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Cancer selective auxotrophs

A nutrient deficiency and model technology, applied in allergic diseases, antibody medical ingredients, medical raw materials derived from bacteria, etc., can solve problems such as the nature of mutations that are not described

Inactive Publication Date: 2007-08-01
ANTICANCER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zhao, M., et al., Proc.Am.Assoc.Cancer Res.(2004) 45:869 (No.3765) disclose an abstract, which describes the effect of Salmonella typhimurium prepared according to the present invention , but does not describe the nature of the mutation

Method used

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  • Cancer selective auxotrophs
  • Cancer selective auxotrophs
  • Cancer selective auxotrophs

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Preparation of Salmonella typhimurium A1

[0059] A single clone of Salmonella typhimurium (ATCC 14028) (Stratagene, San Diego CA) was picked from the LB agar plate, cultured in 5 ml of liquid LB medium, and shaken at 37° C. at 300 rpm overnight. The overnight culture was diluted 1:10 with LB medium and incubated at 37°C. For each time point, the cultured bacteria were at OD 600 down to measure.

[0060] At mid-log phase, cells were harvested at 4°C, washed three times with ice-cold glycerol (10% V / V), and then resuspended in approximately 1 / 100 of the original culture volume in ice-cold glycerol (10% V / V). 40 μl of 2.0 x 10 8 Cells were mixed with 2 μl of pGFP (Clontech) vector, kept on ice for 5 minutes, and then electroporated with a Gene Pulser device (BioRad Labs) according to the manufacturer's instructions: electroporation was performed at a setting of 1.8 kv with a pulse controller, 1000 Ω parallel resistor. Immediately after the electric pulse, 1 ml of SOC ...

Embodiment 2

[0071] In vitro bacterial invasion and intracellular replication of tumor cell lines

[0072] Tumor cells were grown in 24-well tissue culture plates to a density of approximately 10 4 cells / well. Bacteria were grown to mid-log, late-log and stationary phase (OD 600= 0.3, 0.6, 1.2, 2.0). Bacteria were diluted in cell culture medium and added to tumor cells (1 × 10 5 / ml), and then placed in a 37°C incubator. After 1 hour, cells were washed and then cultured in medium containing gentamicin sulfate (20 μg / ml) to kill external, but not internal, bacteria. Intracellular bacteria were monitored at different time points under a fluorescence microscope.

[0073] Salmonella typhimurium-GFP virulence in tumor cells is affected by the growth state of the bacteria: bacteria in mid-log phase are minimally virulent, resuming their invasiveness in late-log and stationary phase. Bacteria in late logarithmic phase had the greatest virulence to kill PC-3 tumor cells. These 24 hour pos...

Embodiment 3

[0077] Preparation of in vivo tumor models

[0078] Tumors were labeled with RFP by retroviral transduction, PT67, a packaging cell line derived from NIH3T3, expressing the 10A1 viral envelope, purchased from CLONTECH Laboratories Inc. PT67 cells were cultured in DME (Irvne Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gemini Bio-products Calabasas, CA). For vector production, 70% confluent packaging cells (PT67) use DOTAp TM Precipitated mixture of reagents (Boehringer Mannheim) and saturation pLNCX 2 - DsRed-2-RFP plasmid was incubated for 18 hours. Host pLNCX purchased from CLONTECH Laboratories Inc (Palo Alto, CA) 2 The vector contains a neomycin resistance gene for antibiotic selection in eukaryotic cells. The RFP gene (DsRed2, CLONTECH Laboratories Inc., Palo Alto, CA) was inserted into pLNCX 2 Eg1II and NotI sites of the vector. Fresh medium was supplemented, and cells were viewed with a fluorescent microscope 48 hou...

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Abstract

Bacteria that are auxotrophic for at least two amino acids found in at least one tumor are effective antitumor treatments, labeling agents, and vaccines against infection. Improved antitumor effects can also be provided such strains by passage through an appropriate tumor model.

Description

[0001] related application [0002] This application claims the benefit of US Provisional Application Serial No. 60 / 584,301, filed June 29, 2004, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to antitumor bacteria which have been modified to require at least two amino acids as nutrients and which may further exhibit enhanced tumor adhesion and virulence. As an example, Salmonella typhimurium containing a double auxotrophic mutation to require arginine and leucine was shown to be avirulent in vivo and grow selectively in tumors. Background technique [0004] It is well known that anaerobic bacteria grow selectively in tumors, and indeed large numbers of bacteria are found in tumors resected from patients. In view of this, bacteria have been suggested as tumor therapeutic agents. For example, Yazawa K., et al., Breast Carc. Res. and Treat. (2001) 66: 165-170 demonstrated that Bifidobacterium longum (Bifidoba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/00C12N1/00C12N1/20A61K35/74
CPCA61K39/00A61K39/0275A61K35/74A61K38/2013A61K38/51A61P31/00A61P35/00A61P37/04C12N1/00C12N1/20
Inventor 赵明杨萌
Owner ANTICANCER
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