Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli

Journal of Immunological Methods 242 (2000) 101–114 www.elsevier.nl / locate / jim

Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli Andreas Schmiedl a ,2 , Frank Breitling b,2 , Christoph H. Winter a ,1 , Iris Queitsch a , ¨ a ,* Stefan Dubel a

¨ Heidelberg, Institut f ur ¨ Molekulare Genetik, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany Universitat b German Cancer Research Centre, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received 3 March 2000; received in revised form 10 May 2000; accepted 12 May 2000

Abstract New E. coli vectors based on the pOPE / pSTE vector system [Gene 128 (1993) 97] were constructed to express a single-chain Fv antibody fragment (scFv), a scFv–streptavidin fusion protein and two disulfide bond-stabilized Fv antibody fragments (dsFvs) utilizing different side chain positions for disulfide stabilization. All of these constructs encoded fusion proteins carrying five C-terminal histidine residues preceded by an unpaired cysteine. The influence of this cysteine, which was originally introduced to allow the chemical modification of the fusion proteins, was assessed by exchanging the two amino acids CysIle in front of the carboxy terminal His-tag to SerHis in all constructs. Yield and antigen-binding activity of the antibody constructs were compared after standard lab-scale periplasmic expression in Escherichia coli. The removal of the unpaired cysteine resulted in a significant increase in antigen-binding activity of the crude periplasmic extracts. Further, a three–five fold increase of yield and a significantly improved purity were observed after immobilized metal affinity chromatography (IMAC) with all four constructs.  2000 Elsevier Science B.V. All rights reserved. Keywords: Antibody engineering; scFv; dsFv; Bispecific antibodies; Streptavidin

Abbreviations: Fv, antibody fragment consisting of the variable region of light and heavy chain; VH , variable region of heavy chain; VL , Variable region of light chain; scFv, Single-chain Fv antibody fragment; dsFv, Disulfide-stabilized Fv antibody fragment; phOx, 4-Ethoxymethylene-2-phenyl-2-oxazoline-5-one *Corresponding author. Tel.: 149-6221-54-5638; fax: 1496221-54-5678. E-mail address: [email protected] (S. ¨ Dubel). 2 Both authors have contributed equally. 1 Current address: Yale University, Department of Molecular Biophysics and Biochemistry, 333 Cedar Street, New Haven, CT 06520-8024, USA.

1. Introduction The variable region (Fv) of an antibody is the smallest antibody fragment containing a complete antigen-binding site. It is comprised of the antibody variable heavy (VH ) and the variable light chain (VL ) ¨ domain (reviewed in Breitling and Dubel, 2000). To stabilize the association of these domains, they can be linked by a short peptide that connects the carboxy-terminus of one domain with the aminoterminus of the other to form a single-chain Fv

0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759( 00 )00243-X

102

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

antibody fragment (scFv) (Huston et al., 1988; Bird et al., 1988; Whitlow and Fipula, 1991). Alternatively, the two domains can be produced as separate chains and posttranslationally linked via genetically engineered cysteines in complementary framework positions of the VH and the VL domain to form a disulfide-bond-stabilized Fv antibody fragment (dsFv, Reiter et al., 1995). In vivo, scFvs and dsFvs have lower retention times in non-target tissues, a more rapid blood clearance and better tumor penetration than the larger Fab9 and F(ab9) 2 antibody fragments or complete IgGs (Milenic et al., 1991; Yokota et al., 1992; Adams et al., 1993; Colcher et al., 1999). Therefore, they represent potentially useful molecules for the targeted delivery of drugs, toxins and radionuclides to a tumor site. For most of these therapeutic applications, however, reagents have to be coupled to the antibody fragments. To achieve site-directed covalent coupling, an unpaired cysteine residue has been genetically engineered near the C-terminus of the antibody fragments in various constructs to allow their chemical modification (Liberatore et al., 1995; Debinski and Pastan, 1995; Wang et al., 1997; Verhaar et al., 1996). Unpaired cysteine residues were further employed for the production of bivalent homo- and heterodimers of antibody fragments by site-specific dimerization in vitro as shown for recombinant Fab9 (Carter et al., 1992; Better et al., 1993; Rodrigues et al., 1993) and Fv fragments (Cumber et al., 1992), or in vivo, as shown for scFv antibody molecules (Kipriyanov et al., 1995). More recently, strategies have been developed for some of these applications which allow to substitute chemical cross-linking by genetical fusion of the compounds (Brinkmann et al., 1993; Friedman et al., 1993; Goshorn et al., 1993). These strategies are not restricted to proteinaceous compounds. For example, site-specific covalent radioactive labelling using a cysteine-free carboxy terminal peptide tag has been recently demonstrated (Waibel et al., 1999). In these cases, however, the effect of unpaired cysteines introduced by various standard vectors should be evaluated carefully. Unpaired cysteines have been shown to play a significant role in the quality control of antibodies produced in eukaryotic cells (Sitia et al., 1990; Fra et al., 1993). In Escherichia coli, the formation of disulfide bonds is catalyzed by periplas-

