We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy. Bacterial expression can be used to express small recombinant antibodies on a large scale; however, their overexpression often results in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography. Here, we propose a novel method for preparing refined, functional, tag-free bi-specific diabodies from IgG-like bi-specific antibodies (BsAbs) in a mammalian expression system. We created an IgG-like BsAb in which bi-specific diabodies were fused to the human Fc region via a designed human rhinovirus 3C (HRV3C) protease recognition site. The BsAb was purified by protein A affinity chromatography, and the refined tag-free hEx3-Db was efficiently produced from the Fc fusion format by protease digestion. The tag-free hEx3-Db from the Fc fusion format showed a greater inhibition of cancer growth than affinity-tagged hEx3-Db prepared directly from Chinese hamster ovary cells. We also applied our novel method to another small recombinant antibody fragment, hEx3 single-chain diabody (hEx3-scDb), and demonstrated the versatility and advantages of our proposed method compared with papain digestion of hEx3-scDb. This approach may be used for industrial-scale production of functional tag-free small therapeutic antibodies.
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humanized bi-specific diabody that targets epidermal growth factor receptor and CD3
hEx3 single-chain diabody
human rhinovirus 3C
3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt
single chain Fv
lymphokine-activated killer cells with the T-cell phenotype
tandem single-chain diabody
Bi-specific antibodies (BsAbs) are attractive formats for recombinant antibodies that can bind to two different epitopes on antigens. This bi-specificity can be used in cancer immunotherapy by cross-linking tumor cells to immune cells such as cytotoxic T cells, natural killer cells and macrophages. This linkage accelerates the destruction of the tumor cells by immune cells, so that the dose of therapeutic antibodies can be reduced from that required in the case of mono-specific antibodies [1,2].
Conventionally, BsAbs are produced by chemical conjugation or somatic fusion of two hybridomas, forming a quadroma that can produce bi-specific IgG molecules [1,3]. Clinical studies of these BsAbs have been performed, and some impressive local anti-tumor responses have been reported; however, these trials have also been limited by the occurrence of human anti-mouse antibody and/or Fc-mediated side-effects such as the induction of a cytokine storm [4,5]. Furthermore, these methods cannot be utilized for large-scale production, and a quadroma cannot control the heterogeneity of the antibodies produced; for instance, ten possible variants of antibodies can be generated when two heavy and two light chains are randomly associated. Therefore, steady production of homogeneous BsAbs requires the use of a host-vector system.
Advances in antibody engineering techniques and host-vector expression systems have facilitated the generation of recombinant BsAbs with improved properties. A variety of recombinant BsAbs have been developed from two antibody fragments such as single-chain Fv fragments (scFv; 25 kDa) [6,7], and diabodies (Db; 55 kDa)  that recognize different antigens. The most common BsAb formats that have been produced from these fragments are tandem scFv (taFv) , tandem single-chain diabodies (tandem scDb, tanDb)  and mini-bodies (dimeric scDb–CH3 fusion protein) . Compared with classic BsAbs prepared by chemical conjugation or production of a quadroma, small antibody molecules, such as diabodies, are of a suitable size for rapid tissue penetration, high target retention and rapid clearance [12,13]. Their smaller size also enables expression of BsAbs in bacteria, and as the structure is composed only of antibody variable regions, this eliminates the Fc-mediated side-effects of BsAbs. Although the rapid blood clearance and monovalency of bi-specific diabodies, scDbs and taFv (all approximately 55 kDa) may limit their therapeutic application, engineering the length and amino acid composition of the middle linker in scDb, for example, may enable them to assemble into multimers, such as tanDb (114 kDa), with higher molecular weight and bivalency for each target antigen [14,15].
Small bi-specific antibody fragments prepared in bacteria are often expressed as insoluble aggregates in the cytoplasmic or periplasmic space [10,16–18], and require fusion of artificial affinity peptide tags, such as a polyhistidine tag, hemagglutinin tag or FLAG tag, at the N- or C-terminus of the BsAbs to allow complete removal of the vast amount of host-derived proteins by affinity chromatography [16,19]. The requirement for such tags raises concerns about immunogenicity. We have previously reported significant anti-tumor activity in vitro and in vivo for a humanized bi-specific diabody targeting epidermal growth factor receptor (EGFR) and CD3 (hEx3-Db) . However, even though the yield of hEx3-Db was over 10 mg·L−1 culture, it was also expressed as insoluble aggregates, and fusion of an affinity tag was necessary for purification before the re-folding process.
