Methods, compositions, and kits for detection of aspergillosis

Patent metadata

Field	Value
Publication number	US-10648982-B2
Application number	US-201615236988-A
Country	US
Kind code	B2
Filing date	Aug 15, 2016
Priority date	Mar 15, 2013
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

Provided herein are methods for detecting an Aspergillus protease in a sample, diagnosing a subject with aspergillosis caused by an Aspergillus infection based on the presence of an Aspergillus protease in a sample, and methods of aspergillosis treatment that incorporate these diagnostic methods. In certain embodiments, the Aspergillus protease is Asp f2, and the Aspergillus infection is caused A. fumigatus, A. flavus, A. versicolor, A. niger, or A. terreus. Also provided herein are antibodies and kits for use in these methods, including novel antibodies specific for Asp f2.

First claim

What is claimed is:

1. A method for detecting Aspergillus Asp f2 in a biological sample comprising contacting the sample with an Asp f2 protease substrate that can be cleaved by Asp f2,
wherein cleavage of the protease substrate indicates the presence of Asp f2 in the sample; and
wherein the protease substrate comprises the amino acid sequence of SEQ ID NO:5.

2. The method of claim 1 , wherein the sample is enriched for Asp f2 prior to contact with the Asp f2 protease substrate by contacting the sample with an Asp f2-specific antibody or antigen binding fragment, the Asp f2-specific antibody or antigen binding fragment comprising one or more CDRs selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16 and 18.

3. The method of claim 2 , wherein the Asp f2-specific antibody is immobilized on an enrichment matrix.

4. The method of claim 1 , wherein the protease substrate is conjugated to one or more fluorophores.

5. A method for diagnosing aspergillosis in a subject comprising:
(a) obtaining a biological sample from the subject;
(b) detecting the presence of Aspergillus Asp f2 in the sample by contacting the sample with an Asp f2 protease substrate that can be cleaved by Asp f2, wherein cleavage of the protease substrate indicates the presence of Asp f2 in the sample; and
(c) diagnosing the subject with aspergillosis if Asp f2 is present in the sample,
wherein the protease substrate comprises the amino acid sequence of SEQ ID NO:5.

6. The method of claim 5 , further comprising (d) administering one or more therapeutic agents for the treatment of aspergillosis.

7. The method of claim 6 , wherein the sample is enriched for Asp f2 prior to contact with the Asp f2 protease substrate by contacting the sample with an Asp f2-specific antibody or antigen binding fragment the Asp f2-specific antibody or antigen binding fragment comprising one or more CDRs selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16 and 18.

8. The method of claim 7 , wherein the Asp f2-specific antibody is immobilized on an enrichment matrix.

9. The method of claim 5 , wherein the protease substrate is conjugated to one or more fluorophores.

10. The method of claim 5 , wherein the aspergillosis is the result of infection with an Aspergillus species selected from the group consisting of A. fumigatus, A. flavus, A. versicolor, A. niger , and A. terreus.

11. A method for the treatment of aspergillosis in a subject in need thereof comprising:
(a) obtaining a biological sample from the subject;
(b) detecting the presence of Aspergillus Asp f2 in the sample by contacting the sample with an Asp f2 protease substrate that can be cleaved by Asp f2, wherein cleavage of the protease substrate indicates the presence of Asp f2 in the sample; and
(c) administering one or more therapeutic agents for the treatment of aspergillosis if Asp f2 is detected in the sample, and
wherein the protease substrate is selected from the group consisting of a protein or peptide comprising the amino acid sequence of SEQ ID NO:5, an elastin protein, and a collagen protein.

12. The method of claim 11 , wherein the sample is enriched for Asp f2 prior to contact with the Asp f2 protease substrate by contacting the sample with an Asp f2-specific antibody or antigen binding fragment the Asp f2-specific antibody or antigen binding fragment comprising one or more CDRs selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16 and 18.

13. The method of claim 12 , wherein the Asp f2-specific antibody is immobilized on an enrichment matrix.

14. The method of claim 11 , wherein the protease substrate is conjugated to one or more fluorophores.

15. The method of claim 11 , wherein the aspergillosis is the result of infection with an Aspergillus species selected from the group consisting of A. fumigatus, A. flavus, A. versicolor, A. niger , and A. terreus.

16. A kit for performing the method of claim 1 or 5 comprising an Asp f2 protease substrate that can be cleaved by Asp f2, wherein the protease substrate comprises the amino acid sequence of SEQ ID NO:5.

17. A kit for performing the method of claim 11 comprising an Asp f2 protease substrate that can be cleaved by Asp f2, wherein the protease substrate is selected from a group consisting of a protein or peptide comprising the amino acid sequence of SEQ ID NO:5, an elastin protein, and a collagen protein.

18. An Asp f2 protease substrate that can be cleaved by Asp f2, the protease substrate comprising the amino acid sequence of SEQ ID NO:5.

19. The protease substrate of claim 18 , wherein the protease substrate is conjugated to one or more fluorophores.

Assignees

• Hope City

Inventors

• Kalkum Markus
• Bagramyan Karine
• Diaz-Arevalo Diana
• Ito James I
• Dadwal Sanjeet

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K16/40

  against enzymes · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• G01N33/573 Primary

  for enzymes or isoenzymes · CPC title

• A61P31/10

  Antimycotics · CPC title