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Optimized monoclonal antibodies against tissue factor pathway inhibitor (tfpi)

A monoclonal antibody, tissue factor technology, applied in the direction of antibodies, anti-peptide structure protease inhibitor immunoglobulin, antibody medical components, etc., can solve the problem of long duration

Inactive Publication Date: 2013-02-20
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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  • Optimized monoclonal antibodies against tissue factor pathway inhibitor (tfpi)
  • Optimized monoclonal antibodies against tissue factor pathway inhibitor (tfpi)
  • Optimized monoclonal antibodies against tissue factor pathway inhibitor (tfpi)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Example 1: Cloning, Expression and Quantification of Antibody Expression Levels

[0130] The heavy and light chains of wild-type Fabs 2A8 and 4B7 carrying a c-myc tag and a hexahistidine tag at the C-terminus of the heavy chain were subcloned into the pET28a bacterial expression vector (Novagen / Merck Chemicals Ltd., Nottingham, UK), and transformed into Top 10F cells (Invitrogen GmbH, Karlsruhe, Germany). Alternatively, other bacterial expression vectors (eg, pQE vector system, Qiagen GmbH, Hilden, Germany) and strains (eg, DH5α, Invitrogen GmbH, Karlsruhe, Germany) can be used. Variants were generated by standard oligomer-based site-directed mutagenesis and confirmed by DNA sequencing. Specifically, amino acid residues in or around the complementarity determining regions are modified within the heavy and / or light chains.

[0131] To be able to use wild-type or mutant antibodies as competitors, standard PCR-based techniques were used to remove or replace the epitope t...

Embodiment 2

[0134] Example 2: Determination of activity and cross-species cross-reactivity of generated antibody variants

[0135] To determine the activity of mutated antibody variants against human or mouse TFPI (American Diagnostica, 4900B and R&D Systems, 2975-P1, respectively), equilibrium or competitive ELISA assay formats were used. Briefly, MTP plates (Mesoscale Discovery, L21XA-4 or Nunc maxisorp black (black), 460518), and incubated overnight at 4°C. After washing, plates were blocked with 3% bovine serum albumin (Sigma, A4503) in PBST for 1 hour at room temperature and the washing step was repeated. For antibody binding, 10-25 μl of culture supernatant normalized to their corresponding antibody expression levels (if not indicated otherwise) was added to the plate for 1 hour at RT, followed by washing with PBST. Bound wild-type and variants are then detected by epitope tag-specific antibodies or a competition step is included. For competition, 50-300 nM competitor or free ant...

Embodiment 3

[0136] Example 3: Single and Multiple Amino Acid Substitutions

[0137] Several examples of single amino acid substitutions introduced in the 2A8 heavy or light chain are provided in Table 1. Expression levels of variants were analyzed in quadruplicate in a quantification ELISA. Human and murine TFPI were analyzed for performance in quadruplicate in a competition ELISA after normalization to the corresponding expression levels, and the variant to wild type (wt) ratio was determined. Calculates the error from the standard deviation through error propagation.

[0138] Table 1: Analysis of single amino acid substitutions within 2A8.

[0139]

[0140]

[0141] Some examples of amino acid substitutions combined in the 2A8TFPI antibody are provided in Table 2. Although not every combination is provided in Table 2, it is contemplated that the TFPI antibody may comprise any combination of the modifications provided. Expression levels of variants were analyzed in quadruplicat...

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Abstract

Isolated monoclonal antibodies that bind human tissue factor pathway inhibitor (TFPI) are provided. Isolated nucleic acid molecules encoding monoclonal antibodies that bind TFPI are also contemplated. 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] Zhuozhi Wang, Junliang Pan, Joanna Grudzinska, Christian Votsmeier, Jan Tebbe, Joerg Birkenfeld, Nina Wobst, Simone Brückner, Susanne Steinig, Peter Scholz [0002] Sequence Listing Submission [0003] 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. 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 a stable clot to stop bleeding. This 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) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/395
CPCC07K2319/21C07K2317/565C07K2317/92C07K16/38A61K2039/505C07K2317/76C07K2317/55A61K39/00A61K39/395A61P43/00A61P7/02A61P7/04
Inventor 王卓智潘俊亮J.格鲁德津斯卡C.沃特斯梅尔J.特贝J.伯肯菲尔德N.沃布斯特S.布吕克纳S.施泰尼希P.肖尔茨
Owner BAYER HEALTHCARE LLC
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