RAGE fusion proteins, formulations, and methods of use thereof
View Patent ↗Disclosed are RAGE fusion proteins comprising RAGE polypeptide sequences linked to a second, non-RAGE polypeptide. The RAGE fusion protein may utilize a RAGE polypeptide domain comprising a RAGE ligand binding site and an interdomain linker directly linked to the N-terminus of an immunoglobulin C H 2 domain. Also disclosed are RAGE fusion protein formulations and the use of the RAGE fusion proteins and RAGE fusion protein formulations as therapeutics for RAGE-mediated pathologies.
1. A formulation comprising a lyophilized mixture of a lyoprotectant, a RAGE fusion protein, and a buffer, wherein the RAGE fusion protein comprises the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56 or a sequence at least 90% identical thereto, wherein the lyoprotectant comprises a non-reducing sugar, and wherein the amount of buffer is sufficient to provide a reconstituted formulation having a pH ranging from 6.0 to 6.7 when the concentration of the RAGE fusion protein in the reconstituted formulation is within the range from 1 mg/mL to 400 mg/mL.
2. The formulation of claim 1 , wherein the non-reducing sugar comprises at least one of sucrose, mannitol, or trehalose.
3. The formulation of claim 1 , wherein the buffer comprises histidine.
4. The formulation of claim 1 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 32, or a sequence 98% identical thereto.
5. The formulation of claim 1 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 33, or a sequence 98% identical thereto.
6. The formulation of claim 1 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34, or a sequence 98% identical thereto.
7. The formulation of claim 1 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 56, or a sequence 98% identical thereto.
8. The formulation of claim 1 , wherein the RAGE fusion protein is the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56.
9. The formulation of claim 1 , wherein the sequence at least 90% identical to SEQ ID NOs: 32, 33, 34, or 56 comprises the polypeptide of SEQ ID NOs: 32, 33, 34, or 56 without the C-terminal lysine.
10. The formulation of claim 1 , further comprising at least one of a surfactant, a chelating agent, or a bulking agent.
11. A reconstituted formulation comprising a lyophylized RAGE fusion protein reconstituted in a diluent, wherein the RAGE fusion protein concentration in the reconstituted formulation is within the range from about 1 mg/mL to about 400 mg/mL, wherein the reconstituted formulation comprises a lyoprotectant, the RAGE fusion protein, and a buffer, wherein the RAGE fusion protein comprises the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56 or a sequence at least 90% identical thereto, and wherein the lyoprotectant comprises a non-reducing sugar, and wherein the pH is in the range from 6.0 to 6.7.
12. The reconstituted formulation of claim 11 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 32, or a sequence 98% identical thereto.
13. The reconstituted formulation of claim 11 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 33, or a sequence 98% identical thereto.
14. The reconstituted formulation of claim 11 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34, or a sequence 98% identical thereto.
15. The reconstituted formulation of claim 11 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 56, or a sequence 98% identical thereto.
16. The reconstituted formulation of claim 11 , wherein the RAGE fusion protein is the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56 or a sequence at least 90% identical thereto.
17. The reconstituted formulation of claim 11 , wherein the sequence at least 90% identical to SEQ ID NOs: 32, 33, 34, or 56 comprises the polypeptide of SEQ ID NOs: 32, 33, 34, or 56 without the C-terminal lysine.
18. The reconstituted formulation of claim 11 , wherein the diluent comprises water for injection (WFI).
19. The reconstituted formulation of claim 11 , wherein the reconstituted formulation is suitable for subcutaneous or intramuscular administration.
20. The reconstituted formulation of claim 11 , wherein the reconstituted formulation is isotonic.
21. The reconstituted formulation of claim 11 , wherein the formulation comprises about 1-400 mg/mL of RAGE fusion protein; about 1 mM to about 100 mM histidine buffer; about 60 mM to about 65 mM sucrose; about 0.001% to about 0.05% Tween 80; and a pH of about 6.0 to 6.5.
22. The reconstituted formulation of claim 11 , wherein the formulation comprises about 40-50 mg/mL of RAGE fusion protein; about 10 mM histidine; about 65 mM sucrose; about 0.01% Tween 80; and a pH of about 6.0.
23. The reconstituted formulation of claim 11 , wherein the formulation exhibits less than 5% decomposition after 1 week at 40 degrees Centigrade.
24. The reconstituted formulation of claim 11 , wherein less than 10% of the RAGE fusion protein is present as an aggregate in the formulation upon reconstitution.
25. The reconstituted formulation of claim 11 , wherein the non-reducing sugar comprises at least one of sucrose, mannitol, or trehalose.
26. The reconstituted formulation of claim 11 , wherein the lyophylized formulation of the RAGE fusion protein comprises at least one of a surfactant, a chelating agent or a bulking agent.
