Derivation of RNA aptamer inhibitors of human complement C5

Abstract

Specific aptamer inhibitors of the human complement C5 component were produced by the SELEX methodology of directed evolution of nucleic acid ligands. The SELEX procedure started with a pool of random-sequence, 2′F-pyrimidine-modified nuclease-stabilized RNA, and after twelve rounds of iterative C5 binding and nucleic acid amplification an evolved RNA pool was obtained which contained the highest affinity binders to the C5 protein. The evolved RNA pool was then cloned and sequenced, and individual clones were analyzed for binding and function. Twenty-eight clones (out of sixty) were identified which bound C5 (termed aptamers). Seven of these aptamers formed a closely related sequence homology family; these aptamers bound C5 with a K_d 20–40 nM and also inhibited human serum hemolytic activity. In addition, these aptamers inhibited zymosan-induced generation of C5a. Aptamer inhibition of both C5b and C5a suggests that aptamer binding inhibits cleavage of C5 by the C5 convertase of both pathways. One of the inhibitory aptamer sequences was truncated to yield a 38-mer 2′F RNA aptamer which retained C5 binding and inhibitory activity. The structure of this aptamer is predicted to be a stem-loop containing thirteen base pairs, and also containing two bulges. The affinity of this aptamer was improved by performing a second biased SELEX experiment, where the randomized starting RNA pool uses a template where the individual base compositions are biased toward a specific sequence. This second SELEX experiment produced an aptamer with a K_d of 2–5 nM which retained functional activity. Another SELEX to rat C5 produced an aptamer with binding and inhibitory properties virtually identical with the human aptamer. The human and rat aptamers are being evaluated for complement inhibition in vitro and in vivo as potential therapeutics for treatment of human disease.

Introduction

RNA aptamers which bind with high affinity to proteins or other targets can be made by the SELEX™ technology for directed evolution of nucleic acid ligands (Tuerk and Gold, 1990). As employed here, the SELEX combinatorial chemistry technique uses a starting pool of >10¹⁴ unique RNA sequences and selects, through an iterative process of binding and amplification, for the few molecules which bind with highest affinity to the protein target. RNA aptamers are selected whose specificity is based on shape complementary to a target, as distinct from RNA specificity involving base-hybridization. The RNA aptamers are stabilized to nuclease digestion (Jellinek et al., 1995) through incorporation of 2′F-ribose modified cytosine and uridine residues during SELEX, and through post-SELEX procedure for 2′OMe modification of purines, and can be employed for in vivo inhibition in therapeutic applications.

Aptamers were produced to the C5 component of human complement. Complement C5 is a 210-kDa serum glycoprotein which during complement activation is cleaved into a 9-kDa chemotactic and vasoactive C5a fragment and a 200-kDa C5b fragment (reviewed in Morgan, 1995). The C5a polypeptide binds to a 7TM-G-protein receptor which was originally described on leukocytes and is now known to be expressed on a variety of tissue including hepatocytes (Haviland et al., 1995) and neurons (Gasque et al., 1997). C5a is the primary chemotactic component of human complement, stimulates neutrophil-endothelial adhesion (Mulligan et al., 1997), cytokine and lipid mediator release, oxidant formation, and is thus a central component of inflammation. The larger C5b fragment is transiently able to bind C6 and to initiate formation of the C5b-9 membrane attack complex (MAC). The MAC is lytic for erythrocytes, and in greater quantities is lytic for leukocytes and damaging to tissue such as muscle, epithelial and endothelial cells (Stahl et al., 1995). The MAC can also in sublytic amounts stimulate upregulation of adhesion molecules, intracellular calcium increase and cytokine release (Ward, 1996). The actions of the MAC thus overlap with those of C5a, however, the MAC functions through lipid bilayer perturbation and does not bind a specific receptor.

C5a and MAC both have been implicated in acute and chronic inflammation associated with human disease, and their role in disease has been confirmed in animal models. C5a is required for complement- and neutrophil-dependent lung vascular injury (Ward, 1997; Mulligan et al., 1998), and is associated with neutrophil and platelet activation in shock and in burn injury (Schmid et al., 1997). MAC mediates muscle injury in acute autoimmune myasthenia gravis (Biesecker and Gomez, 1989), organ rejection in transplantation (Baldwin et al., 1995; Brauer et al., 1995; Takahashi et al., 1997), and renal injury in autoimmune glomerulonephritis (Biesecker et al., 1981; Nangaku et al., 1997). Both are implicated in acute myocardial ischemia (Homeister and Lucchesi, 1994), acute (Bednar et al., 1997) and chronic CNS injury (Morgan et al., 1997), leukocyte activation during extracorporeal circulation (Spycher and Nydegger, 1995; Sun et al., 1995), and in tissue injury associated with autoimmune diseases including arthritis and lupus (Wang et al., 1996). Modalities for preventing complement and C5-dependent injury, either directly through C5 blockade or by preventing earlier complement activation, are actively pursued for treatment of inflammatory disease (Kirschfink, 1997; Spencer et al., 1997).

Aptamers have unique properties which enhance their utility as therapeutic agents. First and probably most important, aptamers can be selected to functionally important regions on a target protein, and aptamer–protein interactions are high affinity and highly specific. Second, aptamers can be chemically modified to provide additional functions or to alter properties, and without reducing aptamer solubility, stability or production protocols. Third, aptamers are not intrinsically immunogenic and can be used chronically. Fourth, aptamer synthesis is ultimately a traditional chemistry procedure, with all the advantages of batch-to-batch reproducibility, economy of scale, and process improvements inherent in chemical vs. biological production. Results presented here show that aptamers can be derived to the complement C5 component, these aptamers inhibit C5 activation in serum, and are suitable for development as therapeutic molecules.