Review Building better drugs: developing and regulating engineered therapeutic proteins 1* 1* 1* Chava Kimchi-Sarfaty , Tal Schiller , Nobuko Hamasaki-Katagiri , 2* 3,y 1* Mansoor A. Khan , Chen Yanover , and Zuben E. Sauna 1 Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA 2 Division of Product Quality Research, Center for Drugs Evaluation and Research, Food and Drug Administration, White Oak, Silver Spring, MD 20993, USA 3 Program in Computational Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA Most native proteins do not make optimal drugs and small molecule drugs (see [2] for a classification of thera- thus a second- and third-generation of therapeutic pro- peutic proteins). Therapeutic proteins represented 17% of teins, which have been engineered to improve product new drugs approved by the USA Food and Drug Adminis- attributes or to enhance process characteristics, are tration (FDA) in 2005 but increased to 32% by 2011 [3]. The rapidly becoming the norm. There has been unprece- influence of these medications on the practice of modern dented progress, during the past decade, in the devel- medicine is, however, deeper than these numbers alone, opment of platform technologies that further these because protein drugs often address unmet medical needs, ends. Although the advantages of engineered therapeu- provide cures or permit the management of complex tic proteins are considerable, the alterations can affect diseases, and improve quality of life. For example, the the safety and efficacy of the drugs. We discuss both the introduction of Factors VIII and IX as replacement therapy key technological innovations with respect to engi- for hemophilia has significantly extended the life expectan- neered therapeutic proteins and advancements in the cy of patients and, equally importantly, created a profound underlying basic science. The latter would permit the improvement in quality of life. In the absence of treatment, design of science-based criteria for the prediction and recurring bouts of bleeding and inflammation occur in the assessment of potential risks and the development of joints resulting in a loss of joint cartilage and bone destruc- appropriate risk management plans. This in turn holds tion eventually leading to deformity and crippling arthritis promise for more predictable criteria for the licensure of [4]. Intracranial hemorrhage is a less common complication a class of products that are extremely challenging to of untreated hemophilia but has even more severe conse- develop but represent an increasingly important com- quences with death rates exceeding 20% and even survivors ponent of modern medical practice. experience long-term consequences such as seizures and neurological impairment [5]. Historically, these products Therapeutic proteins were purified from animal and human sources but have Although a reliable count of functionally distinct proteins almost completely been superseded by proteins manufac- in humans is lacking, estimates suggest that the number tured by recombinant DNA technology. runs to at least several tens of thousands [1]. Abnormali- The recombinant proteins pipeline now includes a new ties in one or more of these proteins can manifest as disease generation of therapeutics that involves engineering the conditions. However, these molecules can also be developed protein (i.e., altering the genetic sequence) to achieve as therapeutics for replacement therapy, augmenting an desirable therapeutic outcomes or manufacturing efficien- existing pathway, providing a novel function or targeting cies [6]. Technological innovations and scientific progress have also presented manufacturers with the opportunity to Corresponding authors: Kimchi-Sarfaty, C. ([email protected]); develop so-called ‘bio-betters’ which are also the result of Sauna, Z.E. ([email protected]). Keywords: drug development; immunogenicity; drug safety; risk mitigation; quality altered sequences. by design. Proteins are engineered for numerous reasons but in- * Our contributions are an informal communication and represent our own best volve three principal strategies: (i) genetically or chemi- judgment. These comments do not bind or obligate the FDA. y Current address: Machine Learning Group, IBM Research Laboratory, Haifa, cally linking the therapeutic protein to another protein or Israel. polymer; (ii) the introduction of one or several mutations or 0165-6147/$ – see front matter. deletions in the primary sequence of the therapeutic pro- Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tips.2013.08.005 tein; and (iii) modifying the genetic code of the recombinant protein for codon optimization. The first two of these involve a change in the primary