mic protein disulfide-isomerase (DsbA, Wunderlich and Glockshuber, 1993a,b). It could be possible that, besides folding efficiency, unpaired cysteines might also be an important factor in lowering yields of soluble periplasmic products to very unsatisfactory levels, as observed (Li et al., 1999). Therefore, we evaluated the effects of the presence of an unpaired cysteine on the production of four different recombinant antibody constructs in E. coli: a single-chain Fv antibody fragment, a scFv–streptavidin fusion protein and two disulfide-bond-stabilized Fv antibody fragments. The variable regions were either derived from the mouse monoclonal antibody mAb 215 (or ‘clone 3’) directed against the largest subunit (215 kDa) of Drosophila melanogaster RNA polymerase ¨ II (Kramer et al., 1980; Kontermann et al., 1995) or from a single-chain Fv antibody fragment specific for the hapten 2-phenyl-oxazoline-5-one (phOx), which has been isolated from a phage library and improved by chain shuffling (Marks et al., 1992). To acknowledge a possible influence of the location of the engineered interface cysteines in disulfide bondstabilized Fv fragments, two different dsFvs were constructed utilizing two different pairs of complementary framework positions for disulfide stabilization. Each of the four constructs has initially been designed to carry five C-terminal histidine residues preceded by an unpaired cysteine, which was originally introduced to allow the chemical modification of each fusion protein (Kipriyanov et al., 1994). To analyze the effect of the unpaired cysteine on the bacterial expression, a derivative of each of the four constructs was generated by substitution of SerHis for the two amino acids CysIle in front of the carboxy terminal His-tag. Yield and antigen-binding activity of the generated antibody constructs were compared after standard lab-scale periplasmic expression in E. coli as well as after a single-step purification by immobilized metal affinity chromatography (IMAC).

2. Materials and methods

2.1. Vector construction All standard cloning procedures were carried out as described by Sambrook et al. (1989). The con-

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

struction of the vector pOPE51-215(GS) was described previously (Kontermann et al., 1995). pOPE51-215(Yol) was obtained from pOPE51215(GS) in various steps. The PCR fragment ob¨ tained from pOPE Lys (Dubel et al., 1992) using the oligonucleotide primers GCA AGT TCA GCT GCA GGA GAG CGG TCC AGG TCT TGT GAG ACC TAG and CTG CTT GGG CTT TGG GTG AGC TC GATA TCT ACG CGT was digested with EcoRV (New England Biolabs, Schwalbach, Germany) and ligated to the EcoRV digested PCR fragment obtained from pOPE51-215(GS) using the oligonucleotide primers GGT GAT ATC GTG CTG ACC CAA TCT CCA CTC T and GTA TGC ATA GAT CTT CTT CTG AGA TCA GCT TTT GTT C. The ligation product was further used as a template for a PCR using the oligonucleotide primers GGT CAG GGC TCA TTG GTC ACC GTA TCC TCA and GTA TGC ATA GAT CTT CTT CTG AGA TCA GCT TTT GTT C. The PCR fragment was digested with BstEII and NotI (New England Biolabs) and the 419-bp containing insert was ligated to the vector fragment obtained from pOPE51-215(GS) digested with the same enzymes thus resulting in pOPE51215(Yol). To generate pOPE101-215(Yol) (Genebank accession no. Y14585) from pOPE51215(Yol) the region coding for the carboxy terminal tags of the VL domain was modified. A new linker insert was generated by overlap PCR using the oligonucleotide upstream primers 59-GAG TGT TGA CTT GTG AGC and 59-GAA GAT CTA TCC CAT CAT CAC CAT C and the downstream primers 59-GAT GGT GAT GAT GGG ATA GAT CTT C and 59-CCG CCA GTC TTT CGA CTG. The resulting PCR fragment was digested with NotI and NheI (New England Biolabs) and ligated to the vector fragment of pOPE51-215(Yol) digested with the same enzymes. The vector pOPE511-215 was obtained from pOPE51-215(Yol) in three steps. First, the Ser(44) from the VH domain was mutated to cysteine by PCR mutagenesis using the oligonucleotide primers 59-AGG CGT ATC ACG AGG CCC T and 59-TCC AAT CCA CTC TAG ACA CTT TGC ATG A. The resulting PCR fragment was digested with XhoI and XbaI (New England Biolabs) and ligated to the vector pOPE51-215(Yol) digested with the same enzymes. The resulting vector pOPE51a215(Yol) was further digested with HpaI (New

103

England Biolabs) and ligated to the HpaI-digested PCR product obtained from pOPE51-215(Yol) using the oligonucleotide primers 59-GTC GAC GTT AAC CGA CAA ACA ACA GAT AA and 59-AAC GTT CGG TTG GGC ACC AAG CT. Thereby the Ala(100) from the VL domain was mutated to cysteine. In a third step, the resulting vector pOPE51b-215(Yol) was digested with EcoRV and blunt end ligated to a PCR-fragment coding for a stop codon, an additional RBS and a second pelB signal sequence that was amplified from the vector pOPE51-215(Yol) using the oligonucleotide primers 59-TAA TTC ATT AAA GAG GAG AAA TTA ACC A and 59-ATC CGC CAT CGC CGG CTG AGC T. To obtain pOPE111-215, the 39-terminally deleted dsFv gene was cleaved out of pOPE511-215 with EcoRI and BglII (New England Biolabs) and ligated to the vector fragment of pOPE101-215(Yol) cut with the same restriction enzymes to remove its 39-terminally deleted scFv gene. The construction of the vector pSTE-215(GS) (Genebank accession no. ¨ Y14583) was described previously (Dubel et al., 1995). pSTE2-215(Yol) (Genebank accession no. Y18290) was obtained by introducing the core-streptavidin coding region of pSTE-215(GS) behind the VL coding region of pOPE101-215(Yol) into the BamHI (New England Biolabs) site. pOPE51phOx(GS) was obtained by exchanging the VH and VL coding region of pOPE51-215(Yol) with the respective coding insert of the scFv antibody recognising 4-ethoxy-methylene-2-phenyl-2-oxazoline-5one (phOx; Marks et al., 1992) using the NcoI (New England Biolabs) and NotI site. pOPE511-phOx was obtained from pOPE51-phOx(GS) in two steps. First the Ala(43) from the VL domain was mutated to a cysteine by PCR mutagenesis. Therefore, the PCR product obtained from pOPE51-phOx(GS) using the oligonucleotide primers 59-ATC GTG TTG ACT CAG CCG CCC TCA GT and 59-TTG GGA CAT GTT CCT GGG AGT TGA ACG TAC CA was blunt-end ligated to the PCR product obtained from the same vector with the oligonucleotide primers 59-ACT CCT CAT TTA TGA CAA TAA TAA GCG A and 59 GTC GAC GTT AAC CGA CAA ACA ACA GAT AA. The ligation product was gel purified and further used as a template for a PCR amplification using the oligonucleotide primers 59ATC GTG TTG ACT CAG CCG CCC TCA GT and

