Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display

Abstract

There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement 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 10 10 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.

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

“…For the expression of DARPins, selected DARPin genes were cloned into pDST67 (20). To generate biotinylated DARPins E40 and pE59, coding sequences were cloned into pBD001 (45).…”

“…From the enriched DARPin DNA pools, N2C and N3C DARPin ORFs were amplified by PCR as described in ref. 12 and cloned into pDST67 (20). DARPin pools were screened by a crude extract ELISA according to previous studies (12,47).…”

“…Members of these libraries show very favorable biophysical properties, are expressed in soluble form with high yields in the cytoplasm of Escherichia coli, and do not contain disulfide bonds, allowing for intracellular applications (12,(15)(16)(17)(18). Furthermore, using complex DARPin libraries, specific binders with high affinity and selectivity for their target antigen could be isolated (12,19,20).…”

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

“…We previously generated high-affinity DARPins targeting various tumor-associated antigens, including members of the EGF receptor family (11,12) and the epithelial cell adhesion molecule (EpCAM; ref. 13).…”

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…Such target receptor-specific DARPins with affinities similar or better than those of antibodies can be obtained from libraries by selection approaches against essentially any molecule 12 and fold correctly even in the context of being fused to a virus capsid protein. For our proof-of-concept, we had employed a DARPin specific for Her2/neu 13 . Combining insertion of the Her2/neu-specific DARPin-9.29 as fusion to AAV's capsid protein VP2 (viral protein 2) with ablation of natural receptor binding through mutagenesis of two arginine residues in each of the 60 capsid monomers resulted in an AAV vector preparation that was by far more specific for its target cells than any AAV vector developed so far 11 .…”