Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris
© Schoonooghe et al; licensee BioMed Central Ltd. 2009
Received: 8 April 2009
Accepted: 11 August 2009
Published: 11 August 2009
Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.
Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli.
Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.
Monoclonal antibodies (MoAbs) targeted at tumour associated antigens (TAAs) are a promising new therapeutic option for treating cancer patients. MoAbs such as Rituximab  and Trastuzumab  are already becoming part of the standard treatment regimen, targeting TAA+ tumours specifically and with fewer side effects than chemotherapeutics. However, a number of problems that diminish antibody efficacy in a therapeutic setting still need to be addressed. The large size of a full sized antibody slows vascular diffusion and prevents its penetration deep into solid tumours[3, 4]. Moreover, radionuclide or cytotoxin coupled molecules persist longer in the general circulation and can thus cause more toxic side-effects . An equally important, yet sometimes overlooked issue is the production of sufficient quantities of MoAb. MoAb therapies involve high doses, usually more than 1 g per patient per year, and can only be generated in relatively expensive mammalian cell fermentors[6, 7].
In order to address the poor tumour penetration of full-sized antibodies, MoAb derivatives, such as Fab and scFv fragments, have been generated that, due to their smaller size, penetrate more readily in tissues and can deliver a rapid peak dose at the tumour site. However, molecules smaller than 60 kDa are generally cleared too rapidly from the body to allow sufficient tumour accumulation. Intermediate sized recombinant antibodies, such as the 80 kDa minibodies, still demonstrate good tissue penetration, while not being cleared as rapidly from the blood[9–12]. Most of these molecules lack an Fc effector domain but can be used as blocking agents for growth factor receptors, inducers of apoptosis or as carriers of radiotherapeutic isotopes, toxins, cytokines or other biologically active proteins[13–15]. Several ways of making intermediate sized antibody derivatives have already been described which mainly involve the usage of different dimerisation motifs such as leucine zippers or Fc chain interactions[16–19]. As with MoAbs, obtaining sufficient correctly folded or heterodimerised product for large-scale application often is a bottleneck for these recombinant antibody derivatives. Major efforts are therefore being made in the development of cheap and efficient heterologous expression systems. Over the last few years, yeasts, like Pichia pastoris, have gained a significant interest for the production of recombinant antibody fragments using cheap mineral defined media and requiring shorter process times as compared to mammalian cell culture[20, 21]. The possibility to grow yeasts to high cell densities of up to 100 g/l dry biomass, along with the availability of strong, inducible promoters, such as the alcohol oxidase gene (AOX1) promoter, are further advantages of heterologous expression in P. pastoris .
We previously described a novel model to engineer bi- or trivalent antibody derivatives (bi- or tribodies) of intermediate size (75–100 kDa), based on fusion of single-chain variable fragments (scFv) to the C-terminus of one or each of the Fd and L chains of a Fab fragment. Fab-scFv BsAb are efficiently produced in mammalian cells, with 90% of the product being in the correctly heterodimerised form. In this study, we describe the production and purification of the human PH1 Fab-scFv bivalent bibody and Fab-(scFv)2 trivalent tribody directed against the MUC1 TAA. This tumour antigen is an under-glycosilated form of MUC1, and is associated with poor prognosis upon overexpression in breast and pancreas cancer[24, 25]. Using the Fab-sFv template, we formatted the αMUC1 bi- and tribodies for expression in P. pastoris, and compared the P. pastoris production system to transient expression in human HEK293T cells and stable expression in mouse NS0 cells. The binding functionalities of both mammalian and yeast derived preparations are gauged demonstrating the functional equivalence of the respective preparations. Combined, these results demonstrate P. pastoris to be a viable alternative expression system for Fab-scFv molecules.
Construction of PH1 bibody and tribody expression vectors
The pES31 vector was constructed to facilitate cloning and expression of bi- and tribody genes in mammalian cells (Figure 1C). To this end, the βactin-βglobulin promoter and CMV enhancer from pCAGGS were introduced in the pCDNA3- plasmid carrying the zeocin resistance gene. To facilitate expression and excretion, a kozak sequence and a consensus excretion signal sequence were introduced in front of the expression cassette. Eco47III, BspEI and BamHI restriction sites were introduced to allow modular exchange of antibody expression cassettes. The PH1heavy-scFv and PH1light-scFv expression cassettes were subsequently cloned into pES31 for expression in HEK293T and NS0 mammalian host cells.
For the expression of PH1 bi-and tribodies in P. pastoris, a single vector based on pKai51 was constructed, with a bicistronic expression cassette for both the light and heavy chain (Figure 1D). pKai51 originated from pGAPZalfaA in which several additional features were introduced, including an AOX1 promotor from pPICZA. The PH1 bibody and tribody light and heavy chains were first introduced into separate pKai51 derived vectors, after which the expression modules for the heavy and light chain were combined in one vector, resulting in the pKai61-BiBody-PH1 and pKai61-TriBody-PH1 vectors.