structure of the protein whereas the third does not. 534 Trends in Pharmacological Sciences, October 2013, Vol. 34, No. 10 Review Trends in Pharmacological Sciences October 2013, Vol. 34, No. 10 There has been considerable progress in the engineering serum half-life. Most platform technologies that do this of therapeutic proteins during the past decade that has the take advantage of mechanisms used by native proteins potential for noteworthy improvements in product quality, such as antibodies, serum albumin and transferrin, which therapeutic outcomes, and patient convenience. Conse- persist in the serum for up to 25 days [10,11]. The Fc region quently, engineered therapeutic proteins are rapidly be- of antibodies as well as human serum albumin are pro- coming the norm rather than the exception as are more tected from degradation by pH-dependent recycling medi- extensive alterations to the wild type sequence [7]. These ated by interaction with the neonatal Fc receptor, FcRn changes present difficult regulatory decisions as to how [12]. More recently, unstructured recombinant polypep- much deviation from a wild type or parent molecule may be tides called XTEN have been successfully used to generate permitted in terms of protein engineering and the manip- fusion proteins with improved pharmacokinetic properties ulation of the DNA sequence. Moreover, these decisions [13]. have to be made even as the technology and the underlying The proteins or polypeptides may be fused to the biolog- basic science are both evolving rapidly. In the past year ically active protein either by genetic engineering or by alone the development of two bioengineered recombinant chemical crosslinking. The latter often involves the fusion factor VIIa (rFVIIa) analogs, Vatreptacog alpha (http:// of polymers other than proteins/polypeptides such as the www.novonordisk.com/include/asp/exe_news_attachment. covalent attachment of the polyethylene glycol (PEG) moi- asp?sAttachmentGUID=b7ff4a52-53ab-4a97-875c-156 ety (PEGylation) [14,15]. Moreover, platforms that rely on dedaf427a) and BAY86-6150 (http://www.investor.bayer. chemical crosslinking may also include genetic engineering de/no_cache/en/news/investor-news/investor-news/show- of the therapeutic proteins to improve the efficiency of NewsItem/1567) was discontinued in Phase III clinical crosslinking or reduce the heterogeneity of the product. trials due to safety issues. Similarly, peginesatide, a novel For instance, earlier attempts at chemical attachment of functional analog of erythropoietin, had to be recalled PEG moieties to proteins resulted in complex product (http://www.fda.gov/Safety/Recalls/ucm340893.htm) less mixtures which were difficult to characterize and monitor than a year after its March 2012 approval due to hyper- [16]. Subsequent advances addressed this problem by con- sensitivity reactions, including fatal anaphylaxis in some trolled PEGylation only at specific sites following site- patients. These examples illustrate the risks associated directed mutagenesis [17]. Although this strategy reduces with this class of drug products. In this review, our aim is to product heterogeneity, additional genetic modifications of provide an overview of the emerging technologies that the protein are required which can potentially trigger other involve protein engineering and discuss these in the con- unintended safety concerns (see below). text of the underlying discoveries that are essential for Table 1 lists fusion proteins (excluding monoclonal anti- science-based regulatory decisions. The review addresses bodies, antibody–drug conjugates, and fusion proteins with these issues for all therapeutic proteins other than thera- peptides and aptamers) in different stages of drug devel- peutic antibodies, which constitute a separate class with opment. Table 1 has been classified on the basis of platform distinctive properties. technologies used for bioengineering and then on the basis of the therapeutic area. As illustrated, engineered proteins Engineered proteins: therapeutics by design have potential in many therapeutic areas. Also evident is Experience with first-generation recombinant therapeutic the robust development of diverse technologies to modify proteins suggests that most proteins do not make optimal properties of proteins to overcome difficulties in their drug products. Most therapeutic proteins are rapidly clinical use or address different clinical needs. cleared from circulation and have a very short serum As seen in Table 1, multiple technologies are in use to half-life and poor bioavailability [8]. They can elicit forma- circumvent the same clinical predicament. Thus site-di- tion of antibodies with an inhibitory