Fragments of CR1 and their use

Details Fragments of CR1 and their use Fragments of CR1 and their use

1998-12-01

2004-09-28

Kunz, Gary (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S380000, C530S395000, C530S402000, C530S387300, C424S185100, C424S195110

Reexamination Certificate

active

06797806

ABSTRACT:

The present invention relates to polypeptides and their use in the diagnosis and therapy of disorders involving complement activity and various inflammatory and immune disorders.
Constituting about 10% of the globulins in normal serum, the complement system is composed of many different proteins that are important in the immune system's response to foreign antigens. The complement system becomes activated when its primary components are cleaved and the products alone or with other proteins, activate additional complement proteins resulting in a proteolytic cascade. Activation of the complement system leads to a variety of responses including increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, opsonization of foreign particles, direct killing of cells and tissue damage. Activation of the complement system may be triggered by antigen-antibody complexes (the classical pathway) or, for example, by lipopolysaccharides present in cell walls of pathogenic bacteria (the alternative pathway).
Complement activation (CA) is known to occur in a wide variety of acute inflammatory processes particularly those associated with ischaemia and reperfusion injury (Rossen et al., 1985 Circ. Res., 57, 119,; Morgan B. P., 1990 The biological effects of complement activation. In ‘
Complement, Clinical Aspects and Relevance to Disease
’, Academic Press, London.).
It is generally accepted that at least some of the components of the classical complement cascade can be detected by immunohistochemical methods in close association with senile plaques in AD brain (Eikelenboom et al., 1994, Neuroscience, 59, 561-568). There is good evidence for the involvement of C1, C3 and C4, but evidence for the presence of the C5-C9 membrane-attack complex (MAC) is not yet evident (Veerhuis et al, 1995, Vichows Arch. 426, 603-610). Cells of the CNS have been shown to synthesise complement components (for review see Barnum, 1995 Crit. Rev. Oral. Biol. Med 6, 132-146), and production of C3 is enhanced in response to incubation with bA4 peptide (Haga et al., 1993 Brain Res., 601, 88-94). Thus complement can be induced locally in the brain itself and is not necessarily derived solely from the plasma compartment.
Of particular interest is the fact that the bA4 peptide has been found to bind directly to the initial component of the complement cascade (C1q) and to initiate the whole of the classical complement system in vitro (including MAC) by an antibody-independent mechanism (Rogers et al., 1992, Proc. Nat. Acad. Sci. USA., 89, 10016-10020; Jianh et al., 1994, J. Immunol., 152, 5050-5059). This interaction appears to involve region 6-16 of &bgr;A4 and 14-26 of the collagen-like tail region of the C1q A chain. The lamer site is separate from the IgG-immune complex binding site located on the globular head domain of C1q. There is some evidence that fibrillar bA4 binds with higher affinity to C1q than monomeric peptide, potentially providing a rational basis for activation of complement in the disease process (Jiang et al., 1994, J. Immunol., 152, 5050-5059; Snyder et al., 1994, Exp. Neurol., 128, 136-142).
Complement receptor type 1 (CR1) has been shown to be present on the membranes of erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. CR1 binds to the complement components C3b and C4b and has also been referred to as the C3b/C4b receptor. The structural organisation and primary sequence of one allotype of CR1 is known (Klickstein et al., 1987, J. Exp. Med. 165:1095-1112, Klickstein et al., 1988, J. Exp. Med. 168:1699-1717; Hourcade et al., 1988, J. Exp. Med. 168:1255-1270, WO 89/09220, WO 91/05047). It is composed of 30 short consensus repeats (SCRs) that each contain around 60-70 amino acids. In each SCR, around 29 of the average 65 amino acids are conserved. Each SCR has been proposed to form a three dimensional triple loop structure through disulphide linkages with the third and first and the fourth and second half-cystines in disulphide bonds. CR1 is further arranged as 4 long homologous repeats (LHRs) of 7 SCRs each. Following a leader sequence, the CR1 molecule consists of the N-terminal LHR-A, the next two repeats, LHR-B and LHR-C, and the most C-terminal LHR-D followed by 2 additional SCRs, a 25 residue putative transmembrane region and a 43 residue cytoplasmic tail.
