Bioactive purified hspe7 compositions

A technology of biological activity and composition, applied in the field of immunology, can solve problems such as poor immunogenicity

Inactive Publication Date: 2009-07-22
NVENTA BIOPHARMACEUTICALS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the success of this treatment is often limited by several deficiencies inherent in immunotherapy regimens, for example, poor immunogenicity of selected cytotoxic T lymphocyte (CTL) epitopes

Method used

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  • Bioactive purified hspe7 compositions
  • Bioactive purified hspe7 compositions
  • Bioactive purified hspe7 compositions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Preparation of HspE7

[0101] The Hsp65-E7 fusion (HspE7) was obtained as described in WO99 / 07860 (which is incorporated herein by reference). HspE7 is a fusion protein that includes the entire HPV16E7-coding region inserted at the carboxy-terminal end of the Hsp65 gene (pET65H). This HspE7 is called method AHspE7, and can be obtained from Nventa Biopharmaceuticals Corporation as required.

[0102]Before use, HspE7 was purified to a purity higher than 95%. A seed culture of E. coli expressing HspE7 was used to inoculate 250 L of fermentation medium. During the fermentation process, yeast extract and glucose are added as nutrients, and pure oxygen is emitted into the fermentation vessel to provide sufficient ventilation. The expression of HspE7 was induced by adding IPTG (isopropyl-β-D-thiogalactopyranoside). The contents of the fermenter are then cooled to below 20 and the cell paste is collected by centrifugation. The cytoplasm is resuspended in a buffer contain...

Embodiment 2

[0104] Example 2: Determination of the biological activity of HspE7 preparations

[0105] Antigen-specific stimulation of IFN-γ production by splenocytes: ELISPOT analysis

[0106] In the presence of E7 peptide by ELISPOT (Asai, T. et al., 2000, Clin. Diagn. Lab. Immunol. 7(2): 145-154), HspE7 induction of E7-specific CD8-positive T lymphocytes ( The enhancement of the ability of IFN-γ to produce cells): The mice were immunized with HspE7 (with or without adjuvant) subcutaneously at the nape of the neck in a total volume of 200 μl. After five to seven days, the mice were sacrificed, and their spleens were taken and processed into a single cell suspension. The cells were seeded in complete RPMI on a microporous filter plate pre-coated with anti-mouse IFN-γ antibody. The microfiltration plate was incubated at 37 for 20 hours. The cells were washed away, and IFN-γ spots were detected by incubating the microporous filter plate with the biotinylated secondary antibody mouse IFN-γ antib...

Embodiment 3

[0110] Example 3: The effect of TLR9 agonist CpG on HspE7

[0111] The enhancement of the ability of HspE7 to induce E7-specific CD8-positive T lymphocytes was determined in the presence of CpG oligonucleotides (TLR9 ​​agonists). Use separate HspE7 (400μg method A HspE7 or 400μg method L HspE7) or HspE7 (400μg method A HspE7 or 400μg method L HspE7) prepared by two different purification methods plus 30μg of CpG (TCC ATG ACG TTC CTG ATG CT; SEQ ID NO: 1; obtained from Invitrogen, including phosphorothioate backbone and designated as: ZOO FZE FOE ZZO OZE FZE OT) Naive C57B1 / 6 mice were injected subcutaneously as described in Example 2. Five days later, the spleen was removed from the mouse and used E7 specific class I MHC binding peptide E7 49-57 (RAHYNIVTF; Dalton Chemical Laboratories) or control peptide HBCAg 93-100 (MGLKFRQL; Dalton Chemical Laboratories) was used as a recall antigen to measure the number of E7-specific splenocytes by ELISPOT.

[0112] figure 2 The results sh...

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Abstract

A method of increasing the biological activity of a purified Hsp65 - E7 fusion protein (HspE7) is provided. The method comprises admixing the HspE7 along with an immune stimulant selected from the group consisting of CpG, a TLR3 agonist such as PoIyLC, PoIyICLC, mono-phosphoryl-lipid A (MPL), MPL- trehalose 6,6'-dimycolate (MPL-TDM), and anti-CD40. A composition comprising HspE7 and one or more than one of CpG, a TLR3 agonist such as PoIyLC, PoIyICLC, MPL, MPL-TDM, and anti-CD40, and method of reducing a tumor or virus development in a mammal or subject in need thereof by using the composition are also provided.

Description

Technical field [0001] The present invention relates to the field of immunology. Furthermore, the present invention provides a composition comprising HspE7 and a method of use thereof. Background technique [0002] Vaccination and immunotherapy refer to the manipulation of a series of complexly designed cell interaction sequences. Cell interactions include immune surveillance, so that in general, antigen-presenting cells (APC) and specifically dendritic cells (DC) meet and take up the antigen, generate peptide epitopes from the antigen, and load the epitope into the main tissue In the recognition cleft of the molecule encoded by the compatibility complex (MHC). After being exported to the surface of DC, the epitope-loaded MHC molecules present the epitope-MHC complex to T cells and activate T cells. Activated CD4+T helper (Th) cells transmit chemokine and cytokine signals to other DCs, enabling them to activate naive CD8+ T cells, thereby transforming these cells into antigen-spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/12A61P31/20A61P35/00
CPCA61K2039/55572C07K2319/00A61K39/12C12N2710/20034A61K2039/55561A61K2039/55505C12N2710/20022C12N7/00A61K2039/55566C07K14/005A61K39/39A61K2039/545A61K2039/585A61K2039/6043A61P31/12A61P31/20A61P35/00A61P37/04
Inventor G·罗斯J·韦布M·西格尔P·埃姆塔格
Owner NVENTA BIOPHARMACEUTICALS CORP
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