Universal red blood cells, methods of preparing same, and uses thereof
View Patent ↗The present invention is directed to pegylated red blood cells comprising a polyethylene glycol (PEG) attached to a thiolated amino group on a membrane protein, and to compositions comprising the pegylated red blood cells. The invention also provides methods of preparing pegylated red blood cells comprising reacting red blood cells with a compound that produces a thiolated amino group on a red blood cell membrane protein, and reacting the thiolated red blood cell with a PEG. The invention further provides methods of treatment using pegylated red blood cells.
1. A method of preparing pegylated red blood cells comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons and a polyethylene glycol (PEG) having a molecular weight of 20 K daltons, the method comprising the sequential steps of:
(a) introducing a polyethylene glycol (PEG) having a molecular weight of 5 K daltons onto the red blood cells to prepare pegylated red blood cells comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons, wherein either (1) the red blood cells are contacted with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cells and the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide or (2) the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG);
(b) removing excess reagents; and
(c) introducing a polyethylene glycol (PEG) having a molecular weight of 20 K daltons onto the pegylated red blood cells comprising the polyethylene glycol (PEG) having a molecular weight of 5 K daltons to prepare pegylated red blood cells comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons and a polyethylene glycol (PEG) having a molecular weight of 20 K daltons, wherein either (1) the red blood cells are contacted with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cells and the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide or (2) the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG);
wherein the polyethylene glycol (PEG) is a methoxy PEG; and
wherein pegylation of the red blood cells masks a Rh antigen and one or more of A antigen and B antigen.
2. The method of claim 1 , wherein step (a) comprises contacting the red blood cells with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cells and wherein the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide.
3. The method of claim 2 , wherein the chemical thiolating compound is iminothiolane.
4. The method of claim 2 , wherein the chemical thiolating compound is dithio sulfo succinimidyl propinate (DTSSP).
5. The method of claim 1 , wherein in step (a), the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG).
6. The method of claim 1 , wherein step (c) comprises contacting the red blood cells with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cell and wherein the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide.
7. The method of claim 6 , wherein the chemical thiolating compound is iminothiolane.
8. The method of claim 6 , wherein the chemical thiolating compound is dithio sulfo succinimidyl propinate (DTSSP).
9. The method of claim 1 wherein in step (c), the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG).
10. The method of claim 1 , wherein the polyethylene glycol (PEG) in step (a) is a maleimide methoxypolyethylene glycol.
11. The method of claim 10 , wherein the maleimide methoxypolyethylene glycol is a maleimide phenyl methoxypolyethylene glycol.
12. The method of claim 10 , wherein the maleimide methoxypolyethylene glycol is a maleimidophenyl carbamate of methoxypolyethylene glycol.
13. The method of claim 1 , wherein the polyethylene glycol (PEG) in step (a) is a maleimide methoxypolyethylene glycol and wherein the polyethylene glycol (PEG) in step Cc) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG).
14. The method of claim 1 , wherein the Rh antigen is one or more of Rh(D), Rh(C), Rh(c), Rh(E) and Rh(e).
15. The method of claim 1 , wherein pegylation of the red blood cells does not alter the oxygen affinity of the red blood cells by more than 20%.
16. The method of claim 1 , wherein pegylation of the red blood cells does not alter the oxygen affinity of the red blood cells by more than 15%.
17. The method of claim 1 , wherein the method is carried out at a temperature between about room temperature and about 4° C.
18. The method of claim 1 , wherein the method is carried out at a pH of about pH 7.4-9.2.
19. The method of claim 1 , wherein the method is carried out for about 2 hours.
20. A pegylated red blood cell comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons and a polyethylene glycol (PEG) having a molecular weight of 20 K daltons produced by the method of claim 1 .
21. A composition comprising the pegylated red blood cell of claim 20 and a pharmaceutically acceptable carrier.