Personalized protease assay to measure protease activity in neoplasms

• US 10,385,380 B2
• Filed: 10/02/2015
• Issued: 08/20/2019
• Est. Priority Date: 10/02/2014
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Whitney, Mike, Nguyen, Quyen T., Tsien, Roger Y.
• Primary Examiner(s): Lieb, Jeanette M
• Application Number:
  US14/874,297
• Publication Number:
  US 20160160263A1
• Time in Patent Office: 1,418 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2800/7028 Cancer

Abstract

Disclosed herein, the invention pertains to methods and compositions that find use in diagnostic, prognostic and characterization of neoplasia samples based on the ability of a neoplasia sample to cleave a MTS molecule of the present invention. In some embodiments, a MTS molecule disclosed herein has the formula (A-X-B-C), wherein A is a peptide with a sequence comprising 5 to 9 consecutive acidic amino acids, wherein the amino acids are selected from: aspartates and glutamates; B is a peptide with a sequence comprising 5 to 20 consecutive basic amino acids; X is a linker; and C is a detectable moiety.

Claims

1. An ex vivo method for detecting the presence of one or more protease activities in a neoplasia sample from a subject with cancer comprising:

a) combining ex vivo a tissue sample from a subject with cancer with a molecule of the structure A-X-B-C, wherein

B is a peptide portion of about 5 to about 20 basic amino acid residues, which is suitable for cellular uptake,

A is a peptide portion of about 2 to about 20 acidic amino acid residues, which when linked with portion B is effective to inhibit or prevent cellular uptake of portion B, and

X is a cleavable linker of about 2 to about 100 atoms joining A with B, where X is cleavable under physiological conditions, and

C is a detectable moiety; and

b) detecting cleavage of A-X-B-C by detecting a change in said detectable moiety C, wherein said change in C is indicative of cleavage, said cleavage is indicative of the presence of one or more protease activities in said tissue sample, and the presence of the protease activity is indicative that said tissue sample is a neoplasia sample.

2. The method of claim 1, wherein the presence of the protease activity is indicative of metastasis.

3. The method of claim 1, wherein C is a fluorescent detectable moiety.

4. The method of claim 1, wherein C comprises a FRET pair.

5. The method of claim 1, said molecule further comprising a Q moiety, wherein when said Q moiety is present, said molecule has the structure Q-A-X-B-C.

6. The method of claim 5, wherein C and Q comprise a FRET pair.

7. The method of claim 6, wherein the FRET pair is selected from the group consisting of CFP:

YFP;

Cy5;

Cy7;

FITC;

TRITC;

Cy3;

Cy5;

EGFP;

Cy3;

EGFP;

YFP;

6-FAM;

LC Red 640 or Alexa Fluor 546;

fluorescein;

tetramethylrhodamine;

IAEDANS;

fluorescein;

EDANS;

Dabcyl;

fluorescein;

fluorescein;

BODIPY FL;

BODIPY FL; and

fluorescein;

QSY 7 and QSY 9.

8. The method of claim 1, wherein cleavage of A-X-B-C is detected by FRET.

9. The method of claim 6, wherein cleavage of Q-A-X-B-C is detected by FRET.

10. The method of claim 1, wherein said peptide portion A comprises about 5 to about 9 glutamates or aspartates.

11. The method of claim 1, wherein said peptide portion A comprises about 5 to about 9 consecutive glutamates or aspartates.

12. The method of claim 1, wherein said peptide portion B comprises about 9 to about 16 arginines.

13. The method of claim 1, wherein said peptide portion B comprises about 9 to about 16 consecutive arginines.

14. The method of claim 1, wherein said peptide portion A comprises D-amino acids.

15. The method of claim 1, wherein said peptide portion B comprises D-amino acids.

16. The method of claim 1, wherein said peptide portion A consists of D-amino acids.

17. The method of claim 1, wherein said peptide portion B consists of D-amino acids.

18. The method of claim 1, wherein said peptide portions A and B each consist of D-amino acids.

19. The method of claim 1, wherein cleavable linker X is a flexible linker.

20. The method of claim 1, wherein cleavable linker X is a flexible linker about 6 to about 30 atoms in length.

21. The method of claim 1, wherein cleavable linker X is cleavable in an acidic environment.

22. The method of claim 1, wherein cleavable linker X comprises a peptide linkage.

23. The method of claim 1, wherein cleavable linker X comprises aminocaproic acid.

24. The method of claim 1, wherein cleavable linker X is configured for cleavage exterior to a cell.

25. The method of claim 1, wherein cleavable linker X is configured for cleavage by an enzyme.

26. The method of claim 25, wherein said enzyme is selected from the group consisting of a matrix metalloprotease, elastase, plasmin, thrombin, chymase, urokinase-type plasminogen activator and tissue plasminogen activator.

27. The method of claim 1, wherein cleavable linker X comprises an amino acid sequence selected from the group consisting of PLGLAG (SEQ ID NO:

1), PLGC(met)AG (SEQ ID NO;

     2), EDDDDKA (SEQ ID NO;

     3), RS-(Cit)-G-(homoF)-YLY (SEQ ID NO;

     4), CRPAHLRDSG (SEQ ID NO;

     5), SLAYYTA (SEQ ID NO;

     6), NISDLTAG (SEQ ID NO;

     7), PPSSLRVT (SEQ ID NO;

     8), SGESLSNLTA (SEQ ID NO;

     9), RIGFLR (SEQ ID NO;

     10), RLQLA(acetyl)L (SEQ ID NO;

     11), RLQLKL (SEQ ID NO;

     12), DPRSFL (SEQ ID NO;

     13), PPRSFL (SEQ ID NO;

     14), Norleucine-TPRSFL (SEQ ID NO;

     15), GVAY|SGA (SEQ ID NO;

     16), YGRAAA (SEQ ID NO;

     17), YGPRNR (SEQ ID NO;

     18), RSHP(Hfe)TLY (SEQ ID NO;

     19), RSHG(Hfe)FLY (SEQ ID NO;

     20), SNPYK-Y (SEQ ID NO;

     21), SNPKG-Y (SEQ ID NO;

     22), SNPYG-Y (SEQ ID NO;

     23), TLSE-LH (SEQ ID NO;

     24), TIAHLA (SEQ ID NO;

     25), (RLQLK(acetyl)L (SEQ ID NO;

     26), and KLRFSKQ (SEQ ID NO;

     27).

28. The method of claim 1, wherein cleavable linker X comprises a S-S linkage.

29. The method of claim 1, wherein cleavable linker X comprises a transition metal complex, wherein said transition metal complex linker is cleaved when the metal is reduced.

30. The method of claim 1, wherein in said method comprises multiple molecules of the structure A-X-B-C and wherein the cleavable linker X comprises a plurality of cleavable linkers X.

31. The method of claim 30, wherein the plurality of cleavable linkers X linking a portion A to a structure B-C are cleavable by a single protease.

32. The method of claim 30, wherein the plurality of cleavable linkers X linking a portion A to a structure B-C are cleavable by more than one protease.

33. The method of claim 1, wherein cleavable linker X comprises an amino acid sequence selected from the group consisting of RSHP(Hfe)TLY (SEQ ID NO:

19), RSHG(Hfe)FLY (SEQ ID NO;

     20), SNPYK-Y (SEQ ID NO;

     21), SNPKG-Y (SEQ ID NO;

     22), SNPYG-Y (SEQ ID NO;

     23), TLSE-LH (SEQ ID NO;

     24), TIAHLA (SEQ ID NO;

     25), (RLQLK(acetyl)L (SEQ ID NO;

     26), and KLRFSKQ (SEQ ID NO;

     27).