The clinical potential of Affibody-based inhibitors of C5 for therapeutic complement disruption

The awareness of the complement system as an important target area for therapeutic intervention has increased over the last decade, primarily due to the successful introduction of the C5 inhibiting mAb eculizumab (Soliris®, Alexion Pharmaceuticals Inc.) in the treatment of the ultra-rare conditions paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. C5 is a highly tractable target molecule as it is common to all complement pathways and its inhibition thus blocks terminal complement effects, irrespective of the cause of activation. In addition, C5 targeting leaves the proximal complement functions intact, thereby preserving important housekeeping functions such as the opsonization and subsequent clearance of microbial surfaces and debris. This editorial describes how the Affibody® platform, a novel protein targeting scaffold, could be utilized to develop C5 inhibitors for versatile and efficient therapeutic targeting of the terminal complement pathway.

Affibody® molecules (developed by Affibody AB, Stockholm, Sweden) are a class of protein targeting molecules that can be considered as an alternative scaffold to antibodies. The prototype Affibody protein is the engineered, so called, Z domain that was originally derived from the B domain in the immunoglobulin-binding region of staphylococcal protein A [1,2]. This small protein domain is composed of 58 amino acids (approximately 6.5 kDa) that are folded into a three-helical bundle structure with notable biophysical properties, including high melting temperature, reversible, and rapid folding, a binding surface as large as that of an antibody and high solubility in aqueous solutions [1]. Z-based ligands with specific affinity for a protein-of-interest are typically selected using phage display in combinatorial libraries containing different Affibody molecules that have been generated by randomizing 13 surface-exposed amino acids located in helices 1 and 2 of the molecule, i.e. the helices that comprise the original IgG Fc-binding surface of the Z-domain. Furthermore, Affibody molecules are readily and cost efficiently produced in E. coli and can also be chemically synthesized. The above mentioned properties make the Affibody platform attractive for several potential applications and this class of molecules has already been commercialized for certain laboratory and biotechnological usages (e.g. as an affinity ligand for antibody purification), and are under development for imaging/diagnostic use and as therapeutic proteins [2].

Affibody molecules have the ability to block a range of therapeutically relevant target proteins without eliciting any inherent effector function. This contrasts to the IgG Fc-mediated effector mechanisms of antibodies, such as cell- or complement-mediated cytotoxicity. Since Affibody molecules are small in molecular size, below the glomerular filtration threshold, they are rapidly cleared from the plasma [3,4]. However, by using different protein engineering approaches to increase the hydrodynamic radius and/or engaging the neonatal Fc receptor (FcRn)-mediated recycling pathway, prolongation of plasma persistence can be achieved [2,5]. Furthermore, if desired, additional effector functionality could also be engineered by applying various payload strategies [2,6].

Selection of Affibody molecules targeting human complement, component 5 (C5) has been performed using plasma derived human full-length C5 as target protein in phage display selections from libraries consisting of billions of Affibody clones [1,7]. In vitro screening using ELISA and functional complement hemolysis assays were used to identify candidates with appropriate characteristics, including cross-species reactivity in both rodents and primates. After subsequent directed combinatorial mutagenesis-based affinity maturation leads with affinity for human C5 in the sub- to low-nM range were identified. The final selected C5 inhibiting domain has subsequently been used to design candidate therapeutic proteins with tailored properties to meet the needs of specific complement-mediated disorders. In particular, four molecular designs have been thoroughly characterized. Two E. coli expressed recombinant fusion proteins with either an engineered albumin binding domain (ABD) [8–10] or IgG Fc have been developed as long-acting C5 inhibitors to support continuous systemic inhibition of terminal complement activation. In addition, chemically synthesized Affibody domains, alone or conjugated to polyethylene glycol (PEG), are being investigated for acute and/or local administration to specific organs such as the eye. The first candidate drug, denoted SOBI002, comprising an Affibody domain fused to ABD [7] was recently tested in a clinical trial in healthy volunteers (study NCT02083666) [7]. However, the study was discontinued due to transient adverse events in a small number of the enrolled subjects, the cause of which is currently being investigated further.

