Human beta-glucuronidase mutants with elevated enzymatic activity under physiological conditions and method for identifying such
A number of human beta-glucuronidase variants having higher enzymatic activity at physiological pH as compared with wild-type beta-glucuronidase and uses thereof in prodrug therapy. Also disclosed herein is a method for identifying enzyme variants having elevated enzymatic activity using a mammalian surface display system.
1. A polypeptide comprising an amino acid sequence, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs:6-10.
2. The polypeptide of claim 1 , further comprising the amino acid sequence of a single-chain antibody that specifically targets cancer cells.
3. The polypeptide of claim 2 , wherein the single-chain antibody is CC49 scFv.
4. The isolated polypeptide of claim 2 , wherein the single-chain antibody is a human or humanized antibody.
5. The polypeptide of claim 1 , further comprising the amino acid sequence of a peptide that particularly targets cancer cells.
6. The polypeptide of claim 5 , wherein the peptide is selected from the group consisting of AHNP, RGD4C, HN-1, CSNRDARRC (SEQ ID NO: 12), CNGRCVSGCAGRC (SEQ ID NO: 13), Z HER2:342-pep2 , ZEGFR:1907, and SP94.
7. A glucuronidase conjugate, comprising a human beta-glucuronidase mutant associated with a cancer-targeting agent, wherein the beta-glucuronidase mutant has the amino acid sequence of one of SEQ ID NOs:6-10.
8. The glucuronidase conjugate of claim 7 , wherein the cancer-targeting agent is an antibody specifically binding to cancer cells.
9. The glucuronidase conjugate of claim 8 , wherein the antibody is a human or humanized antibody.
10. The glucuronidase conjugate of claim 7 , wherein the cancer-targeting agent is a peptide specifically binding to cancer cells.
11. The glucuronidase conjugate of claim 7 , wherein the cancer-targeting agent is an aptamer.