C9 complement inhibitor

#The complement system is a complex interaction of plasma proteins and membrane cofactors which act in a multi-step, multi-protein cascade sequence in conjunction with other immunological systems of the body to provide immunity from intrusion of foreign cells. Complement proteins represent up to about 10% of globulins in normal serum of man and other vertebrates.

#The classic complement pathway involves an initial antibody recognition of, and binding to, an antigenic site (SA) on a target cell. This surface bound antibody subsequently reacts with the first component of complement, C1q, forming a C1-antibody complex with Ca ++, C1r, and C1s which is proteolytically active. C1s cleaves C2 and C4 into active components, C2a and C4a. The C4b, 2a complex is an active protease called C3 convertase, and acts to cleave C3 into C3a and C3b. C3b forms a complex with C4b, 2a to produce C4b, 2a, 3b, which cleaves C5 into C5a and C5b. C5b combines with C6. The C5b, 6 complex combines with C7 to form the ternary complex C5b, 6, 7. The C5b, 6, 7 complex binds C8 at the surface of the cell, which may develop functional membrane lesions and undergo slow lysis. Upon binding of C9 to the C8 molecules in the C5b, 6, 7, 8 complex, lysis of bacteria and other foreign cells is rapidly accelerated.

#The C5b-9 proteins of the human plasma complement system have been implicated in non-lytic stimulatory responses from certain human vascular and blood cells. The capacity of C5b-9 to modify membrane permeability and to selectively alter ion conductance is thought to elicit these non-lytic responses from human cells. In the case of human blood platelets and vascular endothelium, assembly of the C5b-9 complex initiates a transient and reversible depolarization of the plasma membrane potential, a rise in cytosolic Ca2+, metabolic conversion of arachidonate to thromboxane or prostacyclin, and the activation of intracellular protein kinases. In addition, human platelets exposed to C5b-9 undergo shape changes, secretory fusion of intracellular storage granules with plasma membrane, and the vesiculation of membrane components from the cell surface. Human endothelial cells exposed to the human C5b-9 proteins secrete high molecular weight multimers of the platelet adhesion protein, von Willibrand Factor (vWF), and the intracellular granule membrane protein, GMP140, is translocated from the Weibel-Palade body to the endothelial surface. High molecular weight multimers of vWF have been implicated in the pathogenesis of vaso-occlusive platelet adherence to endothelium and cell surface GMP140 has been implicated in the adherence of inflammatory leukocytes to endothelium.

#FIGS. 1A and 1B are a plot of the inhibitory activity of CD59 towards hu/rb chimeras of complement C9. Bar graph (FIG. 1A) summarizes combined results of all experiments measuring the inhibitory activity of CD59 towards recombinant hu/rb chimeras of C9. In each assay, hemolytic titrations of C9 were performed against C5b-8 chE in the presence and absence of membrane CD59, and the percent reduction of hemolysis due to CD59 (ordinate)was determined, with normalization to that observed for hu C9 (100% inhibition). Error bars denote mean +S.D., parentheses indicate number of independent experiments; asterisks (*) indicate significance (p<0.01) when compared to rb C9; pound signs (#) indicate significance (p<0.01) when compared to hu C9. The protein assayed is depicted (FIG. 1B) so as to designate those portions of the polypeptide containing hu C9 (open) or rb C9 (shaded) sequence. Numbers above each construct indicate the junctional hu C9 residue at each transition between hu and rb protein sequence. Bars designated as human C9 and rabbit C9 denote recombinantly-expressed hu and rb C9, respectively. Recombinant C9 chimeras (designated as #1-12) contain human (H) or rabbit (R) sequence according to the deduced mature primary structure of hu and rb C9. In some C9 chimeras, the numbering appears discontinuous because of gaps in the alignment of the hu and rb sequences: 1, R1-338H334-415R425-536; 2, R1-363H359-538; 3, H1-357R 363-536; 4, R1-363H359-415R425-536; 5, R1-363H359-391R401-536; 6, R1-400H392-415R425-536; 7, R1-363H359-384R394-536; 8, H1-333R339-424H416-538; 9, H1-357R363-424H416-538; 10, H1-357R363-400H392-538; 11, H1-391R401-424H416-538; 12, H1-357R363-393H385-538.

#FIG. 2 is a schematic representation of the segment of hu C9 identified as containing the CD59 binding site, which according to the proposed domain structure includes: thrombospondin type 1 (TS), LDL-receptor (LDLR), hinge (Hinge), membrane binding (MB), and epidermal growth factor precursor (EGFP) domains. Shaded segment indicates residues 334-415 of hu C9, spanning the putative CD59 binding site. The amino acid sequence of this peptide segment Sequence ID No. 3 is given below, and is shown in an alignment with rb C9 Sequence ID No. 4 (alignment done for full-length polypeptides with the PALIGN program in PCGENE). Asterisks indicate sequence identity. Dotted lines indicate the Cys 359/384 disulfide of hu C9 and the assumed corresponding internal disulfide in rb C9. Residue numbers refer to the mature proteins.

#FIG. 3 is a graph showing inhibitory activity of CD59 is unaffected by disruption of the Cys 359/384 disulfide. Recombinant hu C9 was expressed with a Cys.fwdarw.Ala mutation at either residue 384 or at both residues 359/384, and analyzed as described in FIG. 1. Inhibitory activity of CD59 towards the hemolytic function of each recombinant C9 is expressed as a percentage, relative to that measure for wild-type hu C9 (ordinate). Error bars denote mean +S.D., n, indicates number of independent experiments; asterisks indicate significance (p,0.001) compared to hu C9. Hu C9 and rb C9 denote the wild type hu and rb proteins, respectively.

#I. C9 Peptide/CD59 C9 binding site Immunomodulators

#Peptide sequence in human complement protein C9 has been identified that contributes to the recognition of this protein by its naturally occurring inhibitor, CD59 . CD59 is known to bind to neo-epitopes that become exposed in complement C8 and C9 during assembly of the cytolytic membrane attack complex of proteins C5b through C9. Through this interaction, CD59 interrupts assembly of the C5b-9 complex, protecting the target cell from destruction by these complement proteins. Data demonstrates that antibody raised against this human C9-derived peptide sequence is functionally inhibitory towards the lytic activity of the human C5b-9 complex. This permits design of reagents directed specifically at human C9 that mimic or inhibit the complement-inhibitory function of cell-surface CD59.

#Compounds which bind CD59