104

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

59-GTC GAC GTT AAC CGA CAA ACA ACA GAT AA. The resulting PCR fragment was digested with BamHI and ligated into the vector pOPE511215 digested with EcoRV and BamHI. The resulting vector pOPE511c-phOx was digested with NcoI and HindIII (New England Biolabs) and ligated to the NcoI- and HindIII-digested PCR product obtained from pOPE51-phOx(GS) using the oligonucleotide primers 59-AGG CGT ATC ACG AGG CCC T and 59-ACC GCC AAG CTT TGA GGA GAC GGT GAC CGT GGT CCC ACA GCC CCA, thereby mutating the Lys(105) from the VH domain to cysteine. The vector pSTE2-phOx(S-S) was constructed by ligating the dsFv phOx insert obtained from a NotI and NcoI digest of pOPE511-phOx to the vector fragment of pSTE2-215(Yol) digested with the same enzymes. pOPE111-phOx was constructed by removing the core-streptavidin coding region from pSTE2-phOx(S-S) by BamHI digestion and re-ligation of the larger fragment. All vectors and ligation products were electrotransfected into E. coli XL1-Blue competent cells (Stratagene, La Jolla, CA, USA). Clones with open reading frames were identified on colony lifts ¨ (Breitling and Dubel, 1997) on Protran BA 85 nitrocellulose (Schleicher und Schuell, Dassel, Germany) after induction with 100 mM IPTG for 3 h and immunostaining with mouse monoclonal antibody Myc1-9E10 (Calbiochem, Cambridge, UK). The correct insertion of each gene fragment as well as the introduced mutations in productive clones were confirmed by restriction analysis and sequencing of the insert region. The production of antibody constructs in shaker cultures after induction with 20 mM IPTG for 3 h was confirmed by electrophoresis of total cell lysates on 12% polyacrylamide SDS gels followed by immunoblotting as well as by ELISA, as described below.

2.2. SDS–PAGE and immmunoblot analysis SDS–PAGE was carried out according to Laemmli (1970) on 12% polyacrylamide gels stained with Coomassie Brilliant Blue R250 (Serva, Heidelberg, Germany). Immunoblots were performed according to Towbin et al. (1980). Unspecific antibody binding was blocked with 2% (w / v) skim milk powder (Oxoid, Basingstoke, UK) in PBS (137 mM NaCl, 3

mM KCl, 8 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , pH 7.3) containing 0.05% (v / v) Tween 20 (MPBST). The mouse monoclonal antibody Myc1-9E10 (Calbiochem) recognizing the c-myc-tag, the rat monoclonal antibody Yol1 / 34 (Harlan Sera-lab, Sussex, UK) recognizing the Yol-tag or a rabbit serum ‘A’ recognizing exclusively the processed N-terminus of ¨ the VH domain (Dubel et al., 1992) were used as primary antibodies for immunodetection (1 / 1000 in MPBST). Subsequently, HRP-conjugated antibodies to mouse, rat or rabbit immunoglobulins (Dianova, Hamburg, Germany) were applied (1 / 2000 in MPBST) before TMB Stabilized Substrate for HRP (Promega, Madison, USA) was used for visualizing bound enzymatic activity.

2.3. Expression and purification of soluble Fvfragments from the periplasma Overnight cultures of E. coli cells transformed with one of the vectors described in Fig. 1 were diluted 1 / 20 into LB-medium containing 100 mg / ml of ampicillin (Biomol, Hamburg, Germany) and 100 mM glucose and were grown at 378C to an OD 600nm of 0.6. The promotor was induced by the addition of 20 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After an additional 3 h of shaking at room temperature, the bacteria were harvested by centrifugation (50003g, 10 min). The total cell lysate was analyzed by SDS–PAGE followed by immunoblotting using the mouse monoclonal antibody Myc1-9E10 as described in Section 2.2. For the preparation of periplasmic extracts, the bacteria were resuspended in spheroblast solution (1 / 10 volume of the original culture) containing 50 mM tris(hydroxymethyl)-aminomethane / HCl (pH 8.0), 20% (w / v) sucrose and 1 mM EDTA, left for 20 min on ice with occasional shaking and harvested by centrifugation at 48C (62003g, 10 min). The supernatant representing the periplasmic enriched fraction was cleared by centrifugation at 48C (30 0003g, 30 min), dialyzed against PBS and used for functional studies in ELISA. The bacterial pellet was resuspended in 1 / 10 volume of the original culture in a 5 mM MgSO 4 solution and left for 20 min on ice with occasional shaking. After centrifugation at 48C (30 0003g, 30 min), the supernatant

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114 Fig. 1. General organisation of the eight plasmid constructs employed in this study. RBS: ribosome binding site; pelB leader sequence: signal peptide sequence of bacterial pectate lyase, mediating secretion into the periplasmic space; VH and VL : heavy and light chain variable region coding sequences; Cys: nucleotides coding for an additional, unpaired cysteine residue; His-tag: sequence encoding for five or six histidine residues; Amp: b-lactamase gene for ampicillin resistance; ori: origin of DNA replication for ColE1; IR: intergenic region of phage f1. The elements of the insert regions are not drawn to scale. 105

106

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

representing the osmotic shock fraction was dialyzed against PBS.

