Cross-linkers with high reactivity and solubility and their use in the preparation of conjugates for targeted delivery of small molecule drugs
Disclosed is a method of making conjugates of cell binding agents and small molecule drugs comprising reacting a cell binding agent with a bifunctional cross-linking moiety to thereby provide the cell binding agent with a reactive disulfide group and then reacting the modified cell binding agent with a small molecule drug comprising a free thiol group. Bifunctional cross-linking moieties are also disclosed.
1. A method of making a conjugate comprising a cell binding agent and one or more small molecule drugs, wherein said conjugate is represented by formula (V):
wherein CB represents the cell binding agent, A represents the small molecule drug linked by a disulfide moiety, R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer from 1 to 4, and m is an integer of 1 to 10 or more, said method comprising:
(1) reacting the cell binding agent with a cross-linker of the formula (I):
wherein X and Y are the same or different and are H, CONR 4 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, and Z is SO 3 − M + or H, wherein M + represents a metal ion or a tetra alkyl ammonium ion,
to thereby give a compound of the formula (III):
wherein p represents an integer of 1 to 10 or more, and
(2) reacting the compound of the formula (III) with one or more small molecule drugs comprising a free thiol group.
2. A method of making a conjugate comprising a cell binding agent and one or more small molecule drugs, wherein said conjugate is represented by formula (V):
wherein CB represents the cell binding agent, A represents the small molecule drug linked by a disulfide moiety, R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer from 1 to 4, and m is an integer of 1 to 10 or more, said method comprising:
(1) reacting the cell binding agent with a cross-linker of the formula (II):
wherein X and Y are the same or different and are H, CONR 4 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, and Z is SO 3 − M + or H, wherein M + represents a metal ion or a tetra alkyl ammonium ion,
to thereby give a compound of the formula (IV):
wherein p represents an integer of 1 to 10 or more, and
(2) reacting the compound of the formula (IV) with one or more small molecule drugs comprising a free thiol group.
3. The method of claim 1 or 2 , wherein the cell-binding agent is an antibody or an antigen binding fragment thereof.
4. The method of claim 1 or 2 , wherein the cell-binding agent is a monoclonal antibody or an antigen binding fragment thereof.
5. The method of claim 1 or 2 , wherein the small molecule drug is a cytotoxic agent.
6. The method of claim 1 or 2 , wherein the small molecule drug is at least one member selected from the group consisting of a maytansinoid compound, a taxane compound, a CC-1065 compound, a daunorubicin compound, a doxorubicin compound, and analogues or derivatives thereof.
7. The method of claim 1 or 2 , wherein both of R and R 1 are H or methyl, or one of R and R 1 is H and the other is methyl.
8. The method of claim 1 or 2 , wherein n is 1, R 1 is methyl, and R, R 2 and R 3 are H.
9. The method of claim 1 or 2 , wherein n is 1 and R, R 1 , R 2 , and R 3 are H.
10. The method of claim 1 or 2 , wherein n is 1, R and R 1 are both methyl, and R 2 and R 3 are both H.
11. A method of making a conjugate comprising a cell binding agent and one or more small molecule drugs, wherein said conjugate is represented by formula (V):
wherein CB represents the cell binding agent, A represents the small molecule drug linked by a disulfide moiety, R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer from 1 to 4, and m is an integer of 1 to 10 or more, said method comprising:
reacting a compound of the formula (III)
wherein X and Y are the same or different and are H, CONR 4 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, and p represents an integer of 1 to 10 or more,
with one or more small molecule drugs comprising a free thiol group.
12. A method of making a conjugate comprising a cell binding agent and one or more small molecule drugs, wherein said conjugate is represented by formula (V):
wherein CB represents the cell binding agent, A represents the small molecule drug linked by a disulfide moiety, R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer of 1-4, and m is an integer of 1 to 10 or more, said method comprising:
reacting a compound of the formula (IV):
wherein X and Y are the same or different and are H, CONR 4 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, and p represents an integer of 1 to 10 or more,
with one or more small molecule drugs comprising a free thiol group.
