Soluble complement regulatory molecules

Details Soluble complement regulatory molecules Soluble complement regulatory molecules

1992-12-28

2002-10-01

Spector, Lorraine (Department: 1647)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Conjugate or complex

C424S192100, C424S194100, C435S069700, C435S069600, C530S350000, C530S387300, C530S395000, C530S402000

Reexamination Certificate

active

06458360

ABSTRACT:

This invention was made with support provided by National Institutes of Health Grant Nos. AI22833 and AI28191. The U.S. Government retains certain rights in this invention.
FIELD OF THE INVENTION
This invention is directed to soluble, recombinant, fused proteins which contain a recognition site for a target molecule.
BACKGROUND OF THE INVENTION
A wide variety of different molecules are present in the mammalian circulatory system, although the precise components as well as their concentration vary from time to time. These variations in the composition of the serum are in response to a spectrum of stimuli, and by sensing the changes in serum composition and concentration, the various organs of the mammals are able to respond to the stimuli. The cells of the organism recognize changes in the circulatory system by means of cell surface receptors which bind to various molecular components of the serum. It is possible to affect the way that cells of the organism will respond to the stimuli by affecting the binding of these particular components in the circulatory system to cell surface receptors.
The Complement System
One example of a system of serum components which changes in response to environmental stimuli and whose changes are sensed through binding to cell surface receptors is the complement system. The complement system is a mechanism for the recognition of foreign materials, such as microorganisms, that proceeds through two phases: the first being the covalent attachment of two complement proteins, C3 and C4, to proteins and carbohydrates that are part of the complement-activating complex. Depending on the environmental stimuli, one of two separate pathways activates an enzyme called C3-convertase which cleaves C3, releasing the C3a peptide from the alpha polypeptide of C3 and causing a major conformational change in the C3b fragment.
The second phase is the receptor-mediated binding of these complexes by various cell types, such as lymphocytes and phagocytes. In the second phase of recognition by complement, complexes containing covalently-bound fragments of C3 and C4 are bound by cells having receptors specific for these fragments. These receptors are termed complement receptors type 1 (CR1, CD35), type 2 (CR2, CD21), and type 3 (CR3, CD11b/18). The receptors are found on the surfaces of various cell types involved in immune and inflammatory responses. By modulating the response of phagocytes and lymphocytes to microorganisms and their products, this recognition program of the complement system plays a primary role in the host resistance when activation of C3 occurs through the alternative pathway. When the classical pathway has been recruited by antibody directed to the foreign molecules, binding of complement fragments plays an amplifying role.
The SCR Motif of Complement Receptor Type 1
CR1 has been extensively studied, and a structural motif of 60-70 amino acids, termed the short consensus repeat (SCR) has been found. The SCR motif is tandemly repeated 30 times in the F-allotype of CR1 and additional repeat cycles occur in other allotypes. The consensus sequence of the SCR includes 4 cysteines, a glycine and a tryptophan that are invariant among all SCR. Sixteen other positions are conserved, with the same amino acid or a conservative replacement being found in over half of the other 30 SCRs Kliekstein, et al., (1987),
J. Exp. Med.
, (165: 1095-1112, and (1988),
J. Exp. Med.
, 168:1699-1717; Hourcade, et al. (1988)
J. Exp. Med.
, 168:1255-1270). The dimensions of each SCR are estimated to be approximately 2.53.0 nm×2 nm×2 nm.
Tandem repeats of SCRs (with the same invariant residues and similar spacing between cysteines) have been identified in 12 additional proteins of the complement system (Ahearn, et al. (1989),
Adv. Immunol.
