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Human complement c3 derivates with cobra venom factor-like function

This International application claims priority to U.S. Provisional Applications 60/567,069, filed Apr. 30, 2004, 60/653,247, filed Feb. 14, 2005, and 60/667,352, filed Mar. 30, 2005, each of which is incorporated by reference in its entirety.

The invention relates generally to chimeric derivatives of Human Complement C3 having a substitution of a portion of a human C3 protein with a corresponding portion of a Cobra Venom Factor (CVF) protein. Preferably, a portion of the alpha chain of C3 is substituted with the corresponding portion of CVF.

The third component of complement, C3, plays a pivotal role in both the classical and alternative pathways of complement activation, and many of the physiologic C3 activation products have important functions in the immune response and host defense. In the alternative pathway, the activated form of C3, C3b, is a structural subunit of the C3 convertase. This bimolecular enzyme consists of C3b and Bb, the activated form of factor B. This enzyme is formed by the binding of C3b to factor B that is subsequently cleaved by factor D, resulting in the formation of the C3 convertase, C3b,Bb, and the release of the activation peptide Ba. The C3 convertase activates C3 by cleaving the molecule into C3b and the anaphylatoxin, C3a. The C3b molecule will bind to a cell or particle in close proximity to the C3 convertase. Eventually, the bound C3b will allow for the activation of C5 into C5b and the anaphylatoxin, C5a. C5 activation occurs by the same C3b,Bb enzyme that can cleave C5 when it is bound to an additional C3b molecule to produce a trimolecular complex composed of (C3b)2,Bb. This C5-cleaving trimolecular enzyme is called C5 convertase. Inasmuch as the activation of both C3 and C5 occurs at the identical active site in the Bb subunit, the enzyme is also called C3/C5 convertase; and only one EC number has been assigned (EC 3.4.21.47).

Cobra venom contains a structural and functional analog of C3 called cobra venom factor (CVF). This molecule can bind factor B in human and mammalian serum to form the complex, CVF,B, which is also cleaved by factor D into the bimolecular enzyme CVF,Bb and Ba. The bimolecular complex CVF,Bb is a C3/C5 convertase that activates C3 and C5 analogously to the C3/C5 convertase formed with C3b. Although the two C3/C5 convertases, C3b,Bb and CVF,Bb, share the same molecular architecture, the active site-bearing Bb subunit, and the substrate specificity, the two enzymes exhibit significant functional differences. The CVF,Bb enzyme is physiochemically far more stable than C3b,Bb, it is resistant to inactivation by the regulatory proteins factors H and I, it exhibits different kinetic properties, and it does not require additional C3b for C5 cleavage.

CVF and mammalian C3 have been shown to exhibit several structural similarities including immunologic cross-reactivity, amino acid composition, circular dichroism spectra, secondary structure, electron microscopic ultrastructure, and amino acid sequence. Nevertheless, significant structural differences exist between the two molecules. Whereas C3 is a two-chain molecule with an apparent molecular mass, dependent on the species, of 170 to 190 kDa, CVF is a three-chain molecule with an apparent molecular mass of 149 kDa that resembles C3c, one of the physiologic activation products of C3. Another significant structural difference between C3 and CVF lies in their glycosylation: CVF has a 7.4% (w/w) carbohydrate content consisting mainly of N-linked complex-type chains with unusual α-galactosyl residues at the non-reducing termini. In contrast, human and rat C3 exhibit a lower extent of glycosylation with different structures of their oligosaccharide chains.

Whereas CVF,Bb and C3b,Bb are both C3/C5 convertases, they exhibit important differences. The CVF-containing enzyme is far more stable than the C3-containing enzyme. Both convertases will spontaneously decay into their two respective subunits. However, the intrinsic half-life (stability) of the CVF-containing convertase is approximately 7 hours at 37° C., several hundred times longer than the C3-containing enzyme with an intrinsic half-life of approximately 1.5 minutes. Furthermore, the CVF-containing enzyme as well as free CVF are not subject to regulation by the complement regulatory proteins factors H and I. The combination of the long intrinsic half-life and the resistance to regulation of the CVF-containing enzymes allows CVF to continuously activate C3 and C5 (and subsequently other complement components), ultimately resulting in depletion of the serum complement activity.

Based on the involvement of the complement system in multiple diseases, including diseases of major prevalence, the last decade has seen the development of multiple anti-complementary agents to interfere with the unwanted complement activation process in these disease states. All complement-oriented drug development attempts are based on inhibiting the activation of complement, while CVF acts by depleting complement in serum. Of interest for the treatment of diseases of complement activation is a C3-type molecule which combines the non- or low immunogenicity of C3, with the complement-depleting function of CVF.

The following listing of embodiments is a nonlimiting statement of various aspects of the invention. Other aspects and variations will be evident in light of the entire disclosure.

