Calicheamicin derivative-carrier conjugates

1. A method for preparing a composition comprising monomeric calicheamicin derivative/antibody conjugates with reduced low conjugated fraction (LCF) having the formula, Pr(—X—W)m, wherein:

Pr is an anti-CD22 antibody comprising SEQ ID NO: 1 for CDR-H1, SEQ ID NO: 2 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 16 or residues 50-66 of SEQ ID NO: 23 or residues 50-66 of SEQ ID NO: 27 for CDR-H2, SEQ ID NO: 3 for CDR-H3, SEQ ID NO: 4 for CDR-L1, SEQ ID NO: 5 for CDR-L2 and SEQ ID NO: 6 for CDR-L3;

W is a calicheamicin derivative comprising a calicheamicin;

X is a hydrolyzable linker that links the antibody to the calicheamicin derivative and that is capable of releasing the calicheamicin derivative from the calicheamicin derivative/antibody conjugate after binding and entry into target cells;

m is the average loading for a purified conjugation product such that the calicheamicin constitutes 4-10% of the calicheamicin derivative/antibody conjugate by weight;

the method comprising the steps of:

(1) adding the calicheamicin derivative to the antibody wherein the calicheamicin derivative is 4.5-11% by weight of the antibody;

(2) incubating calicheamicin derivatives and antibodies in a non-nucleophilic, protein-compatible, buffered solution having a pH in the range from about 7 to 9 to produce a composition comprising the monomeric calicheamicin derivative/antibody conjugates, wherein the solution further comprises (a) an organic cosolvent, and (b) an additive comprising at least one C6-C18 carboxylic acid or its salt, and wherein the incubation is conducted at a temperature ranging from about 30° C. to about 35° C. for a period of time ranging from about 15 minutes to 24 hours; and

(3) subjecting the produced in step (2) to a chromatographic separation process to separate monomeric calicheamicin derivative/antibody conjugates with a loading in the range of 4-10% by weight calicheamicin and with low conjugated fraction (LCF) below 10 percent from unconjugated antibody, calicheamicin derivative, and aggregated conjugates.

2. The method of claim 1 , wherein the organic cosolvent of step (2)(a) is selected from the group consisting of propylene glycol, ethylene glycol, ethanol, DMF and DMSO.

3. The method of claim 1 , wherein the additive of step (2)(b) is selected from the group consisting of nanonoic acid, decanoic acid, undecanoic acid, and dodecanoic acid or its salt.

4. The method of claim 1 , wherein the additive of step (2)(b) is provided at a concentration of no more than 200 mM.

5. The method of claim 1 , wherein the additive is provided at a concentration of no more than 100 mM.

6. The method of claim 1 , wherein the additive is provided at a concentration of no more than 50 mM.

7. The method of claim 1 , wherein the chromatographic separation process of step (3) is size exclusion chromatography (SEC).

8. The method of claim 1 , wherein the chromatographic separation process of step (3) is HPLC, FPLC or Sephacryl S-200 chromatography.

9. The method of claim 1 , wherein the chromatographic separation process of step (3) is hydrophobic interaction chromatography (HIC).

10. The method of claim 9 , wherein the hydrophobic interaction chromatography (HIC) is carried out using a phenyl-based chromatographic medium, a butyl-based chromatographic medium, an octyl-based chromatographic medium, an ether-based chromatographic medium, or a methyl-based chromatographic medium.

11. The method of claim 10 , wherein the hydrophobic interaction chromatography (HIC) is carried out using a butyl-based chromatographic medium.

12. The method of claim 1 , wherein the antibody is a humanized antibody.

13. The method of claim 1 , wherein the antibody is a CDR-grafted antibody.

14. The method of claim 1 , wherein the anti-CD22 antibody comprises SEQ ID NO: 1 for CDR-H1, residues 50-66 of SEQ ID NO: 27 for CDR-H2, SEQ ID NO: 3 for CDR-H3, SEQ ID NO: 4 for CDR-L1, SEQ ID NO: 5 for CDR-L2 and SEQ ID NO: 6 for CDR-L3.

15. The method of claim 14 , wherein the anti-CD22 antibody comprises a heavy chain variable domain comprising a heavy chain framework region comprising donor residues at positions 1, 28, 48, 72, and 97 of SEQ ID NO: 8 occupied by Glu, Arg, Ile, Ala, and Thr, respectively, wherein the remainder of the heavy chain framework region is occupied by corresponding residues of the human acceptor framework of SEQ ID NOs: 21 or 22.

16. The method of claim 15 , wherein the anti-CD22 antibody comprises a light chain variable domain comprising a light chain framework region comprising donor residues at positions 2, 4, 42, 43, 50, and 65 of SEQ ID NO: 7 occupied by Val, Val, Leu, His, Gln, and Asp, respectively, wherein the remainder of the light chain framework region is occupied by corresponding residues of the human acceptor framework of SEQ ID NOs: 17 or 18.

17. The method of claim 16 , wherein the anti-CD22 antibody comprises a heavy chain variable domain comprising SEQ ID NO: 27 and a light chain variable domain comprising SEQ ID NO: 19.

18. The method of claim 17 , wherein the anti-CD22 antibody comprises a light chain consisting of residues 21-239 of SEQ ID NO: 28 and a heavy chain consisting of residues 20-466 of SEQ ID NO: 30, and wherein the antibody is expressed in a mammalian cell.

19. The method of claim 18 , wherein the anti-CD22 antibody results from the expression of SEQ ID NO: 29 and SEQ ID NO: 31 in the mammalian cell.

20. The method of claim 1 , wherein the calicheamicin is gamma calicheamicin or N-acetyl gamma calicheamicin.

21. The method of claim 1 , wherein the calicheamicin is functionalized with 3-mercapto-3-methyl butanoyl hydrazide.

22. The method of claim 1 , wherein the hydrolyzable linker comprises 4-(4-acetylphenoxy) butanoic acid (AcBut).