2.4. Enrichment of antibody fragments by IMAC The antibody fragments were further enriched from the periplasmic extracts by immobilized metal affinity chromatography (IMAC) utilizing the carboxy terminal His-tag (Lilius et al., 1991). The IMAC column, containing a 2-ml bed volume of chelating Sepharose (fast flow, Pharmacia, Uppsala, Sweden; loaded by equilibriating with NiCl 2 followed by washing with water) per liter of original bacterial culture, was equilibrated with PBS containing 10 mM imidazole and 1 M NaCl (pH 7.5). The dialyzed periplasmic extracts were pooled, adjusted to the same buffer composition and applied to the column. After subsequent washing with ten bed volumes of PBS containing 10 mM imidazole, 1 M NaCl (pH 7.5), ten bed volumes of PBS containing 50 mM imidazole, 1 M NaCl (pH 7.5) and a final washing step with ten bed volumes of PBS, the proteins were eluted from the column using an imidazole gradient from 50 to 500 mM in PBS containing 1 M NaCl (pH 7.5) over ten bed volumes and with another ten bed volumes of PBS containing 500 mM imidazole and 1 M NaCl (pH 7.5). The collected fractions were analyzed by SDS–PAGE followed by immunoblotting using the mouse monoclonal antibody Myc1-9E10 (Calbiochem) for immunodetection. Protein containing fractions were pooled and analyzed by ELISA. Total protein was determined photometrically using Bradford solution ¨ (Biorad, Munchen, Germany). The portion of the Fv fragment was estimated from SDS–PAGE.

2.5. ELISA Ninety-six-well Immuno Plates (MaxiSorp F96; Nunc, Wiesbaden, Germany) were coated overnight at 48C with 100 ml per well of 0.1 M NaHCO 3 buffer (pH 8.0) containing 0.1 mg of antigen (pIII-215 epitope fusion protein, pIII-Yol fusion protein, BSA or BSA-phOx, respectively). The fusion protein pIII215 represents a soluble moiety of phage fd gene product III consisting of the amino-terminal 371 amino acids extended by a tag consisting of the 215-epitope DYGPESRGFVENSYLAGL (Konter-

mann et al., 1995). An analogous construct of pIII fused to the Yol 1 / 34 epitope (pIII-Yol) served as a specificity control. The recombinant pIII fusion proteins were expressed in E. coli and purified by ¨ metal affinity chromatography (Breitling and Dubel, unpublished). Phoxylated BSA was prepared by incubating a 20-fold molar excess of 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx, Sigma, Deisenhofen, Germany) with BSA (Sigma) in 0.1 M NaCO 3 (pH 8.5) for 2 h. To remove uncoupled 4-ethoxy-methylene-2-phenyl-2-oxazoline-5one, the reaction mixture was dialyzed twice against PBS. Unspecific antibody binding was blocked with 400 ml per well of 2% (w / v) skim milk powder (Oxoid, Basingstoke, UK) in PBS (MPBS) for 3 h at RT. A 100 ml volume of crude periplasmic extract containing the antibody constructs (diluted 1 / 2 in MPBS) was added per well. The mouse monoclonal antibody Myc1-9E10 (1 / 1000 in MPBST) was used for immunodetection. Subsequently, HRP-conjugated goat anti-mouse antibodies were applied (1 / 2000 in MPBST), before 3,39,5,59-tetramethylbenzidine (TMB, Sigma-Aldrich, Steinheim, Germany) was used for the detection of bound enzymatic activity. The anti-215 mouse monoclonal antibody ARNA3 (Progen, Heidelberg, Germany) was used as a positive control.

3. Results

3.1. Vector constructs In order to compare the effects of an unpaired cysteine on the expression of different antibody fragments, we generated vector constructs expressing a single-chain Fv antibody, a scFv–streptavidin fusion protein and two disulfide-bond-stabilized Fv antibody fragments (Fig. 1, Table 1). For the disulfide stabilization of the two dsFv antibody fragments, different positions in conserved, complementary framework positions distant from the CDRs were mutated to cysteine, as described by Reiter et al. (1995). The pOPE vector backbone was designed for production of functional antibody fragments in E. ¨ coli (Dubel et al., 1992, 1993). Its derivatives share a strong synthetic promotor, the pelB leader sequence

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

107

Table 1 Vector properties and their effects on yield and purity of expressed antibody fusion proteins. Vector

Fusion protein

VH .VL linker

Tags SerA

Yol

cmyc

pOPE51-215(Yol) pOPE101-215(Yol) pOPE511-215 pOPE111-215 pSTE-215(GS) pSTE2-215 (Yol) pOPE511-phOx pOPE111-phOx

scFv215 scFv215 dsFv215 dsFv215 scFv215–strep scFv215–strep dsFv phOx dsFv phOx

Yol Yol 2 2 GS Yol 2 2

1 1 1 1 1 1 2 2

1 1 1 1 2 1 1 1

1 1 1 1 1 1 1 1

(Lei et al., 1987) for bacterial secretion of the antibody fragments into the periplasmic space and a carboxy terminal sequence tag for detection and ¨ IMAC purification of the fusion proteins (Dubel et al., 1992, 1993). In pOPE51, pOPE511 and pSTE, the sequence tag encodes the peptide ‘EEKLISEEDL’ which is the epitope of the mouse monoclonal antibody Myc1-9E10 (Evan et al., 1985) followed by an unpaired cysteine and five histidine residues. The vector pOPE51-215(Yol) encodes for the single-chain Fv antibody fragment scFv 215(Yol) binding to Drosophila melanogaster RNA Polymer¨ ase II (Kramer et al., 1980; Kontermann et al., 1995), and pOPE511-215 for the disulfide bondstabilized Fv 215 antibody fragment derived thereof. ¨ pSTE-215(GS) (Dubel et al., 1995, Genebank accession no. Y18290) encodes for a scFv 215–streptavidin fusion protein. To obtain pOPE511-phOx encoding for the disulfide-bond-stabilized Fv anti-phOx antibody fragment, the VH and VL domain coding regions of pOPE51215(Yol) were exchanged by the respective coding insert of the scFv phOx antibody fragment (Marks et al., 1992), and Lys(105) of the VH domain and Ala(43) of the VL domain were mutated to cysteine. All of the constructs described so far carry an unpaired cysteine. The influence of this cysteine on antibody activity, solubility and yield was assessed by constructing a derivative of each of the vectors in which the two amino acids CysIle in front of the carboxy terminal His-tag were exchanged to SerHis. A summary of the key features of the eight vectors employed in this study is given in Fig. 1 and Table 1.