Some embodiments include one or more modified human complement C3 proteins, which can have a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein of a sequence substantially related thereto. In some embodiments the substituted portion of the CVF can be within the alpha chain of C3. In other embodiments, the substituted portion of the CVF can be a C-terminal portion of the alpha chain of C3. In some embodiments, the substituted C-terminal portion can include amino acid 1663 of the human C3 protein. In some embodiments, the substituted C-terminal portion can be an internal portion that does not extend through the entire C-terminus of the human C3 protein. In further embodiments, the modified protein can have substantially the same number of amino acid residues as an unmodified human C3 protein. In some embodiments, the substitution can include any positions within amino acid positions 700-1663 of the human C3 protein. Other embodiments are human complement C3 proteins, which can have a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein of a sequence substantially related thereto and which can have at least two substitutions. In some embodiments, the substitution has a selected beginning position and a selected last position; in some such embodiments, the beginning position can be, for example, 749, 874, 936, 994, 1264, 1348, 1496, 1504, 1550, and the like; the last position can be, for example, 784, 921, 970 1324, 1550, 1617, 1663, and the like. In preferred embodiments, the one or more substitutions can include any of amino acids: 1550-1663, 1504-1663, 1348-1663, 1550-1617, 1504-1617, 1496-1663, 1348-1617, 1496-1617, 1264-1324, 749-784, 874-921, 994-1663, 994-1550 and 936-970. In some embodiments, the substituted portion of CVF can be within the beta chain of C3.

In some embodiments, the modified C3 protein can have an affinity for factor B and can support formation of a convertase. In some embodiments, the resulting convertase can cleave C3 and not C5. In further embodiments, the convertase can have an intrinsic half-life between about 1.5 minutes and about 7 hours at 37° C. In some embodiments, the resulting convertase can have an intrinsic half-life of at least about 7 hours at 37° C.

In some embodiments, the modified C3 protein can be expressed as a single chain protein. In some embodiments, the modified C3 protein can be cleaved into at least two chains in a form that resembles C3. In further embodiments, the modified C3 protein can be cleaved to release a C3a portion therefrom. In some embodiments, the modified protein can have an additional 1 to about 19 amino acids at the N-terminus that are not encoded by C3 or CVF. In some embodiments, the modified protein can include a non-C3 signal peptide, such as a Drosophila Bip signal sequence. In some embodiments, the modified C3 protein can have modified affinity for factor B and/or factor D. In some embodiments, the modified protein can show partial or complete resistance to Factor H and/or Factor I. In some embodiments, the modified C3 protein can be substantially non-immunogenic.

Other embodiments can include a method for depleting complement by administering a modified C3 protein to a patient in an amount effective for the depletion of complement. In some embodiments, the administration can be local. In further embodiments, the local administration can be into an organ, subcutaneously, into a cavity, or into a tissue. In other embodiments, the local administration can employ a targeting function capable of concentrating the modified C3 protein in a desired location. In further embodiments, the targeting function can include using an antibody conjugated to the modified C3 protein. In some embodiments, the administration can be a systemic administration, such as intravenous or intraperitoneal.

Further embodiments can be methods for avoiding or ameliorating reperfusion injury in a patient by delivering a modified C3 protein to the patient, sufficient to deplete complement; and permitting reperfusion in the patient. In some embodiments, the delivering step can include injecting the modified C3 protein into an artery. In other embodiments, the delivering step can include a local delivery of the modified C3 protein. In other embodiments, the delivering step can include a systemic delivery of the modified C3 protein. In some embodiments, reperfusion can include opening a blocked artery. In some embodiments, the reperfusion can occur in connection with transplantation of an organ.

Some embodiments can include methods for increasing the efficiency and/or effectiveness of gene therapy by delivering a modified C3 protein in an amount sufficient to deplete complement, providing the gene therapy; and observing an enhanced result therefrom.

Some embodiments can include methods of increasing delivery of a therapeutic or diagnostic agent by delivering a modified C3 protein sufficient to increase blood flow; and providing the therapeutic or diagnostic agent. In some embodiments, the method can include chemically linking the modified C3 protein to an antibody with an affinity for a specific tissue prior to the delivering step. In some embodiments, the antibody can be attached to the modified C3 by recombinant DNA technology. In some embodiments the antibody can be a monoclonal antibody.

Some embodiments include methods of treating an autoimmune disease, comprising administering the modified C3 protein sufficient to deplete complement. In some embodiments, the administration can be episodic and corresponds to periods of at least one elevated disease symptom. In some embodiments, the autoimmune disease can be any of asthma, systemic lupus erythematosus, glomerulonephritis, rheumatoid arthritis, Alzheimer's disease, multiple sclerosis, myocardial ischemia, reperfusion, sepsis, hyperacute rejection, transplant rejection, cardiopulmonary bypass, myocardial infarction, angioplasty, nephritis, dermatomyositis, pemphigoid, spinal cord injury and Parkinson's disease.

Some embodiments include methods of mimicking the properties of CVF in a human protein, by, for example, substituting a portion of a human complement C3 protein with the corresponding portion of a Cobra Venom Factor (CVF) protein. In some embodiments the portion can be within the alpha chain of C3. In other embodiments, the portion can be a C-terminal portion of the alpha chain of C3. In some embodiments, the C-terminal portion can include amino acid 1663 of the human C3 protein. In some embodiments, the substituted C-terminal portion can be an internal portion that does not extend through the entire C-terminus of the human C3 protein.