Pharmaceutical composition comprising a bacterial cell displaying a heterologous proteinaceous compound

A protein compound and heterologous protein technology, applied to the direction of immobilized on or in biological cells, derived from bacterial medical raw materials, bacterial antigen components, etc., can solve life-threatening problems

Inactive Publication Date: 2008-02-06
ALK ABELLO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After each injection, the patient must remain under medical care for 30 minutes, because of the risk of allergic side reactions, which in principle are extremely rare but can be life-threatening

Method used

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  • Pharmaceutical composition comprising a bacterial cell displaying a heterologous proteinaceous compound
  • Pharmaceutical composition comprising a bacterial cell displaying a heterologous proteinaceous compound
  • Pharmaceutical composition comprising a bacterial cell displaying a heterologous proteinaceous compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1. Chemical crosslinking of β-galactosidase with Lactobacillus by glutaraldehyde

[0159] This example demonstrates the use of the bifunctional cross-linking reagent glutaraldehyde (GLA), which is a 5-carbon dialdehyde, to render protein β-galactosidase from Sulfolobus solfataricus (Pisani F.M. et al. 1990 Eur J Biochem., 187:321-8) chemically cross-links with the cell surface of Lactobacillus plantarum UP1. GLA acts as a crosslinker by forming Schiff bases (-H=N-) with amino groups of proteins. Thus, GLA-mediated cross-linking of β-galactosidase to the bacterial surface is expected to occur between lysine or arginine residues present in the β-galactosidase protein and on or near the surface of the bacterial cell. between accessible lysine or arginine residues.

[0160] The β-galactosidase used for cross-linking studies was obtained by recombinant expression in Escherichia coli using the pET-3a vector system (Invitrogen, CA). Briefly, the lacS gene encoding β...

Embodiment 2

[0161] Example 2. Chemical crosslinking of arabinose isomerase with Lactobacillus by glutaraldehyde

[0162] To confirm that chemical crosslinking of proteins to bacterial cell surfaces is not limited to β-galactosidases, we showed the crosslinking of arabinose isomerase from the thermophilic Thermoanaerobacter mathrani to bacterial cells. Arabinose isomerase converts D-galactose to D-tagatose and was obtained by recombinant intracellular expression in E. coli as described by Jorgensen and co-workers (Jorgensen F et al. 2004, Appl Microbiol Biotechnol 64:816-22). After growth and expression in recombinant E. coli, cells were lysed using a French press. This lysed mixture was centrifuged and the supernatant containing arabinose isomerase was used for subsequent crosslinking experiments. L. plantarum UP1 was grown and washed as described in Example 1. The washed cells (10 10 cells) were incubated with varying amounts of arabinose isomerase-containing lysate with a final conce...

Embodiment 3

[0163] Example 3. Chitosan as a spacer molecule increases the level of β-galactosidase cross-linked with Lactobacillus by glutaraldehyde

[0164] Chitosan, a naturally occurring molecule containing multiple reactive groups, can be used as a spacer molecule to increase the amount of protein bound to the bacterial cell surface by chemical cross-linking. Lactobacillus plantarum cells were grown and washed as described in Example 1, and washed in 10 10 Cells / ml were suspended in M9 buffer, to which 0.5% w / v chitosan 500 kDA (Cognis Deutschland GmbH, Germany) and 0.2% GLA were added with 1 ug / mL or 2 ug / mL β-galactosidase . The effect of chitosan on the crosslinking of β-galactosidase to cells was compared to a control crosslinking reaction in which no chitosan was used. L. plantarum cells were harvested and washed as described in Example 1 and the catalytic activity of β-galactosidase in the washed cell fractions and supernatants of the crosslinking reaction mixture was determin...

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Abstract

The present invention relates to a composition for the manufacture of a medicament comprising living or dead bacteria with controlled amounts of surface-coupled proteins or proteinaceous compounds and a method for the preparation of the composition. The bacterium provides a multivalent heterologous protein display vehicle that may be used in the manufacture of vaccines or medicaments for delivery via the mucosa.

Description

Background of the invention [0001] Mucosal vaccination has received increasing attention in recent years because of: i) the new understanding of immune system mechanisms; ii) the principle of mimicking the infection route of most pathogens, but also because of: iii) the ease of administration and the The need for effective vaccines against new and emerging diseases. Furthermore, the threat of global bioterrorism requires effective vaccines that are easy to produce and can be administered rapidly without trained personnel. [0002] The mucosal immune system is ideal for obtaining an effective immune response because induction at one site in the mucosa can generate a specific response throughout the immune system. Induction at mucosal sites can often also generate a systemic immune response (Huang J. et al., 2004 Vaccine 6:794-801; Verdonck F. et al., 2004 Vaccine 31-32:4291-9). Most pathogens infect their hosts through mucosal surfaces. This fact makes it advantageous to pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/16A61K35/74A61K39/02C12N11/06A61K39/385
Inventor J·格伦廷F·约恩森S·M·马德森H·伊斯拉埃尔森
Owner ALK ABELLO SA
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