ARTICLE A Novel Platform For Antibody Library Selection in Mammalian Cells Based on a Growth Signalobody Rie Yoshida,1 Masahiro Kawahara,2 Teruyuki Nagamune1,2 1

Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 2 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; telephone: þ81-3-5841-7290; fax: þ81-3-5841-8657; e-mail: [email protected]

Introduction Abstract: While many antibody-screening methods in vitro have been developed, these methods need repeated cycles of panning or sorting procedures to isolate antigen-specific antibodies. Here we developed a new antibody selection system based on antigen-dependent growth of mammalian cells. In this system, a growth signalobody library, which is a naïve single-chain Fv (scFv) library/cytokine receptor chimera that can transduce a growth signal in response to a specific antigen, is expressed in murine interleukin-3dependent Ba/F3 cells. Simple culture of the cells in an antigen-containing medium results in growing cells with a high-affinity scFv gene, leading to selection of the scFv specific to the target antigen without panning/sorting procedures. To demonstrate this system, we used the SD1D2g signalobody having the signaling domain of gp130 and fluorescein-conjugated BSA as a target antigen, and investigated whether a fluorescein-specific scFv could be selected from a naïve scFv library. As a result, we successfully obtained fluorescein-binding scFv clones, and the scFv clone with the highest affinity was most abundantly selected, having the same sequence as the clone, which had been obtained through phage display. These results demonstrate the utility of our system as an affinity-based scFv selection method based on growth advantage of mammalian cells. Biotechnol. Bioeng. 2014;111: 1170–1179. ß 2013 Wiley Periodicals, Inc. KEYWORDS: antibody selection; mammalian cell; growth; cytokine receptor; signal transduction

Correspondence to: M. Kawahara Contract grant sponsor: Grants-in-Aid for Young Scientists, JSPS Contract grant number: 21686077 Contract grant sponsor: Grants-in-Aid for Challenging Exploratory Research, JSPS Contract grant number: 23656516 Contract grant sponsor: The Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) Received 3 October 2013; Revision received 28 November 2013; Accepted 2 December 2013 Accepted manuscript online 12 December 2013; Article first published online 27 December 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25173/abstract). DOI 10.1002/bit.25173

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Antibodies are widely used for diagnostic and therapeutic applications as well as basic research tools, taking advantage of their high-affinity against specific antigens. There has been growing pressure for a rapid and easy method for isolation of high-affinity antibodies. To date, many antibody-screening methods in vitro have been developed (Hoogenboom, 2005). Ribosome/mRNA display is a cell-free system to enable the coverage of significantly larger library size than other methods (Rothe et al., 2006). However, in ribosome display, the complexes obtained include all elements of the protein synthesis machinery, which not only depletes the translation reaction but also results in a very high background (Lipovsek and Pluckthun, 2004). In mRNA display, there remain concerns in terms of instability of RNA and its fast degradation, together with non-specific binding of proteins (Lipovsek and Pluckthun, 2004). The systems using bacterial cells were also developed, such as phage display, bacterial display, and periplasmic expression with cytometric screening (PECS) (Chen et al., 2001; Daugherty, 2007; Daugherty et al., 1998; Geyer et al., 2012). However, the issue of unfolding and misfolding of antibody fragments is inevitable in the bacterial expression systems (Venturi et al., 2002). For this reason, eukaryotic cells such as yeast and mammalian cells have been recognized as alternative hosts for antibody library selection. In yeast display, antibody fragments are displayed on a cell-surface anchor protein. However, differential glycosylation in yeast compared to mammalian cells may affect the physicochemical properties of antibody fragments (Boder et al., 2012). In this context, mammalian cells provide the most suitable environment in which antibodies are naturally expressed. In mammalian cell display, antibody fragments are displayed on the transmembrane domain of a membrane protein in such cell lines as HEK 293 and CHO cells (Bowers et al., 2013; Forsyth et al., 2013; Ho and Pastan, 2009; Li et al., 2012a,b; Zhou and Shen, 2012; Zhou et al., 2010). While these methods are recognized as powerful tools to obtain antigen-specific antibody fragments, all of these ß 2013 Wiley Periodicals, Inc.