We have also reported the construction of a mammalian expression system for affinity-tagged bi-specific diabodies and their Fc fusion formats . Here, we developed a novel method for the production of highly purified tag-free diabodies using the mammalian expression system. Diagrams of the various gene constructs are shown in Fig. 1. The tag-free hEx3-Db alone was expressed sufficiently to be purified by ion-exchange chromatography. Expression of the hEx3 diabodies fused to the human Fc region via a designed protease recognition site enabled high-efficiency purification by protein A affinity chromatography and increased the yield of tag-free hEx3-Db. We also used our method to produce tag-free small BsAbs to hEx3-scDb. For hEx3-scDb, use of the designed protease recognition site had advantages over papain digestion, which caused unwanted degradation. Both tag-free hEx3-Db and hEx3-scDb prepared by restriction protease digestion from the Fc fusion format showed a greater inhibition of cancer growth in vitro than previously produced affinity-tagged diabodies directly prepared from the supernatant of Chinese hamster ovary (CHO) transfectants . Thus, this approach appears to improve both the yield and efficacy of the bi-specific antibody fragments.
Preparation of tag-free bi-specific diabodies
Tag-free hEx3-Db was directly secreted from mammalian cells and purified by cation-exchange chromatography as described in Experimental procedures. Purified hEx3-Db was applied to a gel filtration column for further analysis and purification (Fig. 2A). The first small peak, second large peak and the shoulder of the major peak seen in the chromatograph were identified as the multimeric, dimeric and monomeric structures of tag-free hEx3-Db, respectively. Equivalent amounts of hOHh5L (humanized OKT3 VH - linker - humanized 528 VL) and h5HhOL (humanized 528 VH - linker - humanized OKT3 VL) were confirmed in the dimeric fraction by SDS–PAGE analysis (Fig. 2B). Thus, purified tag-free hEx3-Dbs were obtained without affinity chromatography at a final yield of approximately 1 mg·L−1 culture.
To prepare the high-quality, tag-free bi-specific diabodies, we fused the hEx3-Db to the human IgG1 Fc region. We inserted a recognition site for HRV3C protease between the diabody fragments and the Fc portion of hEx3-Fc. A schematic illustration of the preparation of tag-free hEx3-Db from its Fc fusion format is shown in Fig. 3A. The expressed IgG-like BsAbs were purified by protein A affinity chromatography and digested using glutathione S-transferase (GST)-fused HRV3C protease. The treated solution was loaded onto a glutathione-immobilized column and then a protein A column to remove added protease and digested Fc. SDS–PAGE analysis of each purification step showed the successful preparation of tag-free hEx3-Db from its Fc fusion format (Fig. 3B). Gel filtration chromatography showed that tag-free hEx3-Db predominantly formed dimers, with a small amount of multimeric forms (Fig. 4A). The homogeneity of tag-free hEx3-Db in the eluted fraction was also confirmed by SDS–PAGE (Fig. 4B). The final yield of tag-free hEx3-Db from the Fc fusion format was approximately 5 mg·L−1 culture, i.e. five times that of the secreted tag-free hEx3-Db. Thus, secretion of BsAbs as the Fc fusion format increased the amount of prepared tag-free diabodies due to the high productivity (approximately 10 mg·L−1) and the efficient purification using protein A.
Mass spectrometry of tag-free bi-specific diabodies
We previously reported that the strong inter-domain interaction between cognate VH and VL domains of hEx3-Db leads to the spontaneous formation of functional heterodimers . In the present study, the molecular weight of the monomorphous heterodimer of the tag-free hEx3-Db prepared from the Fc fusion format was confirmed by MALDI-TOF mass spectrometry (Fig. 4C). The mass spectrum for the diabodies prepared from the Fc fusion format had two peaks, one at m/z 26 424 and another at m/z 25 970, which correspond to the calculated molecular weights of hOHh5L digested from the Fc fusion (26 442) and h5HhOL without the peptide tag (25 991), respectively. These results indicate that Db–3C–Fc fusion proteins can serve as a tool for preparing tag-free diabodies with high yield and purity.
