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Survivin-directed cancer vaccine therapy

A survivin and vaccine technology, applied in the field of survivin-oriented cancer vaccine therapy, can solve the problems of insufficient immunogenicity, incompatibility, and reduced stability of survivin in vivo

Inactive Publication Date: 2016-08-17
MERCK PATENT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0030] One of the problems to be solved must be the inherent incompatibility of the antigen with the adjuvant with regard to their different permanent net charges (positive net charge of R-DOTAP, and negative net charge of survivin), which leads to reduced stability
Another issue is the insufficient in vivo immunogenicity of survivin in each formulation

Method used

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  • Survivin-directed cancer vaccine therapy
  • Survivin-directed cancer vaccine therapy
  • Survivin-directed cancer vaccine therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0234] Example 1: Material

[0235] The phospholipid substances used in the present invention are listed in Table 1. DOTAP was obtained from Merck Millipore and cholesterol was purchased from Sigma Aldrich. All other lipids were obtained from Lipoid AG. dC-survivin was prepared as described below and consisted of the amino acid sequence of human survivin truncated at the C-terminus for better stability characteristics and better manufacturability. It contains amino acids 2-120 of human survivin (SEQ ID NO: 3), and has the following characteristics: molecular weight 13.8 kDa, isoelectric point 6.0 and melting point 48°C.

Embodiment 2

[0236] Example 2: Engineering transformation of source strain Escherichia coli BL21 (DE3) and preparation of Escherichia coli T7E2 bacterial strain as the expression strain of the truncated survivin used in the present invention

[0237] The source strain Escherichia coli BL21 (DE3) (Novaki) was engineered as follows:

[0238] ·Incoming analysis of Escherichia coli BL21(DE3)

[0239] Changing the rpsL gene of E. coli BL21(DE3) genome to obtain streptomycin resistance phenotype

[0240] Substitution of non-essential DE3 prophage regions with the loxP-cmR–loxP cassette in the context of the E. coli BL21(DE3) STR genome

[0241] Replacement of Rac prophage with synthetic pgl gene in E. coli BL21(DE3)STR L'1DE3:loxP-cmR-loxP genetic background

[0242] Removal of loxP-flanked chloramphenicol markers

[0243] The genome sequence of E. coli BL21(DE3) [gb#CPOO 1509.3] and Artemis software (Rutherford et al., Bioinformatics 16(10):944-5, 2000) were used for in-silico strain engin...

Embodiment 3

[0245] Example 3: Transformation of Escherichia coli T7E with expression plasmid pAL37

[0246] The source plasmid was pET42 (Novaki), which was finally designed into the final expression plasmid pAL37 through pAL28. The plasmid DNA of pLA37 is shown entirely in Figure 20 and represented by SEQ ID NO:4. The restriction sites of the corresponding restriction enzymes are shown in Figure 19 . Plasmid AL37 contains DNA encoding the sequence of SEQ ID NO: 2 (1–120 aa of SEQ ID NO: 1) for a truncated survivin that is released into the medium after its expression, the first methionine residue The base was cleaved, thereby obtaining a representative truncated survivin SEQ ID NO:3 (2-120aa of SEQ ID NO:1).

[0247] For transformation, E. coli T7E2#1 cells were lysed on ice, 1 μl of plasmid al37 was added and incubated on ice for 30 minutes, at 42° C. for 30 seconds, and again on ice for 2 minutes. Then, add 250 μl of antibiotic-free LB Vegitone Broth and incubate at 37 °C for ...

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Abstract

The invention relates to a therapeutically effective truncated survivin and its therapeutic use as vaccine in a liposomal preparation, wherein the survivin fragment is preferably gluconoylated, and said liposomal preparation elicits in-vivo anti-tumor activity. The invention is directed, in more detail, to a cancer vaccine comprising a fragment of human survivin that is specifically effective in conjunction with a lipid adjuvant. The invention relates in more detail to a liposomal vaccine delivery system comprising an active truncated survivin molecule as tumor antigen and a chiral cationic lipid, for example R-DOTAP acting as adjuvant, which is part of the liposome preparation, wherein the liposomal drug delivery system is optimized with regard to its lipid and adjuvant components, physical or physicochemical parameters, and the final therapeutic efficacy of the released truncated survivin molecules. The invention is finally related to a method of increasing CD4+CD8+ T-cell responses in-vivo, thus resulting in an increased anti-tumor activity by providing a liposome preparation comprising said truncated preferably gluconoylated survivin.

Description

technical field [0001] The present invention relates to therapeutically effective truncated survivin and its therapeutic use as a vaccine in liposome formulations, wherein said survivin fragments are preferably gluconoylated, and said liposome formulations Induces antitumor activity in vivo. More specifically, the present invention relates to a cancer vaccine comprising a fragment of human survivin which is particularly effective in combination with a lipid adjuvant. More specifically, the present invention relates to a liposomal vaccine delivery system comprising a truncated survivin molecule as a tumor antigen, and a chiral cationic lipid (such as R-DOTAP) as an adjuvant, which is the , wherein the liposomal drug delivery system is optimized with respect to its lipid and adjuvant components, physical or physicochemical parameters, and the ultimate therapeutic effect of the released truncated survivin molecule. Finally, the present invention relates to a method for increasi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435
CPCC07K14/4747C07K14/4748A61K2039/55555A61P35/00A61P35/02A61P37/04A61P43/00A61K39/00115A61K39/39A61K45/06A61K2039/55511A61K2039/572
Inventor S·盖斯勒P·伯尼福特J·普拉什克M·维冈特S·杰克尔R·凯尔纳T·里斯奥克D·穆勒-珀帕拉K·汉斯
Owner MERCK PATENT GMBH
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