Protease assay

Patent metadata

Field	Value
Publication number	US-9682155-B2
Application number	US-201514605039-A
Country	US
Kind code	B2
Filing date	Jan 26, 2015
Priority date	Mar 3, 2008
Publication date	Jun 20, 2017
Grant date	Jun 20, 2017

Abstract

The present invention provides a diagnostic reagent or assay for assessing the activity of a protease in vivo or in vitro and methods of detecting the presence of a cancerous or precancerous cell. The assays are comprised of two particles linked via an oligopeptide linkage that comprises a consensus sequence specific for the target protease. Cleavage of the sequence by the target protease can be detected visually or using various sensors, and the diagnostic results can be correlated with cancer prognosis.

First claim

We claim:

1. An in vitro method for detecting the activity of a protease associated with a cancerous or precancerous cell in a mammal, said method comprising:
(a) contacting a fluid sample from the mammal in vitro with a nanoplatform assembly comprising:
a first particle, wherein said first particle is a core/shell nanoparticle, said core being selected from the group consisting of Au, Ag, Cu, Co, Fe, and Pt, and said shell being selected from the group consisting of Au, Ag, Cu, Co, Fe, Pt, the metal oxides thereof, and combinations thereof;
a second particle, wherein said second particle is selected from the group consisting of nanoparticles, chromophores/luminophores, quantum dots, viologens, and combinations thereof; and
a linkage between said first and second particles, wherein said linkage comprises a protease consensus sequence;

(b) exposing said assembly to an energy source; and
(c) detecting changes in an absorption or emission spectrum of the assembly,
wherein said changes correspond to protease activity, and wherein said linkage separates said first and second particles by a distance such that said first particle quenches an excited state of said second particle prior to said exposing (b).

2. The method of claim 1 , wherein said energy source is selected from the group consisting of a tungsten lamp, laser diode, laser, bioluminescence, and combinations thereof.

3. The method of claim 1 , wherein said exposing occurs at a wavelength of from about 400 nm to about 1000 nm.

4. The method of claim 1 , wherein said fluid sample is selected from the group consisting of urine and blood.

5. The method of claim 1 , wherein a blue-shift in the absorption or emission maximum of the assembly after contact with the fluid sample relative to the absorption or emission spectrum of the assembly prior to contact with said fluid sample indicates the presence of a cancerous or precancerous cell in the mammal.

6. The method of claim 1 , wherein a blue-shift in absorption or emission spectrum maximum between about 5 nm and about 200 nm indicates the presence of a cancerous or precancerous cell in the mammal.

7. The method of claim 1 , wherein said changes comprise the appearance of a new visible color or luminescence band relative to the absorption or emission spectrum of said assembly prior to contact with said fluid sample, said visible color or luminescence band indicating the presence of a cancerous or precancerous cell in the mammal.

8. The method of claim 1 , wherein said changes in the absorption or emission spectrum of said assembly are observed over a time period of from about 1 second to about 30 minutes.

9. The method of claim 1 , wherein said changes in absorption or emission spectrum indicate the activity of a protease selected from the group consisting of uPA, MMP-1, MMP-2, MMP-7, and combinations thereof.

10. The method of claim 9 , further comprising correlating said protease activity with a prognosis for cancer progression.

11. The method of claim 10 , wherein the detection of activity of both uPA and MMP-7, and absence of activity of both MMP-1 and MMP-2, is correlated with a prognosis for angiogensis.

12. The method of claim 10 , wherein the detection of activity of all of uPA, MMP-1, MMP-2, MMP-7 is correlated with a prognosis for cell invasion.

13. The method of claim 1 , wherein said cancerous or precancerous cell is associated with a cancer selected from the group consisting of an AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, extrahepatic bile duct cancer, childhood brain stem glioma, adult brain tumor, childhood malignant glioma, childhood ependymoma, childhood medulloblastoma, childhood supratentorial primitive neuroectodermal tumors, childhood visual pathway and hypothalamic glioma, breast cancer, pregnancy-related breast cancer, childhood breast cancer, male breast cancer, childhood carcinoid tumor, gastrointestinal carcinoid tumor, primary central nervous system lymphoma, cervical cancer, colon cancer, childhood colorectal cancer, esophageal cancer, childhood esophageal cancer, intraocular melanoma, retinoblastoma, adult glioma, adult (primary) hepatocellular cancer, childhood (primary) hepatocellular cancer, adult Hodgkin lymphoma, childhood Hodgkin lymphoma, islet cell tumors, Kaposi Sarcoma, kidney (renal cell) cancer, childhood kidney cancer, adult (primary) liver cancer, childhood (primary) liver cancer, Non-small cell liver cancer, small cell liver cancer, AIDS-related lymphoma, Burkitt lymphoma, adult Non-Hodgkin lymphoma, childhood Non-Hodgkin lymphoma, primary central nervous system lymphoma, melanoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, childhood multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, adult acute myeloid leukemia, childhood acute myeloid leukemia, multiple myeloma, neuroblastoma, non-small cell lung cancer, childhood ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, childhood pancreatic cancer, islet cell pancreatic cancer, parathyroid cancer, penile cancer, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, childhood renal cell cancer, renal pelvis and ureter, transitional cell cancer, adult soft tissue sarcoma, childhood soft tissue sarcoma, uterine sarcoma, skin cancer (nonmelanoma), childhood skin cancer, melanoma, Merkel cell skin carcinoma, small cell lung cancer, small intestine cancer, squamous cell carcinoma, stomach cancer, childhood stomach cancer, cutaneous T-Cell lymphoma, testicular cancer, thyroid cancer, childhood thyroid cancer, and vaginal cancer.

14. The method of claim 1 , wherein said consensus sequence is selected from the group consisting of a serine protease cleavage sequence, an aspartate protease cleavage sequence, a cysteine protease cleavage, and a metalloprotease cleavage sequence.

15. The method of claim 1 , wherein said core/shell nanoparticle is selected from the group consisting of Fe/Au, Fe/Fe 3 O 4 , Fe/Fe x O y , and Au/Fe 2 O 3 .

16. The method of claim 1 , wherein said core/shell nanoparticle is a stabilized nanoparticle comprising an organic monolayer surrounding the nanoparticle.

17. The method of claim 1 , wherein said second particle is a chromophore or luminophore selected from the group consisting of organic dyes, inorganic dyes, fluorophores, phosphophores, light absorbing nanoparticles, combinations thereof, and the metalated complexes thereof.

18. The method of claim 1 , said nanoplatform assembly further comprising multiple of said second particle, each of said second particles being linked to said first particle by respective linkages, wherein each of said linkages comprises a protease consensus sequence.

19. The method of claim 18 , wherein the respective linkages between each of said second particles and said first particle each comprises a different protease consensus sequence.

20. The method of claim 18 , wherein said nanoplatform assembly comprises up to 10 of said second particles linked to said first particle.

Assignees

• Univ Kansas State

Inventors

• Bossmann Stefan H
• Troyer Deryl L
• Basel Matthew T

Classifications

• A61K49/0056

  Peptides, proteins, polyamino acids · CPC title

• A61K49/0036

  Porphyrins (used in photodynamic therapy A61K41/0071 or A61K41/0076; used as targeting group or modifying agent for targeting a therapeutic compound A61K47/546) · CPC title

• C12Q1/37

  involving peptidase or proteinase · CPC title

• C07K7/08

  having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) · CPC title

• Y10S977/774

  Exhibiting three-dimensional carrier confinement, e.g. quantum dots · CPC title