Binding affinity of tag-free bi-specific diabodies and its effect on growth inhibition
The binding affinity of tag-free hEx3-Dbs for CD3-positive lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells) and EGFR-positive TFK-1 cells was measured by flow cytometry using polyclonal antibody to hEx3-Db. Tag-free hEx3-Dbs interacted with each targeted antigen (Fig. 5A), and the binding profiles were comparable with those previously reported for affinity-tagged hEx3-Db [20,22]. These results indicate that the diabody prepared by HRV3C protease digestion from the Fc fusion format retained sufficient binding activity and bi-specificity.
To evaluate the inhibition of cancer growth by tag-free hEx3-Db, an MTS assay was performed for TFK-1 cells by using T-LAK cells at an effector/target ratio of 5 : 1. Tag-free hEx3-Db prepared from the Fc fusion format inhibited cancer cell growth more effectively than did affinity-tagged hEx3-Db (Fig. 5B). Imperceptible differences in purity and local structural perturbations that are dependent on the preparation method might affect these activities.
Application of method to tag-free bi-specific single-chain diabody
To demonstrate the utility of this novel method, we applied it to the preparation of tag-free hEx3-scDb, which is a single-chain form of hEx3-Db (Fig 6A). An HRV3C protease recognition site was inserted between hEx3-scDb and the Fc portion, and the recognition sequence for papain was conserved. Papain is a cysteine protease that is generally used in the preparation of Fab fragments from IgG, because the recognition site for papain naturally exists around the hinge region of intact antibody.
When we digested hEx3-scDb–3C–Fc with HRV3C protease, hEx3-scDb was separated from the Fc portion with no degradation. Similar to the tag-free hEx3-Db, the Fc portion was completely removed by protein A affinity chromatography (Fig. 6B). To confirm the benefit of the design of the HRV3C protease digestion site, we also followed the time course of papain digestion of hEx3-scDb–3C–Fc (Fig. 6C). Although tag-free hEx3-scDb was successfully prepared by papain digestion, especially with an incubation time of 1 h, two unexpected bands corresponding to hOHh5L and h5HhOL caused by a break in the middle linker from scDb also appeared. This digestion proceeded as the incubation time increased, and further degradation of h5HhOL was observed after incubation for 10 h.
The binding affinity of tag-free hEx3-scDb prepared from the Fc fusion format for both targeted cells was confirmed by flow cytometry (Fig. 7A), and its enhanced cytotoxicity was compared with affinity-tagged hEx3-scDb  in the MTS assay with the use of T-LAK cells as effector cells (Fig. 7B). These results strongly support the utility and general applicability of our method for the preparation of homogeneous tag-free small recombinant antibodies.
Recombinant BsAbs have several advantages over classic BsAbs prepared by chemical cross-linkage or fusion of two hybridoma clones [16,23–25]. The IgG-like BsAbs containing human Fc regions are highly effective recombinant antibodies [25–27] because of the antibody-dependent cellular cytotoxicity effect. By comparison, small bi-specific diabodies without Fc have the advantages of rapid tissue penetration, high target retention and a distance between the two antigen-binding sites of the diabodies that is large enough to bring two cells together for recruitment of immune cells [1,2,28].
Large-scale production of bi-specific diabodies in bacterial expression systems would be expected because of their small size; however, the yield is typically only a few mg per L in most cases [10,16,17]. We previously proposed an in vitro refolding system to prepare functional bi-specific diabodies from the insoluble fraction, but solubilizing the expressed proteins from insoluble fraction required purification from the vast amount of host-derived proteins, which forced us to utilize an artificial tag [20,22,29]. The immunogenicity of the artificial peptide tag has not been determined, and preparation of tag-free formats from insoluble fractions may be difficult to achieve . For these reasons, a new preparation method for bi-specific diabodies was needed that required minimal artificial amino acid sequences and produced high yields.
In the present study, we successfully purified tag-free hEx3-Db from the supernatant of transfected CHO cells using cation-exchange and gel filtration chromatography (Fig. 2). However, the final yield of this secreted tag-free hEx3-Db was approximately 1 mg·L−1 culture. We thus developed a novel method using IgG-like BsAb and a restriction protease with high specificity. The fusion of Fc to diabodies resulted in high productivity and enabled affinity purification using protein A. The homogeneous dimer structure and molecular weight of the tag-free hEx3-Db prepared from the Fc fusion format (hEx3-Db–3C–Fc) were confirmed by gel filtration and mass spectrometry, and the yield was approximately five times that of the directly secreted tag-free hEx3-Db (Figs 3 and 4).
