Single chain Fab (scFab) fragment
- Michael Hust†1,
- Thomas Jostock†1, 2,
- Christian Menzel1, 3,
- Bernd Voedisch1,
- Anja Mohr1, 4,
- Mariam Brenneis1, 5,
- Martina I Kirsch1,
- Doris Meier1 and
- Stefan Dübel1Email author
© Hust et al; licensee BioMed Central Ltd. 2007
Received: 13 November 2006
Accepted: 08 March 2007
Published: 08 March 2007
The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.
Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised.
A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.
The production of functional antibody fragments in E. coli was first described by Skerra and Plückthun . Key to this success was production in the periplasm, where the oxidizing environment allows the formation of disulphide bonds. Later, the linkage of the variable regions by a 15–25 amino acid linker of both Fv chains improved the expression of antibody fragments in E. coli [2, 3]. However, these so called single chain fragment variable (scFv) have the tendency to form aggregates and are relatively unstable over longer periods of time . Furthermore, some scFvs show a reduced affinity of up to one order of magnitude compared to the corresponding Fab fragments . Only in rare cases have scFvs with a higher affinity than the associated Fab been found . Because they are double the molecular size, and require the production and connection of two different polypeptides with a disulphide bond, folding and assembly of Fab fragments in the periplasm of E. coli is less efficient than for scFvs . A further disadvantage of Fab fragments is the tendency of light chains to form homo-dimers, which are known as Bence Jones proteins [8, 9]. Advantages of Fab fragments are their high stability in long term storage  and their compatibility with common detection antisera without the need for a re-engineering step . An antibody design combining stability and assay compatibility of Fab fragments with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.
Currently, most recombinant antibody fragments are generated by antibody phage display. Phage display technology is based on the groundbreaking work of Smith . Antibody phage display was first described by Huse et al.  for the phage Lambda and by McCafferty et al.  for the M13 phage. However, practical use was only achieved by uncoupling antibody gene replication and expression from the phage life cycle by locating them on a separate plasmid (phagemid) to improve genetic stability, handling, and screening of antibody libraries [15–18]. So far, naive scFv antibody libraries with a theoretical diversity of up to 1011 independent clones  and Fab antibody libraries with a size of 3.5 × 1010 clones  have been generated as molecular repertoires for phage display selections (overview given by Hust and Dübel ). Antibody phage display is a key technology for the generation of human recombinant antibody fragments for therapy and diagnostics .
Here, we demonstrate, that the introduction of a polypeptide linker between Fd fragment and light chain, resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of antibody fragments. We tested the impact of various linker lengths and the presence of the disulphide bond on both the display efficiency on phage and the yield of soluble antibody fragment production in both prokaryotic and eukaryotic cells.
Construction of the pHAL vectors with different antibody formats
Production of antibody phage
SDS-PAGE analysis of antibody phage
Antigen ELISA of antibody phage
Antigen binding of phage presenting the different antibody formats was determined by ELISA on lysozyme. 5 × 108 phage/well were applied when M13K07 was used for phage rescue, whereas 107 phage/well were used after packaging with Hyperphage to compensate the known differences in antibody presentation efficiency of both systems [9, 26, 27].
Production of soluble antibody variants using the phage display vector pHAL1
Production of soluble antibody variants using the E. coliexpression vector pOPE101-XP
Afterwards, the antibody fragments were purified by IMAC followed by protein L affinity chromatography. Purified samples of scFv, Fab, scFab and scFabΔC were analyzed by surface plasmon resonance (SPR) to determine the affinity of the different antibody variants. Following affinities were measured: scFv: 1,04 × 10-8 M; Fab 1,91 × 10-8 M; scFab: 3,46 × 10-8 M; scFabΔC: 4,45 × 10-8 M. All measured affinities were in a similiar range, indicating that the newly introduced linker does not inhibit or impair binding to the antigen.