27. An article of manufacture comprising a container which holds a lyophylized RAGE fusion protein comprising a lyophilized mixture of a lyoprotectant, a RAGE fusion protein, and a buffer, wherein the RAGE fusion protein comprises the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56 or a sequence at least 90% identical thereto, wherein the lyoprotectant comprises a non-reducing sugar, and wherein the amount of buffer is sufficient to provide a reconstituted formulation having a pH ranging from 6.0 to 6.7 when the concentration of the RAGE fusion protein in the reconstituted formulation is within the range from 1 mg/mL to 400 mg/mL, and instructions for reconstituting the lyophilized formulation with a diluent.
28. The article of manufacture of claim 27 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 32, or a sequence 98% identical thereto.
29. The article of manufacture of claim 27 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 33, or a sequence 98% identical thereto.
30. The article of manufacture of claim 27 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34, or a sequence 98% identical thereto.
31. The article of manufacture of claim 27 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 56, or a sequence 98% identical thereto.
32. The article of manufacture of claim 27 , wherein the RAGE fusion protein is the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56 or a sequence at least 90% identical thereto.
33. The article of manufacture of claim 27 , wherein the sequence at least 90% identical to SEQ ID NOs: 32, 33, 34, or 56 comprises the polypeptide of SEQ ID NOs: 32, 33, 34, or 56 without the C-terminal lysine.
34. The article of manufacture of claim 27 , wherein the diluent comprises water for injection (WFI).
35. The article of manufacture of claim 27 , wherein the reconstituted formulation is suitable for subcutaneous or intramuscular administration.
36. The article of manufacture of claim 27 , wherein the reconstituted formulation is isotonic.
37. The article of manufacture of claim 27 , wherein upon reconstitution according to the instructions, the formulation comprises about 1-400 mg/mL of RAGE fusion protein; about 1 mM to about 100 mM histidine buffer; about 60 mM to about 65 mM sucrose; about 0.001% to about 0.05% Tween 80; and a pH of about 6.0 to 6.5.
38. The article of manufacture of claim 27 , wherein upon reconstitution the formulation comprises about 40-50 mg/mL of RAGE fusion protein; about 10 mM histidine; about 65 mM sucrose; about 0.01% Tween 80; and a pH of about 6.0.
39. The article of manufacture of claim 27 , wherein the instructions are for reconstituting the lyophilized formulation such that the RAGE fusion protein concentration in the reconstituted formulation is within the range from about 40 mg/mL to about 100 mg/mL.
40. The article of manufacture of claim 27 , wherein the lyophilized formulation is sterile.
41. The article of manufacture of claim 27 , further comprising a second container which holds a diluent for reconstituting the lyophilized formulation, wherein the diluent is water for injection (WFI).
42. The article of manufacture of claim 27 , wherein upon reconstitution according to the instructions, the formulation exhibits less than 5% decomposition after 1 week at 40 degrees Centigrade.
43. The article of manufacture of claim 27 , wherein less than 10% of the RAGE fusion protein is present as an aggregate in the formulation upon reconstitution.
44. The article of manufacture of claim 27 , wherein the non-reducing sugar comprises at least one of sucrose, mannitol, or trehalose.
45. The article of manufacture of claim 27 , wherein the lyophylized RAGE fusion protein comprises at least one of a surfactant, a chelating agent or a bulking agent.
46. A method for preparing a stable reconstituted formulation of a RAGE fusion protein comprising reconstituting a lyophilized mixture of the RAGE fusion protein, a lyoprotectant, and a buffer in a diluent such that the RAGE fusion protein concentration in the reconstituted formulation is in a range from about 1 mg/mL to about 400 mg/mL, and the pH is in the range from 6.0 to 6.7, wherein the RAGE fusion protein comprises the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56 or a sequence at least 90% identical thereto, and wherein the lyoprotectant comprises a non-reducing sugar.
47. The method of claim 46 , wherein the lyoprotectant comprises at least one of sucrose, mannitol or trehalose.
48. The method of claim 46 , wherein the lyophilized mixture further comprises at least one of a surfactant, a chelating agent or a bulking agent.
49. The method of claim 46 , wherein the RAGE fusion protein is the amino acid sequence as set forth in one of SEQ ID NOs: 32, 33, 34, or 56 or a sequence at least 90% identical thereto.
50. The method of claim 49 , wherein the sequence at least 90% identical to SEQ ID NOs: 32, 33, 34, or 56 comprises the polypeptide of SEQ ID NOs: 32, 33, 34, or 56 without the C-terminal lysine.
51. The method of claim 46 , wherein the reconstituted formulation comprises about 40-100 mg/mL of RAGE fusion protein; about 2 mM to about 60 mM histidine; about 60 mM to about 65 mM sucrose; about 0.001% to about 0.05% Tween 80; and a pH of about 6.0 to 6.5.
52. The method of claim 46 , further comprising preparing the lyophilized RAGE fusion protein by lyophilizing a mixture comprising a RAGE fusion protein and a lyoprotecting amount of a lyoprotectant.