Preprint Review Version 2 Preserved in Portico This version is not peer-reviewed

The DARPin Encyclopedia: from Basic Research Towards Therapeutics

Version 1 : Received: 2 June 2022 / Approved: 10 June 2022 / Online: 10 June 2022 (04:20:57 CEST)
Version 2 : Received: 14 June 2022 / Approved: 15 June 2022 / Online: 15 June 2022 (02:54:19 CEST)
Version 3 : Received: 21 June 2022 / Approved: 21 June 2022 / Online: 21 June 2022 (10:21:27 CEST)

How to cite: Wild, P.; Eapen, R.; Forrer, P.; Jost, C. The DARPin Encyclopedia: from Basic Research Towards Therapeutics. Preprints 2022, 2022060147. Wild, P.; Eapen, R.; Forrer, P.; Jost, C. The DARPin Encyclopedia: from Basic Research Towards Therapeutics. Preprints 2022, 2022060147.


The adaptive immune system in vertebrates comes in two flavors. Historically, adaptive immunity focused on the study of immunoglobulins (also referred to as Ig-based antibodies herein); more recently, jawless vertebrates were found to use an entirely different class of proteins for their adaptive immunesystem. Specifically, these organisms express variable lymphocyte receptors (so called VLR antibodies) that create diversity based on the rearrangement of leucine-rich repeats. Meanwhile, various, naturally occurring repeat motifs have been used as building blocks for the design of artificial binding scaffolds which, among others, include designed ankyrin repeat proteins (DARPins). Such binding scaffolds are also referred to as non-Ig-based antibodies or non-Ig scaffolds herein). DARPins display several benefits such as a low molecular weight (~15 kDa), high thermal stability, high specificity and affinity, versatility (e.g., in terms of valency and multi-specificity), speedy preclinical development, low production costs and, thus, bear the potential to not only complement existing therapeutic Ig-based antibodies but also open up novel therapeutic strategies. The first generation DARPin therapeutic abicipar pegol has completed two Phase III studies in 2020 while several other DARPin drug candidates are currently undergoing clinical validation. Most recently, the rapid development of tri-specific SARSCoV-2 DARPin therapeutics showcases the immense potential of recombinant repeat proteins in adopting quickly e.g., new forms of target neutralization that a single Ig-based antibody cannot afford. Here, we highlight the design principles of repeat proteins in general and summarize in detail the continuous advancements of the DARPin scaffold that made it one of the most promising antibody mimetics to date. This review provides an overview of current and emerging applications of DARPins as both a research tool and therapeutic drug that can match or even surpass Ig-based antibody applications.


Designed Ankyrin Repeat protein; DARPin; drug development; ensovibep; abicipar pegol


Biology and Life Sciences, Biochemistry and Molecular Biology

Comments (3)

Comment 1
Received: 15 June 2022
Commenter: Christian Jost
Commenter's Conflict of Interests: Author
Comment: formatting issures resolved(first version contained Figures at low resolution and formatting issues on page 1.Please compare compiled version to co-submitted PDF prior to publication)
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Comment 2
Received: 21 June 2022
Commenter: Mattia Deluigi
The commenter has declared there is no conflict of interests.
Comment: Congratulation on this beautiful DARPin encyclopedia! Very interesting!

Here, I’d like to point out a recent, new application of DARPins that is not mentioned in the current preprint version or the referenced reviews. This new application may thus be a valuable addition to the DARPin encyclopedia that the authors of this preprint can consider.

We have recently established a new crystallization design based on the fusion of a DARPin to G protein-coupled receptors (GPCRs) as a tool to facilitate GPCR crystallization and structure determination:

In the first study, the fusion of a DARPin to the neurotensin receptor 1 enabled the crystallization and structure determination of this receptor in the ligand-free state and in complex with several ligands. These structures shed light on mechanistic aspects of ligand-induced receptor activation and inactivation. Such findings may foster drug discovery to treat cancer, metabolic, and neurological diseases.

In the second study, a similar fusion of a DARPin to the α1B-adrenergic receptor enabled the crystallization and structure determination of this receptor. This structure shed light on molecular determinants of ligand selectivity, which may assist the development of drugs to treat cardiovascular, neurological, and inflammatory diseases.

The new DARPin fusion design was vital to structure determination in both studies, as previously described strategies for GPCR crystallization had proven unsuccessful.

All the best,
Mattia Deluigi
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Response 1 to Comment 2
Received: 28 June 2022
The commenter has declared there is no conflict of interests.
Comment: Dear Dr. Deluigi,

Thank you for your comment and appreciation of The DARPin Encyclopedia.
We are of course aware of your most recent and elegant work based on DARPin-GPCR fusions, establishing a new crystallization tool for this challenging class of transmembrane targets.
However, since the focus of the actual and initial version of the excyclopedia lays on the current stage of DARPins as an upcoming new class of therapeutics, we did so far not include an own section on the use of DARPins as a tool to facilitate structure determination.
Instead, we decided to refer the readership interested in that topic to the excellent previous review by Mittl, Ernst and Plückthun (2020) (cf. reference #102 in the excyclopedia).
Since the issue of DARPins as crystallization tools is definitively another important area of huge DARPin-potential, we envision this topic to become a valuable addition to one of the next versions of The DARPin Encyclopedia to come!
We will then of course be happy to provide our readership with an overview of the exiting research that is done in that area, including e.g. the work of Batyuk et al. (2016), Wu et al. (2017) and Deluigi et al. (2022).

Kind regards,
Christian Jost

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