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Anti-tumour agent, marker for tumour detection, and oral vaccine agent

A technology of single-chain antibody and anaerobic Gram, which is applied in the direction of anti-tumor drugs, antibody mimics/stents, measuring devices, etc., and can solve the problems of biological safety, narrow application range, and high invasiveness

Inactive Publication Date: 2015-03-18
TEIKYO HEISEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has the following problems: in addition to the recombinant bifidobacteria, it is necessary to administer a large amount of 5-FC to the living body, so it is cumbersome, and there are various constraints in clinical development in consideration of side effects and the like.
However, this method has the following problems: it needs to pinpoint the position of the tumor accurately, so the scope of application is narrow, and when a tumor exists deep in the body, it is highly invasive, so it lacks versatility
[0005] As a tumor treatment method using other bacteria, Clostridium novyi (Clostridium novyi) described in Patent Document 3 and Non-Patent Documents 4 and 5, or Salmonella typhimurium ( Salmonella typhimurium), etc., but they all come from pathogenic bacteria and secrete harmful exotoxins, so there is a big problem in the safety of the organisms administered

Method used

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  • Anti-tumour agent, marker for tumour detection, and oral vaccine agent
  • Anti-tumour agent, marker for tumour detection, and oral vaccine agent
  • Anti-tumour agent, marker for tumour detection, and oral vaccine agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Embodiment 1: Construction of expression cassette

[0163] Hereinafter, the structure and construction method of the bifidobacterium-introduced expression cassette used in this example as an obligate anaerobic gram-positive bacterium will be described.

[0164] (1) Anti-EGFR antibody / Pseudomonas exotoxin A fusion protein expression cassette (hereinafter referred to as "8C7 toxin") ( figure 1 a; SEQ ID NO: 1 / amino acid sequence: SEQ ID NO: 16)

[0165] It is a fusion protein expression cassette comprising a fusion gene having the structure described in the first aspect of the present invention. This expression cassette is designed according to the HindIII cleavage site, the promoter region (sequence number 4) from the hup gene of Bifidobacterium longum, the usp secretion signal sequence (sequence number 5) from Bifidobacterium longum, DTY (inserted after the signal sequence) sequence), with linker peptide (GGSGG) 2 Two anti-EGFR llama single-chain antibody coding sequ...

Embodiment 2

[0170] Example 2: Cell Proliferation Inhibitory Activity of Fusion Protein 8C7 Toxin

[0171] In this example, the preparation of recombinant Escherichia coli BL21(DE3) expressing the fusion protein such as 8C7 toxin described in Example 1 and the cell growth inhibitory activity of the fusion protein were verified.

[0172] (1) Construction of expression vectors for E. coli expression Ec-8C7 toxin, Ec-toxin control and Ec-8C7 EGFP

[0173] Escherichia coli expression Ec-8C7 toxin expression cassette Using the 8C7 toxin inserted into the pBluescript plasmid as a template, amplification by PCR was performed using the primer kits of SEQ ID NOs: 11 and 12. As for the conditions of PCR, PrimeSTAR GXL DNA Polymerase (TaKaRa Bio, Otsu City, Japan) as a DNA polymerase will be used as a cycle of 1 minute at 95°C, 10 seconds at 98°C, and 1 cycle at 60°C. 25 cycles of 15 seconds, 2 minutes at 68°C were performed. The amplified product was purified using a MinElute column (Qiagen) accor...

Embodiment 3

[0182] Example 3: The combination of tumor cells with 8C7 EGFP and the determination of the dissociation constant with EGFR ECD

[0183] In this example, the binding of Ec-8C7 EGFP described in Example 2 to living cells expressing EGFR was investigated.

[0184] (1) Binding to tumor cells of Ec-8C7 EGFP

[0185] On the Falcon 24-well plate (Becton Dickinson), at 2.5×10 5Cells / 0.5mL medium (DMEM medium containing 0.2% fetal bovine serum) / well were respectively inoculated with EGFR overexpression strain A431 cells and low expression strain HT-29 cells, the next day, in the medium containing Example 2 Purified Ec-8C7 EGFP was exchanged with 10 μg / mL of 0.2% FBS in DMEM medium. After 2 hours of cultivation, remove the above-mentioned medium, and replace with PBS (Mg-free 2+ , Ca 2+ Phosphate-buffered saline) and washed twice, 0.5 mL / well of PBS was added, and observed with a fluorescence microscope (ECLIPS Ti, Nikon). Since Ec-8C7EGFP purified with a HisTrap column was used, ...

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Abstract

The purpose of the present invention is to provide an anti-tumour agent and / or marker for tumour detection which have low invasiveness, few side effects, and high target specificity. Provided are a marker for tumour detection, which enables treatment effects to be monitored over time, and / or an anti-tumour agent, which can deliver functional peptides to target cells, the marker and the anti-tumour agent having said properties as a result of using a recombinant strict anaerobe gram-positive bacterium as an active ingredient, said bacterium containing a nucleic acid which codes a secretory fused protein comprising a signal peptide, a low molecular weight single-chain antibody, and a functional peptide.

Description

technical field [0001] The invention relates to an antitumor agent, a tumor detection marker and an oral vaccine agent, which take recombinant obligate anaerobic Gram-positive bacteria as active ingredients. Background technique [0002] When administered intravenously, obligate anaerobic bacteria mainly including bifidobacteria have very high selectivity and proliferate only in tumors (Non-Patent Document 1). This is considered to be due to the presence of an anaerobic environment in the center of the tumor as a hypoxic region where obligate anaerobic bacteria can survive and proliferate. In addition, there is also an advantage that bifidobacteria are Gram-positive bacteria and there is no risk of releasing endotoxin after death. On the other hand, the anaerobic environment in the center of tumor tissue is considered to be the cause of resistance to conventional chemotherapy (Non-Patent Document 2). Therefore, the use of the above-mentioned characteristics of bifidobacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K35/745A61K39/02A61K48/00A61P35/00C12N15/09C12Q1/04C07K16/00C07K19/00A61K39/00
CPCC07K2319/02C07K2319/00A61K48/00A61K39/02C07K2317/569C07K2317/92C07K16/2863G01N33/574A61K2039/542A61K2039/523C07K2319/40C07K2319/60C07K2317/22A61K38/00G01N2800/52C07K2319/33A61K39/00A61K2039/505C07K2317/73C07K2317/77C07K16/30A61P35/00C07K2319/55C12N9/1077C12Y204/02036A61K35/74C12N15/74A61K2039/62C07K2317/622C07K2317/14C07K16/40
Inventor 平裕一郎石田功
Owner TEIKYO HEISEI UNIVERSITY
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