The EPICC peptides are a family of peptides that have been developed from the sequence of the capsid protein of human astrovirus type 1 and previously shown to inhibit the classical and lectin pathways of complement. The EPICC peptides have been further optimized to increase aqueous solubility and identify additional mechanisms of action. Our laboratory has developed the lead EPICC molecule, PA-dPEG24 (also known as RLS-0071), which is composed of a 15 amino acid peptide with a C-terminal monodisperse 24-mer PEGylated moiety. RLS-0071 has been demonstrated to possess other mechanisms of action in addition to complement blockade that include the inhibition of neutrophil-driven myeloperoxidase (MPO) activity, inhibition of neutrophil extracellular trap (NET) formation as well as intrinsic antioxidant activity mediated by vicinal cysteine residues contained within the peptide sequence. RLS-0071 has been tested in various ex vivo and in vivo systems and has shown promise for the treatment of both immune-mediated hematological diseases where alterations in the classical complement pathway plays an important pathogenic role as well as in models of tissue-based diseases such as acute lung injury and hypoxic ischemic encephalopathy driven by both complement and neutrophil-mediated pathways (i.e., MPO activity and NET formation). Next generation EPICC peptides containing a sarcosine residue substitution in various positions within the peptide sequence possess aqueous solubility in the absence of PEGylation and demonstrate enhanced complement and neutrophil inhibitory activity compared to RLS-0071. This review details the development of the EPICC peptides, elucidation of their dual-acting complement and neutrophil inhibitory activities and efficacy in ex vivo systems using human clinical specimens and in vivo efficacy in animal disease models.
The innate immune system is the body’s first line of defense against microorganisms. It consists of both humoral and cellular mechanisms that are triggered immediately upon infection. Two of the primary components of innate immunity are phagocytic cells (neutrophils and macrophages) and blood proteins (the complement system). While complement and neutrophils are an essential host defense against invasive microbes, dysregulation of the innate immune response plays a prominent role in a variety of inflammatory and autoimmune diseases. In this review article, we describe a novel class of anti-inflammatory peptides (termed “EPICC”) that possess a unique dual-acting mechanism of action: inhibition of both complement and neutrophil-mediated activation. The discovery, in vitro and ex vivo characterization as well as the in vivo activity of these peptides in pre-clinical blood and tissue-based disease models, will be discussed along with their implications for therapeutic use in inflammatory disease processes.
Human astrovirus serotype 1 (HAstV-1) is a non-enveloped, icosahedral virus with a single-stranded, 7 kilobase positive-sense RNA genome that is an endemic global pathogen. HAstV-1 is the prototypic family member and the most well studied of the Astroviridae. Human astroviruses infect young children and unlike other enteric pathogens, cause a non-inflammatory, self-limiting gastroenteritis. The lack of inflammation associated with astrovirus infection led us to hypothesize that the virus capsid may directly interact with components of the host immune system resulting in a blunted inflammatory response as reported in humans and other animal infections. The HAstV-1 capsid is composed of 180 copies of a single capsid protein of 787 amino acid residues and structural predictions using three-dimensional position-specific scoring matrix (3D-PSSM) software revealed that the capsid protein possessed weak homology to human complement regulatory proteins. This led us to examine if the HAstV-1 capsid protein could directly modulate the human complement system.
The complement system constitutes part of the humoral innate immune system and is comprised of over 30 plasma and cell bound proteins. The complement system can be activated through three pathways: classical, lectin, and alternative. These pathways are enzymatic cascades that converge at C3 and are normally tightly controlled by soluble and cell bound regulatory proteins. The main activities of the complement system are to provide a first line of defense against infection, bridge the innate and adaptive immune systems, and maintain immune homeostasis by clearing apoptotic cell debris. The classical complement pathway is activated via antibody bound to the surface of a microbe or immune complexes as well as in an antibody-independent manner, by molecules such as C-reactive protein or pentraxins that directly activate the classical pathway. Similar to the classical pathway, the lectin pathway is composed of soluble pattern-recognition molecules, that activates upon recognition of mannose groups on the surface of microbes. The alternative pathway is activated when C3 directly recognizes microbial surface structures and amplifies the activation of both the classical and lectin pathways. Activation of any of these three pathways results in generation of the anaphylatoxins C3a and C5a as well as the membrane attack complex (MAC) which results in cellular lysis. This cascade of complement activation results in microbial destruction through multiple mechanisms, including the lysis of infected cells and certain pathogens, opsonization and phagocytosis, removal via immune complexes, enhanced priming of T and B cells, and mediation of a robust inflammatory response via leukocyte chemotaxis to the site of infection. However, abnormal activation of any of these pathways can lead to significant tissue damage and is associated with inflammatory and autoimmune diseases.
