Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-10-19
2004-12-21
Spector, Lorraine (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S006120, C435S007210, C435S069100, C435S252300, C435S320100, C436S501000, C514S002600, C530S402000, C536S023500
Reexamination Certificate
active
06833437
ABSTRACT:
This invention relates to novel polypeptides and their derivatives which act as inhibitors or regulators of complement activation and are of use in the therapy of diseases involving complement activation such as 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 also 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 the brains of sufferers from Alzheimer's disease (Eikelenboom et al., 1994, Neuroscience, 59, 561-568) and that complement activation plays a role in the inflammatory component of this condition.
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; Pruitt 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 encephalomyclitis (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 (also known as TP10 or 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 WO94/00571 comprising, in sequence, one to four short consensus repeats (SCR) selected from SCR 1, 2, 3 and 4 of long homologous repeat A (LHR-A) as the only structurally and functionally intact SCR domains of CR1 and including at least SCR3.
Pseudogenes are usually defined as DNA sequences which possess a high degree of homology to genes with identified function but which are not expressed. The origins of the lack of transcription and translation vary but are commonly the presence of accumulated mutations which inactivate inscriptional initiation sites, disrupt RNA splicing or introduce frame-shift mutations and premature termination codons. Pseudogenes are sometimes regarded as genetic relics which have been isolated within the genome through a primary loss of expressability and which have subsequently mutated randomly in situ to highly aberrant forms. There is a frequent presumption that pseudogene sequences, if expressable at all, will not be functionally active because of an accumulation of deleterious in-frame mutations. However, studies of immune system genetics suggest that pseudogenes may act as a source of diversity in somatic mutation processes and that non-expressed sequences may recombine with normally expressed genes to create fu
Cox Vivienne Frances
Mossakowska Danuta Ewa Irena
Smith Richard Anthony Godwin
Adprotech Limited
Brannock Michael
Heller Ehrman White & McAuliffe
Spector Lorraine
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