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Part I
Introduction
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1
Fusion Proteins: Applications and Challenges
Stefan R. Schmidt
Rentschler Biotechnologie GmbH, Laupheim, Germany
1.1 History
Proteins as drugs have a long history. In the beginning, natural proteins were extracted from animal, human sources, or in some rare cases even from plants. Large-scale processing of human plasma became a primary source for the isolation of many proteins [1]. For instance, blood factors became available as a therapy against the different forms of hemophilia or the lack of functional (α-1 antitrypsin. The major serum component, albumin, has now been used for more than 50 years as a treatment for shock, trauma, or burns. Immune globulins isolated from human sources are also used successfully in various immunodeficiency diseases. However, despite the great success of plasma products, contaminations with the HIV or hepatitis virus in the 1970–1980s triggered more intensified efforts to prepare virus-free recombinant therapeutic proteins.
Since its identification in the 1920s until the 1980s, insulin from animals was the only treatment for diabetes patients. Particularly, the porcine insulin was widely used since there is only a single amino acid variation from the human form. Chemical processes were developed to obtain the fully human variant from the pig isoform [2]. Finally, the first recombinant human insulin was manufactured by Eli Lilly & Co in partnership with Genentech, approved in 1982 by the FDA and marketed under the name Humulin® [3]. This was also the first therapeutic recombinant protein for human use.
Since that time, the number of recombinant products and approved biopharmaceuticals has increased considerably. Initially, recombinant copies of proteins were made that replaced the natural protein, which until then was harvested from animal or human sources. With the exception of factor VIII against hemophilia, all these proteins such as human growth hormone (hGH) or follicle stimulating hormone (FSH) belonged to the class of hormones. They were soon accompanied by a growing number of first generation therapeutics that could only be obtained recombinantly such as erythropoietin (EPO), interferon (IFN) or tissue plasminogen activator (tPA), just to name a few. After this first enthusiasm and the success with reproducing natural proteins by recombinant DNA technology, researchers started to consider the de novo design of therapeutic proteins that do not occur in nature. There is one specific class that can be seen as intermediate link between natural and designed proteins, monoclonal antibodies (mABs).
Antibodies, being a major part of the organism's immune defense, are large proteins that exist in all higher animals. In 1975, a method was developed to generate murine cell lines producing antibody molecules of a single specificity, the so-called monoclonal antibodies (mABs) [4]. The first therapeutic monoclonal antibody, orthoclone OKT3, was of murine origin and approved in 1986. From then, this concept was further refined with the help of modern recombinant DNA technology to obtain the first fully human antibody against tumor necrosis factor-α (TNF-α), marketed under the name Humira® in 2002 [5]. The milestones for recombinant therapeutic proteins can be seen in Figure 1.1.
Interestingly, preventing the activity of TNF-α was also the goal for the first fusion protein, Etanercept. It consists of the TNF-α receptor attached to a sequence encoding the Fc portion and hinge region of an IgG1 heavy chain. This drug has been marketed under the name Enbrel® since 1998 and is the best selling fusion protein till date [6]. The two extraordinarily successful drugs Humira and Enbrel can serve as prototype for their molecule classes, exemplifying the different ways to address the same target and present a typical case of competition between antibodies and fusion proteins in the market.
1.2 Definitions and Categories
This book focuses on fusion proteins that are generated by joining two or more genes by genetic engineering that originally code for separate proteins. The result is a single polypeptide with functional properties of both parent proteins. These recombinant proteins are combinations of unrelated domains not occurring in nature. Excluded from the content of this book are multiepitope recombinant vaccines [7], chemical conjugates [8] naturally occurring fusion proteins resulting from chromosomal rearrangements that can be observed in many cancer cells [9] or fusion tags for affinity purification [10].
Bi or multispecific antibodies are special case that do not always represent a single polypeptide chain but usually consist of the combination of heavy and light chains. The Part IIIb of this book discusses some non-natural versions that have more than a single specificity.
The most straightforward classification of these novel proteins can be based on the functions of their incorporated domains. Typically, one part serves molecular recognition or binding, whereas the other part adds certain functionalities such as extending half-life or stability, cytotoxicity, or novel targeting or delivery routes [11].
