Coagulation factor binding proteins and uses thereof

A blood coagulation factor and binding protein technology, applied in the direction of anticoagulation factor immunoglobulin, coagulation/fibrinolytic factor, VII factor, etc., can solve the problem of prolonging and short half-life of blood coagulation factors, and achieve good safety effect

Inactive Publication Date: 2019-07-16
CSL LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite these efforts, prolongation of clotting factor half-life re...

Method used

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  • Coagulation factor binding proteins and uses thereof
  • Coagulation factor binding proteins and uses thereof
  • Coagulation factor binding proteins and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0874] Example 1: Generation and Purification of Recombinant Antibodies

[0875] Expression constructs are generated using standard molecular biology methods. Nucleotides encoding the antibody, Annexin A5 (NP_001145; SEQ ID NO: 14) and GS linker (SEQ ID NO: 20) were synthesized by Geneart (Thermo Fisher Scientific, NY, USA) sequence. The sequences were amplified by PCR, digested by restriction enzymes and cloned into expression vectors by T4 DNA ligase.

[0876] Antibodies were generated according to Table 1 below.

[0877] Table 1: Recombinant Antibodies

[0878]

[0879]

[0880] Recombinant plasmid DNA was purified using the QIAprep Spin Miniprep kit (QIAGEN, Hilden, Germany) and quantified by Nanodrop UV spectrophotometer. Confirm the sequence before transfection.

[0881] According to the manufacturer's instructions, use the Expi293F TM Expression Systems All transfections were performed by transient transfection.

[0882] Proteins were harvested from the res...

Embodiment 2

[0883] Example 2: Annexin A5-linked antibodies are membrane-targeted

[0884] To assess whether the Annexin A5-linked antibody targets the cell membrane, a biosensor analysis of the Annexin A5-linked antibody was performed. Briefly, a phosphatidylserine (PS) / phosphatidylcholine (PC) / phosphatidylethanolamine (PE) (PS / PC / PE-biotinyl 70:25:5) phospholipid mixture was dissolved in TRIS [20mM] pH 8.0, NaCl [150 mM], NOG [2 mM], and vesicles were obtained using sonication. Phospholipids lacking phosphatidylserine (PC / PE-biotinyl 95:5) were prepared in a similar manner as reference surfaces in biosensor studies.

[0885] Phosphatidylserine-containing vesicles were immobilized at low levels on an SA sensor chip-docked On the active flow cell of the T-200 biosensor. Vesicles lacking PS were immobilized on upstream reference cells. Binding to PS / PC / PE was assessed by injecting 3.3, 1.1, 0.37, 0.12 and 0.04 nM aFIX-Annexin A5 (CSL4060) for 5 minutes at 37°C. The data ...

Embodiment 3

[0887] Example 3: Membrane Targeting Anti-Factor IX Monospecific Whole and Half Antibodies Have Enhanced Factor VII Bypassing Activity Compared to Non-Targeting Antibodies

[0888] In order to study the potential factor VIII-bypass activity and coagulation activity of the generated antibodies, the Thrombolyzer Compact X system (Behnk Elektronik, Inc.) with standard assay reagents from Siemens Healthcare (Siemens, Germany) was used according to the manufacturer's instructions. Germany) measures the activated partial thromboplastin time (aPTT). Antibodies were diluted in FVIII deficient plasma (Siemens Healthcare) as indicated to achieve final concentrations ranging from 1000 nM to 1 pM. Briefly, 50 μl of each dilution was mixed with 50 μl of aPTT reagent (Pathromtin SL) on one side of a Thrombolyzer cuvette. 50ul of CaCl 2 [25mM] was added to the other side of the Thrombolyzer cuvette and allowed to equilibrate to 37°C. by adding CaCl 2 The solution was mixed with an antibo...

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Abstract

A membrane targeted binding protein that binds to at least one blood coagulation factor, wherein the binding protein has pro-coagulant activity.

Description

[0001] relevant application data [0002] This application claims Australian Patent Application No. 2016903858, filed 23 September 2016, entitled "Coagulation factor binding proteins and uses thereof" and filed 20 June 2017, entitled "Coagulation factor binding proteins and uses thereof". Priority to Australian Patent Application No. 2017902352 for "Coagulationfactor binding proteins and uses thereof". [0003] sequence listing [0004] This application is filed together with a Sequence Listing in electronic format. The entire content of the Sequence Listing is incorporated herein by reference. technical field [0005] The present disclosure relates to coagulation factor binding proteins and uses thereof. Background technique [0006] Normal blood coagulation is a highly conserved process in mammalian biology that involves complex physiological and biochemical processes, including activation of the coagulation factor (or clotting factor) cascade, culminating in fibrin for...

Claims

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Application Information

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IPC IPC(8): C07K16/36C07K14/755A61K38/37A61K47/28
CPCC07K14/4721A61K38/00A61K2039/505C07K2317/31C07K2317/76C07K2319/035C07K16/36A61P7/02A61P7/04
Inventor P·施密特陈朝广C·帕诺西斯L·法布里
Owner CSL LTD
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