Expression of PH1 bibody and tribody in mammalian cells
Purification of mammalian cell produced PH1 bibody and tribody
Based on colorimetric concentration determination and on the analysis of the Coomassie stained SDS-PAGE gels, the overall efficiency for stably produced PH1 bi- and tribody was estimated at 77% and yielded 130.5 mg bibody and 27 mg tribody per litre NS0 supernatant, starting from 2 l cultures.
Stable production of PH1 bibody and tribody in P. pastoris
A small scale 6 day production of the bi- and tribody was performed in YPNM with antifoam and 1% methanol added every 12 h. The resulting supernatant was purified using IMAC (Figure 4E), preceded by ammonium sulphate precipitation to remove peptides and other interfering substances from the yeast medium. Further purification over a gelfiltration column of the antibody containing eluate resulted in an estimated 95% pure preparation free of light chain homodimers (Figure 4F), with a total estimated product recovery of 22%. In total 36.8 mg pure bibody per litre of supernatant was recovered from a 250 ml shake-flask culture. For the tribody 12 mg pure product per litre supernatant was recovered from an initial culture volume of 500 ml.
Binding characteristics of mammalian and yeast cell produced bi- and tribodies
Alternatively glycosilated MUC1 is overexpressed on a large number of carcinomas and is associated with poor prognosis, making it an interesting target for antibody immunotherapy[26–28]. The human PH1 Fab targeted against the MUC1 TAA was used to develop a human bivalent bibody and trivalent tribody by respectively fusing one or two PH1 scFvs to the C-terminus of the Fab chains. Both molecules are of an intermediate weight, which should slow clearing from the body compared to Fab and scFv fragments, and should also improve tumour penetration compared to MoAbs. As with previously described mammalian expressed bi- and tribodies, the HEK293T and NS0 produced PH1 bi- and tribody were expressed almost exclusively as correctly heterodimerised Fab-scFv products, a characteristic attributed to processing in the endoplasmatic reticulum of the L and Fd chains by the mammalian immunoglobulin heavy chain binding protein (BiP) chaperone . However, also expression in P. pastoris resulted in the majority of bi- and tribody products being correctly heterodimerised. SDS-PAGE analysis of Pichia supernatant revealed, as in mammalian cells, the absence of heavy chains released from the cells. This suggests that the yeast Karp2, a Hsp70-class (78 kDa) heat shock protein and analogue of mammalian BiP, plays the same chaperone role as BiP in the endoplasmatic reticulum[30, 31], retaining the heavy chains until correctly paired with a light chain. Transfection of the light chain resulted in secretion of mainly L-chain monomers with only a minor fraction of dimers, indicating that the affinity between light chains is limited. By removing the His6-tag sequence from the light chain DNA, L-chain homodimers were further excluded during IMAC purification.
In mammalian cells, the use of the strong βactin-βglobulin hybrid promoter and upstream CMV enhancer in pES31 results in increased expression compared to CMV and EF1 promotors . Transient expression of the PH1 bi- and tribody in the pES31 system produced 15.4 mg bibody and 5.77 mg tribody per litre HEK293T medium (not shown). These values are akin to what we described before for mouse bispecific bibodies . The three-fold difference in expression levels between the PH1 bibody and tribody was also observed in the stably transfected mammalian NS0 and yeast cells. Besides the 35% larger size of the tribody, the more complex structure of the molecule and the presence of 3 copies of both the PH1 VH and VL could hamper the correct cellular expression.
Stable production in NS0 cells clearly resulted in increased production compared to transient expression in HEK293T. In addition, the use of protein free medium raised the overall purification efficacy from 56% for HEK293T productions to 77% for NS0 productions. The peak cell concentration in NS0 batch culture was around 3.109 cells/l. This leaves the possibility to further increase production by switching to a fed-batch system in which culture conditions are more finely tuned . Although culture media costs have been significantly reduced in the past years, producing antibodies and their derivatives in mammalian cells still is a relatively expensive procedure . Hence, there is a profound interest in alternative production systems based on more primitive cells with shorter process times and the ability to grow to very high cell densities on cheap media. The commonly used prokaryotic E. coli expression systems do not seem suited for the production of the large heterodimeric bi- and tribody molecules. Bibody production in E. coli cells resulted in a limited expression levels of 50–250 μg product/l. Pichia pastoris has however gained significant interest over the last few years for the production of recombinant antibodies and antibody fragments. This methylotropic yeast can secrete large amounts of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs[20, 21, 36–38]. The non-human glycosilation patterns of P. pastoris are however a problem for the production of complete MoAbs, yet are not an issue for the non-glycosilated bi- and tribodies. Building on this, we successfully produced in P. pastoris PH1 bi -and tribodies in viable quantities. Gelfiltration of the Pichia products demonstrated that very few dimers and nearly no degradation products were present after IMAC purification. Yet, also in P. pastoris, tribody clones were 3 times less productive as compared to bibody clones. However, the surprisingly good production level in Pichia of a trivalent antibody with 12 mg pure product/l supernatant may be an advantage when compared to other trispecific or trivalent intermediate sized antibody models, like trispecific single chains[39, 40] and triabodies[41, 42] that produce in the 0.5 mg/l range in E. coli fermentations. Although the yields obtained in Pichia medium were comparable to NS0 yields, the low recovery rate from Pichia medium of 22% leaves ample room for optimisation of the purification procedures. Replacing the standard ammonium precipitation step, wasting about 50% of the product, with a column based capture step may be a straightforward way to achieve such an increase in recovery. Combined with increases in yield through growing the Pichia clones in closely monitored fermentation conditions, further increases in overall production yield are likely to be achieved.