methods need repeated cycles of panning or sorting procedures in order to concentrate antigen-specific clones over non-specific ones. In fact, the antibody library displayed on phage, ribosome, and mRNA needs to be panned repeatedly against a target antigen immobilized on a solid phase. Even in the case of bacterial display, PECS, yeast display and mammalian cell display, antigens need to be labeled with fluorophore or a tag sequence, and an expensive fluorescenceactivated cell sorter or magnetic beads should be employed to isolate the cells displaying antigen-specific antibody fragments. To overcome these problems, we aimed to develop a new antibody selection system with neither panning nor sorting procedure in mammalian cells. To attain this, we developed the system in which antigen binding to an antibody results in a growth signal of mammalian cells. We have previously developed a growth signalobody, which is a single-chain Fv (scFv)/cytokine receptor chimera that can transduce a growth signal in response to a specific antigen (Kaneko et al., 2012; Kawahara et al., 2004, 2011; Liu et al., 2009; Nakabayashi et al., 2013; Sogo et al., 2009; Tanaka et al., 2009). Since cytokine receptors are activated by dimerization, homooligomeric antigen was employed to activate the growth signalobody. While we have successfully developed functional growth signalobodies using a scFv clone, we have never investigated whether the growth signalobody could function as a platform for scFv library selection. In this study, we propose a new antibody-screening system using a growth signalobody (Fig. 1A). In this system, the scFv region of an antigen-responsive growth signalobody is replaced with a naïve scFv-library, and the resultant growth signalobody library is stably expressed in interleukin-(IL)-3dependent Ba/F3 cells using retroviral gene transduction. Then, the cells are cultured in a medium containing a target antigen but without IL-3. Since Ba/F3 cells are strictly IL-3dependent, growing cells, if any, would express antigenresponsive growth signalobodies. Genomic PCR would easily recover the scFv genes from the growing cells, leading to selection of scFvs specific to the target antigen. As a proof-ofconcept experiment, we used the SD1D2g signalobody (Fig. 1A) (Liu et al., 2008) having signaling domain of gp130 and fluorescein-conjugated BSA (BSA-FL) as a target antigen. We investigated whether FL-specific scFv could be selected from a naïve scFv library.

GCTTTGATTTCCACCTTGGTCCC-30 ) were used to amplify a portion of scFv sequence of pMK-Sg-IG(-NotI). The amplified fragment was digested with XhoI and BspEI, and subcloned into pMK-Sg-IG(-NotI) to produce pMK-SgIG(þNotI). To replace the scFv sequence with a stuffer sequence, pDisplay (Invitrogen, San Diego, CA) was digested with EcoRI and NotI, and subcloned into pMK-Sg-IG(þNotI) to create pMK-stuffer-g-IG. To insert the extracellular D1 and D2 domains of EpoR, pMK-SD1D2g was digested with BspEI and BsiWI, and subcloned into pMK-stuffer-g-IG to produce pMK-stuffer-D1D2g-IG. The final product, pMK-stufferD1D2g-IG, encodes an immunoglobulin kappa chain signal sequence, an HA tag, a SfiI site, a stuffer sequence, a NotI site, the extracellular D1 and D2 domains of human EpoR, the transmembrane domain of human EpoR and the intracellular domain of human gp130.

Materials and Methods

Retroviral Transduction

Plasmid Construction The plasmid pMK-Sg-IG (Liu et al., 2008), which encodes the HA-tagged anti-fluorescein scFv clone 31IJ3, the transmembrane domain of human EpoR and the intracellular domain of human gp130 at the upstream of an IRES-EGFP (IG) cassette, was used as a starting construct. To delete an undesirable NotI site, pMK-Sg-IG was digested with NotI, blunted and self-ligated to produce pMK-Sg-IG(-NotI). To newly create a NotI site (underlined) just downstream of the scFv sequence, two primers (For: 50 -GGGGCCAGGGAACC CTGGTCACCG-30 , Rev: 50 -GGGTCCGGAACCTGCGGCC

