Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-04-15
2001-07-31
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C424S094630, C424S178100, C435S212000, C514S012200, C514S885000, C530S395000
Reexamination Certificate
active
06268485
ABSTRACT:
The present invention relates to novel modified proteins capable of forming C3 convertases resistant to down- regulation, DNA sequences encoding such proteins and the use of such proteins as therapeutic agents, particularly for use in depleting levels of complement pathway proteins or in targeting complement attack (C3b deposition) at specific sites.
The complement system functions in the immune response of humans and other vertebrates, being of major importance in the effector functions such as phagocytosis, cytolysis and recruitment of cells that induce local inflammatory responses [15]. These properties are desirable for elimination of invading pathogens, such as bacteria, but undesirable when triggered to act against host tissues (e.g. in post-ischemic reperfusion injury [3]) or against foreign therapeutic material (e.g. hyperacute rejection of xenografts [7]). There have been attempts to abrogate these undesirable properties by exploiting derivatives of complement regulator proteins whose normal function is to suppress complement activation [10, 18].
The complement system comprises proteins both on the surface of cells, (receptors and regulators) as well as in the fluid-phase (blood plasma and other extracellular environments). The critical step for the generation of responses is the proteolytic conversion of C3 to the fragments C3b and C3a. C3a is an anaphylatoxin that, like CSa, attracts mast cells to the site of challenge, resulting in local release of histamine, vasodilation and other inflammatory effects. The nascent C3b has an ability to bind to surfaces around its site of generation. This C3b then focuses attack by the cytolytic complement components (C5-C9).
Surface-bound C3b, and its degradation products, also function as ligands for C3 receptors mediating, for example, phagocytosis [15]. There are two distinct pathways of complement activation that both result in conversion of C3 to C3b and subsequent responses. The classical pathway is commonly triggered by complexes of antibody with antigen, initiating an enzyme cascade involving the proteins C1q, C1r, C1s, C2 and C4. The alternative pathway depends on an activation loop involving C3 itself and requiring factors B and D.
Conversion of C3 to C3b (or C3i) produces a product that can combine with factor B, giving C3bB (or C3iB). These complexes are acted upon by factor D to generate C3bBb, which is a C3 convertase capable of cleaving more C3 to C3b, leading to more C3bBb and even more C3 conversion. Under certain circumstances the C3bBb complex is stabilised by association with the positive regulator properdin (P). However, this positive-feedback loop is normally limited to a slow tick-over by regulatory proteins, notably factor H and factor I.
Factor H (and structurally related cell-associated molecules) (i) displaces B and Bb from C3b, and (ii) acts as a cofactor for factor I which cleaves C3b into iC3b thereby preventing any recombination with factor B to form more C3 convertases. The pathway is “fired” into amplified generation of C3b in the presence of surfaces, such as many bacterial cell walls, that bind nascent C3b and impede its regulation by factors H and I. Nascent C3b is also able to bind to endogenous cells. Endogenous cell surfaces normally exposed to complement are therefore additionally protected by membrane-bound regulators sucn as MCP, DAF and CRI acting in a similar manner to factor H.
There are a few rare naturally occurring conditions where the normal fluid-phase regulation cannot occur and spontaneous C3 conversion ultimately results in generalised depletion of C3 from the circulation: (i) genetic deficiencies of factor H or I [13], (ii) the presence of antibodies (nephritic factors) that bind to C3bBb and impede dissociation [4], and (iii) contact with a protein in cobra venom, called cobra venom factor (CVF), that combines with factor B and forms a C3, convertase enzyme which does not contain C3b and is not affected by factors H and I [14]. These illustrate the normal physiological importance of down-regulation of complement in the absence of specific activation.
There are also circumstances where specific activation occurs, but is unwanted, particularly when it is directed against tissues of the host (e.g. tissue damaged by ischemia or surgery) or against foreign material deliberately given for therapeutic purposes (such as a xenograft, artificial organ or a dialysis membrane). The complement activation results in undesirable attack and further damage, so in these cases it would be beneficial to block or inhibit the activation and response.
Existing approaches to preventing complement-mediated damage have targeted the use of down-regulatory proteins (CR1, MCP, DAF and factors H and I) to inhibit complement activation. Complement inhibitors like factor I., factor H and soluble derivatives of the membrane-bound proteins CR1, DAF, MCP do suppress the fluid-phase amplification loop of the alternative pathway. Therefore there have been attempts to use these molecules, particularly CR1 (which seems to be the most potent) to reduce complement-mediated damage in models of physiological situations [10, 18].
Factor H is endogenously present in blood plasma in high concentrations (typically 0.3-0.5 mg/ml (153), so even though increased levels of inhibitors do dampen-down fluid-phase reactions, their potency is weak so large amounts of purified proteins would have to be administered in vivo (e.g probably in excess of 5 mg/Kg body weight of soluble CR1). In addition, the alternative pathway is activated by surfaces where the effect of factor H is already impeded. While this does not necessarily concomitantly reduce the activities of other inhibitors, the same factors suggest that they are unlikely to be completely or universally effective.
Cobra Venom Factor(CVF) has the property of generating a stable C3 convertase which can be used experimentally to deplete complement in animals in vivo, and in other samples (e.g. human blood plasma) in vitro. CVF is potent (e.g. 40 &mgr;g/Kg can destroy the complement activity of a mouse [16]). However, there are disadvantages that make it unsuitable for therapeutic use in humans.
Firstly, it is obtained from cobra venom (a difficult source to obtain and dangerous to handle) and must therefore be carefully purified from the venom neurotoxins. There is also the obvious difficulty in obtaining supplies. This problem cannot readily be overcome by cloning and expressing the gene ex vivo, because there are post-translational modifications that occur in the snake (specific proteolytic processing) that may be difficult (or impossible) to reproduce in vitro. In addition, the enzymes and digestion conditions required for this processing are currently unknown. Secondly, the protein is of foreign origin (to humans) and therefore immunogenic. This precludes its repeated therapeutic use, as would be required to decomplement a patient over many weeks (e.g. to allow xenograft survival).
Although CVF has some structural and functional homologies with human C3 [17], it also has major differences in both respects (e.g. chain structure, site of biosynthesis, insensitivity to complement regulators, formation of a stable C3 convertase). It is not derived from the cobra equivalent of C3 which is known, having been cloned and sequenced, and which in gross structure and function resembles human C3 more closely than does CVF [8].
CVF is a venom-specific product of an animal of great evolutionary distance from homo sapiens. It is therefore not practicable to use genetic manipulation to modify this protein into a product that can be used non-immunogenically in humans.
We have now devised an alternative strategy which relies on by-passing the physiological regulation and, instead of inhibiting complement activation, causes the system to be super-activated. This has two applications. Firstly, it can be used in vivo to activate complement until one or more components are exha
Farries Timothy Charles
Harrison Richard Alexander
DeCloux Amy
Furman Diane E.
Imutran Limited
Pfeiffer Hesna J.
Saunders David
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