The complement system is an instrumental part of the innate immune system that provides an immediate line of defense against microorganisms, independently of previous exposures of the infectious agent [11]. This system is activated by exogenous surfaces and danger- and pathogen-associated molecular pattern molecules. Complement activation is typically divided into three separate routes, i.e. the classical, lectin, and alternative pathways, each recognizing specific signals. The three pathways converge at the level of C3 cleavage to C3a and C3b with subsequent cleavage of C5 into C5a and C5b. C5a is a potent anaphylatoxin acting via a distinct C5a receptor (C5aR1, CD88) to recruit leukocytes to areas of complement activation but also acts as a bridge to the adaptive immune system as the receptor is expressed on antigen presenting and other immune cells [12]. C5b, in turn, polymerizes with C6-9 to form a lytic pore named C5b-9 or membrane attack complex (MAC). Dysregulated complement activation is involved in the pathology of a broad range of diseases including various autoimmune- and immune complex driven diseases. Specific complement defects, both inherited and acquired, have been associated with diseases of the kidney and eye as well as certain hematologic thrombotic microangiopathies. Furthermore, complement is involved in several acute and possibly detrimental inflammatory responses to localized ischemic injury as well as systemic inflammatory response syndrome (SIRS).

C5 blockade has proven to be generally safe, as shown by many years of anti-C5 treatment and the fact that complete C5 deficient individuals are reported without significant clinical manifestations [13], the exception being increased susceptibility for infections from encapsulated bacteria, predominantly Neisseria meningitidis, conditions managed by vaccination and/or antibiotics treatment. At present, the two ultra-rare diseases PNH and aHUS are treated with the C5 blocking monoclonal antibody Eculizumab (Soliris®). For these patients, Soliris has transformed the treatment and quality of life but due to the high cost of treatment there is still a significant unmet medical need in these patient populations as access to treatment is limited [14]. Other examples of C5 targeting in various stages of clinical development are Coversin, derived from the tic protein OmCI (Akari Therapeutics); RA101495, which is an inhibitor based a small cyclic peptide scaffold (RaPharma); and ALN-CC5, which features RNAi-mediated gene silencing of C5 production in the liver (Alnylam); see Risitano for an extensive list of complement programs [15]. The number of indications with clinical validation for C5 inhibition is rapidly growing and thus also the space for new drug entities with properties tailored for the specific indication with respect to, e.g. pharmacokinetics, administration, and distribution. For example in autoimmunity, neuromyelitis optica (NMO), Myasthenia gravis (MG), and ANCA associated vasculitis could benefit from either shorter periods of C5 (or alternatively C5a) blockade to induce remission or for chronic treatment [16–18]. Promising phase 2 data for these indications have been presented and registration studies for Soliris are ongoing in NMO and MG [18]. Several late-stage clinical trials with C5 blockade are also ongoing within the area of organ transplantation to prevent antibody-mediated rejection (AMR) but the long-term benefit is still unclear [19]. A long-acting Affibody molecule could possibly enable subcutaneous dosing in these indications, as higher molar doses could be administered in a single injection leading to more convenient treatment for the patients, particularly those who are dependent on life-long treatment. In ophthalmology, complement is strongly implicated in for example age-related macular degeneration (AMD), the most common causes of blindness in the western world [20]. Whereas the wet form of AMD can be treated with VEGF inhibitors, there is no efficacious treatment for dry AMD, but there is evidence for complement involvement in both forms. Complement attenuation, including C5 blockade, to treat AMD is currently being investigated with Ophthotech, Novartis, and Roche/Genentech all having clinical programs with various complement inhibiting antibody fragments or PEGylated aptamers for AMD using intravitreal administration. A small Affibody molecule with its unique properties could be interesting to test as an alternative to the investigational products mentioned above. Finally, complement is also involved in the inflammatory processes of ischemia-reperfusion injury for example in delayed graft function following transplantation, stroke, and myocardial infarction. A free Affibody domain is eliminated very rapidly but will probably have superior tissue penetration compared to, for example, antibodies. These are attributes that could be beneficial for intensive-care therapeutic areas where a short acting drug may be desired [18].

In conclusion, complement inhibition at the C5 level represents a promising approach to treat a multitude of disorders. So far this modality has only gained access to the ultra-rare disease space, with only some thousands of patients treated worldwide. Therefore, other options, such as Affibody based drugs, hold promise to fill significant medical needs where treatments currently are unavailable. In addition, since the currently available product is extremely costly, other options would also be beneficial in for example PNH and aHUS. The engineering of Affibody molecules alone or as a fusion partner to create molecules with optimized drug-like properties for specific therapeutic applications could also result in drugs with superior properties compared to, for example, conventional monoclonal antibodies.

Financial & competing interests disclosure

The authors are employed by Swedish Orphan Biovitrum AB (Sobi; Stockholm, Sweden).The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.