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Coagulation factor-targeting to tlt-1 on activated platelets

a coagulation factor and activated platelet technology, applied in the field of procoagulant proteins and polynucleotides, can solve the problems of threatening bleeding, spontaneous bleeds, and dysfunctional coagulation cascade steps

Inactive Publication Date: 2013-12-19
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a group of proteins that are specifically targeted to activated platelets, which are present at sites of injury. These proteins are made by combining a coagulation factor with an antibody that binds to a specific receptor on the surface of activated platelets. The coagulation factor is either a serine protease or a derivative thereof, and the antibody is specific to the receptor. These proteins can enhance coagulation on the surface of activated platelets and can be used as a medicament to treat coagulopathies.

Problems solved by technology

In subjects with a coagulopathy, such as human beings with haemophilia A, B or C, various steps of the coagulation cascade are rendered dysfunctional due to, for example, the absence or insufficient presence of a functional coagulation factor.
Such dysfunction of one part of coagulation results in insufficient blood coagulation leading to spontaneous bleeds e.g. in joints and potentially life-threatening bleeding.

Method used

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  • Coagulation factor-targeting to tlt-1 on activated platelets
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  • Coagulation factor-targeting to tlt-1 on activated platelets

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning and Expression of hTLT-1 ECD-his Antigen

[0570]Nucleotide sequences encoding the extracellular domain of human TLT-1 (hTLT-1) (FIG. 1) together with a C-terminal His-6 tag were PCR amplified with a forward primer containing a HindIII recognition site together with a kozak sequence, and a reverse primer containing a stop codon and an EcoRI recognition site (FIG. 2). The HindIII- and EcoRI digested PCR fragment was inserted into the HindIII- and EcoRI sites of a pTT-based expression vector. The pTT vector is essentially described in Durocher, Y. et al., (2002) Nucleic Acid Res, 30: E9. The resulting expression plasmid was designated pTT-hTLT-1 ECD-His. The nucleotide and amino acid sequences for hTLT-1 ECD-His is shown in SEQ ID NO: 3 and 4. pTT-hTLT-1 ECD-His was transfected into HEK293-6E suspension cells in order to transiently express hTLT-1 ECD-His. HEK293-6E cells were grown in Freestyle HEK293 medium (GIBCO, cat. no. 12338-018) supplemented with 1% P / S (GIBCO cat. no. 15...

example 2

Purification and Characterisation of hTLT-1 ECD-his Protein

[0571]Purification of the hTLT-1 ECD-His protein was conducted as a 2-step process composed of 1) His-affinity chromatography using the Cobalt-loaded resin TALON (Clontech, cat. no. 635506) and 2) anion-exchange chromatography using the fine-particle resin Source 15Q (GE Healthcare, cat. no. 17-0947). The purifications were conducted using an ÄktaExplorer chromatography system (GE Healthcare, cat. no. 18-1112-41). The buffer systems used for the first purification step was an equilibration buffer composed of 20 mM Hepes, pH 7.0, 150 mM NaCl, a wash buffer composed of 20 mM Hepes, pH 7.0, 0.5 M NaCl and an elution buffer composed of 20 mM Hepes, pH 7.0, 150 mM Imidazole. The cell supernatant was applied directly without any adjustments onto a pre-equilibrated TALON column. The column was washed with 20 column volumes of equilibration buffer, 20 column volumes of wash buffer and last with 20 column volumes of equilibration buf...

example 3

Preparation of Monoclonal TLT-1 Antibodies

[0573]RBF mice were immunized by injecting 50 μg of hTLT-1 ECD-His. FCA subcutaneously followed by two injections with 20 μg of hTLT-1 ECD-His in FIA. High responder mice were boosted intravenously with 25 μg of hTLT-1 ECD-His and the spleens were harvested after 3 days. Spleen cells were fused with the myeloma Fox cell line. Supernatants were screened for hTLT-1 specific antibody production in a specific ELISA and in a FACS assay utilizing hTLT-1- or Mock-transfected CHO cells as positive and negative target cells, respectively. A secondary screen was done on resting versus dual agonistic activated platelets of human, cynomolgous monkey, dog, rabbit or mouse origin.

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Abstract

The current invention relates to: procoagulant proteins which may, for example, be fusion proteins or chemical conjugates; methods of producing said procoagulant proteins; polynucleotides that encode said fusion proteins and cells that expresses them. Furthermore, the current invention relates to procoagulant proteins for use as a medicament. Individuals that have a coagulopathy, such as haemophilia A and B with or without inhibitors, may be treated with the procoagulant proteins of the current invention.

Description

FIELD OF THE INVENTION[0001]The current invention relates to procoagulant proteins, polynucleotides that encode procoagulant fusion proteins, cells that express procoagulant fusion proteins, a process for preparing procoagulant proteins and uses of said procoagulant proteins.BACKGROUND OF THE INVENTION[0002]Normal resting platelets freely flow throughout the blood circulation when the endothelium is intact. When the single-layered endothelial barrier is damaged, resting platelets adhere to subendothelial structures by means of glycoprotein (GP) receptors. For example, GPIaIIa and GPVI bind collagen; GPIcIIa binds fibronectin; GPIcIIa binds laminin and GPIb-V-IX binds von Willebrand Factor (vWF) polymers. Adhesion to the extravascular tissue components exposed following a vessel injury in conjunction with the influence of factors produced locally at the site of injury, e.g. the serine protease thrombin, lead to activation of the platelets. In the complex process of activation, platel...

Claims

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Application Information

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IPC IPC(8): C12N9/96
CPCC07K16/2803C12N9/96C07K2317/34C07K2317/55C07K2317/92C07K2319/00C07K2319/21C07K14/4703C12Y304/21021C12Y304/21022C12N9/6437C12N9/644A61K38/00C07K14/745C07K2299/00A61K47/6815A61K47/6849A61K38/4846A61P7/04A61K38/36C07K2317/21C07K2317/24C07K2317/41C07K2317/51C07K2317/515C07K2317/56
Inventor HILDEN, IDAPESCHKE, BERNDBREINHOLT, JENSKOFOD-HANSEN, MIKAEL
Owner NOVO NORDISK AS
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