Engineered protein scaffolds as next-generation antibody therapeutics
Introduction
Starting with the early work of Paul Ehrlich [1] the era of chemotherapy was tremendously successful for medicine in the 20th century, providing small molecule drugs for the treatment of many infectious diseases, metabolic disorders, cardiac diseases, neuromedicine, and cancer. However, the amount of approved new chemical entities (NCEs) per year has decreased lately, and a growing number of protein drugs, the so-called ‘biologics’, is entering the clinics, among those an increasing fraction of antibodies, especially during the past 10 years [2]. Today, more than 20 different antibodies have been approved in Europe and the USA, providing a considerable market potential for the pharmaceutical and biotech industry [3, 4].
There are several reasons for the remarkable success of antibodies (immunoglobulins, Igs) as a class of biological drugs. First, they can rather quickly be generated against a wide range of target molecules (antigens or haptens), either by classical immunization of animals – followed by protocols for monoclonal antibody preparation – or, more recently, via in vitro selection from cloned or synthetic gene libraries [5]. Second, they usually possess extraordinary specificities for their targets, with affinities often in the low nanomolar to picomolar range, thus surpassing most chemical drugs. These beneficial properties were already noticed by Emil von Behring when he investigated the humoral immune response more than hundred years ago and, in fact, also by his colleague Ehrlich, who postulated the ‘side-chain’ theory in order to explain the formation of antigen-specific antibodies (originally termed ‘antitoxins’ by von Behring) [1].
Since then it took considerable time until gene technology permitted the heterologous production of recombinant antibodies as well as the ‘humanization’ of antibodies from rodents that, in combination with the methods for selection from cloned Ig libraries and also with the availability of transgenic animals carrying a human Ig locus, provides a mature technology today [5, 6]. For antibodies, clinical safety and efficacy has been well established, including aspects of epitope specificity, immunogenicity (human anti-human antibodies, HAHA), pharmacokinetics, and immune-related effector functions, leading to wide acceptance by physicians and patients.
However, with the increasing application of antibodies several disadvantages have become apparent. For example, they have a large size and complicated composition, comprising four polypeptide chains, glycosylation of the heavy chains, and at least one structurally crucial disulphide bond in each of several Ig domains. Thus, full size antibodies require manufacturing in eukaryotic expression systems, usually involving stably transfected mammalian cell lines, whose optimization and fermentation is laborious and costly [7]. Consequently, exploration of alternative protein reagents with the ability to specifically recognize and tightly bind ‘antigens’ has been stimulated, leading to a range of different antibody fragments – most prominently, Fab and single chain Fv, which may simply be prepared by shortening the reading frame of cloned Ig genes – and, ultimately, even to isolated Ig domains [8].
In parallel to the increasingly advanced manipulation of Ig fragments an independent development has focused on recruiting unrelated proteins for analogous applications. In fact, it was demonstrated that several protein families with non-Ig architecture can be equipped with novel binding sites by employing methods of combinatorial engineering, such as site-directed random mutagenesis in combination with phage display or other molecular selection techniques [9, 10]. As result, novel biomolecular binding reagents have become available, thus triggering a paradigm shift in so far as antibodies are no longer considered as the unique and universal class of receptor proteins in biotechnology and medicine [10, 11].
These novel alternative binding reagents are collectively called engineered protein scaffolds [12], illustrating the fact that a rigid natural protein structure is used to modify an existing – or to implement a new – binding site for a prescribed target. Usually, such a scaffold is derived from a robust and small soluble monomeric protein (such as the Kunitz inhibitors or the lipocalins) or from a stably folded extramembrane domain of a cell surface receptor (e.g. protein A, fibronectin or the ankyrin repeat). Compared with antibodies or their recombinant fragments, these protein scaffolds often provide practical advantages including elevated stability and high production yield in microbial expression systems, together with an independent intellectual property situation.
As these novel binding proteins are obtained by means of a biomolecular engineering process in order to achieve tight target-binding activity, they may also be subjected to further selection schemes focused at other desired properties (such as solubility, thermal stability, protease resistance etc.). Consequently, engineered protein scaffolds have become attractive for many applications in biotechnology and biomedical research. However, since the effort to generate such an alternative binding protein with beneficial properties still is higher than the preparation of a conventional antibody (or a recombinant Ig fragment), most of the ongoing activities in this area are directed toward therapeutic use, offering the chance of high return on investment. Here, we review the current state of the art in this field, with special emphasis on biomolecular structure and function as well as on approaches toward clinical application.
Section snippets
Old and new protein scaffolds
More than 50 different protein scaffolds have been proposed over the past 10–15 years and these numerous examples have been summarized in previous reviews (see e.g. [12, 13, 14, 15]). The most advanced approaches in this field comprise the following protein classes:
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Affibodies based on the Z-domain of staphylococcal protein A, a three-helix bundle of 58 residues providing an interface on two of its α-helices (recently reviewed in [16]);
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engineered Kunitz domains based on a small (ca. 58 residues)
Interfaces for molecular recognition
The structural mechanism by which antibodies recognize their antigens is well understood. A set of six hypervariable loops (also known as complementarity-determining regions, CDRs), three in each variable domain of both the light and heavy Ig chain, come together at the tip of the Y-shaped molecule (the so-called Fab arm) und form there an extended contiguous combining site [10]. The extraordinary structural variability of this interface arises from the high sequence diversity of the CDRs in
Targets and medical mode of action
In analogy to antibodies, targets of engineered binding proteins can be generally classified into (i) antigens, that is usually proteins, and (ii) haptens, that is (bio)chemical compounds of low molecular weight including small peptides or peptidomimetics. Up to now, most of the engineered protein scaffolds were directed against protein targets. One reason is their relevance as disease-related biomolecules and the second is that most of the successful non-Ig scaffolds provide extended
Clinical aspects: delivery, half-life, and immunogenicity
The general experience with alternative scaffolds and also with small antibody fragments from the past few years has shown that one of the most crucial aspects for successful application in vivo is the affinity for the target. Owing to their monovalent nature, non-Ig binding proteins – as well as the single Ig domains (see e.g. [76]) – have a certain disadvantage compared with antibodies, whose antigen-binding activity is often boosted by an avidity effect. Attempts to mimic this
Conclusions and outlook
The engineering and practical use of binding proteins derived from non-Ig scaffolds is an established methodology today that is going to boost biological chemistry both toward basic research and applied science. In contrast, antibody technology is about to reach its peak in the biomedical area, with several hundred drug candidates directed against a broad range of targets currently awaiting clinical study and, finally, market approval. Consequently, under the pressure of ongoing innovation
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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