Production of scFab in P. pastoris
So far, two antibody formats, Fabs and scFvs, have dominated antibody phage display and the production of soluble antibody fragments in E. coli. In this study, to test whether it is possible to combine the advantages of both formats, various single chain Fab (scFab) constructs were generated and evaluated. In scFab, the light chain and the Fd fragment of a Fab fragment are connected by a polypeptide linker derived from the pIII of filamentous phage M13. Three scFab variants with different linker length from 32 to 36 amino acids and one 34 amino acid variant with a deleted intermolecular disulphide bond (scFabΔC) were compared to scFv and Fab using the lysozyme binding antibody D1.3 [23, 24] as a model antibody. In mammalian cells, an antibody construct using a 30 aa Gly-Ser linker to combine the HC fragment and the LC has succesfully been produced to generate a single chain IgG . The scFab designs presented in this study, in contrast, were evaluated both for their compatibility with antibody phage display and production of soluble antibody fragments in microbial production systems. For the latter, in the prokaryotic expression system E. coli, both the phage display phagemid pHAL1  and the E. coli expression vector pOPE101  were tested. Additionally, the production in the eukaryotic expression system using the yeast P. pastoris was demonstrated.
The applicability of the new antibody designs for antibody phage display is of major importance, as antibody phage display is currently a major technology for the generation of human antibodies for research, diagnostic and therapy (for reviews see [11, 22, 30]. Display of the different antibody formats on phage was evaluated using the two most frequently used helperphage, the standard M13K07 and the pIII deficient Hyperphage [26, 27, 31], which enforces polyvalent display on filamentous phage. As described before, when the helperphage M13K07 was used, phage yields were higher compared to Hyperphage. This results from the lack of pIII when Hyperphage is used, leaving the less well produced pIII fusion with the antibody fragment as the sole source of this coat protein. The fusion of pIII to different scFab variants did not significantly inhibit phage assembly with both helperphage, indicating that all of the tested antibody designs are compatible with phage assembly. The length of the polypeptide linker used in the scFab variants had no influence on phage packaging and antigen binding. In contrast, significant benefit was achieved by removing the cysteins from the carboxyterminus of Fd and LC, adding to the body of evidence on the adverse effect of cysteins on the production of proteins in E. coli .
The phage display vector pHAL1 can also be used for production of soluble antibody fragments. This facilitates the analysis of individual clones after the panning procedure, because no recloning steps are necessary. Fractions of soluble antibodies are analysed by ELISA on enzyme rating the production and binding of the antibody fragments in combination. Here, the scFv and scFabΔC designs showed superior antigen binding compared to Fab and scFab. Consistent to the results obtained from the antigen ELISA using antibody phage, the length of the polypeptide linker used in the scFab variants had no influence on the antigen binding. Again, the cysteins connecting CH1 and CL chains in Fab and scFabs had also a negative effect on the production of functional antibody fragments. For further analysis, the scFv, Fab, scFab with 34 amino acid linker and the scFabΔC were produced in E. coli using the expression vector pOPE101  and analysed in detail. The measured affinities by surface plasmon resonance (SPR) showed similiar results for all antibody variants and are in contrast to the results of the functional ELISAs. The scFab variants were found as monomers, dimers and multimers, the scFv was only found as dimer, and the Fab occured only as monomer. The multimeric and dimeric antibody fragment fractions showed better antigen binding compared to the monomeric states, indicating the expected avidity benefit. The results obtained for E. coli could be reproduced by analysis of scFabΔC produced in Pichia pastoris. This yeast expression system is of growing importance for antibody production because a glycosylation of full size antibodies identical to that in humans was achieved with glycoengineered P. pastoris lines .
The novel antibody design scFabΔC can be used for the production of soluble antibody fragments in E. coli and Pichia pastoris and is compatible with the phage display technology. In particular, it allows the use of common standard detection secondary sera, thus avoiding additional anti-tag antibody. Further, reagents with high apparent affinity can be created due to the avidity effects of DiFabodies without any subcloning steps. This advantage is of particular value for high throughput proteome binder generation projects.
pHAL scFab phage display vectors
Standard cloning procedures were performed according to Sambrook and Russell . The pHAL vectors containing the different antibody formats were derived from the phagemid vector pHAL1 . This vector allows both antibody phage display and expression of soluble antibody fragments due to an amber stop codon between the antibody coding region and gIII.