Independent of the expression system used, the bi- or tribody molecules bound to their MUC1 targets with equal effectiveness either in a peptide context or on OVCAR3 cancer cells, indicating the mammalian and yeast expression products were of similar specific activity.
Combined with the efficient expression and recovery of the yeast produced bi- and tribodies, our results identify P. pastoris as a valuable alternative to mammalian expression for the production of recombinant antibody derivatives based on the Fab heterodimerisation platform.
Anti-MUC1 antibody and antigen
The human PH1 Fab and the derived PH1 scFv, both directed against the MUC1 epitope PAPGS were a generous gift from H. Hoogenboom (Dyax, Maastricht, NL). The MUC1 PDTRPAPGS peptide was fused to mGroEL. This GroEL-MUC1 protein is efficiently produced in E. coli and purified with IMAC via an N-terminal His6 tag (data not shown).
HEK293T, a human embryonic kidney cell line transfected with the SV40 large T-Ag (SV40TtsA1609), was used for transient eukaryotic expression. NS0 is a mouse myeloma commonly used for hybridoma production or stable recombinant MoAb expression. OVCAR3 cells are derived from a human adenocarcinoma and express high levels of MUC1-TAA. HEK293T and NS0 were grown in DMEM medium containing 10% foetal bovine serum (FBS). Stable NS0 clones were grown in chemically defined hybridoma medium supplemented with synthechol NS0 supplement (Sigma Aldrich, St. Lois, MO). OVCAR3 was cultured in RPMI1640 medium supplemented with 10% FBS and 0.01 mg/ml bovine insulin. Lovo is a human colorectal adenocarcinoma cell-line with low MUC1 expression cultured in Ham's F12K with 10% FBS. The methylotrophic yeast P. pastoris strain GS115(his4) was obtained from Invitrogen (Merelbeke, BE), maintained on YPD plates at 4°C and grown in liquid YPD at 30°C.
Expression plasmids and gene assembly
The pES31 and pES31Hneo expression plasmids were constructed using pCDNA3, pCDNA3.1zeo- (Invitrogen) and the pCAGGS vectors for mammalian expression. Restriction- and DNA modifying enzymes and Vent-DNA polymerase (New England Biolabs, Beverly, MA) were used as recommended by the manufacturers. Gene assembly was conducted by introduction of suitable restriction sites using modifying PCR primers. All PCR-derived fragments were sequence verified after cloning. The XhoI/BsaI fragment of pCAGGS carrying the βactin-βglobulin promoter and CMV enhancer was fused to the EcoRV/BsaI fragment of pCDNA3.1-carrying the zeocin resistance gene. An XhoI/blunt adaptor sequence was inserted 3' of the promoter and 5' of the pCDNA3.1-fragment. This adaptor incorporated a kozak sequence (GCCACCATGG) and a consensus excretion signal sequence (MGWSCIIFFLVATATGVHS). An Eco47III site was created in frame 3' of the signal sequence to allow for easy insertion of genes behind the signal sequence. At the 3' end of the gene insertion point a BspeI restriction site was provided in frame with an E-tag sequence. A BamHI site is present 3' of the E-tag sequence in front of the stop codon when no tag is to be attached. The pES31Hneo vector was constructed by fusing the pES31 BspEI/BsaI fragment carrying the promoter and signal sequence with the BspEI/BsaI fragment of pCDB1E6H2sc2C11Hneo, a derivative of pCDNA3 . This fragment holds a His6 tag sequence for C-terminal fusion to an inserted gene and is accessible for cloning via a BspEI site. A neomycin resistance gene is also present.