Library Construction The Tomlinson I human synthetic naïve scFv library in a pIT2 plasmid (Medical Research Council, Cambridge, UK; library size: 1.47  108) was digested with SfiI and NotI, and subcloned into pMK-stuffer-D1D2g-IG to make pMK-S(TomI)D1D2gIG. Then, MegaX DH10BTMT1RElectrocompTM Cells (Invitrogen) were transformed with pMK-S(TomI)D1D2g-IG by electroporation, and the transformed cells (colony number: 2.0  105) were cultured in a 250 mL scale. The cells were harvested to obtain purified plasmid DNA using QIAGEN plasmid Midi Kit (QIAGEN, Valencia, CA). Cell Culture A murine IL-3-dependent pro-B cell line, Ba/F3 (RCB0805, RIKEN Cell Bank, Tsukuba, Japan), was cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Biowest, Miami, FL) and 1 ng/mL of murine IL-3 (R&D Systems, Cambridge, MA). A retroviral packaging cell line Plat-E was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical) supplemented with 10% FBS, 1 mg/mL puromycin (Sigma, St. Louis, MO) and 10 mg/mL blasticidin (Kaken Pharmaceutical, Tokyo, Japan).

Plat-E cells were inoculated into two 60 mm diameter dishes at 2.5  106 cells/dish, and cultured overnight. Plat-E cells were transfected with pMK-S(TomI)D1D2g-IG using Lipofectamine LTX (Invitrogen) and Plus reagent (Invitrogen). The plasmid (12 mg) was sterilized by ethanol precipitation, and solubilized in 2.5 mL serum-free OPTI-MEM (GibcoBRL, Rockville, MD), with 12.5 mL Plus reagent. After incubation at room temperature for 5 min, 31.2 mL Lipofectamine LTX was mixed with the DNA-Plus reagent solution and incubated at room temperature for 30 min. The medium of the cells was replaced with 5 mL/dish of serumfree OPTI-MEM, and the Lipofectamine-DNA complex

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Figure 1.

Cell growth-based screening of scFvs from a nay¨ve scFv library. A: We use a growth signalobody, which is a scFv/receptor chimera that transduces a growth signal through dimerization induced by a homo-oligomeric target antigen. In this system, simple culture of the cells expressing scFv-library/receptor chimera in an antigen-containing medium would result in growing cells with a high-affinity scFv gene, leading to selection of scFvs specific to the target antigen. The chimeric receptor construct is composed of a scFv library, extracellular D1/D2 and transmembrane domains of EpoR, and the intracellular domain of gp130, and an HA-tag is appended on the extracellular N-terminus of each chimeric receptor. B: Schematic diagram of the expression vector. Retroviral vectors with long-terminal repeats (LTRs) and a packaging signal (C) are used. An immunoglobulin kappa chain signal sequence (S) is located upstream of the HA-tagged chimeric receptor gene to enable their cell surface expression. An EGFP gene was located downstream of an IRES sequence to facilitate detection of gene-transduced cells.

solution was overlaid onto the cells. After incubation of the cells for 4 h at a 37 C/5% CO2 incubator, the medium was replaced with a normal growth medium. The medium was further replaced after 24 h. After additional 24 h incubation, the retroviral supernatant was added to two 60 mm diameter dishes coated with RetroNectin (Takara-Bio, Otsu, Japan) and incubated for 5 h. After discarding the supernatant and washing the dish with PBS, Ba/F3 cells (1.0  107 cells in 10 mL growth medium) were added to the dishes (5 mL/ dish) for retroviral transduction. The transduced cells were

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grown up to sufficient number of cells for subsequent library screening. Library Screening The transduced cells were washed twice with PBS to remove IL-3, and seeded into forty 96-well plates at 2  104 cells/ well in 200 mL of RPMI1640/10% FBS either with or without 1 mg/mL BSA-FL (Sigma). After several days of culture, top 23 of fast-growing colonies observed in the

presence of BSA-FL were expanded separately and used for further analyses. Sequence Analysis The genome of BSA-FL-selected colonies were extracted using DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s protocol, and the DNA region encoding the scFv was specifically amplified with genomic PCR using two primers (For: 50 -GTACTGCTGCTCTGGGTTCC-30 , Rev: 50 CGCCCGTTTGATTTCCACCTTG-30 ) and the genome as a template. The amplified DNA was subjected to sequence analysis.