Recently, a review classified therapeutic proteins according to their pharmacologic activity to (a) replace a deficient or abnormal protein, (b) augment an existing pathway, (c) provide a novel function or activity, (d) interfere with a molecule or organism, or (e) deliver a payload such as a radionuclide, cytotoxic drug, or protein effector [12].
However, this classification is not fully suitable for the scope of this book. Most fusion proteins serve three major purposes that can be summarized under the triple T (T3) paradigm: (a) t1/2 (half-life), (b) targeting (or binding), or (c) toxicity (cell killing). Of these three elements, at least two are simultaneously present in fusion proteins (Figure 1.2). Antibodies as natural molecules combine all three aspects in a single molecule. However, antibody derivatives, fragments, or domains have also been used extensively as building blocks for fusion proteins, hence constituting a large part of the portfolio of proteins discussed here, and thus deserving their own category. The main functionality of antibodies, the binding with high affinity and selectivity to a specific epitope, has been reproduced in a number of nonantibody scaffolds that can either be used as single module or by combining two units with different specificity [13]. These molecules together with other bi- or multifunctional therapeutics that do not fit to the T3 categories are classified into the group of novel artificial molecules that is discussed in Part IIIa of this book.
A very practical classification is proposed by the authors of Chapter 3 about “Structural Aspects of Fusion Proteins Determining the Level of Commercial Success” in this book. They suggest sorting fusion proteins based on one of three different functional groups: activity, targeting, or half-life, of which the latter two can be summarized under delivery agents, thus being able to define a two-dimensional landscape of fusion proteins. This is quite similar to a previous scheme using the combination of an effector fragment together with a molecular recognition part as building blocks for fusion proteins [14].
But why should we deal at all with fusion proteins? Several advantages make them very attractive: the combination of two functionalities in a single molecular entity simplifies manufacture and drug delivery. Two molecules combined into one will automatically have identical biodistribution profiles instead of two separate molecules that might have a very different distribution. Furthermore, new functionalities can be created that are lacking in natural or separate proteins. This includes the modification of half-life or targeting specificity. Even economic opportunities such as life cycle extension of products with expired patents are possible. This includes also the generation of novel intellectual property for new and non-natural combinations of proteins. Therapeutic benefits derived from reduced side effects or longer dosing intervals and improved activity are strong drivers to promote the generation of fusion proteins.
But besides all these important advantages, there are also a number of challenges. The combination of unrelated proteins might prove difficult to manufacture because in some cases, the fusion partners have noncompatible properties. This can cause aggregation or misfolding of one domain while the conditions might be perfect for the other domain. Despite the fact that some modules of fusion proteins are elements of other well-proven molecules such as antibodies, the established platform processes might not be applicable because other features shield the required property. This can go so far that formulation is not possible due to conflicting stability requirements. Furthermore, it will be difficult to control and tune the relative amounts of each component thus complicating dosing for optimal efficacy and safety. Probably most important challenge is the high potential for immunogenicity due to the formation of novel epitopes at the junction between the fusion partners even if only fully human proteins are connected.
1.3 Patenting
The first generation biologics that represented a true copy of human proteins used for therapeutic applications have already lost or are about to lose their patent protection [15]. In many cases second generation molecules, for example, with improved half-life, are taking their place. A number of them are fusion proteins that are patented as well.
To be able to file a patent for an invention, three characteristics must be achieved novelty, nonobviousness, and utility or enablement [16]. In the postgenome era, the discovery of novel proteins, at least of human origin, will be difficult. This challenges the first critical parameter on the way to a patent, the novelty. If we focus on the scope of this book, the fusion proteins, novelty still seems to be easy to reach. As described in the paragraph about definitions, “joining two or more genes by genetic engineering that originally code for separate proteins,” so the generated fusion protein will be novel if nobody did the same earlier. Therefore, novelty is given on one hand through the composition of matter (the new construct, e.g., long-acting human growth hormone [hGH], consisting of hGH fused to human serum albumin [HSA]) or on the other hand by the use that results from combined features of the new molecule (e.g., to treat dwarfism with longer administration intervals). This is in contrast to a natural polypeptide with multiple functions [17].
Taking the example of antibodies as molecules, their individual characteristics, for example, target or captured epitope, affinity, half-life, or sequence of the variable part should be sufficient to enable patenting based on novelty [18...
