Oklahoma Medical Research Foundation

University of Illinois

Howard Hughes Medical Institute

Naomi L. Esmon

Charles T. Esmon

Fletcher B., Jr. Taylor

Charles T Esmon

Jun Xu

Xiaomei Zhang

Kenji Fukudome

Naomi L Esmon

Shinichiro Kurosawa

Mikhail D Smirnov

Philip C Comp

Fletcher B Taylor, Jr

Deborah J Stearns-Kurosawa

Craig W Carson

Alireza Rezaie

James H Morrissey

Donald S Houston

Patricia C Y Liaw

Gary L Ferrell

Deborah J Stearns

Jian-Ming Gu

Jr Taylor Fletcher B

Hyper-inflammatory responses can lead to a variety of diseases including sepsis. It is now shown that extracellular histones released in response to inflammatory challenge are mediators contributing to endothelial dysfunction, organ failure and death during sepsis. As such, they can be targeted pharmacologically by inhibitors, as well as used as biomarkers for prognosis of sepsis and other diseases.

Extracellular histones as biomarkers for prognosis and molecular targets for therapy

The present invention provides monoclonal antibodies that selectively bind to and inhibit activated protein C without binding to or inhibiting unactivated protein C. Other antibodies inhibit both activated protein C and activation of unactivated protein C. Methods of treatment employing these antibodies are described herein as are methods of screening for and detecting these antibodies.

Methods of treating hemophilia using monoclonal antibodies against activated protein C

Monoclonal antibodies against activated protein C

A Cadependent recombinant antibody that specifically binds to a specific twelve peptide sequence (E D Q V D P R L I D G K) in the activation region of the Protein C has been constructed. The antibody does not bind to Activated Protein C and can be used to inhibit activation of Protein C by thrombin-thrombomodulin, in purification of Protein C, and in treatment of tumors.

Calcium binding recombinant antibody against protein C

Human protein C and activated protein C were shown to bind to endothelium specifically, selectively and saturably (Kd=30 nM, 7000 sites per cell) in a Cadependent fashion. Expression cloning revealed a 1.3 kb cDNA that coded for a novel type I transmembrane glycoprotein capable of binding protein C. This protein appears to be a member of the CD1/MHC superfamily. Like thrombomodulin, the receptor involved in protein C activation, the endothelial cell protein C receptor (EPCR) function and message are both down regulated by exposure of endothelium to TNF. Identification of EPCR as a member of the CD1/MHC superfamily provides insights into the role of protein C in regulating the inflammatory response, and determination of methods for pharmaceutical use in manipulating the inflammatory response.

Cloning and regulation of an endothelial cell protein C/activated protein C receptor

A highly specific monoclonal antibody, produced by a hybridoma cell has been found. A new assay, highly specific for activated protein C (APC) has been developed which will permit rapid determination of APC levels in clinical situations.

Assay for rapid detection of human activated protein C and highly specific monoclonal antibody therefor

Endothelial protein C receptor (EPCR) is found primarily on endothelial cells of large vessels. EPCR translocates from the plasma membrane surface to the nucleus. Molecules which bind to EPCR can be carried from the plasma membrane surface to the nucleus. These molecules include antibodies to EPCR and activated protein C. Protein C, which also binds to EPCR, can be internalized by endothelial cells, but does not enter the nucleus. Thus, EPCR translocation from the plasma membrane to the nucleus provides a means of delivering nucleic acid such as DNA, proteins such as transcription factors, diagnostic agents or other types of drugs to the nucleus of endothelial cells, particularly those on large blood vessels. Conjugates of the materials to be delivered to the nucleus can be formed by ionic or covalent coupling. For example, proteins, including fusion proteins, can be directly conjugated to an anti-EPCR monoclonal antibody. Covalent attachment of positively charged polymers, such as polylysine, to an anti-EPCR antibody allows nucleic acid to bind by ionic charges. Steptavidin and biotin can also be used to conjugate molecules to anti-EPCR antibodies. These conjugated antibodies are transported to the nucleus by EPCR. Eamples demonstrate selective transport to the nucleus which is mediated by EPCR. Molecules transported include activated protein C, antibodies to EPCR, and steptavidin-biotin conjugates. Modification of anti-EPCR monoclonal antibodies by covalently coupling to polylysine allows binding of an expression vector to the modified antibody and translocation to the nucleus.