Primers used in this work, in order of appearance, 5' to 3' notation
Sequence 5' to 3'
Construction of the bi- and trivalent anti-MUC1 mammalian expression vectors
Heavy and light chains of the PH1 Fab were cloned into pES31. The Fd fragment was amplified by PCR using Vent polymerase with primers NM265 and NM263. This PCR fragment was BspEI cut, kinated with T4-kinase and introduced into pES31Hneo, resulting in pES31-PH1heavy_neo. The L chain was cloned in pES31 in a similar fashion using primers NM266 and NM264, resulting in pES31-PH1light_zeo. For the construction of the bi- and tribody vectors, a PH1 scFv was fused to the C-terminus of the PH1-Fd and the PH1-L chain with a flexible linker. First, the DNA fragment coding for the flexible EPSGP(G4S)3 linker  was fused to the 3' end of the PH1 Fd gene with a ApaI/PvuI digest. Similarly, the DVDGGSRGDGPG linker  was fused to the 3' end of the PH1-L chain. Next, the PH1 scFv gene was amplified using NM302 and NM303 primers. After T4 kination, the fragment was introduced after the linker of the PH1-Fd chain using a BspeI digest. In this manner, the His6 tag sequence was retained and fused to the C-terminus of the PH1 scFv, resulting in pES31-PH1heavy-scFv_neo. For cloning the PH1 scFv to the linker behind the PH1-L chain, a SmaI/BamHI fragment was used. This was fused to an Eco47III/PvuI fragment and a BamHI/PvuI fragment from pES31, yielding pES31-PH1light-scFv_zeo.
Construction of antibody expressing plasmids for Pichia pastoris
Using the mammalian vectors, the PH1 light chain was amplified from pES31-PH1light_zeo with NM266 and NM938 and cloned into pKai51.2 with an EcoRV/SpeI cut, resulting in pKai51.2-PH1light. The heavy chain PH1 was amplified from pES31PH1heavy_zeo with NM265 and NM938 and cloned into pKai61 with a EcoRV/SpeI cut, resulting in pKai61-PH1heavy. pKai51.2-PH1light-scFv, containing the light chain PH1-scFv PH1, was made by cloning the EcoRV/SpeI digested amplification fragment from pES31-PH1light-scFv_zeo with primers NM266 and NM937. Similarly, the PH1 heavy chain-scFv PH was amplified from pES31-PH1heavy-scFv_neo with NM265 and NM937, cut with EcoRV/SpeI and cloned in pKai61, resulting in pKai61-PH1heavy-scFv. Finally, vectors containing 2 expression modules for the heavy and light chain were assembled as follows: pKai61-BiBody-PH1 is the ligation product of pKai61-PH1heavy-scFv cut with BamHI/BsrGI and pKai51.2-PH1light cut with BglII/BsrGI. pKai61-TriBody-PH1 is a combination of pKai61-PH1heavy-scFv cut with BamHI/BsrGI and pKai51.2-PH1light-scFv cut with BglII/BsrGI.
Transient Expression in HEK293T cells
For transient antibody expression, HEK293T cells were transfected according to the Ca3(PO4)2 precipitation method . In brief, cells were seeded at 4 × 106 cells/175 cm2, 20 h before transfection, after which 14 μg DNA of each expression plasmid was added to the cells for 24 h. For expression of the bibody pES31-PH1heavy-scFv_neo and pES31-PH1light_zeo were used. The tribody was expressed by combining the pES31-PH1heavy-scFv_neo and pES31-PH1light-scFv_zeo plasmids. The cells were then covered with supplemented DMEM containing 5 mg/l bovine insulin, 5 mg/l transferrin and 5 μg/l selenium (ITS). Medium was harvested every 48 h. For both the bi- and tribody production, 1600 ml HEK293T supernatant was collected.
Stable expression in NS0 cells
NS0 cells were transfected by electroporation with pES31-PH1heavy-scFv_neo and pES31-PH1light_zeo for the bibody, pES31-PH1heavy-scFv_neo and pES31-PH1light-scFv_zeo for the tribody. 2.5 μg of each plasmid was used with Nucleofector technology (Amaxa Biosystems, Cologne, Germany) and program settings G16 and T27 in buffer T as indicated by the manufacturer. Prior to transfection, DNA was sterilised on a 0.22 μm filter. Linearised DNA was obtained through a ScaI digest, followed by a Wizard DNA cleanup procedure (Promega, Leiden, NL). Control transfections were performed using 5 μg pmaxGFP plasmid. After 24 h–48 h transfected cells were evaluated for GFP expression and put on selective medium containing 0.6 mg/ml G418 (Invitrogen) and/or 0.6 mg/ml zeocin (Invitrogen). Mock transfections were performed using H2O instead of DNA. After 1 month of selection, subclones were generated using limiting dilution. ELISAs were performed to ascertain bi- and tribody clone production levels. The best bibody and tribody clone were adapted to protein free hybridoma medium and grown in roller bottles for 7 days. For each clone 3 roller bottles of 670 ml NS0 supernatant/bottle were collected.