His (aa)

Unpaired Cys

Yield (mg / l?OD 600 )

5 6 5 6 5 6 5 6

1 2 1 2 1 2 1 2

0.07 0.32 0.21 0.92 ,0.01 0.03 0.06 0.31

Increase of yield (x-fold) 4–5 4–5 .3 5

Purity after Ni-NTA-affinity chromatography Poor Excellent Poor Excellent Poor Poor Poor Excellent

3.2. Expression To confirm the expression of the different antibody constructs, their production in E. coli XL1-blue cells transformed with one of the plasmids described in Fig. 1 were induced under optimized conditions for cleavage of the pelB signal sequence and secretion of functional antibody fragments into the perip¨ lasmic space (Dubel et al., 1992). Total cell lysates of the induced and non-induced samples were analyzed by electrophoresis on 12% polyacrylamide SDS gels (Fig. 2A) followed by immunoblotting. Under induced conditions, two bands corresponding to the complete translation product (calc. mm 31.7 kDa) and the processed Fv fragment of scFv 215(Yol) (calc. mm 29.6 kDa) were detected with mAb Myc1-9E10 (Fig. 2B). Diluted samples of the cell lysates demonstrate that in case of the scFv fragment carrying the C-terminal cysteine, the main part of scFv fragments remained unprocessed. Under the same conditions, the main portion of the respective scFv 215 antibody fragment without the unpaired cysteine were processed to lower molecular weight (Fig. 2B, insert). Two bands were also detected for the VL domain of dsFv 215 carrying an unpaired cysteine (non-processed VL domain: calc. mm 17.0 kDa; processed VL domain: calc. mm 14.8 kDa). In this case, however, the major fraction of this protein appeared to be processed. All of the expressed VL domains of the dsFv 215 fragment without an unpaired cysteine as well as the VL domains of the two dsFv phOx fragments (nonprocessed VL domain: calc. mm 18.3 kDa; processed VL domain: calc. mm 16.1 kDa) seem to be com-

108

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

Fig. 2. E. coli expression of the antibody fragments. (A) SDS–PAGE (12%, coomassie stain) of total bacterial cell lysates before and after 3-h induction with IPTG. (B) Immunoblot of total bacterial cell lysates before and after 3-h induction with IPTG stained with mAb Myc1-9E10. (C) Immunoblot of total bacterial cell lysates before and after 3-h induction with IPTG stained with rabbit serum ‘A’.

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

pletely processed since only one band corresponding to these proteins could be detected. Two bands of almost equal intensity were detected representing the complete translation products and the processed proteins of the two scFv–streptavidin fusion proteins scFv 215(GS)–strep carrying the C-terminal cysteine (non-processed scFv–strep: calc. mm 44.2 kDa; processed scFv–strep: calc. mm 42.0 kDa) and scFv 215(Yol)–strep without the unpaired cysteine (nonprocessed scFv–strep: calc. mm 45.3 kDa; processed scFv–strep: calc. mm 43.1 kDa). In both cases, additional fragments with lower molecular mass were detected. To confirm the observations in respect of the processing, immunoblots were further analyzed with rabbit serum ‘A’, which recognizes the sequence QVQLQ located at the N-terminus of the VH (215) ¨ domain (Dubel et al., 1992). Serum ‘A’ does not react with QVQLQ when presented as an internal epitope and can therefore be used to monitor the proteolytic removal of the signal sequence from the antibody fragments. The VH domain of the dsFv

109

phOx antibody fragments (processed VH domain: calc. mm 15.3 kDa) does not contain the QVQLQ epitope and cannot be detected in this approach. For the VH domain of each Fv 215 derivative, expression and the correct processing of a substantial fraction of the protein could be confirmed (Fig. 2C). For the scFv 215 derivatives, the amounts of processed antibody fragments without C-terminal cysteine were significantly increased when compared to the amounts detected for antibody fragments carrying a C-terminal cysteine, in particular in the case of the scFv 215–streptavidin fusion proteins (Fig. 2C). These observations confirm the results obtained from immunoblotting using mAb Myc1-9E10. The expression and the processing of the VH domains of each dsFv 215 construct (processed VH domain: calc. mm 14.7 kDa) does not seem to be significantly affected by the different processing of their corresponding VL domains (Fig. 2C). To prove the production of soluble antibody fragments, periplasmic extracts have been prepared and analyzed by SDS–PAGE followed by immunoblotting using mouse monoclonal antibody Myc19E10 (Fig. 3). Under reducing conditions, the detected amounts of antibody fragments without an unpaired cysteine were significantly higher than for the respective antibody fragments carrying the unpaired cysteine (Fig. 3A). A distinct band was detected under non-reducing conditions in samples containing the Fv constructs without an unpaired cysteine. No antibody bands were visible in samples containing the constructs carrying an unpaired cysteine. When compared to the data obtained under reducing conditions from the same samples, this might be an indication that the antibody fragments carrying an additional, unpaired cysteine residue have formed disulfide-bond-stabilized aggregates with E. coli proteins and are thus too diluted to be detected as a distinct band under non-reducing conditions (Fig. 3B).