FL-Binding Assay FL-binding to the BSA-FL-selected clones was measured by flow cytometry. In brief, cells (2  105) were incubated with mouse monoclonal anti-Flag (clone M2, 1:100, Sigma) or its FITC conjugate (1:100, Sigma) in PBS on ice for 30 min. The cells were washed twice with PBS, and incubated with Rphycoerythrin (PE)-conjugated donkey F(ab’)2 anti-mouse IgG secondary antibody (1:100) in PBS on ice for 30 min. The cells were washed twice with PBS and analyzed with a FACSCalibur flow cytometer with excitation at 488 nm and fluorescence detection at 585  21 nm. Affinity Evaluation

Surface Expression of HA-Tagged Growth Signalobodies Surface expression levels of HA-tagged growth signalobodies were measured by flow cytometry. In brief, cells (2  105) cultured in IL-3 were incubated on ice for 30 min with mouse monoclonal anti-HA antibody (1:100, Covance, Princeton, NJ) in PBS, washed twice with PBS, and incubated with R-phycoerythrin (PE)-conjugated donkey F(ab’)2 anti-mouse IgG secondary antibody (1:100, Jackson ImmunoResearch, West Grove, PA) in PBS on ice for 30 min. The cells were washed twice with PBS and analyzed with a FACSCalibur flow cytometer (Becton-Dickinson, Lexington, KY) with excitation at 488 nm and fluorescence detection at 585  21 nm. Western Blotting Cells (1  106) were washed with PBS, lysed with 100 mL of lysis buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mg/mL aprotinin, 10 mg/mL leupeptin) and incubated on ice for 10 min. After centrifugation at 21,500 g for 10 min, the supernatant was mixed with Laemmli’s sample buffer and boiled. The lysate was resolved by SDS–PAGE and transferred to a nitrocellulose membrane (Millipore, Bedford, MA). After the membrane was blocked with 5% skim milk (Wako, Osaka, Japan), the blot was probed with rabbit anti-gp130 (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-btubulin (Santa Cruz), followed by HRP-conjugated antirabbit IgG (Biosource, Camarillo, CA), and detection was performed using the ECL system (GE Healthcare, Buckinghamshire, UK). Cell Proliferation Assay Cells were washed twice with PBS and seeded in 96-well plates at 4  104 cells/mL containing various concentrations of BSA-FL. After 3 days of culture, the cells were mixed with Flow-Count Fluorospheres (Beckman Coulter, Brea, CA), stained with 1 mg/mL of propidium iodide (PI, Sigma), and analyzed on a FACSCalibur flow cytometer with excitation at 488 nm and PI fluorescence detection using a 670 nm longpass filter.

Affinity of the FL-binding clones for FL was measured by flow cytometry. Cells (2  105) were incubated on ice for 1 h with various concentrations of FITC-conjugated mouse monoclonal anti-Flag (clone M2) in PBS, washed twice with PBS and incubated with R-phycoerythrin (PE)-conjugated donkey F(ab’)2 anti-mouse IgG (1:100) in PBS on ice for 30 min. Cells were washed twice with PBS and analyzed with a FACSCalibur flow cytometer with excitation at 488 nm and fluorescence detection at 585  21 nm. From the data, the total mean fluorescence from the PE channel (MFUtot) was plotted against the concentration of FITC ([FITC]). Since the fluorophore/protein (F/P) ratio of the FITC-conjugated mouse antibody ranges from 3 to 6 according to the manufacturer’s datasheet, the average F/P ratio (4.5) was used for calculating [FITC]. Then, the sum of the square of the differences was computed between MFUtot measured and MFUtot calculated from the following equation: MFUtot ¼ where MFUmin þ (MFUrange  [FITC])/([FITC] þ Kdapp), MFUmin is the mean fluorescence from the PE channel without FITC-conjugated mouse antibody, and MFUrange is “MFUtotMFUmin.” To determine apparent dissociation constant (Kdapp), curve fitting was performed by the solver tool in the program Excel using a nonlinear optimization method to minimize the sum.