Construction of pHAL1-D1.3scFv (Fig. 1) was done by assembly PCR. VL fragment and VH fragment DNA were amplified from pHAL1-D1.3Fab  and assembled by a third PCR. The PCR product was cloned into the NheI and NotI site of pHAL1. For the construction of pHAL1-D1.3scFab, the coding region of the 34 amino acids glycine-serine linker (Fig. 1b) was amplified from the M13 geneIII (gIII) of pHAL1-D1.3Fab, by assembly PCR. The PCR product was cloned into the BssHII and PstI site of pHAL1-D1.3Fab. Two codons were deleted by PCR primer directed mutagenesis for the construction of pHAL1-D1.3scFab-2 and the PCR product was cloned into the BssHII and PstI site of pHAL1-D1.3scFab. For the construction of pHAL1-D1.3scFab+2, two codons were added and the PCR product was cloned into the BssHII and PstI site as described. Both cysteines which connect both antibody chains were deleted to construct pHAL1D1.3 C. The cysteine of the LC was deleted by PCR primer directed mutagenesis and assembled with by a second PCR in which the cysteine of the Fd fragment was deleted. The PCR-product was cloned into the BssHII and NotI site. Transformations of XL1-Blue MRF' (Stratagene, Amsterdam, Netherlands) with the constructs were done by electroporation according to the manufacturers' instruction. All steps in vector construction and antibody cloning were confirmed by sequencing of the affected regions.
pOPE101 scFab production vectors
The D1.3scFv, D1.3Fab and D1.3scFabΔC DNA was were cloned from the vectors pHAL1-D1.3scFv, pHAL1-D1.3Fab and pHAL1-D1.3scFabΔC into the E. coli expression vector pOPE101-XP using the restriction sites EcoRI and NotI. pOPE101-XP is a modification of pOPE101-215yol  with the scFv coding sequence replaced by the short DNA sequence containing stop codons in all three reading frames. This vector contains a myc-tag and a his-tag for detection and purification.
pPICZ-α scFab vectors for the expression in P. pastoris
D1.3scFabΔC DNA was cloned from pOPE101-D1.3Fab into the P. pastoris expression vector pPICZaA (Invitrogen, Karlsruhe, Germany) using the restriction sites XbaI and XhoI.
Antibody phage production
50 mL 2xTY medium  + 100 μg/mL ampicillin + 100 mM glucose were inoculated with an overnight culture to O.D.600 = 0.1. Bacteria were grown to O.D.600 = 0.4 – 0.5 at 37°C and 250 rpm. 2 mL XL1-Blue MRF' (Stratagene) (~1 × 109 bacteria) were infected with 2 × 1010 helper phage M13K07 (Stratagene) or Hyperphage [26, 27, 31], incubated at 37°C for 30 min without shaking, followed by 30 min at 250 rpm. The infected cells were harvested by centrifugation for 10 min at 3220 × g and the pellet was resuspended in 30 mL 2xTY + 100 μg/mL ampicillin + 50 μg/mL kanamycin. Phage were produced at 30°C and 250 rpm for 16 h. Cells were pelleted for 10 min at 10000 × g. Phage in the supernatant were precipitated with 1/5 volume of 20% PEG/2.5 M NaCl solution for 1 h on ice with gentle shaking before being pelleted 1 h at 10000 × g at 4°C. The precipitated phage were resuspended in 10 mL phage dilution buffer (10 mM TRIS, 20 mM NaCl, 2 mM EDTA, the pH was adjusted to pH 7.5 with HCl), precipitated again with 1/5 volume of PEG solution as given above for 20 min on ice and pelleted 30 min at 10000 × g at 4°C. The precipitated phage were resuspended in 300 μL phage dilution buffer and cell debris was pelleted by additional centrifugation for 5 min at 15400 × g at 20°C. The supernatant containing the antibody phage were stored at 4°C. Phage titration (cfu/mL) was done according to Koch et al.  with one modification: the infected bacteria were pipetted directly onto LB agar plates, omitting nitrocellulose sheets.