3.3. Effect of unpaired cysteines on antigen binding

Fig. 3. Immunoblot of periplasmic extracts stained with mAB Myc1-9E10 after SDS–PAGE (12%) under reduced (A) and nonreduced (B) conditions.

To assess the influence of the unpaired cysteine on antigen-binding activity and specificity of the antibody constructs, crude periplasmic extracts were prepared, diluted in PBS and subjected to ELISA. The results showed that all of the different antibody

110

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

constructs retained the antigen-binding specificity of the original antibodies (Fig. 4). The scFv–streptavidin fusion proteins showed a significant lower antibody activity than the constructs without streptavidin domain, which was expected due to their lower periplasmic expression. All four periplasmic extracts containing the antibody constructs carrying the unpaired cysteine showed a significant lower antibody activity in comparison to the extracts containing the respective constructs without an unpaired cysteine (Fig. 4). Despite the amount of total product does not vary accordingly within each pair of constructs (Fig. 2A, B), the yield of processed protein (Fig. 2C) is increased for the constructs without unpaired cysteine. This suggests that the differences in the antigen-binding ELISA signal are rather a result of the increase in the yield of functional antibodies than affinity changes introduced by the two amino acid exchanges at the carboxy-terminus of the antibody constructs.

3.4. Comparison of total yields after standardized IMAC enrichment The previous results have underlined the signifi-

cance of the unpaired cysteine in rapid assays using crude periplasmic extracts. The observed advantage of constructs without unpaired cysteines were expected to be due to the total amount of soluble products. To test this hypothesis, the overall yields of the eight antibody constructs were determined after standard lab scale expression and enrichment from periplasmic extracts by IMAC (Lilius et al., 1991). The total yields varied over a wide range, from lower than 0.01 mg / l?OD 600 for the scFv–streptavidin fusion protein carrying the additional cysteine to up to 0.92 mg / l?OD 600 ) of culture medium for the dsFv 215 antibody fragment without an unpaired cysteine (Table 1). For each of the four different antibody constructs, an at least three-fold increase in yield of soluble protein was observed after mutation of the unpaired cysteine. The SDS–PAGE analysis of eluted fractions obtained after IMAC (Fig. 5) further revealed that the mutation of the unpaired cysteine had a significant effect on the purity of the antibody preparations. The comparison of typical fractions obtained after IMAC showed that the preparations containing antibody constructs carrying an unpaired cysteine were significantly contaminated by E. coli proteins. These

Fig. 4. ELISA for antigen-binding activity of crude periplasmic extracts before and after 3-h induction with IPTG. Fv 215-antigen binding activity was determined using the pIII–215 epitope fusion protein as antigen. An analogous construct of pIII fused to the Yol 1 / 34 epitope (pIII-Yol) served as a specificity control. Phoxylated BSA was used to determine phOx binding activity. BSA was used as a negative control. Bound antibody constructs were detected using mAb Myc1-9E10 and anti-mouse immunoglobulin–HRP conjugates.

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

Fig. 5. SDS–PAGE analysis (12%, coomassie stain) of IMACfractions containing the different antibody constructs.

contaminant proteins were found to be dramatically less abundant in the preparations of the respective homologues without the unpaired cysteine. This effect is less pronounced only in the case of the scFv–streptavidin fusion proteins. The presence of several different contaminating bands suggests that these proteins might form disulfide-bond-stabilized aggregates with the antibody fragments carrying an additional, unpaired cysteine and are therefore coenriched by IMAC. These results explain why the improvement in yield by removal of the unpaired cysteine is even more pronounced after IMAC purification when compared to the differences of activity found when comparing the crude periplasmic extracts.

4. Discussion The removal of unpaired cysteines close to the carboxy-terminus of four different designs of antibody fragments expressed in E. coli resulted in a significant improval of (i) functional signal in an-

111

tigen ELISA using crude periplasmic preparations, (ii) purity after IMAC enrichment and (iii) overall yield. These factors are of particular importance to facilitate small scale analysis of clones, but might be of further value for fermenter production as well. Interestingly, the amount of improvement was almost identical for all four protein designs employed in this study. This holds true independent of the presence or absence of additional cysteines engineered for disulfide stabilisation of the dsFv fragments, thus indicating that the cysteines at the VH –VL -Interface were much more rapidly oxidised in the process of secretion and folding. Further, the improvement factor achieved by the removal of the unpaired cysteine was similar for different fusion proteins despite differences in total yield of more than an order of magnitude. Similar results were obtained with the same constructs transfected to another strain of E. coli (TG1, data not shown), despite significant differences in overall yield and growth rate between E. coli TG1 and E. coli XL1-blue. For another pair of closely related vectors with / without unpaired ¨ cysteine (pOPE40 vs. pOPE90, Dubel et al., 1993), a similar effect was observed when using E. coli JM109 (data not shown). However, a careful evaluation of various other E. coli strains and the use of culture media with modified redox conditions might allow the decrease of the influence of the unpaired cysteine, which would be of particular value for the production of antibody constructs where the unpaired cysteine cannot be omitted. Taken together, the observations suggest a general limiting step underlying the decrease of secretion by an unpaired cysteine. The major factor in this process seems to be an elimination of the possibility to form covalent aggregates with other proteins containing cysteines which are accessible in the periplasmic space. This is indicated by the significant number of contaminating bands observed in the respective preparations after IMAC under identical conditions, when compared to the constructs without the unpaired cysteine. The improvement in purity after IMAC seems to be related to this effect rather than to the additional histidine in the constructs without the unpaired cysteine, since IMAC conditions were not stringent and the column material was employed in a vast stoichiometrical excess. This is supported by the observed improvement in the