Results Construction of Growth Signalobody Library In order to investigate whether an antigen-specific scFv could be selected from a naïve library based on a growth signalobody, we chose SD1D2g among previously constructed growth signalobodies. SD1D2g consists of an HAtagged scFv, the extracellular D1/D2 and transmembrane domains of EpoR, and the intracellular domain of gp130 (Fig. 1A). We previously reported that SD1D2g having FLspecific scFv clone 31IJ3 transduced a strict BSA-FLdependent growth signal without any background cell growth when expressed in Ba/F3 cells (Liu et al., 2008). The 31IJ3 clone was obtained from a human synthetic naïve scFv library, Tomlinson I þ J, through phage display. Based on this context, we first replaced the scFv region of SD1D2g

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with a stuffer sequence which is completely irrelevant to scFv. The resultant plasmid encoding stuffer-D1D2g was cut and ligated with the naïve Tomlinson I scFv library to yield S(TomI)D1D2g (colony number: 2.0  105). In this experimental design, contamination of the stuffer plasmid due to insufficient restriction enzyme cutting, if any, would result in an out-of-frame product, leading to no signalobody expression. Therefore, functional signalobodies are solely derived from the inserted library. Library Selection With BSA-FL A murine IL-3-dependent pro-B cell line, Ba/F3 (1.0  107 cells), was transduced with the retroviral vector encoding the growth signalobody library, S(TomI)D1D2g (Fig. 1B). Subsequently, we measured a growth signalobody-positive cell ratio by staining against an HA tag. The result showed that the ratio was 11% (data not shown), indicating that 1.1  106 cells of the parental cells were transduced with the signalobody library, which would theoretically cover the original S(TomI)D1D2g library size (2.0  105). Then, the cells (mixture of untransduced and transduced cells) were washed to remove IL-3, and seeded into total forty 96-well plates at 2  104 cells/well either with or without 1 mg/mL BSA-FL (20 plates for each condition) for growth-based selection. Consequently, cell growth was observed only in the presence of BSA-FL, and background cell growth was not observed in the absence of BSA-FL. The number of growth-observed wells in the presence of BSA-FL was only 95 out of 1,920 wells, suggesting that the cells were growing as independent clones. In summary, we seeded total 4.224  106 transduced cells (¼2.2  103 cells/well  1,920 wells) for each condition, and 95 growing clones were obtained in the presence of BSA-FL, which means that 2.2  105 (¼0.0022%) of the library gave antigen-dependent growth. To characterize the clones in detail, we picked up top 23 fast-growing clones. Genomic PCR followed by DNA sequencing analyses revealed that all of the 23 clones gave single sequences, demonstrating that these clones are singlecopy integrants. This is consistent with the previous report that multiple integrations can be avoided in retroviral transduction efficiency less than 20%, which is suitable for library transduction and subsequent selection/cloning (Onishi et al., 1996). From these clones, 7 unique scFv clones were obtained (Fig. 2A). Interestingly, 17 out of the 23 clones had the same sequence as 31IJ3 which had been obtained through phage display (Fig. 2B, clone #4). Cell Proliferation Assay To examine whether the 7 unique clones were surely selected by growth induction in response to BSA-FL, a cell growth assay was performed. The cells were washed and cultured in serial concentrations of BSA-FL for 3 days, and viable cell concentrations were measured by flow cytometry. As a result, all clones showed BSA-FL concentration-dependent cell growth (Fig. 3). These results clearly indicate that the cells

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Figure 2. Sequencing of the selected scFv clones. A: CDR sequences of the identified unique clones. The randomized amino acids are indicated as asterisks. B: Number of clones with the same sequence.

expressing FL-responsive growth signalobodies were positively selected during the selection process. Expression Levels of Growth Signalobodies We analyzed the expression levels of the 7 unique growth signalobodies by Western blotting (Fig. 4). The result confirms that full-length growth signalobodies were expressed in all of the clones. To measure the expression levels of the 7 unique growth signalobodies on the cell surface, we stained the HA tag in each growth signalobody with mouse anti-HA antibody and PE-labeled secondary antibody. As a result, every clone expressed the growth signalobody stably on the cell surface, although the expression levels varied among clones (Fig. 5). FL-Binding Assay To investigate FL-binding of the scFv clones, the 7 unique growth signalobody clones were stained at the cell surface with FITC-labeled mouse monoclonal antibody or unlabeled one as a control, and PE-labeled anti-mouse secondary antibody to facilitate analysis by flow cytometry (Fig. 6). Whereas the unlabeled antibody did not bind to any scFv clones, the FITC-labeled antibody bound to 6 out of the 7 clones. Of note, the S(TomI)D1D2g transductant before selection showed an undetectable level of FL-binding, which excludes the possibility that the original library was biased to have abundant FL-binding clones.

