Calicheamicin conjugates
Anti-Lewis Y antibodies are described. Methods for preparing monomeric cytotoxic drug/carrier conjugates with a drug loading significantly higher than in previously reported procedures and with decreased aggregation and low conjugate fraction (LCF) are described. Cytotoxic drug derivative/antibody conjugates, compositions comprising the conjugates and uses of the conjugates are also described. Specifically, monomeric calicheamicin derivative/anti-Lewis Y antibody conjugates, compositions comprising the conjugates and uses of the conjugates are also described.
1 . A process for preparing a calicheamicin conjugate comprising reacting at a pH of about 7 to about 9 (i) an activated calicheamicin-hydrolyzable linker derivative and (ii) an IgG1 antibody in the presence of a member of the deoxycholate family or a salt thereof.
2 . The process of claim 1 , wherein the deoxycholate family member has one of the following structures:
wherein
two of X 1 through X 5 are H or OH and the other three are independently either O or H;
R 1 is (CH 2 ) n where n is 0-4 and
R 2 is OH, NH(CH 2 ) m COOH, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) m PO 3 H 2 where m is 1-4.
OR
wherein
one of X 1 through X 4 is H or OH and the other three are independently either 0 or H;
R 1 is (CH 2 ) n where n is 0-2 and
R 2 is OH, NH(CH 2 ) m COOH, or NH(CH 2 ) m SO 3 H, where and m is 2.
OR
wherein
one of X 1 through X 4 is OH and the other three are H;
R 1 is (CH 2 ) n where n is 0-2 and
R 2 is OH, NH(CH 2 ) 2 SO 3 H.
3 . The process of claim 1 , wherein the deoxycholate family member is chenodeoxycholic acid, hyodeoxycholate, urosodeoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, tauroursodeoxycholic, or taurochenodeoxycholic.
4 . The process of claim 1 , wherein the deoxycholate family member is deoxycholic acid at a concentration of about 10 mM.
5 . The process of claim 1 , wherein the calicheamicin derivative is about 3 to about 9% by weight of the IgG1 antibody.
6 . The process of claim 5 , wherein the calicheamicin derivative is about 7% by weight of the IgG1 antibody.
7 . The process of claim 1 , wherein the IgG1 antibody is an anti-Lewis Y antibody.
8 . The process of claim 7 , wherein the anti-Lewis Y antibody is G193 or Hu3S193.
9 . The process of claim 1 , wherein the calicheamicin derivative is an N-acyl derivative of calicheamicin or a disulfide analog of calicheamicin.
10 . The process of claim 9 , wherein the calicheamicin derivative is N-acetyl gamma calicheamicin dimethyl hydrazide (N-acetyl calicheamicin DMH).
11 . The process of claim 1 , wherein the hydrolyzable linker is 4-(4-acetylephenoxy)butanoic acid (AcBut).
12 . The process of claim 1 , wherein the pH is about 8.2.
13 . The process of claim 1 , wherein the process further comprises purifying the calicheamicin conjugate.
14 . The process of claim 13 , wherein purification comprises chromatographic separation and ultrafiltration/diafiltration.
15 . The process of claim 14 , wherein the chromatographic separation is size exclusion chromatography (SEC) or hydrophobic interaction chromatography (HIC).
16 . The process of claim 13 , wherein following the purification step, the average loading of the conjugate is from about 5 to about 7 moles of calicheamicin per mole of IgG1 antibody.
17 . The process of claim 13 , wherein following the purification step, the low conjugated fraction (LCF) of the conjugate is less than about 10%.
18 - 100 . (canceled)