The specific binding affinity and bi-specificity of the tag-free hEx3-Db for T-LAK and TFK-1 cells were observed by flow cytometry (Fig. 5A). Interestingly, the result of the MTS assay showed that growth inhibition by tag-free hEx3-Db from the Fc fusion was more intense than that by affinity-tagged hEx3-Db (Fig. 5B). Although it is unclear why the tag-free diabodies prepared from the Fc fusion format had such high activity, imperceptible differences in purity and local structural perturbations that are dependent on the preparation method might have affected the activity of the diabodies. The reasons for this difference in activity are now under investigation. Furthermore, we were able to reproduce our results with tag-free hEx3-scDb, which indicates the utility and applicability of our method for the preparation of tag-free small recombinant antibodies (Figs 6 and 7). The single-chain format has additional advantages: scDbs can be produced from a single expression vector and are expected to have improved stability in vivo because the two chains in the diabody are connected to each other via a linker [14,30].
In general, papain and pepsin have been used in the preparation of antibody fragments from IgG-like antibodies, and successful preparation of scFv from scFv–Fc has also been reported . However, for hEx3 single-chain diabodies fused with Fc, papain digestion led to undesired degradation (Fig. 6C). Thus, the advantages of using the designed protease recognition site were confirmed, especially in recombinant antibodies that included a number of artificial sequences.
To date, several different small BsAb formats have been proposed to increase efficacy and availability, such as scDbs , taFv [9,32] and mini-bodies . Further, dimeric scDbs known as tanDbs, with bivalency for each target antigen, can be produced by engineering the length and amino acid composition of middle linker of scDb . Here, we selected diabodies and scDb monomers with a 20-amino-acid middle linker [(GGGGS)4] as small BsAbs, because they are one of the simplest construction formats [20,22]. Use of our preparation method for other BsAbs formats is currently in progress.
We previously reported for BsAbs with affinity peptide tags that hEx3-scDb has comparable function to that of hEx3-Db in vitro . In this work, we have shown that tag-free formats behave quantitatively similarly in in vitro cell growth inhibition studies (Figs 5B and 7B). Therefore, regardless of the presence or absence of an affinity tag, the activity of hEx3-Db is comparable to that of hEx3-scDb. Several reports have demonstrated a higher stability of scDb than other formats such as Db and taFv [14,33–35]. Although hEx3-Db and hEx3-scDb showed similar activities in vitro, there is a possibility the hEx3-scDb may exhibit a higher activity in vivo because of higher stability. Stability tests under physiological conditions between hEx3-Db and hEx3-scDb are currently in progress.
Issues such as rapid blood clearance and the relatively low affinity caused by low molecular weight and monovalent binding may limit the therapeutic application of bi-specific diabodies . In such cases, conversion into more effective formats such as tanDb may be required. The approach described here is also expected to be applicable for convenient preparation of such antibody fragments.
In conclusion, we prepared tag-free bi-specific diabodies in a mammalian expression system and developed a novel method using IgG-like antibodies and protease digestion to prepare highly purified, tag-free bi-specific diabodies. Our method may allow industrial-scale production of functional tag-free small biological agents such as small recombinant antibodies.
Preparation of secreted Ex3 diabodies
In accordance with the convention used in a previous report, we describe the two hetero scFvs of hEx3-Db as h5HhOL and hOHh5L . The gene constructs (Fig. 1) were inserted into pcDNA3.1/Neo or pcDNA3.1/Hygro mammalian expression vectors (both from Invitrogen, Groningen, Netherlands). The leader peptide sequences for protein secretion were derived from the mouse OKT3 IgG . The methods for expression and purification of the affinity-tagged hEx3-Db and hEx3-scDb have been described previously . For production of tag-free hEx3-Db, CHO cells were co-transfected with pcDNA-h5HhOL(−) and pcDNA-hOHh5L(−) (Fig. 1), and cell clones expressing tag-free hEx3-Db were established in the presence of neomycin (G418) and hygromycin as described previously . CHO clones that stably expressed tag-free hEx3-Db were selected by screening for a growth inhibition effect of each individual clone. The established CHO clone was cultured as previously described . The secreted tag-free hEx3-Db was purified from pooled supernatants using a 5 mL HiTrap SP XL column (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) with a 5–250 mm gradient of NaCl in 50 mm phosphate solution (pH 6.0).
