J. Biochem. 2015;157(6):497–506

doi:10.1093/jb/mvv008

Domain structure of growth signalobodies critically affects the outcome of antibody library selection Received May 14, 2014; accepted December 27, 2014; published online January 22, 2015

Rie Yoshida1, Masahiro Kawahara2,* and Teruyuki Nagamune1,2 1 Department of Bioengineering and 2Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

Wide applications of antibodies have demanded rapid and easy methods for isolating high-affinity antibodies. We recently developed an antibody screening system in mammalian cells using a growth signalobody, which is a single-chain Fv (scFv) library/cytokine receptor chimera that can transduce a growth signal in response to a target oligomeric antigen. However, we have never investigated how the domain structure of signalobodies affects the outcome of library screening. In this study, we screened naı¨ ve scFv library-inserted signalobodies having distinct extracellular and transmembrane (TM) domains. Although the previously constructed signalobody with the extracellular D1/D2 domains of erythropoietin receptor had recovered the clones with high affinity against a target antigen and with low background cell growth, its D1/D2-deficient variant which was tested in this study recovered the clones with low affinity against a target antigen and with considerable background cell growth. In addition, mutagenesis in the TM domain lowered the level of the background cell growth. These results suggest that the D1/D2 domains increase a threshold to activate signalobodies, thereby selecting clones with high affinity against a target antigen and that the TM domain could be engineered to minimize background growth signalling. Keywords: antibody selection/chimeric protein/cytokine receptor/growth signalobody/single-chain Fv. Abbreviations: BSA-FL, fluorescein-conjugated BSA; EpoR, erythropoietin receptor; FL, fluorescein; HA, hemagglutinin; IL-3, interleukin-3; PE, phycoerythrin; scFv, single-chain Fv; TM, transmembrane.

Antibodies have been used for basic research tools, disease diagnosis and therapy. These wide applications of antibodies have led to a demand for developing a method for isolating antibodies, which have high affinity against target antigens. Among various systems

ß The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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*Masahiro Kawahara, Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel: +81-3-5841-7290, Fax: +81-3-5841-8657, email: [email protected]

that have been developed for antibody screening in vitro (1), ribosome/mRNA display and bacterial/ phage display systems are useful for dealing with a large library (2—6). However, these systems may result in unfolding or misfolding of antibody fragments due to the expression in cell-free or bacterial environment (7, 8). As alternative methods for these systems, yeast display (9—12) and mammalian cell display (13—19), which are eukaryotic expression systems, were developed. However, these systems should employ a target antigen that is conjugated with a fluorophore or a tag sequence to concentrate antigen binders through fluorescence-activated or magnetic sorting, and such conjugation would structurally perturb the target antigen. We previously developed a new mammalian-cell display platform based on a growth signalobody (20). A growth signalobody is a single-chain Fv (scFv)/cytokine receptor chimera that transduces a cognate homooligomeric antigen-responsive growth signal (21—27). In our antibody selection system, a scFv library is genetically fused with a cytokine receptor, which is introduced into interleukin (IL)-3-dependent cell line, followed by culturing the transductant with a target antigen in an IL-3-deficient condition. As resultant growing cells, in principle, are antigen binders, scFv genes specific to the target antigen can be easily cloned from the growing cells by genomic polymerase chain reaction (PCR), which attains coupling of genotype and phenotype. Among a series of growth signalobodies with different domain structures, SD1D2g comprising scFv, the extracellular D1/D2 and transmembrane (TM) domains of erythropoietin receptor (EpoR) and the intracellular domain of gp130 was one of the best signalobodies that can strictly transduce a growth signal in response to a specific antigen when we incorporated anti-fluorescein (FL) scFv clone 31IJ3 (28). Therefore, we chose SD1D2g as a platform for scFv library screening in our previous study. Consequently, we successfully obtained FL-binding scFv clones from scFv library-inserted SD1D2g (20). However, we have never investigated how the overall domain structure of the signalobodies affects the outcome of library selection. We previously reported that not only SD1D2g but also its D1/D2-deficient variant Sg (Fig. 1) incorporating FL-specific scFv clone 31IJ3 transduced a strict BSA-FL-dependent growth signal when expressed in Ba/F3 cells (28). On the basis of this result, we considered that Sg might be another candidate for scFv library selection. Thus, we constructed a naı¨ ve scFv library-inserted Sg and investigated the influence of EpoR D1/D2 domains for library selection.

