Methods of preparation of conjugates
The present invention is directed to methods of preparing a conjugate of a cell-binding agent and a drug (such as a cytotoxic compound). The methods comprise the use of an imine reactive compound to enable efficient conjugations of cytotoxic compounds with cell binding agents.
1. A method for preparing a conjugate represented by the following formula:
or a pharmaceutically acceptable salt thereof, the method comprising:
a) reacting a modified cytotoxic compound with a bifunctional crosslinking agent to form a second modified cytotoxic compound, wherein the modified cytotoxic compound is represented by the following formula:
or a pharmaceutically acceptable salt thereof; the bifunctional crosslinking agent is N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate (sulfo-SPDB); and the second modified cytotoxic compound is represented by the following formula:
or a pharmaceutically acceptable salt thereof; and
b) reacting the second modified cytotoxic compound with an antibody at a pH of about 4 to about 9, to form the conjugate, wherein r is an integer from 1 to 10, Y is —SO 3 M, and M is H, Na or K.
2. The method of claim 1 , wherein the second modified cytotoxic compound is not purified before reacting with the antibody in step b).
3. The method of claim 1 , wherein the antibody is huMy9-6.
4. The method of claim 2 , wherein the antibody is huMy9-6.
5. The method of claim 1 , wherein the modified cytotoxic compound is produced by reacting an imine-containing compound represented by the following formula:
or a pharmaceutically acceptable salt thereof, with an imine reactive reagent, wherein the imine reactive reagent is NaHSO 3 or KHSO 3 .
6. The method of claim 5 , wherein the method further comprising purifying the modified cytotoxic compound prior to step a).
7. The method of claim 2 , wherein the modified cytotoxic compound is produced by reacting an imine-containing compound represented by the following formula:
or a pharmaceutically acceptable salt thereof, with an imine reactive reagent, wherein the imine reactive reagent is NaHSO 3 or KHSO 3 .
8. The method of claim 7 , wherein the method further comprises purifying the modified cytotoxic compound prior to step a).
9. The method of claim 3 , wherein the modified cytotoxic compound is produced by reacting an imine-containing compound represented by the following formula:
or a pharmaceutically acceptable salt thereof, with an imine reactive reagent, wherein the imine reactive reagent is NaHSO 3 or KHSO 3 .
10. The method of claim 9 , wherein the method further comprises purifying the modified cytotoxic compound prior to step a).
11. The method of claim 4 , wherein the modified cytotoxic compound is produced by reacting an imine-containing compound represented by the following formula:
or a pharmaceutically acceptable salt thereof, with an imine reactive reagent, wherein the imine reactive reagent is NaHSO 3 or KHSO 3 .
12. The method of claim 11 , wherein the method further comprises purifying the modified cytotoxic compound prior to step a).