Monoclonal antibodies against tissue factor pathway inhibitor (TFPI)

A monoclonal antibody, tissue factor technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, anti-peptide structure protease inhibitor immunoglobulin, etc.

Inactive Publication Date: 2014-05-14
BAYER HEALTHCARE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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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
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1. Expression and purification of recombinant TFPI (Kunitz domain 2) from Escherichia coli.

[0111] expression system

[0112] A destination vector named pD Eco5 N (according to Gateway nomenclature) was employed. pD Eco5 N is based on pET-16 b (Novagen) and additionally encodes His 10 and NusA tags and a Gateway cloning cassette for expressing fusion proteins composed of His 10 / NusA and target protein composition.

[0113] The TFPI construct encoding the thrombin cleavage site and Gateway binding site (attBl-5#, attB2-3#, Invitrogen) fused to the N-terminus of Kunitz domain 2 (Lys93 to Phe154, see Uniprot 10646) was cloned into the pD Eco5 N vector, resulting in an expression vector designated pD Eco5 N TFPI KD2. The BL21 DE3 (Novagen) expression strain was used.

[0114] Amino acid sequence of the fusion protein expressed using pD Eco5 N TFPI KD2, 600 AA

[0115]

[0116] sequence component

[0117]

[0118] Express

[0119] BL21 DE3 strain t...

Embodiment 2

[0122] Example 2. Production of recombinant monoclonal antibody Fab A against TFPI, expression and purification in Escherichia coli

[0123] Express

[0124] Fab A was co-expressed using expression vector pET28a and E. coli strain BL21 Star DE3. The light and heavy chain regions encoded on the expression vector were fused to the periplasmic signal sequence at their N-termini, respectively. The heavy chain region also encodes His at its C-terminus 6 Tags are used to purify Fabs. Transformed E. coli strains grown overnight in TB-Instant expression medium were used to self-induce recombinant protein expression (#71491, Novagen). Briefly, 10 mL of transformed E. coli culture (in a 50 mL Falcon tube) was grown as a preculture in LB medium with 30 μg / mL kanamycin for 14 h at 37 °C with agitation. The speed is 180 rpm. Subsequently, 4 Erlenmeyer flasks with 500 mL of TB-Instant overnight expression medium were each inoculated with 2 mL of the pre-culture, and incubated at 30 °C ...

Embodiment 3

[0130] Example 3. Crystallization and X-ray structure determination of the TFPI-Fab A complex

[0131] crystallization

[0132] Co-crystals of TFPI Kunitz domain 2 and monoclonal antibody Fab A were grown at 20°C using the sessile drop method. The protein complex was concentrated to 9 mg / mL and crystallized by mixing equal volumes of protein solution and well solution (15% PEG8000, Tris HCl pH 7.5) as precipitant. Crystals appeared after a day.

[0133] Data Acquisition and Processing

[0134] Crystals were snap frozen in 30% glycerol in crystallization buffer in liquid nitrogen for cryoprotection. Data were collected at beamline BL14.1, BESSY synchrotron (Berlin) on a MAR CCD detector. Index the data and communicate with XDS

[0135]

[0136] Integrate, prepare for scale with POINTLESS (P.R. Evans, (2005) Acta Cryst. D62, 72-82) and scale with SCALA (P.R. Evans, (2005) Acta Cryst. D62, 72-82). The crystal diffracts up to 2.6 ? and has space group P2 1 2 1 2 ...

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Abstract

Isolated monoclonal antibodies that bind to specific epitopes of 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.

Description

[0001] Zhuozhi Wang, John Murphy, Tobias Marquardt, Dieter Moosmayer [0002] Sequence Listing Submission [0003] The Sequence Listing associated with this application is submitted electronically via the EFS web and is hereby incorporated by reference into this specification in its entirety. field of invention [0004] Isolated monoclonal antibodies and fragments thereof that bind human tissue factor pathway inhibitor (TFPI) are provided. Background of the invention [0005] Blood coagulation is the process by which blood forms stable clots to stop bleeding. The process involves many zymogens and cofactors (or "clotting factors") circulating in the blood. Those zymogens and cofactors interact via several pathways that convert them into active forms 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 calcium, and platelets. Activated thrombin in turn...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K39/395
CPCC07K16/38C07K2317/55C07K2317/565C07K2317/34C07K2317/92C07K2317/31C07K2317/21C07K2317/622A61K39/395C07K16/18A61P7/00A61P7/04
Inventor 王卓智J.墨菲T.马夸特D.莫斯迈尔
Owner BAYER HEALTHCARE LLC
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