Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening Yariv Mazor 1,2 , Thomas Van Blarcom 1,2 , Sean Carroll 1,2 and George Georgiou 1,2 1 Institute for Cellular and Molecular Biology, University of Texas at Austin, TX, USA 2 Department of Chemical Engineering, University of Texas at Austin, TX, USA Introduction Recombinant antibodies have made a tremendous impact on biomedical research, and are increasingly being used as clinical diagnostic and therapeutic reagents [1,2]. Consequently, the demand for new tech- nologies that aid in the discovery and selection of novel therapeutic antibodies has never been greater. During the past two decades, several display technolo- gies and other library screening techniques have been developed for the isolation of antigen-specific antibod- ies from large collections of recombinant antibody genes [3]. Phage display is the most prevalent method for the display of large ensembles of antibody frag- ments, and is currently considered to be the standard procedure in many molecular biology laboratories for antibody discovery and evolution [3]. Unique antibod- ies are isolated from immune [4–7], naı ¨ ve [8–13] or synthetic [14–21] repertoires, and are further engi- neered for improved affinities for their antigens by using the selected antibody gene as the basis for subse- quent libraries and screening [22–26]. Humira [27,28] Keywords fluorescence-activated cell sorting (FACS); full-length IgG; fUSE5–ZZ phage; protective antigen (PA); spheroplasts Correspondence G. Georgiou, Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA Fax: +1 512 471 7963 Tel: +1 512 471 6975 E-mail: gg@mail.che.utexas.edu (Received 7 February 2009, Revised 4 March 2010, accepted 9 March 2010) doi:10.1111/j.1742-4658.2010.07645.x Phage display of antibody libraries is a powerful tool for antibody discov- ery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab¢) 2 .We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluores- cence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563–565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity >10 8 is technically challenging. We report here a system that takes advan- tage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5–ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demon- strate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without un- dersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process. Abbreviations CFU, colony-forming units; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; IPTG, isopropyl thio-b- D-galactoside; PA, protective antigen; RU, response units; V H , variable heavy; V L , variable light. FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2291 was the first fully human mAb discovered using phage display to receive FDA approval, and at least 16 human mAbs derived from phage display are currently in advanced clinical trials for a wide range of human diseases [29,30]. Phage display is based on encoding the gene of interest in-frame with one of the phage coat proteins (phenotype), and encapsulates the fusion gene within the phage particle (genotype). Recombinant antibodies have been displayed on phage particles as scFv [31] or Fab [7,32] fragments. These monovalent proteins, although relatively easy to produce in Escherichia coli, are typically devoid of avidity effects that allow the recovery of low-affinity binders [33–36]. Polyvalent display of scFv and Fab can be readily achieved, par- ticularly by using phage-based vectors [37]. The resul- tant avidity effects allow for the recovery of low affinity-binders, but these same avidity effects make it difficult to select stringently on the basis of intrinsic affinity. More recently, bivalent Fab [F(ab¢) 2 ] has been dis- played on phage particles in a manner that effec- tively resembles the binding behavior of natural IgGs [36]. Nevertheless, for the vast majority of diagnostic and therapeutic applications, antibody fragments iso- lated from most existing display technologies must be converted to full-length IgG, the format of choice in the clinics. This process requires additional cloning steps and the expression of the reformatted antibody gene in mammalian cells. A conspicuous drawback of the scFv format is that reformatting to IgG can result in loss of activity (TVB, YM, SAC, SK, BLI and GG, unpublished observations). Yet another dis- advantage of most existing phage display systems is that the antibody gene is expressed as a fusion pro- tein with one of the phage coat proteins. As a result, many of the antibodies isolated through library screening can only fold in the context of a fusion protein and cannot be expressed independently, a phenomenon that many laboratories do not report [38]. We recently reported the development of an E. coli- based technology, termed E-clonal, for the successful production of soluble full-length IgGs in bacteria and for screening of combinatorial IgG libraries using bac- terial periplasmic display [39,40]. Library cells express- ing intact IgGs specifically labeled with fluorescently conjugated antigen are readily distinguished and iso- lated by fluorescence-activated cell sorting (FACS). Unlike phage display, FACS has the distinct advantage of relying on real-time quantitative multiparameter analysis of individual cells, allowing single-cell resolu- tion for selection. Although FACS is a very powerful high-throughput screening methodology, sorting a library > 10 9 cells using FACS is time-consuming and challenging [40–43]. To reduce the initial library to a size that is manage- able by FACS and to demonstrate that full-length IgG libraries can be displayed on phage particles and undergo selection, we sought to develop a display sys- tem that will effectively display intact IgGs on filamen- tous phage particles. The system can efficiently downsize very large libraries by employing an initial round of phage biopanning that specifically pre- enriches target cells from the library prior to subse- quent rounds of FACS. Specifcally, E. coli ⁄ F¢ cells expressing soluble IgGs in the periplasm (E-clonal cells) are simultaneously infected with the fUSE5–ZZ phage [44]. These phage particles allow polyvalent dis- play of the Fc-binding ZZ protein [45] on all five cop- ies of the gene-3 minor coat protein of filamentous bacteriophages [44]. The fUSE5–ZZ phage in this sys- tem serves not only as a helper phage, but also as the IgG-capturing surface via the surface-displayed ZZ protein (Fig. 1). Rescued fUSE5–ZZ–IgG phage parti- cles harboring the IgG phagemid are then selected for antigen binding by standard phage biopanning. We describe here the feasibility of propagating phage parti- cles that stably display functional full-length IgGs, and demonstrate that an initial round of phage biopanning followed by FACS facilitates the isolation of a diversi- fied collection of antigen-specific binders from very large antibody libraries. Results Model system validation As a model for our studies and for validation of the display of full-length IgGs on phage particles, we chose two well-characterized antibodies, M18 and 26.10, which are specific for the protective antigen (PA) from Bacillus anthracis (K d =30pm) and digoxin (K d = 1.7 nm), respectively [39]. The full-length heavy and light chain genes were expressed from a dicistronic operon and secreted into the periplasm, where they assembled into aglycosylated IgGs that were fully func- tional for antigen binding [39]. To display the full- length IgG noncovalently on phage particles, we uti- lized fUSE5–ZZ, which displays the Fc-binding ZZ protein [45] on all five copies of the gene-3 minor coat protein, but maintains its ability to infect and propa- gate in E. coli ⁄ F¢ cells [44] (Fig. 1). As the pMAZ360– IgG expression vector contains the packaging signal of f1 bacteriophage that enables the packaging of the plasmid as ssDNA in the presence of a helper phage, Selection of IgG by tandem phage panning-FACS Y. Mazor et al. 2292 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS rescued fUSE5–ZZ–IgG particles harbor the high copy number pMAZ360–IgG phagemid preferentially over the very low copy number and replication-defective fUSE5–ZZ genome [44]. Initially, fUSE5–ZZ particles were evaluated for their ability to capture purified IgGs in solution. The phage particles were mixed with purified M18 IgG or 26.10 IgG, washed to remove any unbound antibodies, and analyzed by ELISA. Phage that had captured the purified IgG via the ZZ protein showed strong ELISA signals with the respective antigens but not with unre- lated antigens (data not shown). We then evaluated whether fUSE5–ZZ is able to capture IgG within the periplasm and form a noncovalent complex that is sta- ble upon extrusion of the phage from the bacteria. E. coli K91K ⁄ F¢ cells transformed with phagemid pMAZ360–M18–IgG or pMAZ360–26.10–IgG were grown under conditions permissive for phage infection. Following infection with fUSE5–ZZ, the cultures were allowed to grow overnight under conditions favorable for phage production. On the following day, phage particles were precipitated and evaluated for specific binding by direct ELISA (Fig. 2). As expected, fUSE5–ZZ propagated in cells expressing M18 IgG bound specifically to PA (Fig. 2A), whereas phage par- ticles produced in cells expressing 26.10 IgG bound specifically to digoxin (Fig. 2B). Competition of the bound IgG by an excess of standard human IgGs resulted in a small reduction ( 15%) of ELISA signal (Fig. 2A,B), indicating that the phage ZZ–IgG com- plex is kinetically very stable, presumably owing to the polyvalent display of the ZZ protein on all copies of the phage p3 coat protein. To determine the number of IgG molecules dis- played on fUSE5-ZZ–IgG particles, we employed the technique described by Junutula et al. [46]. Purified fUSE5-ZZ–M18 IgG phage was applied at varying concentrations to ELISA plates coated with anti-M13 IgG, anti-Fc IgG, or PA. Following incubation, the ELISA was developed with horseradish peroxidase (HRP)-conjugated anti-M13 IgG. The number of IgG molecules displayed on each phage particle was deter- mined by the ratio of the linear range of the ELISA signals obtained with anti-Fc IgG ⁄ anti-M13 IgG or PA ⁄ anti-M13 IgG (Fig. 2C). Analysis of the results indicated that there is an average of 0.6–0.7 IgG mole- cules per phage particle. To assess the efficacy of the IgG phage display sys- tem for selections, we tested the ability to enrich fUSE5–ZZ–M18 IgG phage particles from a 1 000 000-fold excess of phage particles displaying the control 26.10 IgG. The mixture was subjected to three rounds of phage biopanning against PA, and the enrichment after each round of selection was moni- tored by ELISA (Fig. 2D). The increase in ELISA sig- nal for PA in parallel with the decrease in ELISA signal for digoxin clearly indicated a significant enrich- ment of the fUSE5–ZZ–M18 IgG phage population at the expense of a reduction in the number of phage par- ticles displaying the control 26.10 IgG. Sequence anal- ysis revealed that, following the third round of phage panning, seven of 20 randomly selected clones carried + fUSE5-ZZ g3 ZZZZ V L C L P pMAZ360-IgG V H C H1 C H2 C H3 pelB pelB PLAC Fd ori Tet R Amp R F1ori pMAZ360-IgG Fig. 1. Schematic diagram of the IgG phage display format. Left: map of phagemid pMAZ360–IgG for expression of soluble intact IgGs in the E. coli periplasm. This vector facilitates convenient cloning of V H and Vj domains linked to human c1 and j constant domains, respec- tively, as a bicistronic operon downstream of the lac promoter. Center: map of fUSE5–ZZ phage for polyvalent display of the Fc-binding ZZ protein on all copies of the gene-3 minor coat protein. Right: infection of E. coli cells carrying phagemid pMAZ360–IgG with fUSE5–ZZ leads to the production of fUSE5–ZZ–IgG phage particles that stably display functional full-length IgGs. Y. Mazor et al. Selection of IgG by tandem phage panning-FACS FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2293 the M18 mAb sequence, corresponding to over 300 000-fold enrichment. Library selection by tandem phage biopanning and FACS To evaluate the utility of tandem phage panning– FACS for library screening, we used an anti-PA mouse immune IgG library [39]. This library was constructed by cloning pools of variable heavy (V H ) and variable light (V L ) genes from spleens of mice that were immu- nized with PA from B. anthracis into the pMAZ360– IgG vector. Library DNA was transformed into E. coli K91K ⁄ F¢ to generate a total of 10 7 independent transformants. Cells carrying phagemid pMAZ360– IgG were infected with fUSE5–ZZ, and the culture was allowed to grow under conditions favorable for phage production. Phage particles were purified and subjected to an initial cycle of panning by incubation with soluble biotinylated PA in solution, before being captured on streptavidin-coated magnetic beads. Unbound phage particles were removed by washing, and bound phage particles were eluted, neutralized, and used for infection of E. coli Jude-1⁄ F¢ cells har- boring plasmid pBAD33–NlpA–ZZ for subsequent rounds of FACS screening in cells expressing an NlpA–ZZ fusion that allows capture of the IgG on the inner membrane. Cells were converted to spheroplasts by disruption of the outer membrane with Tris ⁄ EDTA and lysozyme treatment, to allow exposure of the membrane-bound IgG to the extracellular fluid. Two color flow cytometry steps, using PA63–fluorescein iso- thiocyanate (FITC) and Alexa Fluor 647–anti-(human IgG) to monitor for affinity and expression of full- length IgG, were employed, and fluorescent clones 100 25 50 75 0 fUSE5-ZZ-26.10 IgG fUSE5-ZZ-26.10 IgG % binding to antigen% binding to antigen 50 75 100 0 25 fUSE5-ZZ-M18 IgG fUSE5-ZZ-M18 IgG fUSE5-ZZ-M18 IgG + hIgG competitor fUSE5-ZZ-26.10 IgG + hIgG competitor 2 2.5 PA anti-M13 1 1.5 2 A 450 nm 0 0.5 Phage concentration (CFU·mL –1 ) 1.5 2 2.5 fUSE5-ZZ-M18 IgG fUSE5-ZZ-26.10 0.5 1 1.5 A 450 nm 0 1E+07 3E+07 1E+08 3E+08 1E+09 3E+09 1E+10 3E+10 Library Cycle 1 Cycle 2 Cycle 3 A B C D Fig. 2. Characterization of fUSE5–ZZ–IgG phage. Binding analysis of fUSE5–ZZ–IgG phage in ELISA was tested with plates coated with PA (A) or digoxin ⁄ BSA (B). For analysis of the stability of the ZZ–IgG complex on phage particles displaying either M18 or 26.16 mAb, fUSE5–ZZ–IgG was incubated with 1 l M standard human IgG as a competitor before being applied to the ELISA plates. (C) Deter- mination of IgG molecules per phage. fUSE5–ZZ–M18 IgG phage particles were serially diluted and applied to ELISA plates coated with either anti-M13 IgG or PA. The number of IgG molecules per phage particle was determined by the phage concentration derived from PA ⁄ anti-M13 IgG in the linear range of the ELISA signals. (D) Enrichment of fUSE5–ZZ–M18 IgG from a 1 000 000-fold excess of fUSE5–ZZ–26.10 IgG. Phage biopanning against PA was performed as described in Experimental procedures. Evaluation of the enrich- ment following each round of phage selection was monitored by phage ELISA on plates coated with PA or digoxin ⁄ BSA. ELISA plates were developed with HRP-conjugated anti-M13 IgG; values at 450 nm represent three independent experiments. Selection of IgG by tandem phage panning-FACS Y. Mazor et al. 2294 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS falling into the double-positive quadrant were sorted (Fig. 3). For better selectivity and for the isolation of clones exhibiting improved dissociation rates, cells col- lected after the first sort were immediately resorted on the flow cytometer. As no additional probe was pro- vided, only clones exhibiting low dissociation rates sur- vive the second sorting cycle. IgG genes in the sort mixture were rescued by PCR amplification, recloned into pMAZ360–IgG, transformed into fresh NlpA–ZZ cells, and induced for IgG expression. Following two rounds of FACS selection, it was clear that IgGs spe- cific for PA had been enriched (Fig. 3), and IgG genes from the second round of FACS selection were ligated into the pMAZ360–IgG expression vector and trans- formed into fresh E. coli Jude-1 cells not carrying plas- mid pBAD33–NlpA–ZZ for expression of soluble antibodies. Individual clones were grown in 96-well plates and induced for expression of soluble IgG, and PA-specific clones were identified by ELISA. Forty- nine of 192 of the screened clones gave a PA-specific signal. Sequence analysis of 25 clones revealed the iso- lation of six unique clones that were subjected to addi- tional characterization. The selected clones were expressed and purified by protein A affinity chroma- tography, and IgG in yields of 0.5–3 mgÆL )1 were obtained. Biacore analysis of binding kinetics revealed that the affinities of the IgGs derived from the immu- nized library ranged from the low to high nanomolar (Table 1). The highest-affinity and lowest-affinity IgGs were determined to have K D values of 1 and 440 nm, respectively. Enrichment of high-affinity and moderate-affinity IgGs To evaluate the utility of tandem phage biopanning followed by FACS for the isolation of IgGs with dif- ferent affinities from very large libraries, we used fUSE5–ZZ–IgG phage displaying either M18, YMF10 or VA IgG, displaying high-affinity binding (30 pm), moderate-affinity binding (30 nm) and no binding to the PA antigen. The high-affinity and moderate-affinity IgGs displayed on the fUSE5-ZZ–IgG phage were each diluted 1 : 10 8 in 10 10 copies of VA IgG displayed on the fUSE5-ZZ–IgG phage. The phage mixture was subjected to one round of phage biopanning, using sol- uble biotinylated PA antigen, and, following neutral- ization and infection of E. coli Jude-1 ⁄ F¢ cells carrying plasmid pBAD33–ZZ, yielded an output of 1.5 · 10 6 cells harboring phagemid pMAZ360–IgG. The cells were induced for the expression and display of IgG, and subjected to three rounds of FACS following labeling with fluorescently conjugated PA63 protein. After the third round of FACS, plasmids encoding the isolated IgG gene inserts were transformed into fresh E. coli cells not carrying plasmid pBAD33–NlpA–ZZ for expression of soluble IgG and antigen specificity screening by ELISA. Twenty-six of 186 of the screened clones were PA-specific binders. Sequence analysis of the positive clones confirmed the selection of the high- affinity M18 and the moderate-affinity YMF10. To assess the enrichment of M18 and YMF10, phagemid was isolated from 2.5 · 10 9 cells from the pre-sort (post-phage biopanning) and after three rounds of Table 1. Binding kinetics of isolated IgG determined by Biacore. Antibody k on (M )1 Æs )1 ) k off (s )1 ) K D (nM) R17 5.3 · 10 7 5.2 · 10 )2 1 R12 3.2 · 10 6 2.6 · 10 )2 8 R10 7.9 · 10 5 2.1 · 10 )2 26 R21 4.9 · 10 5 1.5 · 10 )2 31 R15 3.7 · 10 5 4.3 · 10 )2 120 42R 3.2 · 10 4 1.4 · 10 )2 440 Alexa fluor 647 (IgG expression) 0.5% 2% 7% 29% Library 1 st round phage panning 1 st round FACS 2 nd round FACS PA-63-FITC (antigen binding) 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 Q4Q3 Q2Q1 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 Q4Q3 Q2Q1 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 Q4 Q3 Q2Q1 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 Q4 Q3 Q2Q1 Fig. 3. Library selection using sequential phage biopanning and FACS. Cells were labeled with PA–FITC and Alexa Fluor 647–anti-(human IgG) probes, and flow cytometry was used to monitor the progress of library selection by quantifying the percentage of fluorescent cells fall- ing into the double-positive quadrant, indicating both expression and affinity. The values in quadrant 2 refer to the percentage of double-posi- tive cells as a proportion of cells that express IgG (total cells minus quadrant 3). Y. Mazor et al. Selection of IgG by tandem phage panning-FACS FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2295 sorting. Five nanograms of purified phagemid from each population was subjected to PCR amplification with gene-specific primers, and the intensities of the PCR products were determined by DNA electrophore- sis (Fig. 4). The densities of the respective bands indi- cated significant enrichment of both clones. Notably, following one round of phage biopanning, a noticeable PCR band was identified for clone YMF10 but not for clone M18, despite there being equal numbers of cop- ies of each clone in the initial mixture and in spite of the much higher affinity of M18. This further empha- sizes that library selection based on bivalent IgG dis- play is not dictated by intrinsic affinity. Discussion Recombinant antibodies are routinely displayed on phages as scFv or Fab fragments. The scFv is a mono- mer consisting of the V H and V L gene fragments con- nected by a peptide linker [47]. This small protein of 25 kDa is displayed very efficiently on phages, both in monovalent (single-copy) format when fused to the gene-3 minor coat protein in a phagemid-based system [31], and in multivalent (multiple-copy) format when the scFv gene is fused to all five copies of gene-3 in a phage-based system [37]. Monovalent display systems are more popular, as they allow the selection of anti- bodies of higher affinity, and because it is far easier to create large libraries in phagemids than in phages [48]. Nevertheless, scFvs often oligomerize, both when dis- played on phages and as soluble proteins in solution, thus making it difficult to select stringently on the basis of intrinsic affinity; furthermore, they can be dif- ficult to express and purify in soluble form [36,49–51]. Fab is a heterodimer consisting of the entire light chain (V L –C L ) paired with the variable and first con- stant domain of the heavy chain (V H –C H1 ) [52,53]. As opposed to scFv, the Fab molecule with a total size of 50 kDa is displayed on phages in a monovalent format [54,55]. However, Fab display is not suitable for anti- gens for which high-affinity binders cannot be obtained, either because of limitations in the library diversity, or because of the physicochemical properties of the target (e.g. carbohydrates). Furthermore, the display of Fab on phages is far less efficient than that of scFv [31,36]. To address some of the limitations associated with scFv and Fab phage display, Lee et al. [36] recently reported the development of a system for the display of bivalent Fab [F(ab¢) 2 ] on the gene-3 coat protein of a single phage particle in a manner that effectively resembles the binding behavior of natural IgGs. This display system was successfully employed for the isolation of specific F(ab¢) 2 from synthetic libraries [56,57]. Bivalent display results in an avidity effect that reduces the off-rates of phage bound to immobilized antigen or to cell surface antigens. Yet, at the same time, the display valency is not high enough to influence binding to soluble antigen, and thus biva- lent phage bind to solution-phase antigen with appar- ent affinities close to intrinsic monovalent affinities [36]. Consequently, bivalent display systems can aid in the recovery of antibodies with moderate affinities, and also in selections that require dimerization for activity. In recent years, the significance of bivalent display for the selection of a broader spectrum of antibodies has led to the development of several display systems that display dimers of scFvs or Fabs to effectively mimic the natural IgG [36,58]. Display of IgGs in their natural conformation expands the sequence diversity that can be encoded, and therefore increases the functional library size for screening [38]. For this reason, we recently developed an E. coli-based technology for the isolation of full- length IgGs from combinatorial libraries using FACS [39,40]. FACS is a very powerful and reliable high- throughput screening methodology. However, sorting of a library greater than 10 9 clones using only FACS is time-consuming and challenging. Commercially available flow cytometers are capable of sorting rates of up to 40 000 s )1 , permitting the screening of approximately 10 8 cellsÆh )1 [40]. Therefore, the screen- ing of very large naı ¨ ve ⁄ synthetic libraries comprising more than 10 9 clones would require 1 day of continu- ous operation of the instrument, which is clearly very challenging. This is particularly impractical if one con- siders that at least 10 times the initial library size should be screened to obtain efficient coverage of the library diversity. To reduce the initial library to a size manageable by FACS, we describe here a method that capitalizes on the display and selection of full-length IgGs on fila- Pre-sort M18 Pre-sort YMF10 R3 YMF10 R3 M18 Fig. 4. Enrichment of high-affinity and moderate-affinity IgGs through FACS, determined using PCR with antibody-specific prim- ers. The amounts of M18 (30 p M) and YMF10 (30 nM) DNA present on this agarose gel increase following three rounds (R3) of FACS on the phage output (Pre-sort). This indicates FACS-dependent enrichment of these antibodies. Selection of IgG by tandem phage panning-FACS Y. Mazor et al. 2296 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS mentous phage particles. For the establishment of an IgG phage display system, we took advantage of the fUSE5–ZZ phage. These phage particles display the Fc-binding ZZ protein on all five copies of the phage p3 coat protein, and are exploited as both the helper phage and for capturing the soluble IgG via the ZZ protein. Even though the ZZ domain has a relatively moderate affinity for Fc IgG in solution (10 nm) [45], its display on all copies of p3 gives rise to multivalent display of the ZZ domain that sufficiently diminishes the functional dissociation of the IgG. We showed that phage particles displaying M18 IgG were efficiently enriched from a 1 000 000-fold excess of phage dis- playing the control 26.10 IgG. This significant enrich- ment of specific binders from an excess of nonbinders validated the competency of our display system, and also provided the fundamental basis for selection of fUSE5–ZZ–IgG from combinatorial libraries. We took advantage of the IgG phage display system as a prescreening step prior to selection by FACS. The phage system provides an elegant means for the effi- cient downsizing of very large libraries by employing an initial round of phage selection that specifically pre- enriches target cells from the library. The downsized library can subsequently be subjected to rounds of FACS, a technique that enables very precise control of the selection process as compared with phage display and, importantly, enables the isolation of clones exhib- iting high affinity and selectivity. Using an anti-PA mouse immune IgG library with an estimated size of 2 · 10 7 as a model library, we showed that, following an initial round of phage selec- tion and two tandem rounds of FACS, specific anti- PA IgGs were isolated with affinities ranging from the low-nanmolar to mid-nanomolar range. This spectrum of affinities emphasizes the advantages of employing bivalent IgG display, as it facilitates the selection of both moderate-affinity clones (R17 and R12) with affinities in the single-digit nanomolar range and very rare binders with modest binding kinetics (R15 and R42) that otherwise could probably not be detected by monovalent display. When desired, low-affinity anti- bodies can be further engineered for improved affini- ties by using the selected antibody gene as the basis for subsequent mutagenesis libraries. To demonstrate the utility of this technology with respect to the screening of large libraries, we demon- strated the enrichment of both a high-affinity IgG and a moderate-affinity IgG that recognize the protective antigen of B. anthracis from a 1 : 10 8 dilution of con- trol antibody. Such a dilution represents only 100 mem- bers of each specific clone in a library of 10 10 clones, which is near the upper limit of the diversity currently available in synthetic libraries [17,20]. We showed that the use of sequential IgG–phage panning followed by FACS allowed the simultaneous selection of both anti- bodies despite the fact that they display a 1000-fold dif- ference in affinity and each was present at a frequency of only 1 : 100 million in the initial population. To conclude, the significance of the methodology described here is illustrated by the fact that it provides the first demonstrated approach permitting the selection of full-length IgGs from libraries displayed on phage. We believe that sequential selection by phage display and then FACS enables the efficient screening of very large IgG libraries by sufficiently oversampling to cover diversity and by simultaneously utilizing the superior technique of FACS for final enrichment. Furthermore, with the bivalent IgG format, it should be possible to both select moderate-affinity antibodies from large naı ¨ ve ⁄ synthetic repertoires, and also to affinity mature low-affinity binders using stringent solution-phase selec- tions that discriminate on the basis of intrinsic affinity. Experimental procedures Cell lines and plasmids Phagemid pMAZ360–IgG for production of full-length IgG has been described previously [39]. Phage fUSE5–ZZ [44] was kindly provided by I. Benhar (Tel Aviv University, Israel). E. coli K91K ⁄ F¢ [59] cells were used for propaga- tion of fUSE5–ZZ and production of fUSE5–ZZ–IgG. E. coli JUDE-1 ⁄ F¢ cells [39] (DH10B harboring the F¢ factor derived from XL1-blue) were used for expression and purification of soluble IgG molecules. Production of fUSE5–ZZ phage Escherichia coli K91K ⁄ F¢ cells carrying fUSE5–ZZ DNA were inoculated overnight at 30 C and 250 r.p.m. in 5 mL of 2 · YT medium supplemented with 20 lgÆmL )1 tetra- cycline and 50 lgÆmL )1 kanamycin. On the following day, the culture was diluted into 500 mL of 2 · YT medium supplemented with 20 lgÆmL )1 tetracycline and grown overnight at 30 °C and 250 r.p.m. Cells were pelleted at 4500 g for 15 min at 4 °C, and the supernatant was filtered through a 0.22 lm filter. The phages were precipitated by addition of 20% (w ⁄ v) poly(ethylene glycol) 6000 and 2.5 m NaCl, and this was followed by centrifugation at 8000 g for 30 min at 4 °C. The phages were suspended in sterile and filtered NaCl ⁄ P i at a concentration of 10 13 colony-forming units (CFU) ⁄ mL and stored at 4 °C. To titer the phage stock, 10-fold serial dilutions of the phages were made in sterile 2 · YT medium. A logarithmic E. coli K91K ⁄ F¢ cul- ture was infected with the diluted phages, and the mixed culture was incubated for 60 min at 37 °C without shaking Y. Mazor et al. Selection of IgG by tandem phage panning-FACS FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2297 and then for 30 min with gentle shaking at 110 r.p.m. Infected cells were plated on 2 · YT plates supplemented with 20 l gÆmL )1 tetracycline and 50 lgÆmL )1 kanamycin, and grown overnight at 37 °C. Preparation of fUSE5–ZZ–IgG For preparation of the fUSE5–ZZ–IgG phage particles, cul- tures of E. coli K91K ⁄ F¢ cells transformed with phagemid pMAZ360–IgG were grown overnight at 30 °C and 250 r.p.m. in 5 mL of 2 · YT medium supplemented with 100 lgÆmL )1 ampicillin, 50 lgÆmL )1 kanamycin, and 2% glu- cose. On the following day, the cultures were diluted 1 : 100 into 10 mL of 2 · YT medium supplemented with 100 lg ⁄ mL ampicillin and 2% glucose, and grown at 37 °C and 250 r.p.m. until 0.6 £ A 600 nm £ 0.8. Cultures were infected with fUSE5–ZZ helper phage at a ratio of 1 : 20 (number of bacterial cells ⁄ number of helper phage particles, assuming that A 600 nm = 1.0–5 · 10 8 bacteriaÆmL )1 ). The cultures were incubated at 37 °C for 60 min without shaking, and then with gentle shaking at 110 r.p.m. for an additional 30 min. The infected cells were collected by centrifugation at 4000 g for 10 min at 4 °C and suspended in 40 mL of 2 · YT medium supplemented with 100 lgÆmL )1 ampicillin and 20 lgÆmL )1 tetracycline, and grown overnight at 30 °C and 250 r.p.m. On the following day, the resulting fUSE5– ZZ–IgG phage particles were precipitated with poly(ethylene glycol) ⁄ NaCl as described above; the supernatant was care- fully aspirated off, and the phage particles were suspended in sterile and filtered NaCl ⁄ P i at a concentration of 10 11 CFUÆmL )1 , and kept at 4 °C. Binding analysis of fUSE5–ZZ–IgG in ELISA ELISA plates were coated with 5 lgÆmL )1 PA from B. anthracis (List Biological Labs, Campbell, CA, USA) or 5 lgÆmL )1 digoxin ⁄ BSA [60] in NaCl ⁄ P i at 4 °C overnight, and then blocked with 2% (v ⁄ v) nonfat milk in NaCl ⁄ P i (NaCl ⁄ P i -M) for 2 h at room temperature. Next, 50 lLof 10 11 CFUÆmL )1 fUSE5–ZZ–IgG phage particles was added in a three-fold dilution series to plates already containing 100 lL of NaCl ⁄ P i -M and incubated for 1 h at room tem- perature. The plates were washed three times with NaCl ⁄ P i containing 0.05% (v ⁄ v) Tween-20 (NaCl ⁄ P i -T), and bound phage particles were detected with HRP-conjugated goat anti-M13 IgG (antibody against pVIII) (Amersham-Pharma- cia Biosciences, Piscataway, NJ, USA). The ELISA was developed using the chromogenic HRP substrate TMB+ (DAKO, Glostrup, Denmark), and color development was terminated with 1 m H 2 SO 4 . The plates were read at 450 nm. To determine the number of IgG molecules per phage, ELISA plates were coated with either 5 lgÆ mL )1 goat anti- M13 IgG (Amersham-Pharmacia Biosciences), 5 lgÆmL )1 chicken anti-(human IgG) Fc-specific (GeneTex, Irvine, CA, USA), or 5 lgÆmL )1 PA in NaCl ⁄ P i at 4 °C for 20 h, and blocked with 2% NaCl ⁄ P i -M for 2 h at room temperature. Then, 50 lLof10 11 CFUÆmL )1 fUSE5–ZZ–M18 IgG phage particles were added in a three-fold dilution series to plates already containing 100 lL of NaCl ⁄ P i -M and incubated for 1 h at room temperature. The plates were washed three times with NaCl ⁄ P i -T, and the ELISA was developed as above. The A 450 nm values were plotted against the phage concentration and used as a standard curve. fUSE5– ZZ–M18 IgG was tested for binding to plates coated with anti-M13 IgG, anti-Fc IgG, and PA. The number of IgG molecules per phage was determined by calculating the ratio of the phage concentration derived from anti-Fc IgG ⁄ anti- M13 IgG or from PA ⁄ anti-M13 IgG in the linear range. To evaluate the stability of the ZZ–IgG complex, freshly pro- duced 10 11 CFUÆmL )1 fUSE5–ZZ–IgG in NaCl ⁄ P i was incubated for 1 h at room temperature with 1 lm standard human IgG (Jackson Immunolaboratories, West Grove, PA, USA) as a competitor prior to being applied to the ELISA plates. The plates were washed three times with NaCl ⁄ P i -T, and bound phage particles were detected with HRP-conju- gated goat anti-M13 IgG (Amersham-Pharmacia Bioscienc- es). The ELISA was developed using the chromogenic HRP substrate TMB+ (DAKO), and color development was ter- minated with 1 m H 2 SO 4 . The plates were read at 450 nm. Enrichment of fUSE5–ZZ–M18 IgG by phage biopanning Anti-PA IgG-displaying fUSE5–ZZ–M18 phage particles were enriched from a 1 000 000-fold excess of phage display- ing the control 26.10 IgG. A 35 mm tissue culture six-well plate was coated overnight at 4 °C with 75 lgÆ mL )1 PA in NaCl ⁄ P i in a total volume 0.7 mL. After the excess solution had been discarded, the wells were washed once with NaCl ⁄ P i and blocked with 3 mL of NaCl ⁄ P i containing 0.25% (w ⁄ v) gelatin (NaCl ⁄ P i -G) for 2 h at room tempera- ture. Then, the wells were washed five times with NaCl ⁄ P i , incubated with a phage mixture consisting of a 1 000 000- fold excess (10 10 fUSE5–ZZ–26.10 IgG and 10 4 fUSE5–ZZ– M18 IgG) in a total volume of 0.7 mL, and rocked gently at room temperature for 2 h. Unbound phage particles were rinsed away, and the plate was washed extensively 10 times with NaCl ⁄ P i -T, and then five times with NaCl⁄ P i . Bound phages were eluted by the addition of 400 lL of 0.1 m HCl adjusted to pH 2.2 with glycine and 1 mg ÆmL )1 BSA for 10 min at room temperature with gentle agitation. The eluted phage particles were transferred into a 1.5 mL microfuge tube, and immediately neutralized with 75 lLof1m Tris ⁄ HCl (pH 9). The selected phage particles were used for reinfection of E. coli K91K ⁄ F¢ cells for subsequent rounds of phage selection. The neutralized eluted phage particles (0.5 mL) were mixed with 4.5 mL of 2 · YT medium and 5 mL of logarithmic K91K ⁄ F¢ cells, and infection was per- formed as described above. The infected cells were spread on 2 · YT plates supplemented with 100 lgÆmL )1 ampicillin, Selection of IgG by tandem phage panning-FACS Y. Mazor et al. 2298 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 50 lgÆmL )1 kanamycin, and 2% glucose, and grown over- night at 30 °C. On the following day, the plates were scraped and subcultured into 50 mL of 2 · YT medium supple- mented with 100 l g ÆmL )1 ampicillin, 50 lgÆmL )1 kanamycin and 2% glucose to give a starting A 600 nm of 0.1. The culture was grown at 37 °C and 250 r.p.m. until 0.6 £ A 600 nm £ 0.8, and 10 mL of the culture was used for infection with fUSE5– ZZ, as described above. Evaluation of the enrichment in each round of selection was performed by phage ELISA as described above. Library selection by phage biopanning Electrocompetent E. coli K91K ⁄ F¢ cells were transformed with DNA phagemid of the anti-PA mouse immune library [39] to generate a final library of 10 7 independent transfor- mants. Library cells carrying phagemid pMAZ360–IgG were inoculated in 500 mL of 2 · YT medium supple- mented with 100 lgÆmL )1 ampicillin and 2% glucose to give a starting A 600 nm of 0.1, and grown at 37 °C and 250 r.p.m. until 0.6 £ A 600 nm £ 0.8. Then, the culture was infected with fUSE5–ZZ helper phage at a ratio of 1 : 20, as described above. The infected cells were collected by centrifugation at 4500 g for 10 min at 4 °C, suspended in 2000 mL of 2 · YT medium supplemented with 100 lgÆmL )1 ampicillin and 20 lgÆmL )1 tetracycline, and grown overnight at 30 °C and 250 r.p.m. On the following day, fUSE5–ZZ–IgG library phages were precipitated with poly(ethylene glycol) ⁄ NaCl as described above; the super- natant was carefully aspirated off, and the phages were sus- pended in sterile and filtered NaCl ⁄ P i at a concentration of 10 12 CFUÆmL )1 and kept at 4 °C. An initial cycle of phage panning was performed in solu- tion with biotinylated PA, using streptavidin-coated para- magnetic beads (Invitrogen, Carlsbad, CA, USA), essentially as previously described [61]. For negative selection, the library phages were incubated with 150 lL of streptavidin- coated beads for 30 min at room temperature for depletion of nonspecific fUSE5–ZZ–IgG phage particles. The phage– bead mixture was then applied to a magnet apparatus, and the unbound library phage particles in the supernatant were removed to a new tube. For positive selection, depleted library phage particles were incubated with 500 nm biotiny- lated PA in 1 mL of NaCl ⁄ P i -M for 1 h at room tempera- ture. Then, the phage–antigen complex was incubated with 150 lL of streptavidin magnetic beads for 30 min at room temperature, and the phage–antigen–bead complex was applied to the magnet. The beads were washed vigorously, and bound phage particles were eluted with 1 mL of 100 mm triethylamine (pH 13) for 10 min while being rotated. The eluted phage particles were separated from the beads and immediately neutralized with 200 lLof1m Tris ⁄ HCl (pH 7.4). Library-selected phages were used for infection of E. coli Jude-1 ⁄ F¢ cells harboring plasmid pBAD33–NlpA– ZZ [39] for subsequent rounds of FACS selection. Neutral- ized eluted phage particles were mixed with 4.5 mL of 2 · YT medium and 5 mL of logarithmic Jude-1–NlpA–ZZ cells, and infection was carried out as above. The infected cells were plated on 2 · YT plates supplemented with 100 lgÆmL )1 ampicillin, 30 lgÆmL )1 chloramphenicol, and 2% glucose, and grown overnight at 30 °C. Library selection by FACS Selection by FACS was performed essentially as previously described [40]. Briefly, E. coli Jude-1 ⁄ F¢ cells carrying plas- mids pBAD33–NlpA–ZZ and pMAZ360–IgG were inocu- lated in TB medium supplemented with 100 lgÆmL )1 ampicillin, 30 lgÆmL )1 chloramphenicol, and 2% glucose, and grown at 30 °CtoanA 600 nm of 1.0. Then, cells were collected by centrifugation at 4500 g for 10 min at 4 °C, induced for IgG expression by resuspension in TB medium supplemented with 100 lgÆmL )1 ampicillin, 30 lgÆmL )1 chl- oramphenicol, and 1 mm isopropyl thio-b-d-galactoside (IPTG), and grown overnight at 25 °C and 250 r.p.m. On the following day, cells were induced with 0.2% arabinose for an additional 3 h at 25 °C and 250 r.p.m. for NlpA–ZZ expression. Then, the cellular outer membrane of A 600 nm 5.0 freshly induced library cells was permeabilized by Tris ⁄ EDTA and lysozyme treatment. For two-color FACS based on antigen binding (affinity) and expression of the displayed antibody, cells (10-fold excess of phage-bio- panning-output) were labeled with 500 nm PA63–FITC (List Biological Labs) and 100 nm Alexa Fluor 647-conju- gated chicken anti-(human IgG) Fc-specific (GeneTex) for 2 h at 4 °C. Highly fluorescent cells falling into the double- positive quadrant, indicating both expression and affinity, were sorted on a FACSAria droplet deflection flow cytome- ter (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with both a 488 and a 633 nm laser. For better selectivity, cells captured after the first sort were immediately resorted on the flow cytometer, using the same collection gate as used for the initial sort. Subsequently, a DNA fragment corresponding to the V L –C K –V H sequence of the IgG gene was amplified from DNA plasmid pMAZ360–IgG of sorted cells using the following primers: V L library amplifier, 5¢- CGGATAACAATTTCACACAGG-3¢; and V H library amplifier, 5¢ -AGTTCCACGACACCGTCACCG-3¢. The PCR product was recloned into the pMAZ360–IgG vector, retransformed into fresh Jude-1–NlpA–ZZ cells, and grown overnight on agar plates at 30 °C. The resulting clones were grown, induced for expression of IgG, and subjected to an additional round of sorting. Screening of selected clones by ELISA Following two rounds of FACS selection, single selected cells were screened for antigen binding in ELISA, essen- tially as previously described [40]. Briefly, PCR-recovered IgG genes were ligated into the pMAZ360–IgG expression Y. Mazor et al. Selection of IgG by tandem phage panning-FACS FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2299 vector and transformed into fresh E. coli Jude-1 cells (not carrying plasmid pBAD33–NlpA–ZZ) for expression of sol- uble, noncaptured IgGs. Randomly selected colonies were inoculated into round-bottomed 96-well plates containing 200 lL of LB medium supplemented with 100 lgÆmL )1 ampicillin and 2% glucose, and grown overnight at 30 °C and 150 r.p.m. on a shaker platform. On the following day, the cultures were diluted 1 : 20 for inoculation on fresh round-bottomed 96-well plates containing 200 lLofTB medium supplemented with 100 lgÆmL )1 ampicillin and 2% glucose, and grown for 3 h at 30 °C and 150 r.p.m. Then, the plates were centrifuged for 10 min at 4500 g , and pellets were resuspended in 200 lL of TB medium supplemented with 100 lgÆmL )1 ampicillin and 1 mm IPTG. Cells were induced overnight at 25 °C and 150 r.p.m. for expression of soluble IgG antibodies. On the following day, the plates were centrifuged, and the cells were lysed in 200 lL of 20% BugBuster HT Protein Extraction Reagent (Novagen, Gibbstown, NJ, USA) in NaCl ⁄ P i for 1 h at room tempera- ture. The plates were centrifuged as above, and soluble cell extracts were tested for direct binding to the PA antigen as follows. ELISA plates were coated with 5 lgÆmL )1 PA in NaCl ⁄ P i at 4 °C overnight, and blocked with 2% NaCl ⁄ P i - M for 2 h at room temperature. Then, 25 lL volumes of the cell extracts were applied to plates containing 75 lLof NaCl ⁄ P i -M, and incubated for 1 h at room temperature. The plates were washed three times with NaCl⁄ P i -T, and bound IgG was detected with HRP-conjugated goat anti- (human IgG) (Jackson Immunolaboratories). ELISA plates were developed as above. Expression and purification of soluble IgGs Expression and purification of soluble full-length IgG was performed essentially as previously described [40]. Briefly, E. coli Jude-1 cells carrying plasmid pMAZ360–IgG were grown at 30 °C in 200 mL of TB medium supplemented with 100 lgÆmL )1 ampicillin and 2% glucose until the A 600 nm was 1.0. Subsequently, cells were collected by centrifugation at 4500 g, for 10 min at 4 °C and IgG expression was induced by resuspension in TB medium supplemented with 100 lgÆmL )1 ampicillin and 1 mm IPTG; cells were then grown for 16 h at 25 °C. Induced cultures were lysed as above, and IgGs were purified from the soluble fraction of total cell extracts using protein A Sepharose (Amersham Biosciences, Sweden) chromatography with final yields of 0.2–1 mgÆL )1 of cells. Bound antibody was eluted with 0.1 m citric acid (pH 3) and neutralized with 1 m Tris ⁄ HCl (pH 9). Protein-containing fractions were combined, dialyzed against 5 L of NaCl ⁄ P i , sterile filtered, and stored at 4 °C. Biacore analysis The antigen-binding kinetics of purified IgGs were deter- mined by surface plasmon resonance analysis, using a Biacore 3000 (Biacore-GE Healthcare, NJ, USA) instru- ment, essentially as previously described [39]. Both a direct binding method and a capture method were utilized to determine kinetic parameters. Briefly, PA63 was coupled to a CM5 chip to an equivalent of 750 response units (RU) by using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide ⁄ N-hydroxysuccinimide chemistry as recommended by the manufacturer. Human transferrin (Jackson Immunolabora- tories) was similarly coupled, and used for in-line subtrac- tion. Various concentrations of purified IgG in NaCl ⁄ P i were injected in duplicate at a flow rate of 50 lLÆmin )1 for 1 min at 25 °C, and the surface was regenerated using one pulse of 50 mm NaOH and 1 m NaCl. The data were ana- lyzed using biaevaluation software with appropriate sub- traction methods, and the bivalent analyte model was used to account for avidity effects associated with the IgG (Biacore-GE Healthcare). For the capture method, goat anti-(human IgG 1 ) Fc-specific (Jackson Immunolaborato- ries) was coupled to a CM5 chip to an equivalent of 10 000 RU by using 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide ⁄ N-hydroxysuccinimide chemistry as recom- mended by the manufacturer. Various concentrations of purified IgG in Hepes-buffered saline⁄ EP buffer (Biacore, Pittsburgh, PA, USA) were injected at a flow rate of 5 lLÆ min )1 at 25 °C to achieve  100 RU of captured IgGs. Buffer and antigen were then injected serially through in-line flow cells at a flow rate of 50 lLÆmin )1 (5 min of sta- bilization, 1 min of association, and 5 min of dissociation), and the surface was regenerated using two pulses of 100 mm H 3 PO 4 . A three-fold dilution series of PA-63, starting at 15 nm, was analyzed in duplicate using biaevaluation soft- ware (Biacore) with appropriate subtraction methods. Enrichment of high-affinity and moderate-affinity IgG by tandem phage FACS Three fUSE5–ZZ–IgG phages displaying M18, YMF10 and VA IgGs were produced and purified as described above. The M18, YMF10 and VA IgGs display high affinity (30 pm) or moderate affinity (30 nm) for B. anthacis PA, and the VA IgG binds to an unrelated antigen, the V pro- tein of Yersinia pestis. The high-affinity and moderate-affin- ity IgGs displayed on fUSE5–ZZ–IgG phage particles were each diluted 1 : 10 8 in 10 11 copies of the VA IgG displayed on fUSE5–ZZ–IgG phage particles. Phage biopanning using biotinylated PA antigen was performed as described above. The library was further subjected to three rounds of FACS screening, performed essentially as described above, using 500 nm PA63–FITC and 100 nm Alexa Fluor 647-conju- gated chicken anti-(human IgG) Fc-specific. The top 3%, 2% and 1% of fluorescent cells were collected, respectively, in rounds 1, 2, and 3. After each round of sorting, the insert DNA was rescued by PCR amplification, recloned into vector pMAZ360–IgG as described above, and trans- formed into Jude-1 cells harboring the NlpA–ZZ plasmid Selection of IgG by tandem phage panning-FACS Y. Mazor et al. 2300 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... distinct mechanisms of protease inhibition by antibodies Proc Natl Acad Sci USA 104, 19784–19789 58 Becerril B, Poul MA & Marks JD (1999) Toward selection of internalizing antibodies from phage libraries Biochem Biophys Res Commun 255, 386–393 59 Smith GP & Scott JK (1993) Libraries of peptides and proteins displayed on filamentous phage Methods Enzymol 217, 228–257 Selection of IgG by tandem phage panning-FACS... on phage J Mol Biol 222, 581–597 9 Sheets MD et al (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens Proc Natl Acad Sci USA 95, 6157–6162 10 Nissim A et al (1994) Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents EMBO J 13, 692–698 Selection of IgG by tandem phage. .. CP, Robinson RR & Horwitz AH (1988) Escherichia coli secretion of an active chimeric antibody fragment Science 240, 1041–1043 Horwitz AH, Chang CP, Better M, Hellstrom KE & Robinson RR (1988) Secretion of functional antibody and Fab fragment from yeast cells Proc Natl Acad Sci USA 85, 8678–8682 Baca M, Presta LG, O’Connor SJ & Wells JA (1997) Antibody humanization using monovalent phage display J Biol... After the third round of sorting, recovered clones were transformed into Jude-1 cells and screened by ELISA as described above Phagemid was isolated from 2.5 · 109 cells from the pre-sort (post-panning) and after each of the three rounds of sorting Five nanograms of DNA phagemid from each population was subjected to PCR with gene-specific primers for either M18 or YMF10 The amount of DNA encoding M18... novel display method for efficient selection of high-affinity antibodies J Mol Biol 376, 1182–1200 22 Schier R et al (1996) Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection J Mol Biol 255, 28–43 23 Schier R et al (1996) Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in... M & Marks JD (2002) Phage versus phagemid libraries for generation of human monoclonal antibodies J Mol Biol 321, 49–56 38 Hayhurst A et al (2003) Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis J Immunol Methods 276, 185–196 39 Mazor Y, Blarcom TV, Mabry R, Iverson BL & Georgiou G (2007) Isolation of engineered, full-length antibodies... Phage- displayed antibody libraries of synthetic heavy chain complementarity determining regions J Mol Biol 338, 299–310 15 de Kruif J, Boel E & Logtenberg T (1995) Selection and application of human single chain Fv antibody fragments from a semi-synthetic phage antibody display library with designed CDR3 regions J Mol Biol 248, 97–105 16 Hoet RM et al (2005) Generation of high-affinity human antibodies by. .. of the antibody binding site J Mol Biol 263, 551–567 24 Thompson J et al (1996) Affinity maturation of a highaffinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity J Mol Biol 256, 77–88 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2301 Selection. .. Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains Nature 348, 552–554 32 Hoogenboom HR et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains Nucleic Acids Res 19, 4133–4137 33 Milenic DE et al (1991) Construction, binding properties, metabolism, and tumor targeting of a single-chain... large non-immunized phage display library Nat Biotechnol 14, 309–314 12 de Haard HJ et al (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies J Biol Chem 274, 18218–18230 13 Souriau C & Hudson PJ (2003) Recombinant antibodies for cancer diagnosis and therapy Expert Opin Biol Ther 3, 305–318 14 Sidhu SS et al (2004) Phage- displayed . Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening Yariv. demonstrated approach permitting the selection of full-length IgGs from libraries displayed on phage. We believe that sequential selection by phage display and