Proaerolysin containing protease activation sequences and methods of use for treatment of prostate cancer
View Patent ↗Disclosed herein are modified proaerolysin (PA) peptide. In some examples, the proteins include a prostate-specific protease cleavage site and can further include a prostate-tissue-specific binding domain which functionally replaces the native PA binding domain. In other examples, the proteins include a furin cleavage site and a prostate tissue-specific binding domain which functionally replaces the native PA binding domain. Methods of using such peptides to treat prostate cancer are also disclosed.
1. A purified peptide comprising a variant proaerolysin amino acid sequence, wherein the variation of the proaerolysin amino acid sequence consists of the replacement of the furin cleavage site corresponding to the sequence of amino acids at positions 427-432 of SEQ ID NO:2 by a prostate-specific protease cleavage site, wherein administration of 2.5-25 μg of the peptide to a mouse having a prostate tumor xenograft reduces the volume of the tumor by 30% to 100%.
2. The peptide of claim 1 , wherein the prostate-specific protease cleavage site comprises a prostate-specific antigen (PSA) cleavage site, a prostate specific membrane antigen (PSMA) cleavage site, or a human glandular kallikrein 2 (hK2) cleavage site.
3. The peptide of claim 2 , wherein the PSA cleavage site comprises SEQ ID NO: 5, 8, 11, 14, 15, 16, 17, 18, 19, 20, or 21.
4. The peptide of claim 3 , wherein the PSA cleavage site comprises SEQ ID NO: 5.
5. The purified peptide of claim 1 , wherein the variant proaerolysin amino acid sequence is encoded by the nucleic acid sequence shown in SEQ ID NO: 3, 6, 9, or 12.
6. The purified peptide of claim 1 , wherein the variant proaerolysin amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 4, 7, 10, or 13.
7. The purified peptide of claim 1 , wherein the variant proaerolysin amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 4, 7, 10, or 13.
8. The purified peptide of claim 1 , wherein the variant proaerolysin amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 4.
9. The peptide of claim 1 , wherein the peptide is immobilized to a surface.
10. The peptide of claim 9 , wherein the surface is a bead.
11. The peptide of claim 10 , wherein the bead further comprises a prostate-specific ligand.
12. A purified peptide comprising a variant proaerolysin amino acid sequence, wherein the variations of the proaerolysin amino acid sequence consist of
(i) the replacement of the furin cleavage site corresponding to the sequence of amino acids at positions 427-432 of SEQ ID NO:2 by a prostate-specific protease cleavage site and
(ii) a modification of the amino acid sequence of the proaerolysin binding domain corresponding to the sequence of amino acids at positions 1-83 of SEQ ID NO: 2.
13. The peptide of claim 12 , wherein the proaerolysin binding domain is modified by deletion of the amino acids at positions 1-83 of SEQ ID NO:2 or 4.
14. The peptide of claim 12 , wherein the proaerolysin binding domain of SEQ ID NO:2 or 4 is modified by at least one amino acid replacement selected from the group consisting of W45A, 147E, M57A, Y61A, and K66Q.
15. The peptide of claim 12 , wherein the proaerolysin binding domain is replaced with a prostate-tissue specific binding domain.
16. The peptide of claim 12 , wherein a luteinizing hormone releasing hormone sequence is linked to an amino acid at position 215 or position 300 of SEQ ID NO:2 or 4, wherein the amino acid at position 215 or position 300 has been replaced by a cysteine.
17. The peptide of claim 15 , wherein the prostate-tissue specific binding domain comprises a luteinizing hormone releasing hormone (LHRH) sequence.
18. The peptide of claim 17 , wherein the luteinizing hormone releasing hormone sequence comprises SEQ ID NO: 22 or 23.
19. The peptide of claim 17 , wherein the amino acid sequence of the peptide comprises SEQ ID NO:24 or 25.
20. The peptide of claim 13 , wherein the peptide further comprises a luteinizing hormone releasing hormone sequence linked to the N-terminus of the variant proaerolysin.
21. The peptide of claim 15 , wherein the prostate-tissue specific binding domain comprises an antibody that recognizes prostate-specific antigen, human glandular kallikrein 2, prostate-specific membrane antigen, or-luteinizing hormone releasing hormone.
22. The peptide of claim 21 , wherein the antibody is linked to the N-terminus of the variant proaerolysin.
23. The peptide of claim 21 , wherein the antibody is linked to the C-terminus of the variant proaerolysin.
24. A method for treating prostate cancer in a subject, comprising administration of the peptide of claim 1 to the subject.
25. The method of claim 24 , wherein the peptide is administered intratumorally and/or intraprostatically.
26. The method of claim 24 , wherein the peptide is administered intravenously, intramuscularly, subcutaneously, or orally.
27. The method of claim 24 , wherein the subject has a localized prostate tumor.
28. The method of claim 24 , wherein the subject has a metastatic prostate tumor.
29. The method of claim 24 , further comprising administering GM-CSF to the subject.
30. The method of claim 24 , further comprising administering irradiated prostate cancer cells to the subject.
31. The method of claim 24 , wherein the prostate tumor cell volume is reduced by at least 50%.
32. The method of claim 24 , wherein administration further results in a reduction of a metastatic prostate tumor.
33. The method of claim 24 , wherein administration further results in treatment of a metastatic prostate tumor.
34. A method for treating prostate cancer in a subject, comprising contacting prostate cancer cells of the subject with the peptide of claim 1 .
35. A method for treating prostate cancer in a subject, comprising administering the peptide of claim 12 to the subject.
36. A method for treating prostate cancer in a subject, comprising contacting prostate cancer cells of the subject with the peptide of claim 12 .