Targeting of molecules to large vessel endothelium using EPCR

Compositions, and methods of use thereof, for the inhibition of tumor growth and killing of solid tumors wherein the active agent is a compound blocking the Protein C system, preferably anti-Protein C antibody, anti-Protein S antibody, and C4b binding protein. In the most preferred embodiment, the Protein C blocking compound is provided in combination with a cytokine such as tumor necrosis factor (TNF), gamma interferon, interleukin-1, interleukin-2,p or granulocyte-macrophage colony stimulating factor, or a compound eliciting cytokine production such as endotoxin. Examples are provided demonstrating the administration of the Protein C blocking compound, alone or in combination with TNF or endotoxin, to animals having canine veneral tumors, a fibrosarcoma, and an adenocarcinoma, followed by significant tumor reduction.

Inhibition of tumor growth by blockade of the protein C system

Human protein C and activated protein C were shown to bind to endothelium specifically, selectively and saturably (Kd&equals;30 nM, 7000 sites per cell) in a Ca dependent fashion. Expression cloning revealed a 1.3 kb CDNA that coded for a novel type I transmembrane glycoprotein capable of binding protein C. This protein appears to be a member of the CD1/MHC superfamily. Like thrombomodulin, the receptor involved in protein C activation, the endothelial cell protein C receptor (EPCR) function and message are both down regulated by exposure of endothelium to TNF. Identification of EPCR as a member of the CD1/MHC superfamily provides insights into the role of protein C in regulating the inflammatory response, and determination of methods for pharmaceutical use in manipulating the inflammatory response.

Small, buffer soluble polypeptides having amino acid structures corresponding to residues 234-486, 310-486, and 407-486, of thrombomodulin and functionally equivalent analogs thereof inhibiting the clotting activity of thrombin and increasing protein C activation. The polypeptides can be coated onto the surface of articles adapted for contacting mammalian blood to render the surface non-thrombogenic. In pharmaceutical compositions, the polypeptides act as a natural anticoagulant.

Thrombin binding polypeptides

The promoter of the EPCR gene has been isolated from both murine (SEQ. ID No. 1) and human (SEQ. ID No. 2) genomic libraries. The promoter includes a region (nucleotides 3130 to 3350 of SEQ. ID No. 1 which affects selective gene expression in endothelial cells), and a region (nucleotides 2270 to 2840 of SEQ. ID No. 1) which affects selective gene expression in large vessel endothelial cells, as compared to expression in all endothelial cells. The EPCR promoter contains a thrombin responsive element, CCCACCCC (SEQ. ID No. 3), (murine, nucleotides 3007 to 3014 SEQ. ID No. 1 and human, nucleotides 2722 to 2729 SEQ. ID No. 2). The EPCR also contains a serum response element (nucleotides 2990 to 3061 of SEQ. ID No. 1). The regulatory sequences present in the EPCR promoter can be used for thrombin or serum controlled recombinant gene expression specific to either all endothelial cells or primarily endothelial cells of large vessels. Therapeutic strategies include the use of the minimal promoter for expression of therapeutic agents during times of increased thrombin/platelet activation or regional trauma in all endothelial cells or the use of the large vessel specific promoter for regional specific expression in the endothelial cells of large vessels for use in delivery.

Endothelial specific expression regulated by EPCR control elements

Plasma EPCR has been isolated, characterized and shown to block cellular protein C activation and APC anticoagulant activity. Plasma EPCR appears to be about 43,000 daltons and circulates at approximately 100 ng/ml (98.4.+-.27.8 ng/ml, n=22). Plasma EPCR bound activated protein C with an affinity similar to that of recombinant soluble EPCR (Kd.sub.app approximately 30 nM), and inhibits both protein C activation on an endothelial cell line and APC anticoagulant activity in a one-stage factor Xa clotting assay. Soluble plasma EPCR appears to attenuate the membrane-bound EPCR augmentation of protein C activation and the anticoagulant function of activated protein C. Soluble EPCR has also been detected in urine. Levels of soluble EPCR can rise in inflammatory disease associated with vascular injury and appear to be correlated with inflammation and disease states associated with abnormal coagulation. Since EPCR expression is restricted to larger vessels and is usually negative in cappillaries, these observations provide a mechanism for analyzing injury/stimulation of large vessel endothelial cells.

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Oklahoma City, OK, United States of America

Charles T Esmon