Engineered primate cystine/cysteine degrading enzymes for therapeutic uses

Patent metadata

Field	Value
Publication number	US-10865403-B2
Application number	US-201815977246-A
Country	US
Kind code	B2
Filing date	May 11, 2018
Priority date	May 12, 2017
Publication date	Dec 15, 2020
Grant date	Dec 15, 2020

Abstract

Methods and compositions related to the engineering of a protein with L-cyst(e)ine degrading enzyme activity are described. For example, disclosed are modified cystathionine-γ-lyases comprising one or more amino acid substitutions and capable of degrading L-cyst(e)ine. Furthermore, compositions and methods are provided for the treatment of cancer and cystinuria using the disclosed modified enzymes or nucleic acids encoding said enzymes.

First claim

What is claimed is:

1. An isolated, modified primate cystathionine-γ-lyase (CGL) enzyme having the following substitutions relative to a native human CGL amino acid sequence (SEQ ID NO: 1), wherein the modified enzyme has both cystinase and cysteinase activity, and wherein said primate is selected from the group consisting of Homo sapiens, Pongo abelii, Pan troglodytes, Pan paniscus , and Macaca fascicularis ; said substitutions comprising: a glutamic acid at position 55, a threonine at position 59, and a valine at position 339.

2. The isolated, modified CGL enzyme of claim 1 , further comprising an aspartic acid at position 336.

3. The isolated, modified CGL enzyme of claim 1 , wherein the isolated, modified CGL enzyme is a modified Pongo abelii CGL enzyme, wherein said substitutions comprise H55E, V59T, and E339V.

4. The isolated, modified CGL enzyme of claim 3 , further comprising a T336D substitution.

5. The isolated, modified CGL enzyme of claim 1 , wherein the isolated, modified CGL enzyme is a modified Homo sapiens CGL enzyme, a modified Macaca fascicularis CGL enzyme, a modified Pan troglodytes CGL enzyme, or a modified Pan paniscus CGL enzyme, wherein said substitutions comprise H55E, E59T, and E339V.

6. The isolated, modified CGL enzyme of claim 5 , further comprising a T336D substitution.

7. The isolated, modified CGL enzyme of claim 1 , further comprising a heterologous peptide segment or a polysaccharide.

8. The isolated, modified CGL enzyme of claim 7 , wherein the heterologous peptide segment is an XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide.

9. The isolated, modified CGL enzyme of claim 7 , wherein the polysaccharide comprises polysialic acid polymers.

10. The isolated, modified CGL enzyme of claim 1 , wherein the enzyme is coupled to a polyethylene glycol (PEG).

11. The isolated, modified CGL enzyme of claim 10 , wherein the enzyme is coupled to the PEG via one or more lysine residues.

12. A therapeutic formulation comprising an enzyme of claim 1 , in a pharmaceutically acceptable carrier.

13. A method of treating a tumor cell or subject having a tumor cell comprising administering to the tumor cell or the subject a therapeutically effective amount of the formulation of claim 12 .

14. The method of claim 13 , wherein the subject is maintained on a L-cystine and/or L-cysteine restricted diet.

15. The method of claim 13 , wherein the subject is maintained on a normal diet.

16. The method of claim 13 , wherein the tumor cell has been identified as having a decreased expression level of a cystathionine-β-synthase or cystathionine-γ-lyase gene product relative to a non-tumor cell.

17. The method of claim 13 , wherein the tumor cell has been identified as having an increased expression level of a xCT(−) cystine/glutamate antiporter gene product relative to a non-tumor cell.

18. The method of claim 13 , wherein the tumor cell is a prostate cancer cells, a small cell lung cancer cell, or a hematological malignancy cell.

19. The method of claim 18 , wherein the hematologic malignancy cell is a chronic myelogenous leukemia cell, chronic lymphocytic leukemia cell, acute myelogenous leukemia cell, acute lymphocytic leukemia cell, acute monocytic leukemia cell, diffuse large B-cell lymphoma, myelodysplastic syndrome, chronic myelomonocytic leukemia, primary myelofibrosis, Hodgkin's lymphoma cell, non-Hodgkin's lymphoma cell, or multiple myeloma cell.

20. The method of claim 13 , wherein the subject is a human patient.

21. The method of claim 13 , wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intralesionally, intramuscularly, intravesicularlly, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.

22. The method of claim 13 , wherein the formulation is administered to a nutrient medium of the tumor cell.

23. The method of claim 22 , wherein the nutrient medium is blood, lymphatic fluid, or spinal fluid.

24. The method of claim 13 , further comprising administering at least a second anticancer therapy to the subject.

25. The method of claim 24 , wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy.

26. The method of claim 24 , wherein the secondary anticancer therapy is fludarabine.

27. A method of treating a subject having or at risk of developing cystinuria comprising administering to the subject a therapeutically effective amount of a formulation of claim 12 .

28. The method of claim 27 , wherein the subject is maintained on a L-cystine and/or L-cysteine restricted diet.

29. The method of claim 27 , wherein the subject is maintained on a methionine-restricted diet.

30. The method of claim 27 , wherein the subject is maintained on a normal diet.

31. The method of claim 27 , wherein the subject is a human patient.

32. The method of claim 27 , wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intramuscularly, intravascularly, subcutaneously, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.

33. The method of claim 27 , wherein the subject has previously been treated for cystinuria and the enzyme is administered to prevent the recurrence of cystinuria.

34. The method of claim 27 , further comprising administering at least a second cystinuria therapy to the subject.

35. The method of claim 34 , wherein the second cystinuria therapy is a surgical therapy or a shock wave therapy.

36. An isolated, modified human cystathionine-γ-lyase (CGL) enzyme having the following substitutions relative to a native human CGL amino acid sequence (SEQ ID NO: 1), wherein the modified enzyme has both cystinase and cysteinase activity, said substitutions comprising: a glutamic acid at position 55, a threonine at position 59, and a valine at position 339.

Assignees

• Univ Texas

Inventors

• Georgiou George
• Stone Everett
• Cramer Shira

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

• A61K38/38

  Albumins · CPC title

• A61K38/16

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof {(enzyme inhibitors A61K38/005)} · CPC title

• A61K31/7076

  containing purines, e.g. adenosine, adenylic acid · CPC title

• C07K14/001

  by chemical synthesis · CPC title