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In addition to the extracellular domain, the TM domain plays a key role for interchain interaction of receptor chains. Kubatzky et al. (29) reported that a double mutation (L241G, L242P) in a leucine zipper motif of the EpoR TM domain markedly decreased the interchain interaction, which also decreased the intensity of a growth signal. In fact, we previously reported that EpoR TM-mutated signalobodies showed stricter BSA-FL-dependent cell growth (25, 30). Thus, we constructed a Sgm signalobody library, which contains the double mutation of the EpoR TM domain of Sg and investigated the effect of the double mutation for library selection. 498

Taken together, we constructed Sg and Sgm signalobody libraries and investigated how the domain structure of growth signalobodies affects antibody library selection by comparing background cell growth and antigen dependency using FL as a target antigen.

Materials and Methods Plasmid construction The construction of the plasmid pMK-stuffer-g-IG was as described (20). pMK-stuffer-g-IG encodes an immunoglobulin kappa chain signal sequence, an HA tag, a SfiI site, a stuffer sequence, a NotI site, the TM domain of human EpoR and the intracellular domain of human gp130.

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Fig. 1 Library selection using growth signalobodies with different structures. (A) Concept of a growth signalobody. A growth signalobody is a scFv/cytokine receptor chimera that transduces a growth signal when dimerized by a homo-oligomeric antigen. The culture of growth signalobody-expressing cells in the presence of the antigen leads to selection of cells with an antigen-specific scFv gene. (B) The signalobody constructs. Sg is composed of a scFv library, the TM domain of EpoR and the intracellular domain of gp130. Sgm contains a double mutation (L241G, L242P) in the leucine zipper motif of the EpoR TM domain. Each signalobody contains an HA-tag at the extracellular N-terminus. (C) Result of library selection. ‘Transduced cells for screening’ is the number of the total seeded cells that are transduced with each signalobody library, which corresponds to theoretical library size in this experiment.

Signalobody structure affects antibody selection To make the construct containing two point mutations (L241G, L242P) in the TM domain, two primers (STMm For: 50 -CCCTCCG GACTCATCCTGACGCTCTCCCTC-30 , STMm Rev: 50 -CTGTTT AAGTCTAACAAAATC-30 ) were employed to amplify the mutated TM domain in pMK-SEmg-IG (30). The amplified fragment was digested with BspEI and NsiI and subcloned into pMK-stuffer-gIG to produce pMK-stuffer-gm-IG.

by curve fitting to determine apparent dissociation constant (Kdapp) as described (20, 32).

Library construction The Tomlinson I human synthetic naı¨ ve scFv library (Medical Research Council, Cambridge, UK; library size: 1.47  108) (31) was amplified by two primers (forward: 50 -GCAAATTCTATTTC AAGGAGAC-30 , reverse: 50 -GCCCATTCAGATCCTCTTC-30 ), digested with SfiI and NotI. The digested fragment was ligated with pMK-stuffer-g-IG and pMK-stuffer-gm-IG digested with SfiI and NotI to produce pMK-S(TomI)g-IG and pMK-S(TomI)gm-IG, respectively. These library plasmids were expanded by transformation of electrocompetent Escherichia coli cells as described (20). The numbers of transformed colonies were 1.9  106 and 1.5  106 for S(TomI)g and S(TomI)gm, respectively.

To examine how the domain structure of growth signalobodies affects antibody library selection, here we focused on Sg signalobody. Sg comprises an HAtagged scFv, the TM domain of EpoR and the intracellular domain of gp130 (Fig. 1). Our previous study demonstrated that Sg incorporating FL-specific scFv clone 31IJ3 showed a BSA-FL-dependent growth signal (28). To investigate the effect of a TM domain, we also constructed Sgm, which contains a double mutation (L241G, L242P) in a leucine zipper motif of EpoR TM domain, since Kubatzky et al. (29) reported that the double mutation decreased selfassembly of wild-type EpoR, which led to the loss of receptor homodimers, resulting in impaired signal transduction. Thus, we expected that this double mutation might be effective for impairing the interchain interaction of EpoR TM domains in the signalobodies, which lowers antigen-independent background growth during library selection. We first created the signalobody constructs with a stuffer sequence irrelevant to scFv. Next, we replaced the stuffer sequence with a naı¨ ve Tomlinson I library, resulting in Tomlinson I-inserted Sg and Sgm signalobodies.

Retroviral transduction Overall procedure for producing retroviral vectors using Plat-E packaging cells was as described (20), except that the cell culture was scaled up. The values different from those in the previous paper were as follows: Plat-E cell culture, two 100-mm diameter dishes at 5.0  106 cells/dish; the library plasmid and its re-solubilization, 30 mg in 5.0 ml of serum-free OPTI-MEM (GibcoBRL, Rockville, MD); Plus reagent (Invitrogen, San Diego, CA), 25 ml; Lipofectamine LTX (Invitrogen), 62.5 ml and the medium-replacement volume of Plat-E cells, 10 ml/dish. The produced retroviral vectors were mixed with Ba/F3 cells (6.0  107 cells) in the presence of 10 mg/ml protamine sulphate (Wako, Osaka, Japan) and 1 ng/ml IL-3 (R&D systems, Cambridge, MA). After incubation for 5 h in a 37 C/5% CO2 incubator, the normal medium was added to the transduced Ba/F3 cells and further cultivated for forthcoming library screening. Library screening Library screening was performed as described (20). Briefly, cells were washed for depriving IL-3 and inoculated into 96-well plates in the presence or absence of 14 nM ( = 1 mg/ml) BSA-FL (Sigma, St. Louis, MO). Genomic PCR The genomic PCR was performed as described using DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) and two primers (forward: 50 -GTACTGCTGCTCTGGGTTCC-30 , reverse: 50 -CGCCCGTTT GATTTCCACCTTG-30 ) (20). Confirmation of cell-surface expression of growth signalobodies The cell surface expression of growth signalobodies was confirmed as described (20). Briefly, cells were labelled with mouse monoclonal anti-HA antibody (Covance, Princeton, NJ) followed by R-phycoerythrin (PE)-conjugated donkey F(ab’)2 anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and subjected to flow cytometric analysis using FACSCalibur (Becton-Dickinson, Lexington, KY). Growth assay Growth assay was performed as described (20). Briefly, cells were inoculated in 96-well plates at 4  104 cells/ml and cultivated for 3 days. Cell counting was performed using FACSCalibur. FL-binding assay and evaluation of relative binding strength FL binding was evaluated by the procedure as described (20). Briefly, cells were mixed with mouse monoclonal anti-Flag clone M2 (Sigma) or its FL conjugate (Sigma) and further labelled with R-PE-conjugated donkey F(ab’)2 anti-mouse IgG. The cells were subjected to flow cytometric analysis using FACSCalibur. Relative binding strength of the FL-binding clones towards FL was evaluated

Construction of Sg and Sgm growth signalobody libraries

Library selection with antigen

Ba/F3 cells were transduced with the retroviral vectors encoding each growth signalobody library. The previous report demonstrated that transduction efficiency should be less than 20% to achieve single copy integration, which facilitates subsequent selection/cloning (33). Therefore, we measured growth signalobodypositive cell ratios by staining against the HA tag appended at the N-terminus of the growth signalobodies. The result showed that the ratios for both growth signalobody libraries were less than 6% (data not shown), which is low enough to achieve single integration. Then, the cells were subjected to growth-based screening in multiwell plates either with or without 14 nM BSA-FL. As a result, growing colonies were observed more abundantly in the presence of BSA-FL for both signalobodies (Fig. 1), whereas the background growth in the absence of BSA-FL was also observed. With respect to the mutations in the TM domain, Sgm gave significantly lower level of background cell growth than Sg. Overall, Sg and Sgm gave more remarkable cell growth than SD1D2g that was tested in our previous study (20), in which we seeded 4.2  106 transduced cells for screening, and 95 growing clones were obtained in the presence of BSA-FL and no background cell growth was observed in the absence of BSA-FL. For detailed characterization of the clones, top 12 fast-growing clones were picked up from Sg and Sgm, respectively, which were obtained in the presence of BSA-FL. Genomic PCR and DNA sequencing 499

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Cell culture An IL-3-dependent murine pro-B cell line Ba/F3 and a retroviral packaging cell line Plat-E were cultured as described (20).

Results

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demonstrated that five and two unique scFv clones were obtained from Sg and Sgm, respectively (Fig. 2). Examination of antigen-dependent cell growth

To confirm the expression of the growth signalobodies on the cell surface, each growth signalobody was stained using an anti-HA tag antibody. Consequently, we confirmed that all clones expressed their corresponding growth signalobodies (Fig. 3). Then, the cells were subjected to a growth assay, in which the cells were cultured in various concentrations of BSA-FL. Interestingly, all clones grew BSA-FL concentration dependently (Fig. 4). These results suggest that these FL-responsive cells were dominantly selected by the selection. Evaluation of antigen binding

To examine whether the scFv clones bind to FL, the growth signalobody clones were stained with FL-conjugated ([FL] = 330 nM) or unconjugated antibody, followed by staining with PE-conjugated secondary antibody. Flow cytometric analysis revealed that staining with the FL-conjugated antibody, but not the unlabeled antibody, gave peak shifts compared with the unstained control for all of the seven clones (Fig. 5). This peak shift was not observed for parental 500

Ba/F3 cells. Notably, these clones showed much lower PE fluorescence intensity than the 31IJ3 clone. To evaluate the relative binding strength, the seven clones were stained with various concentrations of the FL-conjugated antibody ([FL] = 0—3300 nM) and PE-conjugated secondary antibody, followed by flow cytometry. In the data analysis, the total mean PE fluorescence was plotted against the FL concentration ([FL]) and apparent dissociation constant (Kdapp) was determined by curve fitting (Fig. 6). Intriguingly, Kdapp of each clone was ranged from 167 to 908 nM, of which value was significantly higher than 27 nM for 31IJ3 clone and those for the clones previously selected by the SD1D2g signalobody (Kdapp ranged from 1.8 to 33 nM) (20). These results suggest an important role of the D1 and D2 domains for selecting high-affinity scFv clones.

Discussion In this study, we constructed Sg and Sgm signalobody libraries which have distinct structures of receptor domains from previously tested SD1D2g signalobody to investigate how the domain structures affect the outcome of library selection. As a result, selection with Sg and Sgm signalobodies gave clones with low affinity

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Fig. 2 Sequencing of the selected scFv clones. (A) Complementarity-determining region sequences of the identified unique clones. Asterisks indicate positions that were randomized in the construction of the Tomlinson I library. (B) Number of clones with the same sequence.

Signalobody structure affects antibody selection

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Fig. 3 Expression of signalobodies on the cell surface. Cells were stained with anti-HA tag antibody or its isotype control and subsequently stained by PE-conjugated secondary antibody. Parental Ba/F3 cells and the cells transduced with 31IJ3-inserted Sg were used as negative and positive controls, respectively.

against a target antigen FL but also gave remarkably high background cell growth even in the absence of BSA-FL. This feature is in contrast to SD1D2g signalobody, which gave clones with high affinity against

FL and with no background cell growth in the absence of BSA-FL (Figs 1 and 6) (20). These results suggest that the extracellular D1 and D2 domains not only inhibit the activation of signalobodies in the absence 501

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of a target antigen but also contribute to increasing a threshold to activate signalobodies, which could select clones with high affinity against a target antigen. The double mutation in the TM domain in Sgm effectively reduced background cell growth compared with its non-mutated counterpart, Sg. This result suggests that the TM domain could be engineered to minimize background growth signalling. The clones selected from Sg and Sgm signalobodies exhibited much lower binding strength (almost two orders of magnitude lower) against FL than those from SD1D2g signalobody (Fig. 6). In contrast, the growth assay showed that the growth response to BSA-FL was not so dramatically different among the clones selected from different growth signalobody libraries (Fig. 4) (20). These results indicate that Sg and Sgm are conformationally susceptible to transduce a growth signal in response to low concentrations of a target antigen even though affinity against the target antigen is low. This context is in line with our selection 502

result showing that Sg and Sgm recovered the clones with low affinity against FL, which resulted in more remarkable cell growth than SD1D2g. In wild-type EpoR, the D1 domains derived from two receptor chains take an open scissors-like conformation in the absence of ligand (34), and ligand binding to the D1 domains induces a conformational change to activate subsequent signalling events (35). Therefore, the D1 domain would contribute to keep a switched-off state of the receptor. Given this notion, SD1D2g may readily keep a switched-off state, which could be converted to an active state only if the scFv has strong affinity to a specific antigen. In contrast, Sg and Sgm, lacking the D1 domain, may exhibit leaky phenotype due to the absence of such a switch-off mechanism. When we selected FL-binding clones from SD1D2g, the scFv clones with higher affinity (Kdapp ranged from 1.8 to 33 nM) were obtained without any background cell growth in the absence of BSA-FL (20). On the other hand, here we obtained the scFv clones with

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Fig. 4 Comparison of the growth response induced by signalobodies. Cells were seeded in 96-well plates at 4  104 cells/ml in the presence of serial concentrations of BSA-FL. Viable cell concentrations of triplicate cultures after 3 days were plotted as mean ± SD.

Signalobody structure affects antibody selection

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Fig. 5 FL-binding assay. Cells were stained with FL-conjugated mouse antibody (FL concentration: 330 nM) or its unconjugated one as a negative control and subsequently stained with PE-conjugated secondary antibody. PE fluorescence was detected by flow cytometry. Parental Ba/F3 and the cell transduced with 31IJ3-inserted Sg were used as negative and positive controls, respectively.

lower affinity towards FL from Sg and Sgm. Sg and Sgm may be used if there is no high-affinity binder in a library, which could be the case especially for a naı¨ ve library. In such a case, low-affinity binders are firstly

selected through the Sg or Sgm signalobody library, followed by introducing mutations at complementarity-determining region residues critical for antigen binding. The resulting mutated library is subsequently 503

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incorporated into the SD1D2g signalobody, followed by selection of high-affinity binders. The demonstration of such affinity maturation would significantly extend our system as a platform for robust antibody screening in future. In living cells, weak or transient protein—protein interactions are rather necessary in dynamically regulating cellular events. For example, electron transfer and signal transduction cascades are regulated by transiently formed protein complexes, and this feature facilitates rapid and dynamic alteration of protein—protein interactions that cannot be attained by strong interactions (36—38). Despite the importance, low-affinity interactions are much less well understood than high-affinity interactions due to intrinsic difficulty to discriminate specific from non-specific interactions. Because this study shows that affinity threshold for detecting interactions could be readily tuned by domain engineering of a 504

signalobody, a chimeric receptor approach may be useful for detecting and selecting protein—protein interactions in physiologically relevant context.

Acknowledgements We are grateful to Dr. Toshio Kitamura (The University of Tokyo) for the retroviral expression system and Yuri Teranishi (The University of Tokyo) for plasmid construction.

Funding 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 Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (M.K.) and by the Global COE Program for Chemistry Innovation. Conflict of Interest None declared.

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Fig. 6 Evaluation of relative binding strength of the selected scFv clones. (A) Cells were stained with serial concentrations of FL-conjugated mouse antibody and subsequently stained with PE-conjugated secondary antibody. PE mean fluorescence values were plotted against the logarithm of FL concentrations. The staining was duplicated for each FL concentration, and the data were designated as ‘measured value 1 and 2’. (B) Apparent Kd values (Kdapp) of the clones.

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