Production of soluble antibody fragments using the phage display vector pHAL1
Soluble antibody fragments were produced in shaking flasks or microtiter plates (MTPs). For expression in shaking flasks, 100 mL 2xTY medium + 100 μg/mL ampicillin + 100 mM glucose were inoculated 1:20 with an overnight culture of XL1-Blue MRF' containing the appropriate vector and grown at 37°C and 250 rpm for 2 h. Bacteria were harvested by centrifugation for 20 min at 3900 × g. The pellet was resuspended in 100 mL 2xTY + 100 μg/ml Ampicillin + 20 μM IPTG at 30°C and 250 rpm overnight. 13 mL PBS (phosphate buffered saline ) containing 1% Tween20 were added and incubated at 30°C and 350 rpm for additional 3.5 h. Cells were separated from the antibody containing supernatant by centrifugation for 10 min at 7000 × g. For expression in MTPs 200 μl 2xTY medium + 100 μg/mL ampicillin + 100 mM glucose was inoculated with 10 μL overnight culture and grown at 37°C and 1400 rpm for 2 h. Bacteria were harvested by centrifugation for 10 min at 2500 × g. The pellet was resuspended in 200 μl 2xTY + 100 μg/mL Ampicillin + 20 μM IPTG at 30°C and 1400 rpm overnight. 50 μL PBS with 1% Tween20 were added and incubated at 30°C and 1400 rpm for additional 3.5 h. Cells were separated from the antibody containing supernatant by centrifugation for 10 min at 3200 × g.
Production of soluble antibody fragments using the vector pOPE101-XP
Various D1.3 antibody formats were produced in shaking flasks according to Dübel et al. . Briefly, 300 mL 2xTY + 100 μg/mL glucose + 100 μg/mL ampicillin were inoculated with an overnight culture yield to O.D.600 < 0.1 and cultured at 30°C and 250 rpm. The induction was started by adjusting to 50 μM IPTG at O.D.600 = 0.6 and shaken for 20 h. Bacteria were harvested by centrifugation for 10 min at 4400 × g at RT. Pellets were resuspended in 30 mL ice cold PE buffer, pH 8 (500 mM sucrose, 100 mM Tris, 1 mM EDTA) and incubated for 20 min on ice, interrupted by short vortexing every 5 min. Subsequently the bacteria were pelleted for 30 min at 30000 × g at 4°C. The supernatant (periplasmic fraction) was stored at -20°C. The pellet was resuspended in 30 mL ice-cold dH2O and incubated for 20 min on ice, interrupted by short vortexing every 5 min. Spheroblasts were centrifuged for 30 min at 30000 × g and 4°C. The supernatant (osmotic shock preparation) was stored at -20°C.
Production of scFab in P. pastoris
Transformation of P. pastoris (KM71) (Invitrogen, Karlsruhe, Germany) was done according to the manufacturers recommendations. Positive clones were selected over YPD-Zeocin (100 μg/mL). To further select transformed clones for good antibody secretion a screening by 96 deep well (Greiner, Flacht, Germany) expression was performed using 500 μL BMGY medium, pH 6.0  for 24 h at 30°C and 350 rpm. Protein synthesis was induced by changing the media to 500 μL BMMY, pH 6.0 containing 1% MeOH  and further incubation for 48 hours. Supernatant was harvested by centrifugation for 10 min, 1500 × g and analysed by antigen ELISA (using Protein L-HRP for detection). Identified clones showing high antibody secretion were expressed in 2 L shake flask containing 500 mL BMGY media which was inoculated to an OD600 0.5 and grown overnight. Protein production was induced by exchanging the media after 24 h with BMMY containing 1% MeOH and further incubation for 48 h, before collecting the supernatent by centrifugation for 10 min and 1500 × g.
Protein L purification of soluble antibody fragments
Soluble antibody fractions from E. coli and P. pastoris expression were purified by affinity chromatography using a Protein L column (Pierce, Bonn, Germany). After loading, the column was washed with 10 column volumes PBS, pH 7.4. Antibody fragments were eluted with glycine buffer 0.1 M, 0.15 M NaCl, pH 2.6 with immediate neutralization by TRIS/HCl buffer 1 M, pH 9.0. Fractions were stored at -20°C.