Figure 3. BSA-FL-dependent cell growth induced by chimeric receptors. Cells cultured in IL-3 were washed twice with PBS and seeded in 96-well plates at 4  104 cells/mL containing various concentrations of BSA-FL. Viable cell concentrations of triplicate cultures after 3 days are plotted as the mean  SD. Note: The scales for y-axis are different among the graphs.

To evaluate the binding affinity, the 6 FL-binding clones were stained at the cell surface with serial concentrations of the FITC-labeled mouse antibody ([FITC] ¼ 0–330 nM) and PE-labeled anti-mouse secondary antibody, and analyzed by

flow cytometry. Then, the total mean fluorescence from the PE channel versus the concentration of FITC ([FITC]) was plotted, and a nonlinear least-squares curve fit was used to determine apparent dissociation constant (Kdapp) (Fig. 7). As a result, Kdapp of each clone was ranged from 1.8 to 33 nM, and clone #4, which has the same sequence as 31IJ3, showed the highest affinity. Thus, the scFv clone with the highest affinity was most abundantly selected from the library through the growth signalobody (Figs. 2 and 7).

Discussion

Figure 4. Western blot analysis to confirm expression of the chimeric receptors in a full-length form. Cells were lysed and subjected to Western blot analysis using a polyclonal anti-gp130 C-terminus antibody. Parental Ba/F3 cell lysate was used as a negative control, whereas the cell transduced with 31IJ3-inserted SD1D2g was lysed, which was used as a positive control. The blots for b-tubulin were shown as a loading control.

In this study, we successfully obtained FL-binding scFv clones with high-affinity through the growth signalobody incorporating a naïve scFv library. This new antibody-screening system is based on antigen-dependent growth of factordependent mammalian cells under the factor-deprived condition. This system has several advantages over the conventional methods. First, the antibody-screening process is very simple. In our system, the cells transduced with a growth signalobody library are simply cultured in an antigencontaining medium, which can isolate antigen-specific growing clones without any laborious panning or sorting rounds. Second, since our selection system is not based on panning or sorting, a target antigen can be directly used for

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Figure 5.

Expression of chimeric receptors on the cell surface. Cells were stained with anti-HA tag antibody or an isotype control, and subsequently stained by PE-labeled secondary antibody. Parental Ba/F3 cells were used as a negative control, whereas the cells transduced with 31IJ3-inserted SD1D2g were used as a positive control. The transduced cells were analyzed by gating on EGFP fluorescence.

selection, without any need for fluorophore conjugation and solid-phase immobilization, by which the target protein may be structurally disordered. Third, since our system is a positive-screening method, fast-growing cells should in principle have scFv clones with relatively high-affinity against the target antigen, which enables easy isolation of highaffinity scFv clones based on growth advantage. Fourth, since our system uses a mammalian cell as a host, it provides a desirable selection platform that facilitates correct posttranslational modification and folding of scFv. We obtained the clones with the same sequence as 31IJ3 at the frequency of 17 out of 23 clones through the selection. Although this frequency may seem to be high, 17 cells are still less than the theoretical number of cells to be obtained, because we expanded the transduced cells before selection, and 4.224  106 transduced cells were seeded for selection, which should theoretically contain approximately 21 identical cells if the signalobody library (library size: 2.0  105) is composed of 2.0  105 different clones. We speculate that the clones with the same sequence as 31IJ3 were prominently

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selected possibly because the clone with the highest affinity grew faster (in fact, #4 (¼31IJ3) grew fastest in the growth assay), which mainly constituted the top 23 clones according to a positive-screening nature in our system. In our plasmid construction procedure, we replaced 31IJ3 with a stuffer sequence completely irrelevant to scFv in order to rule out significant likelihood of contamination of 31IJ3 in the library. Since the stuffer plasmid was obtained from an isolated colony of E. coli transformant, contamination of 31IJ3 in the library, if any, would be of a trace amount. Even if this was the case, the selection outcome rather indicates superior feature of our selection system because such a rare positive event was recovered through the library selection. Furthermore, the binding clones other than 31IJ3 were also obtained through the selection, which clearly demonstrates that our selection system works. The sequencing analyses revealed that most of the clones except #8 and #21 have substantial similarity in their sequences. When compared only in the randomized positions, clones #4, #11, and #13 have identical sequences

