSA) was
`added for 1 h at 25ºC. After another washing cycle, detection of
`cell bound antibodies was performed by addition of 100 μl of the
`chromogenic HRP substrate TMB to each well and color develop-
`ment was terminated with 50 μl/well of 1 M H2SO4. Absorbance
`was measured at 450 nm using a microplate reader.
`Flow cytometry. Binding analysis to CD30 expressed on
`A431/CD30 transfected cells18 with bacterial or mamma-
`lian produced chT427 IgG1 was tested by flow cytometry.
`Approximately 5 x 105 cells in immunotubes (5 ml polystyrene
`tubes, Nunc, Sweden) were used in each experiment. After
`trypsinization, cells were washed once in 2% fetal calf serum in
`PBS (FACS buffer). Next, the chimeric IgGs were added at a final
`concentration of 10 nM in PBS + 3% BSA and the cells were
`incubated for 90 min at 4ºC. The cells were then washed three
`times FACS buffer and FITC-labeled goat anti human antibodies
`(x50 dilution in PBS + 3% BSA) were added to the appropriate
`tubes for 45 min at 4ºC. Detection of bound antibodies was done
`by flow cytometry on a FACS-Calibur (Becton Dickinson, CA)
`and results were analyzed with the CELLQuest program (Becton
`Dickinson). To confirm specificity, antibodies were incubated with
`or without a x30 fold excess of competing protein during the 90
`min incubation period.
`Cell-viability assay. The in vitro cell-killing activities of
`chimeric IgG-ZZ-PE38 immunocomplexes and of IgG-PE38
`fusion proteins were measured by an MTT assay. Tested cells were
`seeded in 96-well plates at a density of 1 x 104 cells/well in DMEM
`supplemented with 10% FBS. Immunocomplexes, IgG-PE38
`fusion proteins or control proteins were added (in triplicate) in
`a 10-fold dilution series and the cells were incubated for 48 h at
`37ºC in 5% CO2 atmosphere. After 48 h, the media was replaced
`by fresh media (100 μl per well) containing 1 mg/ml MTT
`(Thiazolyl Blue Tetrazoliam Bromide, dissolved in PBS) reagent
`and the cells were incubated for another 4 h. MTT-formazan crys-
`tals were dissolved by the addition of 20% SDS, 50% DMF, pH
`4.7 (100 μl per well) and incubation for 16 h at 37ºC. Absorbance
`at 570 nm was recorded on an automated microtiter plate reader.
`The results were expressed as percentage of living cells relatively
`to the untreated controls that were processed simultaneously
`
`286
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`mAbs
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`2009; Vol. 1 Issue 3
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`E. coli produced IgGs and IgG-enzyme fusion proteins
`
`using the following equation: (OD570 of treated sample/ OD570
`of untreated sample) x100. The IC50 values were defined as the
`immunocomplexes or the IgG-PE38 fusion protein concentrations
`that inhibited cell growth by 50%.
`Acknowledgments
`We thank Dr. Ira Pastan (LMB, NCI, NIH) for the expres-
`sion vectors of anti CD30 immunotoxin T427(dsFv)-PE38, the
`pRB98-Amp expression vector for recombinant immunotoxins,
`A431-CD30 cells and an expression vector for CD30 extracel-
`lular domain. We thank Prof. Winfried Wels (Georg Speyer
`Haus, Frankfurt, Germany) for the 225 scFv clone. This study
`was supported in part by a research grant from the Israel Cancer
`Research fund (ICRF).
`Note
`Supplementary materials can be found at:
`www.landesbioscience.com/supplement/
`HakimMABS1-3-Sup.pdf
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