112

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

content of functional antibody fragments in samples before IMAC purification. The formation of disulfide bonds with other proteins accessible in the periplasmic space could be an unspecific process or part of a specific quality control. It has been shown that the C-terminal cysteine of the secretory m-heavy chain (residue Cys575) plays a crucial role in retention and degradation of IgM subunits in the endoplasmic reticulum of eucaryotic cells (Sitia et al., 1990). Secretion of m2L2 monomers can be induced in both B and plasma cells by mutating the C-terminal cysteine (Sitia et al., 1990) or by altering the intracellular redox potential with b-mercaptoethanol (Alberini et al., 1990). On the other hand, appending the 20 C-terminal residues of secretory m-chains to unrelated proteins is sufficient to induce Cys575-dependent pre-Golgi retention and degradation of the chimeric molecules (Sitia et al., 1990; Fra et al., 1993). The role of free thiol groups in preventing the unhindered transport of proteins through the secretory pathway has thus been proposed (Alberini et al., 1990; Sitia et al., 1990) and has been further confirmed for the regulation of the intracellular transport of acetylcholinesterase (Kerem et al., 1993) and of several membrane receptors with a cysteinerich motif (Yamamoto et al., 1986; Esser and Russell, 1988; Olson and Lane, 1989; Bauskin et al., 1991; Collesi et al., 1996). Under this scheme, assembly intermediates or misfolded proteins may interact through their thiol groups between themselves and / or with other proteins of the endoplasmic reticulum. An analogous quality control mechanism utilizing unpaired cysteines might be present in the periplasm of E. coli. Despite that the molecular mechanism underlying the process has yet to be elucidated, the strategy of obviating unpaired cysteines may be beneficial for the improvement of yields in other recombinant production systems in E. coli and other bacteria as well.

Acknowledgements A.S. was supported by the Graduiertenkolleg 388 of the Deutsche Forschungsgemeinschaft. C.H.W.

was supported by Grant BA384 / 19 of the Deutsche Forschungsgemeinschaft.

References Adams, G.P., McCartney, J.E., Tai, M.-S., Oppermann, H., Huston, J.S., Stafford, W.F., Bookman, M.A., Fand, I., Houston, L.L., Weiner, L.M., 1993. Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-cerb B-2 single-chain Fv. Cancer Res. 53, 4026. Alberini, C.M., Bet, P., Milstein, C., Sitia, R., 1990. Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents. Nature 347, 485. Bauskin, A.R., Alkalay, I., Ben-Neriah, Y., 1991. Redox regulation of a protein tyrosine kinase in the endoplasmic reticulum. Cell 66, 685. Better, M., Bernhard, S.L., Lei, S.-P., Fishwild, D.M., Lane, J.A., Carroll, S.F. et al., 1993. Potent anti-CD5 ricin A chain immunoconjugates from bacterially produced Fab9 and F(ab9) 2 . Proc. Natl. Acad. Sci. USA 90, 457. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.-M. et al., 1988. Single-chain antigen-binding proteins. Science 242, 423. ¨ Breitling, F., Dubel, S., 1997. Cloning and expression of single chain fragments (scFv) from mouse and rat hybridomas. In: Reischl, U. (Ed.), Molecular Diagnosis of Infectious Diseases. Methods in Molecular Medicine, Vol. 13. Humana Press, Totowa, NY. ¨ Breitling, F., Dubel, S., 2000. Recombinant Antibodies. John Wiley and Sons, New York. Brinkmann, U., Gallo, M., Brinkmann, E., Kunwar, S., Pastan, I., 1993. A recombinant immunotoxin that is active on prostate cancer cells and that is composed of the Fv region of monoclonal antibody PR1 and a truncated form of Pseudomonas exotoxin. Proc. Natl. Acad. Sci. USA 90, 7538. Carter, P., Kelley, R.F., Rodrigues, M.L., Snedecor, B., Covarrubias, M., Velligan, M. et al., 1992. High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Biotech. 10, 163. Colcher, D., Pavlinkova, G., Beresford, G., Booth, B.J., Batra, S.K., 1999. Single-chain antibodies in pancreatic cancer. Ann. NY Acad. Sci. 880, 263. Collesi, C., Santoro, M.M., Gaudino, G., Comoglio, P.M., 1996. A splicing variant of the RON transcript induces constitutive tyrosine kinase activity and an invasive phenotype. Mol. Cell Biol. 16, 5518. Cumber, A.J., Ward, E.S., Winter, G., Parnell, G., Wawrzynczak, E.J., 1992. Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate. J. Immun. 149, 120. Debinski, W., Pastan, I., 1995. Recombinant F(ab9) C242-Pseudomonas exotoxin, but not the whole antibody-based immunotoxin, causes regression of a human colorectal tumor xenograft. Clin. Cancer Res. 1 (9), 1015.