Stable expression in P. pastoris yeast cells
Prior to transformation 10 μg of the bicistronic expression vectors pKai61-BiBody-PH1 and pKai61-BiBody-PH1 were linearised with a PmeI digest. The plasmid DNA (2.5 μg) was transformed into 100 μl competent P. pastoris GS115 cells by electroporation using a Gene Pulser (Bio-Rad) and 0,2 cm gap cooled electroporation cuvettes. The electric pulse parameters were: 1500 V, 40 μF, 200 Ω and 8 ms duration. Immediately after the pulse, 1 ml of 1 M ice-cold sorbitol was added. This mixture was transferred to tubes containing 2 ml YPD medium. These tubes were incubated at 30°C for 1 – 1.5 hour without shaking. 50 μl was subsequently plated on YPD-agar plates containing 100 μg/ml zeocin and incubated at 28°C for 3–4 days. Colonies were screened for expression through small-scale expression experiments in 24 well deep-well plates containing 2 ml YPNM medium. After 48 h of methanol induction, several clones were compared by means of an S-Tag assay. Production of the bibody and tribody was performed in shake flasks containing 250 ml YPNM medium/flask.
The HEK293T derived bi- and tribodies were purified using an adaptation of a previously described protocol for mouse bi- and tribodies . Harvested mammalian medium was centrifuged for 20 min at 13000 × g and filtered over 0.22 μm bottle-top filters (Nalgene, Neerijse, BE). Having a predicted pI of 7.9 and 8.0 respectively, the pH of the bi -and tribody HEK293T samples was adjusted to pH 5.5 with acetic acid before loading on a 200 ml XK50/20 SP Sepharose Fast Flow (GE Health, Uppsala, Sweden) cation exchange chromatography (CEC) column equilibrated with 50 mM NaAc buffer pH 5.5 and run at 9 ml/min. Further purification of the CEC fractions was performed using 1 ml Hitrap columns (GE Health) loaded with Ni2+. The sample was supplemented with 20 mM Imidazole pH 7.5 and pH was adjusted to 7.5. The column was equilibrated using 20 mM phosphate (PP) buffers containing 0.5 M NaCl and run at 2 ml/min. For elution 20 mM PP buffer with 200 mM or 400 mM imidazole and 0.5 M NaCl were used. IMAC of serum free NS0 samples was performed on a 20 ml C16/20 Chelating Sepharose Fast Flow column (GE Health), under the same buffer conditions as the 1 ml Hitrap columns, but using a gradient elution with 20 mM PP buffer containing 500 mM imidazole and 0.5 M NaCl. For size exclusion chromatography (SEC), the sample was concentrated to 2 ml using Centricon centrifugal filter devices (Amicon Bioseperations, Beverly, MA), with a cut-off of 10 kDa. Size exclusion chromatography was performed on a 120 ml C16/60 Sephacryl S-200 high resolution grade column (GE Health) and run at 0.5 ml/min with PBS. The column was calibrated before each run with Gel Filtration Standard (Bio-Rad Laboratories, Hercules, CA).
Harvested yeast medium was precipitated by addition of 70% (NH4)2SO4. After centrifugation, the pellet was dissolved in 10 ml 20 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole pH 7.5. This solution was injected on a 62 ml desalting sephadex G-25 column (XK16/31) with the same buffer. The desalted protein fraction was further purified on a 6 ml Ni sepharose column for the bibody and on a 1 ml HisTrap column for the tribody. The column was equilibrated with 20 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole pH 7.5, washed with 50 mM imidazole and eluted by 400 mM imidazole in the same buffer. The eluted protein fraction was finally purified by 1 ml injections on a Superdex 200 HR10/30 column to PBS. All liquid chromatography (LC) runs were performed at 4°C on Pharmacia FPLC or Akta purifier systems (GE Health).
Analysis of protein fractions
Collected protein fractions were analysed by SDS-PAGE, after precipitation with TCA. Proteins were visualized using Coomassie Brilliant Blue dye or Western blotted to a nitrocellulose membrane. Immunodetection of the proteins on the blot was performed by incubating consecutively with mouse IgG1 anti-human κ serum, mouse IgG1 anti-human Fd serum (Sigma Aldrich, St. Louis, MO) or mouse IgG1 anti-His (Qiagen, Venlo, NL) and alkaline phosphatase (AP) conjugated anti-mouse IgG1 (Becton Dickinson-Pharmingen, Erembodegem, BE). Alternatively, for yeast constructs S-protein-AP (Merck, Nothingham, UK) was used to detect western blots. Antibodies were incubated for 1 h at room temperature (RT) in a solution containing 5% skimmed milk powder, 50 mM Tris pH 8.0, 0.47% NaCl, 0.2% NP40 and 0.02% NaN3. Subsequent visualisation was performed with NBT/BCIP substrate 'ready to use tablets' (Roche, Vilvoorde, BE). Protein recovery and purity were determined on Coomassie gels that were scanned and analysed with Quantity One software (Bio-rad). Protein concentrations of pure protein were measured with the Micro BCA™ Protein Assay Reagent Kit (Pierce, Rockford, IL) with IgG standard protein. Concentrations of PH1 molecules from P. pastoris medium were quantified with the FRETWorks S-tag assay kit (Merck) according to the instructions of the manufacturer. Yields were calculated as: (amount of bi- or tribody withheld in elution)/(total amount of bi- or tribody found before the purification step). Additionally for gelfiltrations, the purity was calculated by measuring the area under the curve for the total run compared to the specific bi- or tribody peak. The overall yield was calculated by dividing the ultimate quantity of pure bi/tribody by the estimated starting quantity before purification.