Classical complement activation is mediated via the multimolecular complex, C1. C1 is comprised of the charge pattern recognition protein, C1q, and serine protease tetramer, C1s-C1r-C1r-C1s. C1q is composed of 6 copies of three protein chains (A, B and C) that together form an 18-chain molecule with a collagen-like region (CLR) and a globular head region (GHR). The CLR has a semi-flexible “hinge” which houses the C1s-C1r-C1r-C1s tetramer. Binding of IgM or clustered IgG to the GHR induces a conformational change in C1q resulting in activation of the C1s-C1r-C1r-C1s tetramer and sequential activation of C4 and C2 of the classical pathway and downstream effector functions of the complement system. Initial studies in our laboratory demonstrated HAstV-1 capsid protein was able to potently inhibit complement activation in a standard hemolytic assay. In this assay, human serum is added to antibody sensitized red blood cells (RBC) which activates the classical pathway resulting in MAC formation on the RBCs and hemolysis. HAstV-1 capsid protein inhibited hemolysis to a similar degree as cobra venom factor (CVF), the gold standard complement inhibitor. We further demonstrated that HAstV-1 capsid protein binds to the hinge region of the CLR of C1q, interrupting the interaction of the cognate serine protease complex (C1s-C1r-C1r-C1s) and thus inhibiting classical complement pathway activation. The astrovirus capsid protein also was found to bind the initiator molecule of the lectin pathway, mannose binding lectin (MBL) protein which shares structural homology to C1q and inhibit activation of the lectin pathway of complement. HAstV-1 capsid protein had no inhibitory activity on the alternative pathway which provides essential immune-surveillance functions against invading pathogens.
The capsids of icosahedral RNA viruses perform multiple functions in the virus life cycle such as self-assembly, viral RNA packaging, host receptor binding, viral particle disassembly and immune evasion. We thus hypothesized that the region of the HAstV-1 capsid that possesses complement inhibitory activity would map to a limited amino acid sequence within the 787 amino acid capsid protein. Based on sequence alignment data, we identified a 60 amino acid region of the HAstV-1 capsid protein that had limited homology to human neutrophil peptide-1 (HNP-1) which had previously been identified as an inhibitor of the classical and lectin complement pathways. This region of the HAstV-1 capsid was subsequently demonstrated to be exposed on the surface of the mature astrovirus particle by X-ray crystallography and thus available to interact with host immune factors. It was also demonstrated that human astrovirus blunts the intracellular C3 autonomous immune response as assessed by reduced NF-kB levels which the authors attributed to HAstV inhibition of C1.
The active region of this peptide was further defined to a peptide of 15 residues within the HAstV-1 capsid protein and the sequence rearranged to improve the amphipathic nature of the molecule by reorganizing the hydrophobic residues to the N terminus and the hydrophilic residues to the C terminus, creating the Polar Assortant (PA) peptide (amino-acid sequence IALILEPICCQERAA). The PA peptide was shown to have superior complement inhibiting activity in the hemolytic assay compared to previous astrovirus-derived peptide derivatives and is unique in nature with no known homologies to other natural proteins or peptides. Increased functionality of PA was not improved by the systematic substitution of positively or negatively charged residues using alanine, arginine, or glutamic acid scans or deletion of a single residue at the N- or C-termini which resulted in decreased ability of PA to inhibit the classical complement pathway. The PA molecule is the base peptide of the EPICC family of peptides, named for the conserved central five amino acids (IALILEPICCQERAA).
PA was subsequently shown to inhibit ABO mediated RBC incompatibility, a classical complement pathway driven disease process, which was demonstrated in an ex vivo hemolytic assay using incompatible human type O serum and human AB RBCs, ex vivo using serum isolated from the blood of rats, and was also shown to inhibit complement activity when administered intravenously into rats. The cross-species activity of PA and other EPICC derivatives have been essential for the pre-clinical development of the EPICC derivatives as it allows the peptides to be tested in animal models of complement and neutrophil-mediated disease. PA is not water soluble and requires solubilization in dimethylsulfoxide (DMSO). To increase solubility in aqueous solution for in vivo administration, PA was synthesized to include a monodisperse, 24-mer polyethylene glycol (PEG) unit linker on either the N terminus (dPEG24-PA), C terminus (PA-dPEG24), or N and C termini (dPEG-24-PA-
