Journal of Immunological Methods 290 (2004) 51–67 www.elsevier.com/locate/jim Review In-vitro evolution by display and mRNA display

Dasa Lipovseka,*, Andreas Plu¨ckthunb,1

a Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA b Department of Biochemistry, University of Zu¨rich, Winterthurerstr. 190, CH-8057 Zu¨rich, Switzerland Accepted 8 April 2004 Available online 31 May 2004

Abstract

In-vitro display technologies combine two important advantages for identifying and optimizing ligands by evolutionary strategies. First, by obviating the need to transform cells in order to generate and select libraries, they allow a much higher library diversity. Second, by including PCR as an integral step in the procedure, they make PCR-based mutagenesis strategies convenient. The resulting iteration between diversification and selection allows true Darwinian protein evolution to occur . We describe two such selection methods, and mRNA display. In ribosome display, the translated protein remains connected to the ribosome and to its encoding mRNA; the resulting ternary complex is used for selection. In mRNA display, mRNA is first translated and then covalently bonded to the protein it encodes, using puromycin as an adaptor molecule. The covalent mRNA–protein adduct is purified from the ribosome and used for selection. Successful examples of high-affinity, specific target-binding molecules selected by in-vitro display methods include peptides, antibodies, enzymes, and engineered scaffolds, such as fibronectin type III domains and synthetic ankyrins, which can mimic antibody function. D 2004 Elsevier B.V. All rights reserved.

Keywords: Ribosome display; mRNA display; PCR

1. Introduction clonal antibodies have served this function. Most of the time, the target of interest is a protein, but other Much modern biological and biomedical research macromolecules, such as specific DNA structures, and is focused on the proteome. Studies of cellular func- localized features, such as those resulting from post- tion, discovery of new therapeutic targets, and de- translational modification, also play a critical role in tailed mechanistic and structural analyses of biological processes. This review discusses two tech- rely on specific binding reagents. Traditionally, mono- nologies—ribosome display and mRNA display—that can be used to generate, select, and evolve such binding reagents completely in vitro. We will describe * Corresponding author. Tel.: +1-617-258 5293; fax: +1-617- the details of these methods as well as a number of 258 7261. E-mail addresses: [email protected] (D. Lipovsek), their successful applications, not only to synthetic [email protected] (A. Plu¨ckthun). antibodies, but also to novel binding reagents that 1 Tel.: +41-1-635-5571; fax: +41-1-635-5712. can fulfill the same roles.

0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2004.04.008 52 D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67

In-vitro selection technologies have two important 1996). Therefore, the emergent in-vitro technologies advantages: the ability to handle very large libraries, have made Darwinian evolution of proteins a reality. and the convenience of evolving proteins through an The greatest challenge remains the design of selection iteration of random mutagenesis and selection. conditions that reward the property of interest. Recent The initial library size is determined by the number publications (discussed below) describe such selection of different DNA molecules present at the beginning schemes for high affinity, specificity, and stability; for of the selection. For the two in-vitro techniques recognition of particular molecular features; for effi- described here, DNA molecules are first transcribed cient folding; and even for catalytic activity. into mRNA in vitro, and then translated into proteins Regardless of the selection technology employed, by a stoichiometric number of . Ultimately, the two most frequently used types of libraries have the functional library size is the number of different been based on peptides and antibodies. Peptides have full-length protein molecules coupled to their encod- the advantages of small size and simple structure, ing mRNA. While the number of different mRNA– which make them easy to randomize synthetically. On protein complexes cannot be larger than the number of the other hand, antibodies have the advantage of a different DNA molecules serving as templates for high-diversity natural repertoire that can be used in transcription, in practice the library size is limited artificial selection systems. Now that selections can by the number of ribosomes or by the number of occur in vitro, it is convenient to work with fully different mRNA molecules present during translation, synthetic libraries of antibodies. To ensure the re- whichever is smaller. In other words, the functional quired and unique linking of phenotype to genotype, library size is limited by the amount of in-vitro the preferred form of the antibody is one in which the translation mixture used, a limit that is identical for two variable domains are covalently linked, as in ribosome and mRNA display. The most significant single-chain Fv fragments (scFv) (Bird et al., 1988; difference between the two methods is in how the Huston et al., 1988; Glockshuber et al., 1990). Since protein is physically connected to the mRNA. the selection technology is completely independent of In most other selection technologies, such as phage the type of protein molecule used, new types of display, the first step in generating diversity is to ‘‘antibody mimics,’’ designed to parallel the function transform a DNA library into cells, usually Escher- of natural antibodies but to take advantage of different ichia coli. This is a fairly laborious undertaking, and in molecular structures, have also been employed. Below this case the starting library size cannot be larger than we discuss designed ankyrin molecules, an example the number of bacteria that contain a correct plasmid. of repeat proteins (Binz et al., 2003, 2004; Forrer et With recent improvements of electroporation protocols al., 2003; Kohl et al., 2003; Stumpp et al., 2003), (Sidhu et al., 2000), large libraries in E. coli can be where the binding surface is generated by a large obtained; this overcomes the problem as long as only surface of stacked repeats, and fibronectin type III the initial library needs to be made. On the other hand, domains (Fn3) (Koide et al., 1998; Xu et al., 2002), if an evolutionary scheme calls for a new library after where three randomized loops mimic the arrangement each in-vitro diversification (e.g., by error-prone PCR in a single antibody domain. With both types of or DNA shuffling) (Leung et al., 1989; Cadwell and molecules, high-affinity binders have been obtained Joyce, 1994; Stemmer, 1994; Zaccolo et al., 1996; by in-vitro selection and evolution technologies. Zaccolo and Gherardi, 1999), more E. coli need to be transformed at every round. Thus, the amount of labor associated with in-vivo technologies still precludes the 2. Ribosome display evolution of proteins over many generations. In contrast, the PCR step inherent in in-vitro 2.1. Description of the method technologies can be used to introduce additional diversity between generations with little additional Ribosome display has a very simple underlying work (e.g., by error-prone PCR). Even the use of a concept. The key idea is to translate a library of non-proofreading polymerase can introduce errors at a mRNA molecules with a stoichiometric quantity of useful rate (Tindall and Kunkel, 1988; Cline et al., ribosomes. If an mRNA molecule has no stop codon, D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 53 the ribosome will frequently run to the very end of their disulfide bonds for stability, such as antibody the mRNA molecule. The corresponding polypeptide fragments. emerges from the ribosome while its end is still Another issue is the stability of the resulting within the ribosomal tunnel, and its last amino acid complexes between mRNA, ribosomes, and protein. is still connected to the peptidyl-tRNA. The release It is now clear that, under appropriate experimental of the polypeptide from the ribosome is normally conditions, the complexes are stable and can be catalyzed by the release factors (RFs), which are maintained for more than 10 days (Jermutus et al., proteins (Poole and Tate, 2000) that bind to the stop 2001), allowing very extensive off-rate selections. codons UGA, UGG, and UAA, taking the place of a This stabilization is possible by cooling the ribosomal tRNA in normal ‘‘sense’’ codons. In the absence of complexes and by adding a high concentration of stop codons, this binding of RFs does not happen. Mg2+ to the buffer, which is thought to ‘‘crosslink’’ Furthermore, the mRNA can only be released from the phosphate groups of the ribosomal RNA, and may the ribosome after the newly synthesized protein and thus prevent the dissociation of the ribosomal com- the tRNA have already dissociated, and this is caused plexes at decreased temperatures. Additional meas- by the ribosome recycling factor (RRF) (Kaji et al., ures to increase stability include the addition of an 1998). Thus, the ribosomes which translate mRNA antisense oligonucleotide (Hanes and Plu¨ckthun, without stop codons will be trapped in a form where 1997) to titrate out the 10Sa RNA, also known as the protein has emerged from the ribosome and the SsrA or tmRNA, which might otherwise help to mRNA is also still connected to the ribosome, release the polypeptide from the ribosome (Gillet thereby connecting phenotype (the protein) and ge- and Felden, 2001). notype (the mRNA). The actual reaction is thus For most practical applications, the stability of the extremely simple: It only consists of a brief in-vitro complexes is thus fully sufficient, and the absence of a translation reaction which is stopped under appropri- covalent bond is not a disadvantage. One can even ate conditions. exploit this absence to elute the encoding mRNA from In order for this technology to allow selection of high-affinity (Jermutus et al., 2001; Zahnd et al., functional proteins, a number of issues had to be 2004) or covalent binders (Amstutz et al., 2002). addressed. Clearly, the protein must fold to its In fact, one unexpected advantage of maintaining correct three-dimensional structure while still at- these ternary complexes is that proteins displayed on tached to the ribosome. This can be achieved by the ribosome appear to be less aggregation-prone introducing an unstructured ‘‘tether’’ or ‘‘spacer’’ (Matsuura and Plu¨ckthun, 2003), expanding the range region to the C-terminal end of a library of proteins, of proteins for which this technology can be applied. which is genetically encoded as a 3V fusion to the While selections have to be carried out at low tem- DNA library of interest. This peptide spacer fills the perature, it is now clear that the molecules selected ribosomal tunnel and provides some extra flexibility, have the same high affinities at high temperatures (as thus allowing the protein of interest to fold as an expected from the generally small temperature depen- independent unit and bind to the target. Furthermore, dence of DG of binding), and even protein stability some proteins involved in folding, such as molecular can be selected by designing the selection pressure chaperones, are present in the translation mix and appropriately (Jermutus et al., 2001). others can be added exogenously (Ryabova et al., Fig. 1 compares the ribosome display (left) and 1997). In particular, even though translation normal- mRNA display (right) cycles. We will first summarize ly occurs in the reducing milieu of the cellular the steps of ribosome display, while the mRNA cycle cytoplasm, and disulfide bonds are normally only is discussed further below: formed after proteins are secreted into the ER (in eukaryotes) or the periplasm (in bacteria), during in- (1) A large DNA library that encodes the polypeptide vitro translation and ribosome display, disulfides can of interest is fused in frame to a C-terminal tether be formed and correctly isomerized by the addition region by DNA ligation. This DNA does not carry of protein disulfide isomerase (Ryabova et al., a stop codon and is transcribed into mRNA. A 1997). This is essential for proteins that depend on typical ribosome display cassette is shown in 54 D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67

Fig. 1. Comparison of two alternative strategies for coupling phenotype and genotype in vitro. (a) Ribosome display (left). DNA encoding the library is transcribed in vitro. The resulting mRNA lacks a stop codon, giving rise to linked mRNA–ribosome–protein complexes during in vitro translation. These can be stabilized and directly used for selection against an immobilized target. The mRNA incorporated in bound complexes is eluted and purified. Reverse transcription-polymerase chain reaction can introduce mutations and yields a DNA pool enriched for binders that can be used for the next iteration. (b) mRNA display (right). Covalent mRNA–protein complexes are generated by ligation of a DNA linker with a small adaptor molecule, puromycin, to the in-vitro-transcribed mRNA, which lacks a stop codon. The mRNA is translated in vitro, and the ribosome stalls at the RNA–DNA junction. Puromycin then binds to the ribosomal A-site, and the nascent polypeptide is thereby transferred to puromycin, as if it were an aminoacyl-tRNA. The resulting covalently linked mRNA–protein complex is isolated, reverse- transcribed and used for selection experiments. The DNA strand is recovered from target-bound complexes by hydrolyzing the complementary mRNA at high pH, then is amplified by PCR. This figure is adapted from Schaffitzel and Plu¨ckthun (2001) with kind permission.

Fig. 2, and the methods by which it can be (3) The target is added, e.g., in biotinylated form, and constructed in Fig. 3. the ribosomal complexes are captured, e.g., by (2) The resulting modified mRNA is used as template streptavidin-coated magnetic beads, and washed for in-vitro translation. After translating the entire to remove library components that bind weakly or coding sequence, the ribosome stops and the non-specifically. protein that has emerged from the ribosome folds (4) To recover the library enriched for target-binding into a 3D structure. sequences, EDTA is added to destabilize the ribo- D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 55

Fig. 2. The PCR product that serves as the linear template for transcription and prokaryotic ribosome display. T7 denotes the T7 promoter, SD the ribosome binding site, spacer the part of the protein construct connecting the folded protein to the ribosome, 5Vsl and 3Vsl the stem loops on the 5V- and 3V-ends of the mRNA, respectively. The arrow indicates the transcriptional start.

somal complexes and mRNA is isolated. Alterna- 2.2. Application to peptides tively, a competitive elution with the target mo- lecule can be carried out before isolating the A decamer random library was used as the starting mRNA. point to select for binding to the monoclonal antibody (5) A reverse transcription reaction followed by D32.39, which had been raised to bind dynorphin B, a PCR (RT/PCR) provides the DNA template for 13-residue opioid peptide, with 0.29 nM affinity the next round. At this stage, error-prone PCR (Mattheakis et al., 1994). A library of 1012 DNA can be used to increase the diversity centered molecules was used for ribosome display using a around enriched sequences. Depending on coupled E. coli in-vitro transcription–translation sys- library complexity, the type of protein scaffold tem. After five rounds of ribosome display, several and on the target, three to six rounds of selec- different antibody-binding peptides, ranging in affin- tion are required to select proteins with low ity from 7.2 to 140 nM, were found. However, none of nanomolar or subnanomolar affinity for the the peptides obtained were similar to dynorphin B in target. To select for even higher affinities (low either sequence or affinity. to mid-pM range), special strategies (off-rate A wheat-germ ribosome display system was used selection) are required (Jermutus et al., 2001; to display a 20-mer random library, which was select- Zahnd et al., 2004). In addition, a variety of other ed for binding to prostate-specific antigen (PSA), a strategies have been developed to select for high tumor marker (Gersuk et al., 1997). After four rounds stability (Jermutus et al., 2001), enzymatic activity of selection, several peptides showing higher affinity (Amstutz et al., 2002; Takahashi et al., 2002), and to PSA than to bovine serum albumin or gelatin were biophysical properties of globular proteins (Mat- isolated, but no quantitative data were reported. suura and Plu¨ckthun, 2003), which will not be A 15-mer library, fused at the N-terminus to fatty- discussed in detail. acid binding protein, was selected against streptavi- din, resulting in new binders, which did not corre- Ribosome display was first described for pepti- spond to the known consensus of streptavidin binding des (Mattheakis et al., 1994). While the advantage peptides. After seven rounds, peptides were charac- of large libraries is valid for peptides and proteins terized, and the best peptide had an affinity of about 4 alike, exploiting iterative cycles of diversification nM (Lamla and Erdmann, 2003). A randomized and selection (Jermutus et al., 2001), by incorpo- region in the CTLA-4 scaffold was also used as the rating error-prone PCR or related techniques such basis for a library which was selected against lyso- as DNA shuffling (Leung et al., 1989; Cadwell and zyme. While binders were found, no quantitative data Joyce, 1994; Stemmer, 1994; Zaccolo and Gherardi, were reported (Coia et al., 2001). 1999; Zaccolo et al., 1996) requires that the open Recently, ribosome display was used to identify the reading frame have a certain length. The number of main antigenic polypeptides of Staphylococcus aureus mutations in the short open reading frame of a (Weichhart et al., 2003). By constructing and selecting small peptide generated by PCR-based techniques is a cDNA library, which had been enriched in the otherwise too small, and the in-vitro display tech- correct reading frame, against anti-staphylococcal nologies are then limited to being mainly a selec- antisera, a number of ORFs could be identified which tion tool. appear to encode those polypeptides that give rise to a 56 D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67

Fig. 3. Generation of the ribosome display construct. For ribosome display, the library of interest has to be flanked by an upstream promoter region and a C-terminal spacer carrying no stop codon (Fig. 2). (A) The library is PCR-amplified with primers carrying restriction sites suitable for ligation into the ribosome display vector (pRDV), which carries the necessary library flanking regions. The PCR product of the library is digested and ligated into pRDV. A second PCR on this ligation reaction with the primers T7B and tolAk yields a PCR product ready for in vitro transcription. (B) Alternatively, the ribosome display construct can be generated by assembly PCR. The library and the spacer are PCR- amplified separately with primers, so that the C-terminal part of the library and the N-terminal part of the spacer share overlapping sequences. An assembly PCR with the library and the spacer DNA, using appropriate primers, finally yields the ribosome display construct. In-vitro transcription of the PCR product of either (A) or (B) yields mRNA carrying 5V- and 3V-stem loops (which make the mRNA more stable by increasing its resistance to exonuclease digestion), a ribosome binding site (RBS), the library of interest and a spacer carrying no stop codon. By stopping the in-vitro translation in ice-cold buffer with high Mg2+concentration, stable complexes of mRNA, ribosome and nascent protein are formed, ready for panning (Fig. 1). This figure is adapted from Amstutz et al. (in press) with kind permission. major fraction of the human antibody response and 2.3. Application to antibodies could thus be interesting vaccine candidates. For this type of application, the library size accessible with As a first validation of the ribosome display ribosome display was important, as was the fact that, technology for whole proteins, a model system of unlike with in-vivo display techniques, apparently two antibody scFv fragments was used. A 109-fold most sequences could be displayed on the ribosomes. enrichment was achieved by five cycles of ribosome D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 57 display, with an average enrichment of about 102 per Next, a very large synthetic antibody scFv library, cycle (Hanes and Plu¨ckthun, 1997). All selected scFvs HUCAL-1, of 2 Â 109 independent members (Knap- had acquired genetic mutations during the five cycles pik et al., 2000), was used directly as the starting of ribosome display; the range was 0–4 amino acid material (Hanes et al., 2000). It should be pointed out changes from the original sequence per clone. that in preparing the starting material, which is a linear Next, immune libraries were used as the starting PCR product (cf. Fig. 2), from the stored plasmids, material, and it was demonstrated that scFv antibody some mutations were probably already introduced, so fragments could be selected and evolved using the E. that the true starting material is likely to be more coli ribosome display system. Only three rounds were diverse than the library stored in plasmid form. necessary to isolate a family of scFv fragments This naive library was applied for six rounds of binding to a peptide variant of the GCN4 leucine ribosome display selection using insulin as the anti- zipper (Hanes et al., 1998). Most of the isolated scFvs gen. In three independent experiments, different scFv had again acquired mutations, with 0–5 amino acid families with different framework combinations were changes from their consensus sequence, which is the isolated. Since the library used was completely syn- sequence present in the library before the diversifica- thetic (Knappik et al., 2000), the starting scFv sequen- tion occurred during selection and the most likely ces were known and any mutations could be directly progenitor scFv. The best scFv obtained by ribosome identified as being generated during the ribosome display had a dissociation constant of 40 pM, as display procedure by non-proofreading polymerases measured in solution. The likely common progenitor in the PCR steps. of the related scFvs bound the antigen with a 65-fold In summary, this procedure thus closely mimics the lower affinity than the best selected binder, demon- process of somatic hypermutation of antibodies during strating that ribosome display and the inherent PCR- secondary immunization. The final products of selec- based mutations can be exploited to select proteins tion were different families of closely related sequen- with higher affinity. ces, which stem from a common progenitor that A small focussed library derived from a scFv started to evolve during ribosome display. A biophys- derivative of an antibody, VH-linker-VL-CL,that ical comparison of the isolated scFvs to their progen- bound to progesterone, was used for selection with a itors revealed that all selected scFvs had mutated due rabbit reticulocyte ribosome display system (He and to errors introduced by the DNA polymerase, and Taussig, 1997). The authors used a coupled in-vitro consequently had improved their affinities to the transcription–translation system in the presence of 2 antigen significantly, by up to 40-fold by these muta- mM DTT, as this particular antibody can fold even in tions. The best scFvs selected had affinities in the low the presence of reducing agents (see below). Human picomolar range (Hanes et al., 2000) and could be antibody scFv fragments that bind progesterone were further evolved by off-rate selection (see below). selected from a library prepared from transgenic mice In another example, specific binders were selected (He et al., 1999) by using a proofreading polymerase from HuCAL against a special DNA structure which for the PCR amplification steps. Thus, in this case, appears in eukaryotic telomers (Schaffitzel et al., ribosome display was used exclusively for selection, 2001). The telomeric repeat of hypotrichous ciliates, and the original library repertoire was maintained. d(T4G4), forms a 16- 3V-overhang. Such Antibody selections have also been reported by Irving sequences can adopt parallel-stranded as well as et al. (2001), but no quantitative data are given. antiparallel-stranded quadruplex conformations in Interestingly, in a direct comparison (Hanes et al., vitro. Of the scFvs selected, one had a high affinity 1999),theE. coli system was found to be more (KD = 125 pM) for the parallel-stranded guanine-quad- efficient for the display of the model scFv constructs ruplex, and could discriminate with at least 1000-fold tested than the rabbit reticulocyte system. This argues specificity between parallel and antiparallel quadru- against the occasionally proposed idea that eukaryotic plex conformations formed by the same sequence proteins would be displayed more efficiently in a motif. A second scFv bound both the parallel and ‘‘more eukaryotic environment,’’ and it seems that antiparallel quadruplex with similar affinity (KD =3– other factors determine the efficiency of selection. 5 nM). Immunofluorescence studies using these scFv 58 D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 molecules suggest that the telomers present in macro- expressed in the cytoplasm), but also increased the nuclei of ciliates indeed adopt a guanine-quadruplex stability in the presence of the disulfide bond by about structure in vivo (Schaffitzel et al., 2001), which at 30 kJ/mol (Jermutus et al., 2001). Interestingly, the least raises the possibility that this may be the case in selected mutants had all used different mutation other species as well. strategies to adapt to the selection pressure and are derived from different ‘‘lineages’’ during the evolu- 2.4. Evolution of affinity and stability tion process in the same experiment, which is most probably due to the large library size in each ribosome So far, two studies have been reported in which a display selection. given antibody was evolved to higher affinity. In both cases, off-rate selection combined with error-prone 2.5. Application to other frameworks as antibody PCR was used. An antibody against fluorescein was mimics: synthetic repeat proteins evolved (Jermutus et al., 2001) using selections in which antibody–antigen complexes needed to last up In antibodies and all alternative scaffolds reported to 10 days, resulting in final dissociation constants of to date (Nygren and Uhlen, 1997; Skerra, 2000), the about 100 pM. The evolved scFvs all contained binding surface is limited by the size of the scaffold. between 4 and 11 mutated amino acids (mean value Repeat proteins, on the other hand, have evolved of 7.2), with the majority unlikely to be in direct another successful binding strategy (Fig. 4). They contact with the antigen. feature repeating structural units (repeats), which In the second study, the dissociation constant of stack together to form elongated protein domains with one of the scFvs described above to a peptide derived a continuous target-binding surface (repeat domains). from the transcription factor GCN4 was further im- Depending on the type of repeat, the target-binding proved from 40 to 5 pM (Zahnd et al., 2004), which surface may comprise the surface of secondary struc- may be the tightest peptide binder to date. Libraries of ture elements and loops (Kobe and Kajava, 2000). mutants were generated with error-prone PCR and Natural repeat proteins, such as ankyrin or leucine- DNA shuffling, and selected for decreased off-rates. rich repeat proteins, occur in nearly all forms of life, Crystallographic analysis of the scFv in its antigen- in most cellular compartments, and are involved in a bound and free states showed that only a few muta- wide range of biological processes, where specific tions, which do not make direct contact to the antigen, binding to a partner is the key step. lead to the final 500-fold affinity improvement over Based on the modularity of this class of proteins, a its potential germ line precursor. These results suggest novel strategy was developed to generate combinato- that the affinity optimization of very high-affinity rial libraries of target-binding polypeptides (Binz et binders is achieved by modulating existing interac- al., 2003b; Forrer et al., 2003; Kohl et al., 2003; tions via subtle changes in the framework rather than Stumpp et al., 2003). This strategy includes the by introducing new contacts. consensus design of self-compatible repeats that dis- Single-chain Fv antibody fragments have also been play variable surface residues, followed by their evolved for stability by using ribosome display (Jer- random assembly into repeat domains. mutus et al., 2001). ScFvs contain two conserved The idea fundamental to the design of repeat disulfide bridges. These are important stability ele- protein libraries is to exploit the excellent statistics ments, and the removal of the intradomain disulfide which can be derived from the alignment of all single bonds usually results in a significant loss of stability repeats. A consensus ankyrin repeat sequence motif (Proba et al., 1998; Wo¨rn and Plu¨ckthun, 1998, 2001). was developed which is self-compatible and displays More stable mutants can be selected by choosing a variable surface residues, with a diversity of 7 Â 107. reducing redox potential during the translation step in By randomly assembling the modules, a very large ribosome display. The selected mutants had three to interaction surface is created, and the theoretical seven mutations in the coding sequence (mean value diversity is potentiated to (7 Â 107)n, where n is the of 4.8). They had not only acquired the ability to fold number of repeats. The modularity of the protein under reducing conditions (and to be functionally allows the development of novel evolutionary strate- D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 59

gies, such as module shuffling, module insertions, or module deletions. Thus, the size of the potential target-binding surface is adaptable as desired. For example, an elongation strategy may be used for affinity maturation of selected binders. Randomly chosen ankyrin library members were found to be expressed in soluble form in the cyto- plasm of E. coli as up to 30% of total cellular protein (corresponding to about 200 mg/l yield of functional protein in shake-flask cultures) and they show high thermodynamic stability, with Tm values ranging from 66 jC to well above 85 jC, and with free energies of unfolding of 10–20 kcal/mol (Binz et al., 2003; Kohl et al., 2003). The crystal structure of one of these library mem- bers was determined to 2.0-A resolution (Kohl et al., 2003), showing highly complementary hydrophobic repeat–repeat interfaces as well as a regular and extended hydrogen bond networks in the h-turn and loop regions. Ankyrin repeat protein libraries were used in selec- tions for binding against target proteins. Specific binding molecules against several globular proteins were selected with affinities in the low nanomolar range (Binz et al., unpublished; Amstutz et al., unpub- lished). The crystal structure of a complex of a selected

Fig. 4. Design of a synthetic ankyrin library. (A) The designed AR sequence motif. X: Any amino acid but not G, C or P. Z: Any of the amino acids H, N or Y. (B, C) Ribbon representation of the third repeat (second synthetic repeat module) of the crystal structure of one unselected library member in two perpendicular views (Kohl et al., 2003). The orientation of the module in (B) corresponds to that in (D). (D) Ribbon representation of the same molecule showing the helices of the N-terminal capping repeat, internal repeat modules, and the C-terminal capping repeat in green, dark blue and light blue, respectively. (B–D) The side chains of amino acids at variable positions are highlighted in red. (E) Surface representation of the synthetic ankyrin molecule, showing the conserved surface in gray and the variable surface in red. (F, G) Schematic representation of the designed repeat domains. (F) Designed capping repeats and designed self-compatible repeat modules are the building blocks of the repeat domains. Compatible module interfaces allow the stacking of repeat modules. (G) The designed repeat domains consist of N- and C-terminal capping repeats flanking a variable number of (here, three) repeat modules. The repeat modules stack together forming a continuous hydrophobic core, which is sealed on both sides by the capping repeats. The variable molecular surfaces, which are potential target binding sites, are shown in red; the hydrophobic core is shown in blue, and the more hydrophilic conserved molecular surface is represented in gray. This figure is adapted from Forrer et al. (2003) with kind permission. 60 D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67

AR protein with its target was recently determined molecules through a small adaptor molecule, typically (Binz et al., 2004), proving that the use of repeat-based puromycin (Fig. 5). consensus design for the generation of repeat protein A typical mRNA-display selection cycle is shown libraries was successful. in Fig. 1 (right panel), side-by-side with the ribosome- Since the designed libraries contain no cysteines, display selection cycle (left panel). The steps involved these molecules may be especially suitable for intra- in the synthesis of covalent mRNA–protein fusion cellular or proteomics applications. Additionally, the molecules and in the selection of target-binding poly- repeat protein libraries are highly valuable sources for peptides are summarized below: novel binding molecules, which may be suitable for a whole range biotechnological, biomedical, and poten- (1) A large DNA library, which encodes the po- tially therapeutic applications. lypeptide of interest and is free of stop codons, is transcribed into mRNA. (2) The 3V-end of each encoding mRNA is ligated to 3. mRNA display a short, synthetic linker, which contains the adaptor molecule at its 3V-end. 3.1. Description of the method (3) The resulting modified mRNA is used as template for in-vitro translation. After translating the entire Like ribosome display, mRNA display (Nemoto et coding sequence, the ribosome proceeds to form a al., 1997; Roberts and Szostak, 1997; Roberts, 1999; peptide bond between the adaptor molecule and Takahashi et al., 2003) uses a complex between the C-terminal amino acid residue in the nascent mRNA and the polypeptide encoded by that mRNA polypeptide chain (Fig. 1) The resulting mRNA– as the basic unit of selection. Since the mRNA– protein fusion is purified away from ribosomes protein complex is generated entirely in vitro, with- and other components. (The in-vitro translation out transforming bacteria with the genetic material, step can be used to introduce unnatural amino large libraries can be constructed with ease. Typical acids into the protein (Li et al., 2002).) mRNA–protein libraries used in selection contain (4) For most applications, a DNA chain complemen- 1012 –1013 unique sequences (Roberts, 1999; Cho et tary to the protein-bonded mRNA is introduced, al., 2000; Keefe and Szostak, 2001; Xu et al., 2002; using reverse transcription, in order to stabilize the Takahashi et al., 2003). nucleic acid component and to facilitate the reco- What distinguishes mRNA display from ribosome very of genetic information after the selection. display is the covalent nature of the linkage between Typically, the temperature and the buffer condi- the mRNA and the protein in the mRNA–protein tions that can be used during affinity selection are complex. This is achieved by bonding the two macro- limited only by the stability of the protein target.

Fig. 5. Model of mRNA–peptide fusion, the unit of selection in mRNA display. Both a molecule of mRNA (gray) and the encoded peptide or protein (teal and red) form covalent bonds to an adaptor molecule, e.g., puromycin (purple). A strand of complementary DNA (green) is reverse- transcribed from the mRNA and hybridizes to this template. The figure is drawn to scale for Ecballium elaterium trypsin inhibitor, EETI-II, a disulfide-constrained, 28-residue (3 kDa) peptide and for its encoding nucleic acid. D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 61

(5) The cDNA/mRNA–protein library is mixed with every possible permutation of a 9-mer peptide, it the target, and cDNA/mRNA–proteinDtarget seems likely that mRNA display can be used routinely complexes are captured using affinity chroma- to identify linear epitopes of existing antibodies. tography or of the target. Another study started with a linear-polypeptide The isolated complexes are washed to remove the library with 88 randomized positions (Cho et al., library components that bind the target weakly or 2000), and identified 20 different sequences that non-specifically. bound streptavidin (Wilson et al., 2001). Nineteen (6) To recover the library enriched for target-binding of the twenty sequences contained at least one copy sequences, mRNA of the captured cDNA/ of histidine–proline–glutamine (HPQ), the strepta- mRNA–protein complex is hydrolyzed at high vidin-binding tripeptide first identified by phage pH, releasing the cDNA strand. The cDNA is display (Devlin et al., 1990). In contrast to the 10- recovered from the supernatant. residue, HPQ-containing peptide optimized by phage (7) The recovered cDNA, amplified by PCR, provides display, which had been shown to have a KD of 13– the DNA template for another round of selection 72 AM (Schmidt et al., 1996; Voss and Skerra, or for sequencing. At this stage, error-prone PCR 1997), a 38-residue fragment of one of the sequences can be used to increase the diversity centered selected by mRNA display bound streptavidin with a around enriched sequences (Xu et al., 2002). KD of 2.5 nM. When used as an affinity tag, the 38- Depending on library complexity and on the residue peptide allowed single-step purification of target, 4–10 rounds of selection are required to protein from bacterial lysate (Keefe et al., 2001). select proteins with low to subnanomolar affinity Low nanomolar affinity is more common in anti- for the target. bodies than in in-vitro-selected linear peptides, prob- ably because significant entropy is lost upon binding mRNA display has been used to select high- a flexible peptide, but less entropy is lost in forming affinity reagents from engineered libraries of linear a protein–protein complex. One possible interpreta- peptides (Barrick and Roberts, 2002; Barrick et al., tion for the selection of high-affinity linear peptides 2001a,b; Keefe et al., 2001; Wilson et al., 2001; is that the high complexity of peptide libraries used Baggio et al., 2002), constrained peptides (Baggio et in in-vitro selections against a target with a pro- al., 2002), single-domain antibody mimics (Xu et al., nounced groove can thoroughly sample the possible 2002), variable heavy domains of antibodies (Chen, binding interactions. 2003), and single-chain antibodies (Chen, 2003).In Affinity reagents derived from constrained pepti- addition, mRNA-display selections from proteomic des may have an advantage over linear peptides in libraries have identified cellular polypeptides that some applications, both because they are more similar bind specific signaling proteins (Hammond et al., in structure to globular proteins, and because of their 2001) and small-molecule drugs, as well as polypep- smaller loss of entropy upon target binding. A disul- tide substrates of v-abl kinase (Cujec et al., 2002). fide-constrained library based on EETI-II, a knottin trypsin inhibitor, was constructed by randomizing the 3.2. Application to peptides six residues of the trypsin-binding site, and mRNA display was used to select new trypsin-binding pep- Early applications of mRNA display used random- tides. The selected peptides were highly homologous ized linear peptides as libraries, and well-character- or identical to wild-type EETI-II; their dissociation ized proteins known to bind peptides as targets. A constants from trypsin ranged from 16 (for the wild peptide library with 27 randomized positions yielded a type) to 82 AM (Baggio et al., 2002). family of sequences that bound the anti-c-Myc anti- Another comparison between the effectiveness of body, 9E10 (Baggio et al., 2002). The selected clones mRNA display and natural evolution focused on the contained sequences that were either homologous or 22-residue, RNA-recognition domain of E N protein identical to a 10-residue stretch of the 32-residue c- (Barrick et al., 2001b). Of the 22 residues in the RNA- Myc antigen (Chan et al., 1987). Given that an recognition domain, 10 were randomized, and the mRNA-peptide library with 1013 members can sample resulting library was selected for binding to several 62 D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67

RNA hairpin loops, including the binding partner of the wild-type domain, boxBR. The resulting sequences bore little similarity to each other or to the wild-type E N domain, but they bound boxBR with similar KD (0.4–3.4 nM for five selected clones vs. 1.0 nM for the wild type). Experimental evidence and sequence anal- ysis (Barrick and Roberts, 2002) suggest a propensity of the selected sequences to form alpha helices, as does the wild-type E N domain upon target binding. A second selection from very long linear libraries appears to have yielded polypeptides with some characteristics of folded proteins. The selection of ATP-binding polypeptides from a library of 80 ran- domized residues isolated a novel family of polypep- tides, each with two invariant CXXC motifs (Keefe and Szostak, 2001). The highest-affinity clone had a KD of 100 nM; the affinity decreased if more than 10 residues were deleted from the sequence. The require- ment for such a long sequence, as well as the observation that the affinity for ATP is lost in the Fig. 6. NMR structure of the tenth human fibronectin type III domain (1TTG, (Main et al., 1992)). Due to its immunoglobulin- presence of EDTA but restored by excess zinc, sug- like fold, 10Fn3 has been used as an alternative to antibody 2+ gest a compact structure dependent on a Zn ion fragments. The solvent-exposed loops, which are structurally coordinated by the four cysteines. A crystal structure analogous to antibody human CDR regions, are shown in blue (Lo Surdo et al., 2004) of this artificial nucleotide- (loop BC), green (loop DE), and red (loop FG). binding protein revealed a novel, compact fold.

3.3. Application to antibodies and antibody mimics yielded numerous Fn3-like proteins that bound the target with 1–24 nM affinity, and affinity maturation As mRNA display is best suited to single-chain using error-prone PCR improved the affinity to 20 pM. proteins, its first applications to the selection of Similar results were obtained with several other human protein affinity reagents used single immunoglobulin targets, including vascular endothelium growth factor or immunoglobulin-like domains, including variable receptor two (VEGF-R2); this selection yielded ‘‘neu- domains of antibody heavy chains (VH) and the10th tralizing antibody mimics,’’ Fn3-like domains that human fibronectin type III domain (10Fn3). More competed for binding to VEGF-R2 with its natural recently, the method was applied to single-chain anti- ligand, VEGF (Parker et al. and Getmanova et al., bodies (scFv). manuscripts in preparation). In addition to mimicking Most of the published work on globular-protein the specific target-binding function of antibody scaffolds focuses on human 10Fn3, a 94-residue do- domains, 10Fn3-derived antibody mimics contain no main found naturally as a component of fibronectin. disulfides, which facilitates their expression in E. coli, Despite its low sequence homology with antibody folding, and purification. domains (f15% identity), 10Fn3 adopts a three-dimen- In contrast to chemically synthesized antibody- sional structure reminiscent of that of a VH or VL mimic libraries, the antibody-fragment libraries that domain (Fig. 6). This similarity was exploited in the have been used in mRNA display derive their diver- design of several antibody-mimic libraries (Koide et sity from the natural human immune system—the full al., 1998; Xu et al., 2002). The library used in mRNA antibody repertoires from bone marrow, spleenocytes, display was made by randomizing the 21 residues in and peripheral blood mononuclear cells of multiple the loops analogous to antibody CDRs (Fig. 6).A healthy human subjects (Chen, 2003) (Fig. 7). The selection against tumor necrosis factor alpha (TNF-a) mRNA-VH and mRNA-VL display libraries contained D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 63

combination of VH and VL. Third, the selected VH and VL domains were introduced into their respective mammalian expression vectors, resulting in full- length monoclonal antibodies. In the case of several cytokines and cytokine receptors, this selection strat- egy yielded VH domains and single-chain antibodies with low to subnanomolar affinities (Chen, 2003).A scFv against VEGF-R2 was used to construct a full- length antibody, which binds specifically to CHO cells that overexpress VEGF-R2 on their surface (Chen et al., manuscript in preparation).

4. Conclusions and perspectives

4.1. Unresolved questions

Despite the very encouraging results from in-vitro selection and evolution using the two techniques described here, several mechanistic challenges remain to be solved in order to make the methods even more powerful. Both methods implicitly depend on a large fraction of the ribosomes translating the protein to the end in Fig. 7. Antibody-selection strategy in mRNA display. (1) cDNA order for it to fold properly. However, in vitro, such libraries from bone-marrow cells, spleenocytes, and human full-length translation rarely happens on all ribo- peripheral-blood mononuclear cells from several healthy humans somes. Therefore, a certain fraction of molecules are used to make separate VH and VL libraries. (2) A cDNA/RNA- displayed are not full-length. This in turn may limit V library is made from the cDNA library, and (3) target-binding H the enrichment factor, as the incomplete synthesis VH domains are selected from the mRNA-display VH library. (4) DNA encoding the selected VH domain (or a selected pool of target- products would be more likely not to fold, and, binding domains) is recombined with the naive VL library to consequently, to bind nonspecifically. While addition- produce a single-chain-antibody (scFv) library. (5) A cDNA/RNA- al pre-screening steps can be employed to maximize scFv library is made from the cDNA library, and (6) exposed to the fraction of full-length molecules, a direct improve- immobilized target to isolate target-binding scFv clones. (7) DNA molecules encoding the selected scFv fragments are cloned into ment of translational efficiency might be more advan- vectors to assemble full-length antibodies. tageous because it would increase the total number of full-length molecules. In mRNA display, the purification of protein– 1012–1013 molecules each, more than enough to puromycin–DNA–mRNA adduct from the ribosome 8 represent the diversity of the human VH (10 ) and presents a topological puzzle. After translation, the 4 VL (10 ) repertoire before somatic mutation. protein folds outside of the ribosomal tunnel to a The first antibody selections were carried out in globular domain. At the other end of the tunnel, the three stages (Fig. 7). First, VH domains that bind to puromycin–mRNA/DNA reagent reacts with the the target were selected as described previously (Fig. polypeptide. Thus a folded domain sits at one end of 1). Second, the selected VH-pool or single-clone DNA the tunnel, while the long mRNA/DNA is connected to was shuffled with the naive VL library, resulting in a the peptide at the other end. Whereas the purification is single-chain-antibody (scFv) library with pre-selected, performed under conditions expected to dissociate the target-binding domains (or domain). mRNA-display ribosome, no direct evidence is yet available that an selection from the scFv library yields a target-binding ‘‘opening’’ of the tunnel takes place. Alternative 64 D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 explanations are that (i) the mRNA/DNA passes stant of 5 pM by ribosome display is particularly through the protein exit tunnel, or (ii) the protein noteworthy (Zahnd et al., 2004). To date, the mRNA- denatures and goes ‘‘backwards’’ through the tunnel. display approach to antibody selection has been If such denaturation of the displayed protein is re- stepwise, selecting first a target-binding VH domain, quired, that might limit the application of mRNA and second the VL domains that preserve its binding display to proteins with robust refolding properties. in scFv context. The resulting variable domains have been incorporated into a full-length antibody while 4.2. Conclusions preserving their affinity for the target. More published mRNA-display selections with a range of targets and Ribosome display and mRNA display are both antibody formats will be needed to properly evaluate robust, entirely in-vitro selection methods, which the performance of mRNA display in this area. make routine the construction of extremely large An interesting recent development are the so-called libraries (1012 –1013 different sequences). In addition, antibody mimics, proteins not closely related to anti- the PCR step that both methods use in library con- bodies that are designed to perform the antibody struction and mRNA recovery provides a convenient function of tight and specific target binding. A fibro- way to further mutagenize the selected pool after each nectin type III domain, which has an immunoglobu- round of selection; this feature is particularly powerful lin-like fold but is considerably smaller than a VH during affinity maturation. The major difference be- domain, was used to make mRNA-display libraries tween the two techniques is in the physical nature of with diversity concentrated in three exposed loops; the linkage between mRNA (the genotype) and protein after selection Fn3-like domains with affinities as low (the phenotype) and in the number of steps in the as 20 pM were selected. An alternative approach cycle. Whereas ribosome display employs a stable but employs proteins larger than antibody domains: non-covalent association between an mRNA strand, a ankyrins, proteins containing several repeats, have ribosome, and the polypeptide encoded by the mRNA, been used to create libraries with extensive random- mRNA display uses a small adaptor molecule to form a ized surfaces, and were subjected to ribosome-display covalent linkage between the mRNA and the protein. selection to obtain high-affinity reagents. Without Fewer reports are available of the application of affinity maturation, low nanomolar KD values are mRNA display than of ribosome display, and fewer normally obtained directly, leaving room for signifi- different binder–target systems have been explored. cant affinity improvement. Neither Fn3 domains nor At the moment it appears that two in-vitro selection ankyrins contain free cysteines or disulfide bonds; as a methods can be applied to the same types of problems consequence, they perform better than most antibody with similar success. fragments in bacterial expression and can be used Both ribosome display and mRNA display have under both oxidizing and reducing conditions. We been used to select linear peptides that bind to protein anticipate that in-vitro-selected antibody mimics and targets. mRNA display has been applied successfully antibody domains will become invaluable tools in to the selection of disulfide-constrained peptides as proteomic research, as well as in biotechnology and long as 80 residues, and to single antibody variable in medical applications. domains. In summary, the unmatched library size and the Whereas both ribosome and mRNA display have convenience of affinity-maturation protocols in the been applied to single-chain antibodies, considerably two in-vitro selection methods, ribosome display and more information is available for ribosome display, mRNA display, has led to their application to the which has been used to select numerous scFvs with entire range of polypeptide libraries, from short, linear low picomolar affinities for their targets. In particular, peptides, to antibodies and to large, modular antibody- ribosome display has affinity-matured tight (mid-pM) mimic proteins. The growing number of reports on the scFvs to their extremely tight (low pM) derivatives success of these methods in the selection of affinity using error-prone PCR and off-rate selection. The reagents with low nanomolar to low picomolar affin- selection of a monovalent scFv fragment of an anti- ity gives us confidence that they will find an even body that binds a peptide with the dissociation con- broader use in the future. D. Lipovsek, A. Plu¨ckthun / Journal of Immunological Methods 290 (2004) 51–67 65

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