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Monoclonal antibodies against tissue factor pathway inhibitor (TFPI)

A monoclonal antibody, tissue factor technology, applied in the direction of protease inhibitors, antibodies, protease inhibitor immunoglobulins with anti-peptide structure, etc., can solve problems such as long duration

Inactive Publication Date: 2011-08-03
BAYER HEALTHCARE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, chronic prophylactic treatment, such as would be required for hemophilia treatment with anti-TFPI monoclonal antibodies, has a high risk of developing an immune response to the treatment if antibodies with murine components or of murine origin are used, which is Due to the frequent dosing and long duration of therapy required

Method used

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  • Monoclonal antibodies against tissue factor pathway inhibitor (TFPI)
  • Monoclonal antibodies against tissue factor pathway inhibitor (TFPI)
  • Monoclonal antibodies against tissue factor pathway inhibitor (TFPI)

Examples

Experimental program
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Effect test

Embodiment 1

[0181] Example 1: Panning and screening of human antibody library against human TFPI

[0182] Panning Human Antibody Libraries Against TFPI

[0183] Anti-TFPI antibodies were selected by panning the phage display combinatorial human antibody library HuCal Gold (Rothe et al., J. Mol. Biol., 2008, 376: 1182-1200) against human TFPI (American Diagnostica). Briefly, 200 μl of TFPI (5 μg / ml) was coated overnight at 4° C. on 96-well Maxisorp plates, and then the plates were blocked with PBS buffer containing 5% milk. After washing the plates with PBS containing 0.01% Tween-20 (PBST), aliquots of the combinatorial human antibody library were added to the TFPI-coated wells and incubated for 2 hours. Unbound phage were washed away with PBST, and antigen-bound phage were diluted with dithiothreitol, infected, and amplified in E. coli strain TG1. Phage are rescued by helper phage for the next round of panning. A total of three rounds of panning were performed and clones from the first...

Embodiment 2

[0195] Embodiment 2: Expression and purification of anti-TFPI antibody

[0196] Anti-TFPI antibodies were expressed (as Fab fragments) and purified from bacterial strain TG1. Briefly, single clones of bacterial strain TG1 containing antibody expression plasmids were picked and grown overnight in 8 ml 2x YT medium in the presence of 34 μg / ml chloramphenicol and 1% glucose. A volume of 7 ml of culture was transferred to 250 ml of fresh 2x YT medium containing 34 μg / ml chloramphenicol and 1% glucose. After 3 hours of incubation, 0.5 mM IPTG was added to induce Fab expression. The incubation was continued overnight at 25°C. Centrifuge the culture to pellet the bacterial cells. The pellet was then resuspended in Bug Buster lysis buffer (Novagen). After centrifugation, filter the supernatant of the bacterial lysate. Fab fragments were affinity purified by Ni-NTA columns (Qiagen) according to the manufacturer's instructions.

Embodiment 3

[0197] Example 3: EC of Anti-TFPI Antibodies 50 and determination of binding affinity

[0198] Use of purified Fab antibodies to determine the EC of anti-TFPI antibodies against human or mouse TFPI 50 . EC was assessed in an ELISA similar to that described above 50 . Use SoftMax to analyze the results. The binding affinity of anti-TFPI antibodies was determined in a Biacore assay. Briefly, antigens (either human or mouse TFPI) were immobilized on CM5 chips using an amine coupling kit (GE HealthCare) according to the manufacturer's instructions. The amount of immobilized TFPI was adjusted relative to the antigen mass to give approximately 300 RU. Antibody Fabs were analyzed in the mobile phase and at least five different concentrations (0.1, 0.4, 1.6, 6.4 and 25 nM) of purified antibody were used in the Biacore assay. Kinetics and binding affinities were calculated using Biacore T100 evaluation software.

[0199] As shown in Table 3, 24 anti-TFPI Fabs showed various ECs...

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Abstract

Isolated monoclonal antibodies that bind human tissue factor pathway inhibitor (TFPI) and the isolated nucleic acid molecules encoding them are provided. Pharmaceutical compositions comprising the anti-TFPI monoclonal antibodies and methods of treating deficiencies or defects in coagulation by administration of the antibodies are also provided. Methods of producing the antibodies are also provided.

Description

[0001] Sequence Listing Submission [0002] The Sequence Listing pertaining to this application is filed electronically via the EFS web and is hereby incorporated by reference in its entirety into the specification. The name of the text file containing the sequence listing is MSB7329PCT_Sequence_Listing_ST25. field of invention [0003] Isolated monoclonal antibodies and fragments thereof that bind human tissue factor pathway inhibitor (TFPI) and related inventions are provided. Background of the invention [0004] Blood coagulation is the process by which blood forms stable clots to stop bleeding. This process involves a number of zymogens and cofactors (or "coagulation factors") circulating in the blood. Those zymogens and cofactors interact via several pathways that convert them to the active form sequentially or simultaneously. Ultimately, the process results in the activation of prothrombin to thrombin by activated Factor X (FXa) in the presence of Factor Va, ionized...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/36C12P21/08
CPCC07K2319/00C07K2317/21C07K2317/92A61K2039/505A61K39/3955C07K2317/56C07K16/38C07K2317/76C07K2317/55C07K2317/52C07K2317/33C07K2317/565A61P33/02A61P43/00A61P7/04A61K2300/00A61K39/00A61K39/395C07K14/811C07K16/00C07K16/36
Inventor 王卓智约翰·E·默菲潘峻亮蒋海燕刘冰
Owner BAYER HEALTHCARE LLC
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