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Efficient Selection of Darpins with Sub-Nanomolar Affinities Using SRP Phage Display

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Efficient Selection of Darpins with Sub-Nanomolar Affinities Using SRP Phage Display doi:10.1016/j.jmb.2008.07.085 J. Mol. Biol. (2008) 382, 1211–1227 Available online at www.sciencedirect.com Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display Daniel Steiner, Patrik Forrer and Andreas Plückthun⁎ Department of Biochemistry, There is an ever-increasing demand to select specific, high-affinity binding University of Zürich, molecules against targets of biomedical interest. The success of such Winterthurerstrasse 190, selections depends strongly on the design and functional diversity of the 8057 Zürich, Switzerland library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs Received 13 May 2008; the cotranslational signal recognition particle (SRP) pathway for the received in revised form translocation of proteins to the periplasm. This system allows efficient 24 July 2008; filamentous phage display of highly stable and fast-folding proteins, such as accepted 30 July 2008 designed ankyrin repeat proteins (DARPins) that are virtually refractory to Available online conventional phage display employing the post-translational Sec pathway. 6 August 2008 DARPins comprise a novel class of binding molecules suitable to comple- ment or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 1010 individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFα, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins. © 2008 Elsevier Ltd. All rights reserved. Keywords: phage display; library generation; SRP phage display; DARPin; Edited by F. Schmid novel scaffold Introduction body and non-antibody scaffolds, binding molecules with high affinity and high specificity against almost The demand for high-quality binding molecules any chosen target protein can be generated in vitro for biotechnological and biomedical applications is using appropriately designed combinatorial libraries in combination with selection technologies such as ever increasing. Antibodies are currently by far the 1–4 most frequently used binding molecules, but pro- phage display or ribosome display. The success of teins based on non-immunoglobin scaffolds have such selections is based mainly on the design and become an appealing alternative.1 From both anti- size of the combinatorial library and the selection technology employed. Together, these factors will determine the functional size of the library that is *Corresponding author. E-mail address: accessible for the selection of binding molecules. [email protected]. Further important factors are the selection strategy Present address: D.S. and P.F. Molecular Partners AG, chosen and the biophysical properties of the target Grabenstrasse 11a, 8952 Zürich-Schlieren, Switzerland. protein; even with the best library and an optimized Abbreviations used: DARPin, designed ankyrin repeat selection protocol it might be very difficult or even protein; EGFR, epidermal growth factor receptor; ErbB, impossible to obtain useful binding molecules erythroblastic leukemia viral oncogene homolog; HER2, against a "sticky" and unstable target protein. human epidermal growth factor receptor homolog 2; POI, The underlying principle of all selection technol- protein of interest; SRP, signal recognition particle; TNFα, ogies is the physical linkage of the phenotype (i.e. the tumor necrosis factor α. displayed protein) and the genotype (i.e. the DNA 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. 1212 Selection of DARPins with Sub-nanomolar Affinities encoding the displayed protein). Different selection challenge is to introduce a level of diversity high technologies use different strategies to achieve this enough to allow successful selection of binders linkage. The success of any selection experiment against any target protein, yet low enough to not depends on the compatibility of the strategy used destabilize library members too much. with the proteins to be displayed. Ribosome display, In the case of designed ankyrin repeat proteins for example, is a complete in vitro display technology (DARPins),12,13 which comprise a novel class of that relies on the formation of a non-covalent ternary binding proteins, we used consensus design to gene- complex of mRNA, ribosome and the displayed rate a very stable scaffold amenable to the introduc- nascent polypeptide.5 The formation of this ternary tion of the needed diversity.14 This consensus design complex by in vitro translation allows the sampling is based on the sequence and structure analysis of of very large libraries, and the ease of introducing natural ankyrin repeat proteins and allows the mutations by error-prone PCR allows efficient affi- delineation of framework residues, which are nity maturation. However, the linkage of the geno- important for stability of the scaffold, and the defini- type and the phenotype is reliably maintained only tion of potential target interaction residues (inspired under non-denaturing buffer conditions, in the by the study of natural ankyrin complexes) suitable absence of RNases and at low temperature. In con- to introduce the needed level of diversity. Indeed, trast, in filamentous phage display,4,6 this linkage is most members of such DARPin libraries have very very robust, allowing a broader range of selection favorable biophysical properties.13 They are very conditions. Filamentous phage display achieves this well expressed, monomeric in solution, highly robustness by physically linking the displayed soluble, thermodynamically stable and show fast protein to the surface of a very stable phage particle,7 cooperative folding behavior.15 which encapsulates the corresponding genotype. From such DARPin libraries, specific binders with Nevertheless, the limiting factor in this selection high affinities have been selected for a broad range of technology is the involvement of an in vivo step to target proteins by using ribosome display.16–20 produce the protein displaying phage particles. The Nevertheless, and much to our initial surprise, the bacterial transformation needed therefore limits the display of DARPins on filamentous phage was high- library size that can reasonably be achieved to ly inefficient using conventional systems, despite the usually much less than 1011 members. Furthermore, good biophysical properties of the proteins. We the in vivo assembly process of the phage particle is identified this problem as one of inefficient transloca- not necessarily compatible with any given protein to tion of the DARPins into the periplasm due to their be displayed, since its successful translocation to the premature folding in the cytoplasm when using the periplasm and its folding are also prerequisites. In an post-translational Sec pathway, and one that can be ideal selection experiment, high-affinity binders solved by using the signal recognition particle (SRP) should be obtained directly from the library, without translocation pathway for phage display.21 further time-consuming affinity maturations. In SRP phage display,21 the proteins to be dis- The success of a phage selection experiment played are directed to the Escherichia coli cotransla- depends strongly, as a first parameter, on the size of tional SRP translocation pathway by using an the transformed library. In general, the larger the appropriate signal sequence, whereas conventional library, the higher the probability of isolating diverse phage display uses the post-translational Sec trans- binders against a given target protein, and the higher location pathway. This simple change in the use of the affinities of the selected binders.8 For example, the translocation route by changing the signal se- libraries with more than 5 × 109 members were em- quence seems to prevent premature folding of stable ployed to isolate antibody fragments with sub-na- and fast-folding proteins in the cytoplasm and there- – nomolar affinities.9 11 Nevertheless, even more fore allows their efficient translocation and subse- important than the size of the transformed library quent display on filamentous phage particles. These (number of independent clones), is the functional results indicated that the use of SRP phage display library size; i.e. the number of different and correct may help to obtain phage DARPin libraries with high (i.e., full length and folded) molecules available for functional diversity, leading to efficient selections. selection, as often only a fraction of library members Here, we describe the generation of a large phage can be functionally displayed. Three main factors DARPin library by using SRP phage display, selec- influence this functional diversity: the above-men- tions on a panel of target proteins using this library tioned compatibility of the selection system with the and analysis of the selected DARPins. In addition, we protein to be displayed; the actual library design; and
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