Preparation of tag-free hEx3 diabodies from the Fc fusion format
To construct the expression vector for preparing tag-free diabodies by using IgG-like BsAbs, we connected the hEx3 diabodies and the human IgG1 Fc region via a recognition site (LEVLFQGP) for human rhinovirus 3C (HRV3C) protease. CHO cells were co-transfected with equal amounts of the pcDNA-h5HhOL-3C-Fc and pcDNA-hOHh5L(−) vectors (Fig. 1), and grown in presence of neomycin (G418) and hygromycin as described previously . A CHO clone that stably expressed the hEx3-Db–3C–Fc fusion protein was selected in a manner similar to that for tag-free hEx3-Db. For tag-free hEx3-scDb, CHO cells were transfected with the pcDNA-hEx3-scDb–3C–Fc vector, and selection for a stably expressed clone was performed in the presence of 500 μg·mL−1 of G418 (Nacalai Tesque, Kyoto, Japan).
IgG-like BsAbs of hEx3–3C–Fc and hEx3-scDb–3C–Fc were first purified by affinity chromatography on a protein A column (GE Healthcare) and then digested by HRV3C protease fused to GST (PreScission protease; GE Healthcare) according to the protocol described by the manufacturer. The protease was removed using a glutathione Sepharose 4B column (GE Healthcare), and the flowthrough was re-loaded onto the protein A column to remove the digested Fc and undigested hEx3-scDb–3C–Fc fusion protein. The presence of the BsAbs in each stage of purification were confirmed by SDS–PAGE under reducing conditions.
To illustrate the applicability of this novel method, papain digestion of hEx3-scDb–3C–Fc was performed by use of an ImmunoPure Fab preparation kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). The influence of papain digestion was confirmed by SDS–PAGE analysis under reducing conditions at 1, 5 and 10 h after digestion.
Gel filtration chromatography
Gel filtration analysis with a Hiload Superdex 200 pg column (26/60; GE Healthcare) was used to evaluate the structure of the bi-specific diabodies. The column was equilibrated using NaCl/Pi, and then 5 mL of purified recombinant antibodies was applied to the column at a flow rate of 2.5 mL·min−1.
Mass spectra were measured using a REFLEX III MALDI-TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) equipped with a nitrogen laser (337 nm). Sinapic acid was applied as a matrix, and was dissolved to saturation in water:acetonitrile (2 : 1 v/v) containing 0.067% trifluoroacetic acid. Sample solutions from each stage were mixed with the sinapic acid-saturated solution in a 1 : 1 v/v ratio, and then 1 μL of the mixed solution was loaded onto the sample target. After co-crystallization on the target, the crystals were washed twice with 2 μL of water containing 0.1% trifluoroacetic acid to remove residual salts. Analysis was performed in positive and linear modes with an accelerating voltage of 27 kV, and 200 scans were averaged. The spectra obtained were calibrated externally using the [M + H+] ions from two protein standards: cytochrome c from horse heart (m/z 12 360.08) and bovine trypsin (m/z 23 311.53) .
Preparation of T-LAK cells
Peripheral blood mononuclear cells were isolated by density-gradient centrifugation of heparin-containing blood from healthy volunteers. To induce proliferation of T-LAK cells, peripheral blood mononuclear cells were cultured for 48 h at a density of 1 × 106 cells per mL in medium supplemented with 100 IU·mL−1 of recombinant human IL-2 (kindly supplied by Shionogi Pharmaceutical Co., Osaka, Japan) in a culture flask (A/S Nunc, Roskilde, Denmark) that had been pre-coated with OKT3 monoclonal antibody (10 μg·mL−1). Proliferated cells were then transferred to another flask, and expanded for 2–3 weeks in a culture medium containing 100 IU·mL−1 IL-2, as reported previously .
Flow cytometric analyses
Test cells (1 × 106) were incubated on ice with 200 pmol of BsAb for 30 min. After washing with NaCl/Pi containing 0.1% NaN3, they were exposed for 30 min on ice to rabbit anti-hEx3-Db serum (kindly supplied by Immuno-Biological Laboratories Co. Ltd, Gunma, Japan) as the second antibody, and fluorescein isothiocyanate-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as the third antibody. The stained cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA) .
In vitro growth inhibition assay
In vitro growth inhibition of TFK-1 (human bile duct carcinoma) was assayed using a 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay kit (CellTiter 96 aqueous non-radioactive cell proliferation assay; Promega, Madison, WI, USA) as reported previously .
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to R.A. and I.K.) and by grants from the New Energy and Industrial Technology Development Organization of Japan. Additional support was provided through the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation.