Application of the Fc fusion format to generate tag-free bi-specific diabodies Ryutaro Asano 1 , Keiko Ikoma 1 , Hiroko Kawaguchi 1 , Yuna Ishiyama 1 , Takeshi Nakanishi 1 , Mitsuo Umetsu 1 , Hiroki Hayashi 2 , Yu Katayose 2 , Michiaki Unno 2 , Toshio Kudo 3 and Izumi Kumagai 1 1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan 2 Division of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan 3 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Introduction Bi-specific antibodies (BsAbs) are attractive formats for recombinant antibodies that can bind to two differ- ent 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 anti- bodies [1,2]. Keywords bi-specific diabody; Fc fusion format; preparation method; small therapeutic antibody; tag-free protein Correspondence I. Kumagai, Aoba 6-6-11-606, Aramaki, Aoba-ku, Sendai 980-8579, Japan Fax: +81 22 795 6164 Tel: +81 22 795 7274 E-mail: kmiz@kuma.che.tohoku.ac.jp (Received 1 May 2009, revised 9 November 2009, accepted 17 November 2009) doi:10.1111/j.1742-4658.2009.07499.x 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 recombi- nant 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 sin- gle-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. Abbreviations BsAbs, bi-specific antibodies; CHO, Chinese hamster ovary; Db, diabody; EGFR, epidermal growth factor receptor; hEx3-Db, humanized bi-specific diabody that targets epidermal growth factor receptor and CD3; hEx3-scDb, hEx3 single-chain diabody; HRV3C, human rhinovirus 3C; MTS, 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; scDb, single-chain diabody; scFv, single chain Fv; T-LAK, lymphokine-activated killer cells with the T-cell phenotype; tanDb, tandem single-chain diabody; taFv, tandem scFv. FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS 477 Conventionally, BsAbs are produced by chemical conjugation or somatic fusion of two hybridomas, form- ing a quadroma that can produce bi-specific IgG mole- cules [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 produc- tion, 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. There- fore, 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 gen- eration of recombinant BsAbs with improved proper- ties. 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) [8] that recognize different antigens. The most common BsAb formats that have been produced from these fragments are tandem scFv (taFv) [9], tan- dem single-chain diabodies (tandem scDb, tanDb) [10] and mini-bodies (dimeric scDb–CH3 fusion protein) [11]. Compared with classic BsAbs prepared by chemi- cal conjugation or production of a quadroma, small antibody molecules, such as diabodies, are of a suit- able size for rapid tissue penetration, high target reten- tion 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 com- plete removal of the vast amount of host-derived proteins by affinity chromatography [16,19]. The requirement for such tags raises concerns about immu- nogenicity. 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) [20]. However, even though the yield of hEx3-Db was over 10 mgÆL )1 culture, it was also expressed as insoluble aggregates, and fusion of an affinity tag was necessary for purifica- tion before the re-folding process. We have also reported the construction of a mam- malian expression system for affinity-tagged bi-specific diabodies and their Fc fusion formats [21]. 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 con- structs 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 purifi- cation 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 pro- tease digestion from the Fc fusion format showed a greater inhibition of cancer growth in vitro than previ- ously produced affinity-tagged diabodies directly pre- pared from the supernatant of Chinese hamster ovary (CHO) transfectants [21]. Thus, this approach appears to improve both the yield and efficacy of the bi-specific antibody fragments. Results Preparation of tag-free bi-specific diabodies Tag-free hEx3-Db was directly secreted from mamma- lian cells and purified by cation-exchange chromatog- raphy as described in Experimental procedures. Purified hEx3-Db was applied to a gel filtration col- umn for further analysis and purification (Fig. 2A). The first small peak, second large peak and the shoul- der of the major peak seen in the chromatograph were identified as the multimeric, dimeric and monomeric structures of tag-free hEx3-Db, respectively. Equiva- lent amounts of hOHh5L (humanized OKT3 VH - linker - humanized 528 VL) and h5HhOL (humanized 528 VH - linker - humanized OKT3 VL) were con- firmed 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 mgÆL )1 culture. Fc fusion for generation of tag-free diabodies R. Asano et al. 478 FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS To prepare the high-quality, tag-free bi-specific dia- bodies, we fused the hEx3-Db to the human IgG1 Fc region. We inserted a recognition site for HRV3C pro- tease between the diabody fragments and the Fc por- tion 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 chromatog- raphy 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 prote- ase and digested Fc. SDS–PAGE analysis of each puri- fication 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 homogene- ity 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 mgÆL )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 produc- tivity (approximately 10 mgÆL )1 ) and the efficient puri- fication using protein A. Mass spectrometry of tag-free bi-specific diabodies We previously reported that the strong inter-domain interaction between cognate V H and V L domains of hEx3-Db leads to the spontaneous formation of func- tional heterodimers [22]. 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 spec- trometry (Fig. 4C). The mass spectrum for the diabod- ies 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 Nhe I Xho I Nhe I Xho I Nhe I Xho Io h5H hOL Neo r hOH h5L Hyg r h5H h5H CH2 CH3 Neo r pcDNA-h5HhOL-3C-Fc Tag-free hEx3-Db pcDNA-h5HhOL(–) pcDNA-hOHh5L(–) hEx3-Db-3C-Fc(tool for tag-free hEx3-Db) hEx3-scDb-3C-Fc(tool for tag-free hEx3-scDb) Nhe I Xho I h5H hOL CH2 CH3 NhOH h5L h5H hOL CH2 CH3 Neo r hOH h5L 5O pcDNA-hEx3-scDb-3C-FcpcDNA hEx3 scDb 3C Fc CMV promoter Kozak sequence Leader peptide Peptide linker (GGGGS) Peptide linker [(GGGGS)4] HRV3C protease recognition site (LEVLFQGP) Hinge Neo Hyg Neomycin resistance Hygromycin resistance Neo r Hyg r hOL Fig. 1. Schematic illustration of the BsAb gene constructs in pCDNA3.1. The V H and V L regions of humanized 528 Fv are desig- nated h5H and h5L, and those of humanized OKT3 Fv are designated hOH and hOL, respectively. The positions of important restriction enzyme sites used and the key components are shown. R. Asano et al. Fc fusion for generation of tag-free diabodies FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS 479 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 previ- ously 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 effec- tively 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. 67 kDa A B 25 kDa43 kDa 47.5 – 5 mAU 32.5 – Tag-free hOHh5L 25 – Tag-free h5HhOL 16.5 – 150 200 250 300 Elution volume (mL) Fig. 2. (A) Gel filtration of tag-free hEx3-Db. The elution volume is shown on the x axis, and the molecular mass (kDa) is shown above. The eluted fractions containing the bi-specific diabody are indicated by the two-headed arrow. (B) SDS–PAGE analysis under reducing conditions of the eluted fraction. Molecular size markers are shown on the left. HRV3C protease site A B Tag-free hEx3-Db hEx3-Db-3C-Fc hEx3-Db-3C-Fc Tag-free hEx3-Db 1 175 – 83 – 62 – 47.5 – h5HhOL-3C-Fc –32.5 Fc Tag-free hOHh5L 25 – Tag-free h5HhOL 16.5 – 234 Fig. 3. (A) Schematic illustration of the hEx3-Db–3C–Fc fusion pro- tein. The HRV3C protease cleavage site used for preparation of tag-free hEx3-Db is indicated. (B) Reducing SDS–PAGE of each purification step for preparation of tag-free hEx3-Db from hEx3-Db– 3C–Fc. Lane 1, protein A chromatography-purified hEx3-Db–3C–Fc; lane 2, after HRV3C protease digestion; lane 3, after removal of HRV3C protease by glutathione Sepharose 4B chromatography; lane 4, purified tag-free hEx3-Db after removal of the Fc region by protein A chromatography. Fc fusion for generation of tag-free diabodies R. Asano et al. 480 FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS Application of method to tag-free bi-specific sin- gle-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 cyste- ine 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 por- tion 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 mid- dle 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 [21] 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. Discussion Recombinant BsAbs have several advantages over clas- sic 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 effec- tive 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 anti- gen-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 150 200 300 20 000 30 000 35 000 m /z25 000 250 AB C Fig. 4. (A) Gel filtration of purified hEx3-Db after removal of HRV3C protease and the Fc fragment. The elution volume is shown on the x axis, and the molecular mass (kDa) is shown above. The eluted fractions containing the bi-specific diabody are indicated by the two-headed arrow. (B) SDS–PAGE analysis under reducing conditions of the eluted fractions. Molecular size markers are shown on the left. (C) MALDI- TOF mass spectra of the tag-free hEx3-Db prepared from hEx3-Db–3C–Fc. R. Asano et al. Fc fusion for generation of tag-free diabodies FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS 481 functional bi-specific diabodies from the insoluble frac- tion, but solubilizing the expressed proteins from insol- uble 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 [16]. For these reasons, a new preparation method for bi-specific dia- bodies 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 T-LAK A B TFK-1 a b a b Counts Fluorescent intensity 100 * E:T = 5 * 50 Affinity-tagged hEx3-Db Tag-free hEx3-Db from Fc fusion 0 0 1 10 100 10000 1 10 100 1000 (T LAK)(T-LAK) Concentration of BsAb (fmol·mL –1 ) Growth inhibition of cancer cells (%) 10 0 0 40 80 120 160 200 0 40 80 120 160 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Fig. 5. (A) Flow cytometry analysis of tag-free hEx3-Db prepared from hEx3-Db–3C–Fc. Cells were incubated with NaCl ⁄ P i as a nega- tive control (a) and with either OKT3 parental IgG (for T-LAK cells) or 528 IgG (for TFK-1 cells), followed by staining with fluorescein isothiocyanate-conjugated anti-mouse IgG as a positive control (b). The shaded areas correspond to the fluorescence intensity distribu- tions of the cells incubated with hEx3-Db. Each mixture was stained with rabbit anti-hEx3-Db serum followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG. (B) Growth inhibition of EGFR-positive TFK-1 cells by tag-free and affinity-tagged hEx3 diabodies. Each bi-specific diabody and T-LAK cells (effectors, E) were added to TFK-1 cells (T) at a ratio of 5 : 1. The tag-free hEx3-Db inhibited growth significantly better (*P < 0.005) than the affinity-tagged hEx3-Db did [21]. Data are mean values ± SD and are representative of at least three independent experiments with similar results. HRV3C protease site A B C Tag-free hEx3-scDb hEx3-scDb-3C-Fc Tag-free hEx3-scDbhEx3-scDb-3C-Fc 1 hEx3-scDb-3C-Fc (monomer) Tag-free hEx3-scDb Fc 1 2 175 - hEx3-scDb-3C-Fc 83 - (monomer) 62 - Tag-free hEx3-scDb 47.5- 32.5- Fc Tag-free hOHh5L Tag-free h5HhOL 25 - - 16.5- 1 h 5 h 10 h 234 3 1 2 3 1 2 3 Fig. 6. (A) Schematic illustration of the hEx3-scDb–3C–Fc fusion protein. The HRV3C protease cleavage site used for preparation of tag-free hEx3-scDb is indicated. (B) Reducing SDS–PAGE of each purification step for preparation of tag-free hEx3-scDb from hEx3- scDb–3C–Fc. Lane 1, protein A chromatography-purified hEx3- scDb–3C–Fc; lane 2, after HRV3C protease digestion; lane 3, after removal of HRV3C protease by glutathione Sepharose 4B chroma- tography; lane 4, purified tag-free hEx3-scDb after removal of the Fc region by protein A chromatography. (C) Reducing SDS–PAGE of hEx3-scDb–3C–Fc incubated with papain for 1, 5 and 10 h. Lane 1, digested hEx3-scDb–3C–Fc; lane 2, flowthrough from protein A chromatography; lane 3, eluted protein from protein A chroma- tography. Fc fusion for generation of tag-free diabodies R. Asano et al. 482 FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS cells using cation-exchange and gel filtration chroma- tography (Fig. 2). However, the final yield of this secreted tag-free hEx3-Db was approximately 1 mgÆL )1 culture. We thus developed a novel method using IgG- like BsAb and a restriction protease with high specific- ity. 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 inhi- bition 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 dia- bodies 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 activ- ity 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 anti- bodies, and successful preparation of scFv from scFv– Fc has also been reported [31]. However, for hEx3 sin- gle-chain diabodies fused with Fc, papain digestion led to undesired degradation (Fig. 6C). Thus, the advanta- ges 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 [30], taFv [9,32] and mini-bodies [11]. Further, dimeric scDbs known as tanDbs, with biva- lency for each target antigen, can be produced by engi- neering the length and amino acid composition of middle linker of scDb [15]. Here, we selected diabodies and scDb monomers with a 20-amino-acid middle lin- ker [(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 pep- tide tags that hEx3-scDb has comparable function to that of hEx3-Db in vitro [22]. In this work, we have shown that tag-free formats behave quantitatively T-LAK A B TFK-1 a b a b Counts Fluorescent intensity 100 E:T = 5 * * 50 Affinity-tagged hEx3-scDb Tag-free hEx3-scDb from Fc fusion 0 0 1 10 100 10000 (T-LAK) Concentration of BsAb (fmol·mL –1 ) Growth inhibition of cancer cells (%) 10 0 0 40 80 120 160 200 0 40 80 120 160 200 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Fig. 7. (A) Flow cytometric analysis of purified tag-free hEx3-scDb. Cells were incubated with NaCl ⁄ P i as a negative control (a) and with either OKT3 parental IgG (for T-LAK cells) or 528 IgG (for TFK- 1 cells), followed by staining with fluorescein isothiocyanate-conju- gated anti-mouse IgG as a positive control (b). The shaded areas correspond to the fluorescence intensity distributions of the cells incubated with hEx3-Db. Each mixture was stained with rabbit anti-hEx3-Db serum followed by fluorescein isothiocyanate- conjugated anti-rabbit IgG. (B) Growth inhibition of EGFR-positive TFK-1 cells by tag-free and affinity-tagged hEx3 single-chain diabod- ies. Each bi-specific diabody and T-LAK cells (effectors, E) were added to TFK-1 cells (T) at a ratio of 5 : 1. The tag-free hEx3-scDb inhibited growth significantly better (*P < 0.005) than the affinity- tagged hEx3-scDb did [21]. Data are mean values ± SD and are representative of at least three independent experiments with similar results. R. Asano et al. Fc fusion for generation of tag-free diabodies FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS 483 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 pro- gress. Issues such as rapid blood clearance and the rela- tively low affinity caused by low molecular weight and monovalent binding may limit the therapeutic applica- tion of bi-specific diabodies [14]. In such cases, conver- sion 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 devel- oped 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. Experimental procedures 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 [20]. 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 [36]. The methods for expression and purification of the affinity-tagged hEx3-Db and hEx3-scDb have been described previously [21]. 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 pres- ence of neomycin (G418) and hygromycin as described pre- viously [21]. 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 [27]. 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) pro- tease. CHO cells were co-transfected with equal amounts of the pcDNA-h5HhOL-3C-Fc and pcDNA-hOHh5L()) vec- tors (Fig. 1), and grown in presence of neomycin (G418) and hygromycin as described previously [21]. 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 lgÆmL )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 Health- care) according to the protocol described by the manufac- turer. The protease was removed using a glutathione Sepharose 4B column (GE Healthcare), and the flow- through 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 col- umn (26 ⁄ 60; GE Healthcare) was used to evaluate the structure of the bi-specific diabodies. The column was equil- ibrated using NaCl ⁄ P i , and then 5 mL of purified recombi- nant antibodies was applied to the column at a flow rate of 2.5 mLÆmin )1 . Mass spectrometry 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) con- taining 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 lL of the mixed Fc fusion for generation of tag-free diabodies R. Asano et al. 484 FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS solution was loaded onto the sample target. After co-crys- tallization on the target, the crystals were washed twice with 2 lL 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) [37]. Preparation of T-LAK cells Peripheral blood mononuclear cells were isolated by den- sity-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 · 10 6 cells per mL in medium supple- mented with 100 IUÆmL )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 lgÆmL )1 ). Proliferated cells were then transferred to another flask, and expanded for 2–3 weeks in a culture medium containing 100 IUÆmL )1 IL-2, as reported previ- ously [38]. Flow cytometric analyses Test cells (1 · 10 6 ) were incubated on ice with 200 pmol of BsAb for 30 min. After washing with NaCl ⁄ P i containing 0.1% NaN 3 , they were exposed for 30 min on ice to rabbit anti-hEx3-Db serum (kindly supplied by Immuno-Biologi- cal 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) [20]. In vitro growth inhibition assay In vitro growth inhibition of TFK-1 (human bile duct carci- noma) 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-radio- active cell proliferation assay; Promega, Madison, WI, USA) as reported previously [39]. Acknowledgements This work was supported by Grants-in-Aid for Scien- tific 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. 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Kumagai I (2002) Antitumor activity of interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli FEBS Lett 528, 70–76 FEBS Journal 277 (2010) 477–487 ª 2009 The Authors Journal compilation ª 2009 FEBS 487 . mgÆL )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 produc- tivity (approximately. 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. bi-specific diabodies and their Fc fusion formats [21]. Here, we developed a novel method for the production of highly purified tag-free diabodies using the mammalian expression system. Diagrams of the