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Proaerolysin containing protease activation sequences and methods of use for treatment of prostate cancer

A technology of aerolysin and prostate, applied in medical preparations containing active ingredients, gene therapy, peptide/protein components, etc., can solve problems such as systemic poisoning

Inactive Publication Date: 2005-07-06
UNIV OF VICTORIA INNOVATION & DEVMENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this cytotoxicity will not be cell type specific and administration of such a generic toxin would involve severe systemic intoxication

Method used

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  • Proaerolysin containing protease activation sequences and methods of use for treatment of prostate cancer
  • Proaerolysin containing protease activation sequences and methods of use for treatment of prostate cancer
  • Proaerolysin containing protease activation sequences and methods of use for treatment of prostate cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] PSA-activated production of aerolysin protoxin

[0135] This example describes the method used to generate the mutant aerolysin protoxins shown in Table 1 activated by PSA. Those skilled in the art will appreciate that the same method can be used to produce other variant aerolysinogen proteins activated by PSA or any other prostate specific protease. These proteins can be produced by replacing the furin sequence of aerolysinogen with a prostate-specific protease cleavage site, eg, a PSA-specific cleavage sequence (see Example 9).

[0136] mutant

(SEQ ID

NO)

changes made

(SEQ ID NO)

Comparison with wild-type Aeromonas lysinogen

ADSKVRRARSVDGAGQGLRLEIPLD

(Amino acids 424-448 of SEQ ID NO: 2)

PSA-PA1

(3 and 4)

(SEQ ID NO: 427-432 amino group of 2

Acid) KVRRAR becomes HSSKLQ(5)

ADSHSSKLQSVDGAGQGLRLEIPLD

(Amino acids 424-448 of SEQ ID NO: 4)

PSA-1K

(6 and 7)

(SEQ ID NO: 427-434 am...

Embodiment 2

[0145] PSA-PA1 specifically lyses PSA-producing cells in vitro

[0146] This example describes the specific method used to determine the Aeromonas lysinogen variants described in Example 1. Such a method can be used to test the specificity of any Aeromonas lysin pro variant that contains a PSA-specific cleavage site.

[0147] PSA-PA1 toxin was tested against PSA-producing LNCaP cells (American Type Culture Collection (ATCC), Manassas, VA) and non-PSA-producing TSU cells (Dr. T. Itzumi, Teikyo University, Japan). cells in the presence of 10 -12 to 10 -6 M toxin was incubated for 24 hours. Cells were then counted based on their ability to exclude Trypan Blue and the percent cell viability was recorded. The concentration of toxin required to kill 50% of cells against LNCaP and TSU cell lines (IC 50 ).

[0148] LD of PSA-PA1 on PSA-producing cells 50 is 10 -10 M. In contrast, the LD50 of TSU for non-PSA-producing cells is approximately 5 × 10 -8 M. This result indicates...

Embodiment 3

[0150] PSA-PA1 is not activated in PSA-containing blood

[0151] The disclosed prostate-specific protease-activated variant aeromonasin pro-peptides, such as those described in Example 1, can be injected intraprostatically (or intratumorally) when locally treating prostate cancer. The toxin can also be injected intravenously (or intramuscularly) when systemically treating metastatic prostate cancer. These PSA site-containing variant PA molecules should not be activated in blood due to the presence of large molar excesses of serum protease inhibitors such as alpha 1 - Anti-chymotrypsin and alpha 2 - Macroglobulin, which inactivates PSA enzymatically in the blood.

[0152] To test the nonspecific activation of PSA-PA1 by other serum proteases and PSA in human serum, the following sensitive hemolysis assay was performed. Red blood cells (RBC, 2% v / v) were added to plasma or buffer containing PSA-PA1 toxin ± PSA. The degree of hemolysis was analyzed by measuring the hemoglobin...

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Abstract

Modified aerolysinogen (PA) peptides are disclosed herein. In certain embodiments, the protein comprises a prostate specific protease cleavage site and may also comprise a prostate tissue specific binding domain which functionally replaces the native PA binding domain. In other embodiments, the protein comprises a felin cleavage site and a prostate tissue specific binding domain that functionally replaces the native PA binding domain. Also disclosed are methods of using these peptides to treat prostate cancer.

Description

[0001] References to related applications [0002] This application claims priority to US Provisional Patent Application No. 60 / 314,613, filed August 24, 2001, which is incorporated herein by reference in its entirety. technical field [0003] The present application relates to novel variant proaerolysin (PA) proteins and methods of using them for the treatment of localized and metastatic prostate cancer. technical background [0004] One out of every three diagnosed cancers in American men is of prostate origin, making prostate cancer the most frequently diagnosed malignancy in American men (Berges et al., Clin. Cancer Res. 1:473- 480, 1995). In the United States, the incidence of prostate cancer has not decreased with lifestyle changes; in fact, the incidence of clinical prostate cancer has increased steadily since 1930 (Pinski et al., Cancer Res. 61:6372-6, 2001). The incidence of prostate cancer increases with age faster than any other type of cancer; less than 1% of ...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K38/00A61K39/00A61K39/395A61K48/00A61P13/08A61P35/00C07K7/06C07K14/195C07K14/32C07K14/325C07K14/33C07K14/47C07K14/705C07K19/00C12N9/52
CPCA61K38/00C07K14/195C07K14/32C07K14/325C07K14/33C07K2319/00C12N9/52A61P13/08A61P35/00A61K39/001194A61K39/001195A61K39/001193
Inventor 萨穆尔·R·登米德约翰·T·伊萨克斯詹姆士·托马斯·巴克利
Owner UNIV OF VICTORIA INNOVATION & DEVMENT
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