96 well plates were coated overnight at RT with 50 μl mGroEL-MUC1 (10 μg/ml) in NaHCO3 buffer pH 9.6. Plates were blocked with PBS buffer + 0.05% Tween20 and 1% BSA (PBS-BSA) for 2 h at 37°C. Dilution series of the Ab-derivatives were added in 50 μl PBS-BSA and incubated for 1 h at 37°C. In between steps, plates were washed 3 times with PBS + 0.05% Tween20. Detection was performed by incubating consecutively with mouse IgG1 anti-human κ serum and AP-conjugated anti-mouse IgG1. After adding the p-nitrophenyl phosphate (PNPP) substrate in 10% diethanolamine + 1 mM MgCl2 pH 9.8, absorbance at 405 nm was measured in a microplate reader (Bio-Rad). Negative controls consisted of wells treated with antibodies but not coated with antigen and wells that were coated and received detection antibodies, but no samples. ELISA binding curves and KD were calculated using Prism 4.0 software (Graphpad, San Diego, CA).
Aliquots of 2 × 105 cells were incubated with PH1 derivatives in PBS + 0.5% BSA and 0.02% NaN3 (PBS-A) on ice for 2 h. After washing, the cells were allowed to subsequently interact with mouse IgG1 anti-human κ and Alexa Fluor 488-conjugated goat anti-mouse antibodies (Invitrogen) on ice for 1 h. Finally, the cells were resuspended in 300 μL PBS-A. For isotype control, 10 nM 9E10 MoAb (mouse IgG1, Becton Dickinson-Pharmingen) was used instead of PH1 derivatives. Flow cytometric analysis was performed using a FACScan flow cytometer (Becton Dickinson). Data analysis based on the collection of 10.000 events per sample was performed using WinMDI 3.7 software (Joseph Trotter).
We thank H. Hoogenboom and P. Hendrickx for providing the anti-MUC1 PH1 Fab.
SS was supported by a grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT). VK, MZ, CD, JH, JG were all supported by the Flanders institute for biotechnology (VIB) and the University Ghent. None of the funding bodies had any role in the collection, analysis, and interpretation of data; nor in the writing of the manuscript; or the decision to submit the manuscript for publication.
- Molina A: A decade of rituximab: improving survival outcomes in non-Hodgkin's lymphoma. Annu Rev Med. 2008, 59: 237-250. 10.1146/annurev.med.59.060906.220345.View ArticleGoogle Scholar
- Nahta R, Esteva FJ: Trastuzumab: triumphs and tribulations. Oncogene. 2007, 26 (25): 3637-3643. 10.1038/sj.onc.1210379.View ArticleGoogle Scholar
- Jain RK: Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer Res. 1990, 50 (3 Suppl): 814s-819s.Google Scholar
- Reilly RM, Sandhu J, Alvarez-Diez TM, Gallinger S, Kirsh J, Stern H: Problems of delivery of monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions. Clin Pharmacokinet. 1995, 28 (2): 126-142. 10.2165/00003088-199528020-00004.View ArticleGoogle Scholar
- Thrush GR, Lark LR, Clinchy BC, Vitetta ES: Immunotoxins: an update. Annu Rev Immunol. 1996, 14: 49-71. 10.1146/annurev.immunol.14.1.49.View ArticleGoogle Scholar
- Holliger P, Winter G: Engineering bispecific antibodies. Curr Opin Biotechnol. 1993, 4 (4): 446-449. 10.1016/0958-1669(93)90010-T.View ArticleGoogle Scholar
- Werner RG: Economic aspects of commercial manufacture of biopharmaceuticals. J Biotechnol. 2004, 113: 1-3. 10.1016/j.jbiotec.2004.04.036.View ArticleGoogle Scholar
- Behr TM, Memtsoudis S, Sharkey RM, Blumenthal RD, Dunn RM, Gratz S, Wieland E, Nebendahl K, Schmidberger H, Goldenberg DM, et al: Experimental studies on the role of antibody fragments in cancer radio-immunotherapy: Influence of radiation dose and dose rate on toxicity and anti-tumor efficacy. Int J Cancer. 1998, 77 (5): 787-795. 10.1002/(SICI)1097-0215(19980831)77:5<787::AID-IJC19>3.0.CO;2-Z.View ArticleGoogle Scholar
- Wu AM, Chen W, Raubitschek A, Williams LE, Neumaier M, Fischer R, Hu SZ, Odom-Maryon T, Wong JY, Shively JE: Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers. Immunotechnology. 1996, 2 (1): 21-36. 10.1016/1380-2933(95)00027-5.View ArticleGoogle Scholar