Based on the mature CR1 molecule having a predicted N-terminal glutamine residue, hereinafter designated as residue 1, the first four SCR domains of LHR-A are defined herein as consisting of residues 2-58, 63-120, 125-191 and 197-252, respectively, of mature CR1.
Hourcade et al., 1988, J. Exp. Med. 168:1255-1270 observed an alternative polyadenylation site in the human CR1 transcriptional unit that was predicted to produce a secreted form of CR1. The mRNA encoded by this truncated sequence comprises the first 8.5 SCRs of CR1, and encodes a protein of about 80 kDa which was proposed to include the C4b binding domain. When a cDNA corresponding to this truncated sequence was transfected into COS cells and expressed, it demonstrated the expected C4b binding activity but did not bind to C3b (Krych et al., 1989, FASEB J. 3:A368; Krych et al. Proc. Nat. Acad. Sci. 1991, 88, 4353-7). Krych et al., also observed a mRNA similar to the predicted one in several human cell lines and postulated that such a truncated soluble form of CR1 with C4b binding activity may be synthesised in humans.
In addition, Makrides et al. (1992, J. Biol. Chem. 267 (34) 24754-61) have expressed SCR 1+2 and 1+2+3+4 of LHR-A as membrane-attached proteins in CHO cells.
Several soluble fragments of CR1 have also been generated via recombinant DNA procedures by eliminating the transmembrane region from the DNAs being expressed (WO 89/09220, WO 91/05047). The soluble CR1 fragments were functionally active, bound C3b and/or C4b and demonstrated Factor I cofactor activity depending upon the regions they contained. Such constructs inhibited in vitro complement-related functions such as neutrophil oxidative burst, complement mediated hemolysis, and C3a and C5a production. A particular soluble construct, sCR1/pBSCR1c, also demonstrated in vivo activity in a reversed passive Arthus reaction (WO 89/09220, WO 91/05047; Yeh et al., 1991, J. Immunol. 146:250), suppressed post-ischemic myocardial inflammation and necrosis (WO 89/09220, WO 91/05047; Weisman et al., Science, 1990, 249:146-1511; Dupe, R. et al. Thrombosis & Haemostasis (1991) 65(6) 695.) and extended survival rates following transplantation (Pruitt & Bollinger, 1991, J. Surg. Res 50:350; Pruit et al., 1991 Transplantation 52; 868). Furthermore, co-formulation of sCR1/pBSCR1c with p-anisoylated human plasminogen-streptokinase-activator complex (APSAC) resulted in similar anti-haemolytic activity as sCR1 alone, indicating that the combination of the complement inhibitor sCR1 with a thrombolytic agent was feasible (WO 91/05047).
In a model of antibody-mediated demyelinating experimental allergic encephalomyelitis (ADEAE), systemic inhibition of CA using sCR1 over 6 days, produced improvements in clinical score and blocked CNS inflammation, demyelination and deposition of complement components (Piddlesden et al., 1994, J. Immunol. 152, 5477). ADEAE can be regarded as a model of acute relapse in multiple sclerosis (MS) and these striking results suggested possible applications for sCR1 in MS therapy despite the high molecular weight (245 kilodaltons) of this agent.
In a rat model of traumatic brain injury, complement inhibitor sCR1 (BRL55730) was shown to reduce myeloperoxidase activity (an indicator of neutrophil accumulation) following traumatic injury (Kaczorowska et al, 1995, J. Cerebral Blood Flow and Metabolism, 15, 860-864). This is suggested as demonstrating that complement activation is involved in the local inflammatory response.
Soluble polypeptides corresponding to part of CR1 having functional complement inhibitory, including anti-haemolytic, activity have been described in WO94

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