, 46:183-219). These proteins share a capacity for interacting with C3, C4, or C5, the set of homologous complement proteins that are subunits of the alternative and classical C3-C5 convertases and the membrane attack complex, respectively. Complement proteins containing SCRs may have activating functions (C1r, C1s, Factor B and C2), negative regulatory roles (Factor H, C4-BP, DAF, MCP, and CR1), serve as cellular receptors capable of eliciting functions of phagocytes and lymphocytes (CR1 and CR2) or promote the formation of the complement channel-forming membrane attack complex (C6 and C7). thus, the SCR is one of the most characteristic structures of the complememt system. The finding of SCRs in non-complement proteins, such as the interleukin-2 receptor alpha chain, beta-2-glycoprotein 1, and factor XIII does not necessarily indicate a complement-related function, although this possibility has not been excluded.
The first 28 SCRs from the N-terminus of CR1 may be grouped into four sequential groups, each containing seven SCRs, called long homologous repeats (LHR) and designated A, B, C, and D. LHR-D is followed by the remaining two SCRs and then by a 25 amino acid transmembrane region and a 43 amino acid cytoplasmic region that serve to anchor CR1 on the cell surface. Three complement binding sites reside in CR1: one in LHR-A specific for C4b and two additional sites in LHR-B and LHR-C specific for C3b (Klickstein, et al., 1988, supra). The two N-terminal SCRs of each LHR are involved in ligand specificity. Because complement-activating substances will bind multiple C4b and C3b molecules to their surfaces, this multivalent CR1 can interact more effectively with them than would a univalent receptor.
Other Complement Receptors
Complement receptor type 2 (CR2, CD21) is a transmembrane phosphoprotein consisting of an extracellular domain which is comprised of 15 or 16 SCRs, a 24 amino acid transmembrane region, and a 34 amino acid cytoplasmic domain (Moore, et al. (1987),
Proc. Nat'l. Acad. Sci. USA
, 84:9194-9198; Weis, et al. (1988),
J. Exp. Med.
, 167:1047-1066, which are incorporated herein by reference). Electron microscopic studies of soluble recombinant CR2 have shown that, like CR1, it is an extended, highly flexible molecule with an estimated contour length of 39.6 nanometers by 3.2 nanometers, in which each SCR appears as a ringlet 2.4 nanometers in length (Moore, et al. (1989),
J. Biol. Chem.
, 34:20576-20582).
CR2 is the B-cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV) and the C3dg protein fragment of complement (Ahearn, et al., 1989, supra). An anti-CR2 monoclonal antibody (OKB7) blocks binding of both C3dg and EBV, suggesting that the natural and viral ligands bind to identical or proximal sites on the receptor (Nemerow, et al. (1985),
J. Virol.
, 55:347-351). By means of recombinant DNA experiments with eukaryotic expression vectors expressing deletion or substitution mutants of CR2 in COS cells, the ligand binding sites of CR2 have ben localized in the two N-terminal SCRs of the molecule (Lowell, et al., (1989)
J. Exp. Med.
, 170:1931-1946). Binding by cell-bound CR2 of the multivalent forms of C3 ligands such as iC3B and C3dg causes activation of B-cells. (Melchers, et al. (1985),
Nature
, 317:264-267; Bohnsack, et al. (1988),
J. Immunol.
, 141:2569-2576; Carter, et al. (1988)
J. Immunol.
, 457-463; and Carter, et al. (1989),
J. Immunol.
, 143:1755-1760).
A third complement receptor, CR3, also binds iC3b. Binding of iC3b to CR3 promotes the adherence of neutrophils to complement-activating endothelial cells during inflammation (Marks, et al. (1989),
Nature
, 339:314). CR3 is also involved in phagocytosis, where particles coated with iC3b are engulfed by neutrophils or by macrophages (Wright, et al. (1982),
J. Exp. Med.
, 156:1149; (1983)
J. Exp. Med.
, 158:1338).
Soluble Complement Receptors
CR1 is a candidate for effective inhibition of complement activation. Only CR1 combines specificity for both C3b and C4b with capabilities for dissociating the C3 convertases of both pathways and for cofactor activity in the proteolytic inactivation of C3b and C4b by factor I. In addition, and probably of critical importance, these functions of CR1 are not restricted by alt

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