Immobilized metal affinity chromatography (IMAC) purification of soluble antibody fragments
Soluble antibody fragments were purified from E. coli derived material by affinity chromatography using IMAC. Chromatography using 1 mL Chelating Sepharose Fast Flow (Amersham Biosciences, Freiburg, Germany) was done according to the manufacturers' instruction using 10 mM imidazol containing buffer (20 mM Na2 HPO4, 0.5 M NaCl, 10 mM imidazol) when loading the beads with the protein solution, using 1× time 10 mM imidazol and 3× times 50 mM imidazol buffer for washing and PBS, pH 7.4 with 100 mM EDTA for the elution of the 6 × histidine tagged proteins.
Antigen binding ELISA
Microtiter plates (Costar, Cambridge, USA) were coated with 100 ng hen egg white lysozyme in 100 μL 0.1 M NaHCO3, pH 9.6 per well over night at 4°C. Coated wells were washed 3× with PBS and blocked with 2% (w/v) skim milk powder in PBS for 1.5 h at RT, followed by 3× washing with PBS. Antibody phage or soluble antibody fragments were diluted in 100 μL blocking solution and incubated for 1.5 h, followed by 5× washing with PBST (PBS + 0.1% (v/v) Tween 20). Bound antibody phage were detected with mAb anti-M13 conjugated with HRP (Amersham Biosciences, Freiburg, Germany) (1:5000), soluble antibody fragments were detected either with mAb mouse anti-Strep-tag (Qiagen, Hilden, Germany) (1:10000) and mAb goat anti-mouse conjugated with HRP (1:50000), with mAb anti-myc tag (9E10 hybridom) (1:25) and mAb goat anti-mouse conjugated with HRP (1:50000) or with protein L conjugated with HRP (Pierce) (1:10000) and visualised with TMB (3,3',5,5'-tetramethylbenzidine) substrate. The staining reaction was stopped by adding 100 μL 1 N sulphuric acid. The absorbances at 450 nm and scattered light at 620 nm were measured and the 620 nm reference was subtracted using a SUNRISE microtiter plate reader (Tecan, Crailsheim, Germany).
SDS-PAGE and Immunoblot
Antibody phage or soluble antibody fragments were separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked with 2% (w/v) skim milk powder in PBST for 1 h at RT. For the detection of antibody phage, the minor coat protein pIII was detected with 1:1000 diluted mouse mAb anti-pIII (Mobitec, Göttingen, Germany) for 1.5 h at RT, followed by 2× washing with PBST. Goat anti-mouse (Fc specific) (Sigma, Taufkirchen, Germany) conjugated with AP 1:1000 diluted was used for detection and visualised by NBT/BCIP. For the detection of soluble antibody fragments the mAb mouse anti-myc (supernatant 9E10 1:25) was used as primary antibody and goat anti-mouse conjugated with AP (Sigma, Taufkirchen, Germany) (1:2000) was used as secondary antibody.
Molecular sizes of purified scFabs were evaluated by size exclusion chromatography on a calibrated Superdex 200 10/300 GL column (Amersham Bioscience) with PBS, pH 7.4, at a flow rate of 1 mL/min. Samples were fractionated and stored for further ELISA analysis at 4°C.
Affinity measurements were done using surface plasmon resonance (SPR). The analysis was performed with a Biacore2000 (Biacore, Sweden) using a CM5 chip. Lysozyme and BSA were bound on flow cells via amine coupling. The flow rate was 30 μl/min at 25°C using HEPES buffer . Sample concentration of D1.3 antibody variants loaded on flow cells ranged from 6 × 10-8 to 5 × 10-9 M. The curve fitting was performed and the kinetics were analyzed using the Biacore software package.
We would like to thank Bronwyn M. Kenny, Michael Mersmann and Thomas Schirrmann for corrections and discussion on the manuscript and Laila Al-Halabi for the construction of pOPE101-XP. We also gratefully acknowledge Arne Skerra for providing the vector pASK99-D1.3 encoding the anti-lysozyme antibody fragment. We gratefully acknowledge the financial support by the German ministry of education and research (BMBF, SMP "Antibody Factory" in the NGFN2 program) and of the German Research Foundation (DFG, SFB 578).
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