Figure 6. FL-binding assay. Cells were stained with FITC-conjugated mouse antibody (FITC concentration: 330 nM) or its unconjugated one as a negative control, and subsequently stained with PE-labeled secondary antibody. Parental Ba/F3 cells were used as a negative control, whereas the cells transduced with 31IJ3-inserted SD1D2g were used as a positive control. The transduced cells were analyzed by gating on EGFP fluorescence.

in CDR-H2 and CDR-L2, and similar sequences in CDR-H3 (2 or 3 out of 4 randomized positions have identical amino acids), while #4/#11 and #13 have no apparent sequence similarity in CDR-L3. In addition, clones #3 and #9 have identical sequences in CDR-H2, CDRH3 and CDR-L2, and quite similar sequences in CDR-L3 (4 out of 5 randomized positions have identical amino acids). These results imply that the sequences in CDR-H2 and CDR-H3 may be responsible for the affinity against the target antigen, FL. However, the results also suggest that the affinity may be also influenced by the mutations in the positions other than those randomized, which may be due to errors generated by reverse transcription of retroviral genome during transduction into Ba/F3 cells, and/or errors in PCR amplification. Among the 7 unique signalobody clones we analyzed, clone #8 showed no FL-binding in the binding assay using FL-

conjugated IgG (Fig. 6), but induced BSA-FL concentrationdependent cell growth (Fig. 3). This carrier-specific binding property implies that clone #8 recognizes not only the FL molecule but also its surrounding amino acid residues of BSA. This property may be due to the unique amino-acid sequence of #8, which includes four cysteines in the randomized positions. On the other hand, the other 6 clones specifically recognize the FL molecule, regardless of the carrier proteins. Taken together, all of the 7 unique clones were BSA-FL-responsive clones, and no false-positive clones were obtained. Moreover, in this library selection, background cell growth was not observed in the absence of BSAFL. Considering that most of the existing antibody-screening methods need panning or sorting cycles to minimize falsepositive clones, the results in this study demonstrate superior features of our system.

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Figure 7.

Affinity determination of the selected scFv clones. Cells were stained with FITC-conjugated mouse antibody and PE-labeled secondary antibody. PE fluorescence intensity was measured by flow cytometry, and PE mean fluorescence values were plotted versus logarithm of FITC concentrations. The staining was duplicated for each FITC concentration, and designated as measured value 1 and 2. Apparent Kd values (Kdapp) were determined by fitting the data with a nonlinear least squares. Note: The scales for y-axis are different among the graphs.

The results of the growth assays and the affinity measurements indicate that the growth response and the affinity against FL were almost correlated. In addition, the scFv clone with the highest affinity (Kdapp ¼ 1.8 nM) was most abundantly selected (Figs. 2 and 7), and it has the same sequence as the 31IJ3 clone, which had been obtained through phage display. Moreover, the other 6 unique clones, which were newly isolated in this study, also had high-affinity. These results clearly demonstrate the utility of our system as an affinity-based scFv selection method based on growth advantage of mammalian cells. Although versatility of our system should be evaluated using other target antigens, our system would provide a new direction for antibodyscreening. We are grateful to Dr. Toshio Kitamura (The University of Tokyo) for the retroviral expression system, and Dr. Wenhai Liu (The University of Tokyo) for plasmid construction. This work was supported by Grants-in-Aid for Young Scientists (A) 21686077 (M.K.) and for Challenging Exploratory Research 23656516 (M.K.) from the JSPS, Japan, by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) (M.K.) and by the Global COE Program for Chemistry Innovation.

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Yoshida et al.: Antibody Selection Using Growth Signalobody Biotechnology and Bioengineering

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A novel platform for antibody library selection in mammalian cells based on a growth signalobody.

While many antibody-screening methods in vitro have been developed, these methods need repeated cycles of panning or sorting procedures to isolate ant...
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