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Therapeutic uses of fibrinogen gamma prime variants

a fibrinogen and prime variant technology, applied in the field of fibrinogen variants comprising a prime chain, can solve the problem that it is not likely that the amount of fibrinogen variants is sufficien

Inactive Publication Date: 2020-06-25
FIBRIANT BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent is about using fibrinogen variants, specifically the pFib γ′ variant, to prevent or treat infections. The technical effect of this invention is that it provides a readily available alternative to the normal fibrinogen of the host that can still have an effect on bacterial virulence in the presence of WT fibrinogen. This is important because while several pathogens have evolved ways to interact with host fibrinogen in order to enhance pathogen virulence, the pFib γ′ variant has been shown to have an effect on bacterial cell wall-anchored clumping factor A (ClfA) and to induce fibrinogen-dependent clumping of bacteria. The invention also addresses the issue of limited availability of the pFib γ′ variant in plasma.

Problems solved by technology

It is not likely that sufficient amounts of this fibrinogen variant for medical use may be obtained from plasma.
In addition, the effect of the modified fibrinogen in Fib γΔ5 / Δ5 mice on bacterial binding, clumping and virulence is established without the presence of any WT fibrinogen and therefore does not demonstrate that this approach would be useful as a therapeutic or prophylactic product that is able to compete with the normal WT fibrinogen of the host and still introduce a clinically meaningful effect in the presence of WT fibrinogen.

Method used

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  • Therapeutic uses of fibrinogen gamma prime variants
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  • Therapeutic uses of fibrinogen gamma prime variants

Examples

Experimental program
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Effect test

example 1

Isolation of Fibrinogen Variants from Plasma

[0093]Total plasma fibrinogen mixture (pFib total) comprising wild type (WT) fibrinogen (pFib γ411 / 411) and fibrinogen gamma prime (pFibγ427 / 411) was obtained from Enzyme Research Labs, Swansea, United Kingdom (FIB 3). This was used as starting material to separate the plasma fibrinogen with two WT gamma polypeptide chains (pFib γ411 / 411) from the plasma fibrinogen variant with one gamma prime polypeptide chain and one WT gamma polypeptide chain (pFib γ427 / 411) using anion exchange chromatography as described in Lawrence et al. (Blood 1993, vol 82, no 8, pp 2406-2413). pFib total, pFib γ411 / 411 and pFib γ427 / 411 were analysed on SDS-PAGE (Example 3) and used in further experiments as described below.

[0094]Mouse fibrinogen from WT mice (mFib γWT / WT) and from homozygous delta5 transgenic mice (mFib γΔ5 / Δ5) was isolated as described by Flick et al. (2013) Blood (121): 1783-1794.

example 2

Recombinant Production of WT Fibrinogen and Homodimer Fibrinogen Gamma Prime

[0095]cDNA sequences encoding the fibrinogen Aα610 chain (Alpha wild type, SEQ ID NO. 4), Bβ chain (SEQ ID NO. 5), 7411 (gamma) chain (SEQ ID NO. 6) and γ427 (gamma prime) chain (SEQ ID NO. 7) were cloned in the pCDNA 3.1 plasmid (Invitrogen, Carlsbad, Calif., USA) to construct expression vectors for the different fibrinogen chains. Combinations of expression plasmids containing cDNA's encoding Aα, Bβ and γ chains were used to produce fully assembled recombinant human WT fibrinogen (rhFib γ411 / 411) and recombinant fibrinogen gamma prime homodimer (rhFib γ427 / 427) by transient expression in HEK 293 cells (Life technologies EXPI293 system) according to the manufacturer's instructions. These recombinant fibrinogen variants were purified using affinity purification on a GPRP column as described in Kuyas C et al. (Thrombosis Haemostasis 1990 Jun. 28; 63 (3): 439-444) and analysed on SDS-PAGE (as described in Exam...

example 3

SDS-PAGE Analysis of Fibrinogen Isolated from Plasma and Recombinantly Produced Fibrinogen

[0096]pFib γ411 / 411 and pFib γ427 / 411 isolated from total plasma fibrinogen (pFib total) were subjected to SDS-PAGE analysis under reducing conditions. About 0.5 to 1.0 microgram of the fibrinogen samples was loaded on the SDS-PAGE gel, run for 1 hour at 200 Volt and stained with Coomassie blue stain. The result is shown in FIG. 4.

[0097]Lane 1: pFib total (total plasma fibrinogen) showed as dominant bands the Aα610, Bβ 461 and γ411. A slightly degraded Aαband is seen just below the Aα610 band and a faint band at the γ427 position is present. Lane 2: pFib γ411 / 411 only contains only the γ411 band at the gamma polypeptide position. Lane 3: pFib γ427 / 411 isolated from pFib total contains wild type Aα610 and Bβ461 polypeptides, but the γ chain polypeptide consists of approximately equal amounts of γ411 and γ427, demonstrating that pFib γ427 / 411 is a heterodimer with respect to the gamma polypeptide...

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Abstract

The present invention relates to compositions comprising fibrinogen gamma prime variants for use in the treatment or prevention of an infection. The fibrinogen gamma prime variants in the composition comprise at least one fibrinogen gamma prime polypeptide chain. The compositions for use according to the invention may also comprise other fibrinogen variants. Compositions comprising fibrinogen gamma prime variants according to the invention improve survival time after infection up to more than 200 percent compared to WT fibrinogen. They may be used both therapeutically and prophylactically.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The subject application is a continuation of PCT Patent Application No. PCT / EP2019 / 060546, filed Apr. 24, 2019, which claims priority to European Patent Application No. 18168996.9, filed on Apr. 24, 2018, and European Patent Application No. 18168993.6, filed on Apr. 24, 2018, all of which are incorporated by reference herein in their entirety for all purposes.TECHNICAL FIELD[0002]The present invention generally relates to the use of fibrinogen variants comprising a gamma prime chain, in particular to their use to prevent or treat infections.BACKGROUND OF THE INVENTION[0003]Fibrinogen is a soluble plasma glycoprotein and a dimeric molecule, consisting of two pairs of three polypeptide chains designated Aα, Bβ and γ, which are connected by disulfide bridges. The three polypeptide chains are encoded by three separate genes and synthesized individually from 3 mRNAs. Assembly of the three polypeptide chains (Aα, Bβ, and γ) into its final form ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/75A61P31/04
CPCA61K45/06A61P31/04A61K9/0019C07K14/75A61K38/00A61K38/36
Inventor WEGGEMAN, MIRANDAGRIMBERGEN, JOSEPHKOOPMAN, JACOB
Owner FIBRIANT BV
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