13. The method of claim 11 or 12 , wherein the cell-binding agent is an antibody or an antigen binding fragment thereof.
14. The method of claim 11 or 12 , wherein the cell-binding agent is a monoclonal antibody or an antigen binding fragment thereof.
15. The method of claim 11 or 12 , wherein the small molecule drug is a cytotoxic agent.
16. The method of claim 11 or 12 , wherein the small molecule drug is at least one member selected from the group consisting of a maytansinoid compound, a taxane compound, a CC-1065 compound, a daunorubicin compound, a doxorubicin compound, and analogues or derivatives thereof.
17. The method of claim 11 or 12 , both of R and R 1 are H or methyl, or one of R and R 1 is H and the other is methyl.
18. The method of claim 11 or 12 , wherein n is 1, R 1 is methyl, and R, R 2 and R 3 are H.
19. The method of claim 11 or 12 , wherein n is 1 and R, R 1 , R 2 , and R 3 are H.
20. The method of claim 11 or 12 , wherein n is 1, R and R 1 are both methyl, and R 2 and R 3 are both H.
21. A cross-linker of formula (I):
wherein R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer from 1 to 4, X an Y are the same or different and are H, CONR 4 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, an Z is SO 3 31 M + or H, wherein M 31 represents a metal ion or a tetra alkyl ammonium ion, provided that when X and/or Y is NO 2 , Z is not H.
22. A cross-linker of formula (II):
wherein R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer from 1 to 4, X an Y are the same or different and are H, CONR 4 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, an Z is SO 3 31 M + or H, wherein M 30 represents a metal ion or a tetra alkyl ammonium ion, provided t at when X and/or Y is NO 2 , Z is not H.
23. The cross-linker of claim 21 or 22 , wherein both of R and R 1 are H or methyl, or one of R and R 1 is H and the other is methyl.
24. The cross-linker of claim 21 or 22 , wherein n is 1, R 1 is methyl and R, R 2 and R 3 are H.
25. The cross-linker of claim 21 or 22 , wherein n is 1 and R, R 1 , R 2 , and R 3 are H.
26. The cross-linker of claim 21 or 22 , wherein n is 1, R and R 1 are both methyl, and R 2 and R 3 are both H.
27. A method of making a compound of formula (III):
wherein CB represents a cell binding agent, R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer from 1 to 4, X and Y are the same or different and are H, CONR 4 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, and p represents an integer of 1 to 10 or more, comprising reacting the cell binding agent, CB, with a cross-linker of the formula (I):
wherein Z is SO 3 − M H , wherein M 30 represents a metal ion or a tetra alkyl ammonium ion.
28. A method of making a compound of formula (IV):
wherein CB represents a cell binding agent, R, R 1 , R 2 and R 3 are the same or different and are H, methyl, ethyl, or linear, branched or cyclic alkyl having 3 to 6 carbon atoms, n is 0 or an integer from 1 to 4, X and Y are the same or different and are H, CONR 1 R 5 or NO 2 , provided that X and Y are not both H at the same time, R 4 and R 5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, and p represents an integer of 1 to 10 or more, comprising reacting the cell binding agent with a cross-linker of the formula (II):
wherein Z is SO 3 31 M + or H, wherein M + represents a metal ion or a tetra alkyl ammonium ion.
29. The method of claim 27 or 28 , wherein the cell-binding agent is an antibody or an antigen binding fragment thereof.
30. The method of claim 27 or 28 , wherein the cell-binding agent is monocloanal antibody or an antigen binding fragment thereof.
31. the method of claim 27 or 28 , wherein both of R and R 1 are H or ethyl, or one of R and R 1 is H and the other is methyl.
32. The method of claim 27 or 28 , wherein n is 1, R 1 is methyl, and R, R 2 and R 3 are H.
33. The method of claim 27 or 28 , wherein n is 1 and R, R 1 , R 2 , and R 3 are H.
34. The method of claim 27 or 28 , wherein n is 1, R and R 1 are both methyl, and R 2 and R 3 are both H.