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114 ¨ Dubel, S., Breitling, F., Fuchs, P., Klewinghaus, I., Little, M., 1993. A family of vectors for surface display and production of antibodies. Gene 128, 97. ¨ Dubel, S., Breitling, F., Klewinghaus, I., Little, M., 1992. Regulated secretion and purification of recombinant antibodies in E. coli. Cell. Biophys. 21, 69. ¨ Dubel, S., Breitling, F., Kontermann, R., Schmidt, T., Skerra, A., Little, M., 1995. Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J. Immunol. Meth. 178, 201. Esser, V., Russell, D.W., 1988. Transport-deficient mutations in the low density lipoprotein receptor. Alterations in the cysteinerich and cysteine-poor regions of the protein block intracellular transport. J. Biol. Chem. 263, 13276. Evan, G.I., Lewis, G.K., Ramsay, G., Bishop, J.M., 1985. Isolation of monoclonal antibodies specific for human c-myc protooncogene product. Mol. Cell Biol. 5, 3610. Fra, A.M., Fagioli, C., Finazzi, D., Sitia, R., Alberini, C.M., 1993. Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation. EMBO J. 12, 4755. Friedman, P.N., Chace, D.F., Trail, P.A., Siegall, C.B., 1993. Antitumor activity of a single-chain immunotoxin BR96 sFvPE40 against established breast and lung tumor xenografts. J. Immun. 150, 3054. Goshorn, S.C., Svensson, H.P., Kerr, D.E., Somerville, J.E., Senter, P.D., Fell, H.P., 1993. Genetic construction, expression, and characterization of a single-chain anti-carcinoma antibody fused to b-lactamase. Cancer Res. 53, 2123. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.-S., Novotny, J., Margolies, M.N. et al., 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879. Kerem, A., Kronman, C., Bar-Nun, S., Shafferman, A., Velan, B., 1993. Interrelations between assembly and secretion of recombinant human acetylcholinesterase. J. Biol. Chem. 268, 180. ¨ Kipriyanov, S.M., Dubel, S., Breitling, F., Kontermann, R.E., Heymann, S., Little, M., 1995. Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines. Cell. Biophys. 26, 187–204. ¨ Kipriyanov, S., Dubel, S., Breitling, F., Kontermann, R.E., Little, M., 1994. Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: ]Production of bivalent and biotinylated mini-antibodies. Molec. Immun. 31, 1047. Kontermann, R.E., Liu, Z., Schulze, R.A., Sommer, K.A., ¨ Queitsch, I., Dubel, S. et al., 1995. Characterization of the epitope recognised by a monoclonal antibody directed against the largest subunit of Drosophila RNA polymerase II. Biol. Chem. Hoppe-Seyler 376, 473. ¨ Kramer, A., Haars, R., Kabisch, R., Will, H., Bautz, F.A., Bautz, E.K.F., 1980. Monoclonal Antibody Directed Against RNA Polymerase II of Drosophila melanogaster. Molec. gen. Genet. 180, 193. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680. Lei, S.-P., Lin, H.-C., Wang, S.-S., Callaway, J., Wilcox, G., 1987.

113

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J. Bacteriol. 169, 4379. ¨ Li, J.Y., Sugimura, K., Boado, R.J., Lee, H.J., Zhang, C., Dubel, S. et al., 1999. Genetically engineered brain drug delivery vectors: cloning, expression, and in vivo application of an anti-transferrin receptor single chain antibody–streptavidin fusion gene and protein. Protein Eng. 12, 787. Liberatore, M., Neri, D., Neri, G., Pini, A., Iurilli, A.P., Ponzo, F. et al., 1995. Efficient one-step direct labelling of recombinant antibodies with technetium-99m. Eur. J. Nucl. Med. 22, 1326. ¨ Lilius, G., Person, M., Bulow, L., Mosbach, K., 1991. Metal affinity precipitation of proteins carrying genetically attached polyhistidine tails. Eur. J. Biochem. 198, 499. Marks, J.D., Griffiths, A.D., Malmqvist, M., Clackson, T.P., Bye, J.M., Winter, G., 1992. By-passing immunization: building high affinity human antibodies by chain shuffling. Biotech. 10, 779. Milenic, D.E., Yokota, T., Filpula, D.R., Finkelman, M.A.J., Dodd, S.W., Wood, J.F. et al., 1991. Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49. Cancer Res. 51, 6363. Olson, T.S., Lane, M.D., 1989. A common mechanism for posttranslational activation of plasma membrane receptors? FASEB J. 3, 1618. Reiter, Y., Brinkmann, U., Jung, S.H., Pastan, I., Lee, B., 1995. Disulfide stabilization of antibody Fv: computer predictions and experimental evaluation. Protein Eng. 8, 1323. Rodrigues, M.L., Snedecor, B., Chen, C., Wong, W.L.T., Garg, S., Blank, G.S. et al., 1993. Engineering Fab9 fragments for efficient F(ab9) 2 formation in Escherichia coli and for improved in vitro stability. J. Immun. 151, 6954. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sitia, R., Neuberger, M., Alberini, C., Bet, P., Fra, A., Valetti, C. et al., 1990. Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine. Cell 60, 781. Towbin, H., Staehelin, T., Gordon, I., 1980. Electrophoretic transfer of protein from polyacrylamid gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. Verhaar, M.J., Keep, P.A., Hawkins, R.E., Robson, L., Casey, J.L., Pedley, B. et al., 1996. Technetium-99m radiolabeling using a phage-derived single-chain Fv with a C-terminal cysteine. Nucl. Med. 37, 868. Waibel, R., Alberto, R., Willuda, J., Finnern, R., Schibli, R., Stichelberger, A. et al., 1999. Stable one-step technetium-99m labeling of his-tagged recombinant proteins with a novel Tc(I)-carbonyl complex. Nat. Biotechnol. 17, 897. Wang, D., Li, Q., Hudson, W., Berven, E., Uckun, F., Kersey, J.H., 1997. Generation and characterization of an anti-CD19 singlechain Fv immunotoxin composed of C-terminal disulfidelinked dgRTA. Bioconjug. Chem. 8, 878. Whitlow, M., Fipula, D., 1991. Single-chain Fv proteins and their fusion proteins. Methods: A Companion to Meth. Enzym. 2, 97.

114

A. Schmiedl et al. / Journal of Immunological Methods 242 (2000) 101 – 114

Wunderlich, M., Glockshuber, R., 1993a. Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli. Protein Sci. 2, 717. Wunderlich, M., Glockshuber, R., 1993b. In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA). J. Biol. Chem. 268, 24547. Yamamoto, T., Bishop, R.W., Brown, M.S., Goldstein, J.L.,

Russell, D.W., 1986. Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit. Science 232, 1230. Yokota, T., Milenic, D.E., Whitlow, M., Schlom, J., 1992. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res. 52, 3402.