Antibodies capable of specifically binding two epitopes on tissue factor pathway inhibitor

A bispecific antibody and tissue factor technology, applied in the direction of antibodies, protease inhibitor immunoglobulins with anti-peptide structure, antibody medical components, etc., can solve the problem of not being able to completely neutralize the activity of TFPIα

Active Publication Date: 2015-12-30
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, targeting TFPI with monoclonal antibodies or fragments thereof may cause TFPI to accumulate in the circulation as a result of reduced renal clearance of the TFPI-mAb complex or as a result of other clearance mechanisms due to reduced TFPI-mAb complex formation
Third, it may be necessary to target specific TFPI libraries
However, known antibodies specifically targeting the C-terminal region of TFPIα are unable to completely neutralize TFPIα activity, especially at elevated TFPI concentrations

Method used

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  • Antibodies capable of specifically binding two epitopes on tissue factor pathway inhibitor
  • Antibodies capable of specifically binding two epitopes on tissue factor pathway inhibitor
  • Antibodies capable of specifically binding two epitopes on tissue factor pathway inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0507] Example 1: Immunization, Fusion and Screening

[0508] RBF mice are immunized with human TFPIα (SEQ ID NO: 1) or a fragment of TFPI (eg, corresponding to residues 1-161 of SEQ ID NO: 1). Mice were injected subcutaneously: 20 μg of human TFPI mixed with complete Freund's adjuvant was used for the first injection. For subsequent immunizations, incomplete Freund's adjuvant with the same antigen concentration was used. Ten days after the final immunization, human TFPI-specific antibodies were screened from mouse eye blood by ELISA. Mice with positive serum titers were boosted with 10 [mu]g human TFPI[alpha] or TFPI(1-161) by intravenous injection and sacrificed 3 days later. The spleen was aseptically removed and dispersed into a single cell suspension. Fusion of splenocytes and myeloma cells was performed by the PEG method or by electrofusion. The resulting hybridoma cells were cloned by limiting dilution into microtiter plates. Supernatants from each hybridoma were i...

Embodiment 2

[0510] Example 2: Cloning and sequencing of mouse anti-TFPIKPI-3 / C-terminal specific mAbs TFPI4F110, TFPI22F66 and TFPI22F71

[0511] The heavy and light chain sequences of murine TFPI antibodies: TFPI4F110, TFPI22F66 and TFPI22F71 were cloned and sequenced as described in WO2012 / 001087.

[0512]Total RNA was extracted from hybridoma cells using the RNeasy-MiniKit from Qiagen and used as a template for cDNA synthesis. cDNA was synthesized in a 5'-RACE reaction using the SMART™ RACE cDNA Amplification Kit from Clontech. Subsequent target amplification of HC and LC sequences was performed by PCR using PhusionHotStart polymerase (Finnzymes) and the complete primer mix (UPM) included in the SMART™ RACE kit as forward primers. Reverse primers with the following sequences were used for HC (VH domain) amplification:

[0513] 5'-CTTGCCATTGAGCCAGTCCTGGTGCATGATGG-3' (SEQ ID NO: 17)

[0514] Reverse primers with the following sequences were used for TFPI22F66 and TFPI22F71LC (VL domai...

Embodiment 3

[0520] Example 3: Cloning and sequencing of mouse anti-TFPIKPI-3 / C-terminal specific mAbs 22F74, 41F41, 41F30, 22F132, and 22F79 and cloning and sequencing of mouse anti-TFPIKPI-1 mAbs 2F3, 2F22, 2F45, 1F91, and 2F35.

[0521] The murine heavy and light chain sequences of the anti-TFPI antibody were cloned and sequenced as follows.

[0522] Total RNA was extracted from hybridoma cells using the RNeasy-MiniKit from Qiagen and used as a template for cDNA synthesis. cDNA was synthesized in a 5'-RACE reaction using the SMART™ or SMARTer™ RACE cDNA amplification kit from Clontech. Subsequent target amplification of HC and LC sequences was performed by PCR using PhusionHotStart polymerase (Finnzymes) and the complete primer mix (UPM) included in the SMART™ / SMARTer™ RACE kit as forward primers. Reverse primers with the following sequences were used for HC (VH domain) amplification:

[0523] 5'-CCCTTGACCAGGCATCCCAG-3' (SEQ ID NO: 20)

[0524] Reverse primers with the following sequ...

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Abstract

The application discloses a combination of two monospecific TFPI antibodies, wherein one antibody is capable of specifically binding TFPI (1-181) and the other antibody is capable of specifically binding TFPI (182-276), as well as bispecific anti-TFPI antibodies derived from two such monospecific antibodies. Both the combination of the two monospecific antibodies and the bispecific antibody strongly enhance thrombin generation by neutralising full length TFPI[alpha], even where the concentration of TFPI is abnormally elevated.

Description

field of invention [0001] The present invention relates to epitopes capable of interacting with the N-terminal region (residues 1-181) present on both tissue factor pathway inhibitor alpha (TFPIα) and TFPIbeta (TFPIβ) and the C-terminal portion present only on full-length TFPIα Antibodies and compositions thereof that bind to another epitope within (residues 182-276). The invention also relates to pharmaceutical and therapeutic uses of said antibodies. Background of the invention [0002] In bleeding individuals, tissue factor / factor Vila (TF / FVIIa) complexes initiate coagulation when extravascular TF is exposed to FVIIa in the blood. TF / FVIIa complex formation results in the activation of Factor X (FX) to FXa, which together with activated Factor V (FVa) generates limited amounts of thrombin. Small amounts of thrombin activate platelets, which in turn lead to surface exposure of platelet phospholipids, which supports the assembly and binding of the tenase complex consisti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/38C07K16/46
CPCC07K16/468C07K2317/34C07K2317/55A61K2039/507C07K2317/24C07K2317/53C07K2317/565C07K2317/567C07K2317/76C07K2317/92C07K2317/526A61K2039/505C07K2317/56C07K16/38A61P7/04A61K39/3955C07K2317/31
Inventor B.奧森克罗格M.科福德-汉森I.希登H.海布罗奇彼得森L.C.彼得森
Owner NOVO NORDISK AS
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