- Hu S, Shively L, Raubitschek A, Sherman M, Williams LE, Wong JY, Shively JE, Wu AM: Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res. 1996, 56 (13): 3055-3061.Google Scholar
- Adams GP, Schier R, McCall AM, Crawford RS, Wolf EJ, Weiner LM, Marks JD: Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu. Br J Cancer. 1998, 77 (9): 1405-1412.View ArticleGoogle Scholar
- Viti F, Tarli L, Giovannoni L, Zardi L, Neri D: Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis. Cancer Res. 1999, 59 (2): 347-352.Google Scholar
- Sharkey RM, Goldenberg DM: Targeted therapy of cancer: new prospects for antibodies and immunoconjugates. CA Cancer J Clin. 2006, 56 (4): 226-243. 10.3322/canjclin.56.4.226.View ArticleGoogle Scholar
- Schrama D, Reisfeld RA, Becker JC: Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov. 2006, 5 (2): 147-159. 10.1038/nrd1957.View ArticleGoogle Scholar
- Wu AM, Senter PD: Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol. 2005, 23 (9): 1137-1146. 10.1038/nbt1141.View ArticleGoogle Scholar
- Kostelny SA, Cole MS, Tso JY: Formation of a bispecific antibody by the use of leucine zippers. J Immunol. 1992, 148 (5): 1547-1553.Google Scholar
- Pack P, Pluckthun A: Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry. 1992, 31 (6): 1579-1584. 10.1021/bi00121a001.View ArticleGoogle Scholar
- Chang HC, Bao Z, Yao Y, Tse AG, Goyarts EC, Madsen M, Kawasaki E, Brauer PP, Sacchettini JC, Nathenson SG, et al: A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments. Proc Natl Acad Sci USA. 1994, 91 (24): 11408-11412. 10.1073/pnas.91.24.11408.View ArticleGoogle Scholar
- Kipriyanov SM, Little M, Kropshofer H, Breitling F, Gotter S, Dubel S: Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion. Protein Eng. 1996, 9 (2): 203-211. 10.1093/protein/9.2.203.View ArticleGoogle Scholar
- Freyre FM, Vazquez JE, Ayala M, Canaan-Haden L, Bell H, Rodriguez I, Gonzalez A, Cintado A, Gavilondo JV: Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris. J Biotechnol. 2000, 76 (2–3): 157-163. 10.1016/S0168-1656(99)00183-2.View ArticleGoogle Scholar
- Ning D, Junjian X, Qing Z, Sheng X, Wenyin C, Guirong R, Xunzhang W: Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation. J Biochem Mol Biol. 2005, 38 (3): 294-299.View ArticleGoogle Scholar
- Cregg JM, Vedvick TS, Raschke WC: Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (N Y). 1993, 11 (8): 905-910. 10.1038/nbt0893-905.View ArticleGoogle Scholar
- Schoonjans R, Willems A, Schoonooghe S, Fiers W, Grooten J, Mertens N: Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives. J Immunol. 2000, 165 (12): 7050-7057.View ArticleGoogle Scholar
- Hollingsworth MA, Swanson BJ: Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer. 2004, 4 (1): 45-60. 10.1038/nrc1251.View ArticleGoogle Scholar
- Wesseling J, Valk van der SW, Hilkens J: A mechanism for inhibition of E-cadherin-mediated cell-cell adhesion by the membrane-associated mucin episialin/MUC1. Mol Biol Cell. 1996, 7 (4): 565-577.View ArticleGoogle Scholar
- Tamada S, Goto M, Nomoto M, Nagata K, Shimizu T, Tanaka S, Sakoda K, Imai K, Yonezawa S: Expression of MUC1 and MUC2 mucins in extrahepatic bile duct carcinomas: its relationship with tumor progression and prognosis. Pathol Int. 2002, 52 (11): 713-723. 10.1046/j.1440-1827.2002.01414.x.View ArticleGoogle Scholar
- Hinoda Y, Ikematsu Y, Horinochi M, Sato S, Yamamoto K, Nakano T, Fukui M, Suehiro Y, Hamanaka Y, Nishikawa Y, et al: Increased expression of MUC1 in advanced pancreatic cancer. J Gastroenterol. 2003, 38 (12): 1162-1166. 10.1007/s00535-003-1224-6.View ArticleGoogle Scholar
- Fujita K, Denda K, Yamamoto M, Matsumoto T, Fujime M, Irimura T: Expression of MUC1 mucins inversely correlated with post-surgical survival of renal cell carcinoma patients. Br J Cancer. 1999, 80 (1–2): 301-308. 10.1038/sj.bjc.6690355.View ArticleGoogle Scholar
