Oklahoma Medical Research Foundation

Yale University

Bloodcenter Research Foundation

Oklahoma Medical Research

Peter J Sims

Therese Wiedmer

Alfred L Bothwell

Richard A Flavell

Scott A Rollins

Stephen P Squinto

Leonard Bell

Joseph A Madri

Eileen A Elliot

Alfred L M Bothwell

Compounds modulating CD59 mediated complement activity, compositions including these compounds, and methods of making and using the compounds are disclosed, which are based on the identification of the hu CD59 amino acid residues which serve as the binding site for CD59-C9 interactions. These residues correspond to amino acid residues 42&#x2013;58, and bind to the region of C9 corresponding to human 334&#x2013;418, more specifically, between amino acid residues 359 and 384. Compounds can be derived using this basic amino acid sequence and corresponding three dimensional structure within the protein using any of several techniques known to those skilled in the art, including rational drug design using computer data bases and modeling of peptide/protein-ligand binding, antibodies and anti-idiotypic antibodies generated to the proteins or peptides containing this peptide sequence, and modified peptides. Those compounds imitating the structure and/or function of the peptide region are referred to herein as 'peptidomimetics', and include small molecules which present the surface exposed side chains in these amino acids in the same relative positions, compounds identified by combinatorial chemistry techniques which bind to the active portions of human C9, as well as modified peptides. The compounds can be used to inhibit complement by binding to C9 analogously to CD59, or to maintain complement inhibition, by blocking CD59 binding to C9. The compounds can be administered locally or systemically in any suitable carrier in an amount effective to either inhibit complement or block the inhibition of complement, in a patient in need of treatment thereof.

Compositions and methods to inhibit formation of the C5b-9 complex of complement

Genetically engineered cells are provided which can serve as universal donor cells in such applications as reconstruction of vascular linings or the administration of therapeutic agents. The cells include a coding region which provides protection against complement-based lysis, i.e., hyperacute rejection. In addition, the cell's natural genome is changed so that functional proteins encoded by either the class II or both the class I and the class II major histocompatibility complex genes do not appear on the cell's surface. In this way, attack by T-cells is avoided. Optionally, the cells can include a self-destruction mechanism so that they can be removed from the host when no longer needed.

Universal donor cells

A protein preparation that mediates Ca transbilayer movement of phospholipid is disclosed. Additionally, a modified or mutated protein preparation, wherein the protein has a reduced ability to mediate transbilayer movement, is disclosed. In a preferred form of the invention, the protein has been modified such that post-translational modification can no longer occur.

Methods and compositions to alter the cell surface expression of phosphatidylserine and other clot-promoting plasma membrane phospholipids

A protein preparation that mediates Ca,transbilayer movement of phospholipid is disclosed. Additionally, a modified or mutated protein preparation, wherein the protein has a reduced ability to mediate transbilayer movement, is disclosed. In a preferred form of the invention, the protein has been modified such that post-translational modification can no longer occur.

An protein preparation that mediates Ca,transbilayer movement of phospholipid is disclosed. A recombinantly engineered DNA sequence encoding the protein, an inhibitor of the protein activity, genetically engineered cells with altered protein activity, and therapeutic methods are also disclosed.

DNA encoding phospholipid scramblase

A method and means for protecting cells and transplanted organs from the effects of activated complement proteins generated in blood serum or plasma by introducing the gene for CD59 into the cells to be protected is described. In an example of the method, protection against the pore-forming activity of the human C5b-9 proteins was conferred on CHO cells by transfection with cDNA encoding the human complement regulatory protein CD59.

Genetic inhibition of complement mediated inflammatory response

Pharmaceutical compositions are designed based on the criticality of a portion of C9 for assembly of the C5b9 complex, which specifically modulate binding of CD59 to C9, either molecules structurally mimicking C9 amino acid residues 359 to 384 which bind to CD59 or molecules binding to C9 amino acid residues 359 to 384. Molecules which inhibit CD59 binding include peptides containing residues 359-384 which compete for binding with the other components of the C5b9 complex and anti-idiotypic antibodies immunoreactive with C9 amino acid residues 359 to 384. Molecules which prevent assembly of the C5b-9 complex include antibodies and antibody fragments immunoreactive with amino acid residues 359 to 384 of C9, peptides that bind to amino acid residues 359 to 384 of C9, and nucleotide molecules that bind to amino acid residues 359 to 384 of C9.

C9 complement inhibitor

A composition and methods for use thereof relating to polypeptides having the ability to act as an inhibitor of complement C5b-9 complex activity. The compositions contain an 18 kDa protein found on the surface of human erythrocytes, a 37 kDa protein found on the surface of human platelets, a 37 kDa protein found on the surface of human endothelial cells, active derivatives or fragments thereof which act to inhibit the activity of C5b-9, anti-idiotypic antibodies mimicking the action of the inhibitor proteins or antibodies against C7 or C9 which block the formation of the C5b-9 complex. The compositions can be used in vitro to inhibit C5b-9 related stimulatory responses of platelets and vascular endothelium of perfused organs and tissues, thereby preventing the C5b-9 initiated cell necrosis or stimulated secretion of proteolytic enzymes and the exposure of the procoagulant membrane receptors during collection and in vitro storage. Further, immune disease states can be treated by administering an effective amount of a C5b-9 inhibitor to suppress C5b-9 mediated platelet activation in vivo.

Inhibition of complement mediated inflammatory response

Method of inhibition of complement mediated inflammatory response

Compositions and methods for use thereof relating to monoclonal antibodies, and fragments thereof, having inhibitory activity towards the cell-activating function of the complement C5b-9 complex. The compositions can be used in vitro to inhibit C5b-9 related stimulatory responses of platelets and/or endothelial cells, thereby preventing C5b-9-initiated cell necrosis or stimulated secretion of proteolytic enzymes and the exposure of the procoagulant membrane receptors during collection and in vitro storage. Further, disease states can be treated by administering to platelets and/or endothelial cells in vivo an effective amount of a monoclonal antibody, or fragment thereof, which has inhibitory activity towards the cell-activating function of the C5b-9 complex, in a pharmaceutically acceptable carrier.

Inhibition of complement mediated inflammatory response using monoclonal

A method and means for protecting cells and transplanted organs for the effects of activated complement proteins generated in blood serum or plasma by introducing the gene for CD59 into the cells to be protected is described. In an example of the method, protection against the pore-forming activity of the human C5b-9 proteins was conferred on CHO cells by transfection with cDNA encoding the human complement regulatory protein CD59.

Cells expressing high levels of CD59

A composition and methods for use thereof relating to polypeptides having the ability to act as an inhibitor of complement C5b-9 complex activity. The compositions contain an 18 kDa protein found on the surface of human erythrocytes, active derivatives or fragments thereof which act to inhibit the activity of C5b-9, anti-idiotypic antibodies mimicking the action of the inhibitor proteins or antibodies against C7 or C9 which block the formation of the C5b-9 complex. The compositions can be used in vitro to inhibit C5b-9 related stimulatory responses of platelets and vascular endothelium of perfused organs and tissues, thereby preventing the C5b-9 initiated cell necrosis or stimulated secretion of proteolytic enzymes and the exposure of the procoagulant membrane receptors during collection and in vitro storage. Further, immune disease states can be treated by administering an effective amount of a C5b-9 inhibitor to suppress C5b-9 mediated platelet activation in vivo.

A composition and methods for use thereof relating to polypeptides having the ability to act as an inhibitor of complement C5b-9 complex activity. The compositions contain an 18 kDa protein found on the surface of human erythrocytes, a 37 kDa protei

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Mequon, WI, United States of America

Peter J Sims