- Mayer M, Kies U, Kammermeier R, Buchner J: BiP and PDI cooperate in the oxidative folding of antibodies in vitro. J Biol Chem. 2000, 275 (38): 29421-29425. 10.1074/jbc.M002655200.View ArticleGoogle Scholar
- Matlack KE, Misselwitz B, Plath K, Rapoport TA: BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane. Cell. 1999, 97 (5): 553-564. 10.1016/S0092-8674(00)80767-9.View ArticleGoogle Scholar
- Liu YY, Woo JH, Neville DM: Overexpression of an anti-CD3 immunotoxin increases expression and secretion of molecular chaperone BiP/Kar2p by Pichia pastoris. Appl Environ Microbiol. 2005, 71 (9): 5332-5340. 10.1128/AEM.71.9.5332-5340.2005.View ArticleGoogle Scholar
- Willems A, Leoen J, Schoonooghe S, Grooten J, Mertens N: Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives. J Chromatogr B Analyt Technol Biomed Life Sci. 2003, 786 (1–2): 161-176.View ArticleGoogle Scholar
- Sauer PW, Burky JE, Wesson MC, Sternard HD, Qu L: A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies. Biotechnol Bioeng. 2000, 67 (5): 585-597. 10.1002/(SICI)1097-0290(20000305)67:5<585::AID-BIT9>3.0.CO;2-H.View ArticleGoogle Scholar
- Farid SS: Process economics of industrial monoclonal antibody manufacture. J Chromatogr B Analyt Technol Biomed Life Sci. 2007, 848 (1): 8-18. 10.1016/j.jchromb.2006.07.037.View ArticleGoogle Scholar
- Lu D, Jimenez X, Zhang H, Bohlen P, Witte L, Zhu Z: Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments. J Immunol Methods. 2002, 267 (2): 213-226. 10.1016/S0022-1759(02)00148-5.View ArticleGoogle Scholar
- Cregg JM, Cereghino JL, Shi J, Higgins DR: Recombinant protein expression in Pichia pastoris. Mol Biotechnol. 2000, 16 (1): 23-52. 10.1385/MB:16:1:23.View ArticleGoogle Scholar
- Gasser B, Mattanovich D: Antibody production with yeasts and filamentous fungi: on the road to large scale?. Biotechnol Lett. 2007, 29 (2): 201-212. 10.1007/s10529-006-9237-x.View ArticleGoogle Scholar
- Lange S, Schmitt J, Schmid RD: High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris. J Immunol Methods. 2001, 255 (1–2): 103-114. 10.1016/S0022-1759(01)00351-9.View ArticleGoogle Scholar
- Wang XB, Zhao BF, Zhao Q, Piao JH, Liu J, Lin Q, Huang HL: A new recombinant single chain trispecific antibody recruits T lymphocytes to kill CEA (carcinoma embryonic antigen) positive tumor cells in vitro efficiently. J Biochem. 2004, 135 (4): 555-565. 10.1093/jb/mvh065.View ArticleGoogle Scholar
- Liu J, Zhao Q, Zhao B, Cheng J, Wang X, Song L, Zhong Z, Lin Q, Huang H: A new format of single chain tri-specific antibody with diminished molecular size efficiently induces ovarian tumor cell killing. Biotechnol Lett. 2005, 27 (22): 1821-1827. 10.1007/s10529-005-6732-4.View ArticleGoogle Scholar
- Atwell JL, Breheney KA, Lawrence LJ, McCoy AJ, Kortt AA, Hudson PJ: scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies. Protein Eng. 1999, 12 (7): 597-604. 10.1093/protein/12.7.597.View ArticleGoogle Scholar
- Bayly AM, Kortt AA, Hudson PJ, Power BE: Large-scale bacterial fermentation and isolation of scFv multimers using a heat-inducible bacterial expression vector. J Immunol Methods. 2002, 262 (1–2): 217-227. 10.1016/S0022-1759(02)00021-2.View ArticleGoogle Scholar
- Henderikx P, Coolen-van Neer N, Jacobs A, Linden van der E, Arends JW, Mullberg J, Hoogenboom HR: A human immunoglobulin G1 antibody originating from an in vitro-selected Fab phage antibody binds avidly to tumor-associated MUC1 and is efficiently internalized. Am J Pathol. 2002, 160 (5): 1597-1608.View ArticleGoogle Scholar
- DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, Calos MP: Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol. 1987, 7 (1): 379-387.View ArticleGoogle Scholar
- Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991, 108 (2): 193-199. 10.1016/0378-1119(91)90434-D.View ArticleGoogle Scholar
- O'Mahoney JV, Adams TE: Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection. DNA Cell Biol. 1994, 13 (12): 1227-1232. 10.1089/dna.1994.13.1227.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.