Calicheamicin derivative-carrier conjugates

Abstract

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. Monomeric calicheamicin derivative/anti-CD22 antibody conjugates, compositions comprising the conjugates and uses of the conjugates are also described.

1 . A method for preparing monomeric cytotoxic drug/carrier conjugates with reduced low conjugated fraction (LCF) having the formula, Pr(—X—W)m, wherein:
 Pr is a proteinaceous carrier, 
 X is a linker that comprises a product of any reactive group that can react with a proteinaceous carrier, 
 W is a cytotoxic drug; 
 m is the average loading for a purified conjugation product such that the cytotoxic drug constitutes 7-9% of the conjugate by weight; and 
 (—X—W)m is a cytotoxic drug derivative, 
 said method comprising the steps of: 
 (1) adding the cytotoxic drug derivative to the proteinaceous carrier wherein the cytotoxic drug derivative is 4.5-11% by weight of the proteinaceous carrier; 
 (2) incubating the cytotoxic drug derivative and a proteinaceous carrier in a non-nucleophilic, protein-compatible, buffered solution having a pH in the range from about 7 to 9 to produce a monomeric cytotoxic drug/carrier conjugate, wherein solution further comprises (a) a suitable 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 conjugate produced in step (2) to a chromatographic separation process to separate monomeric cytotoxic drug derivative/proteinaceous carrier conjugates with a loading in the range of 4-10% by weight cytotoxic drug and with low conjugated fraction (LCF) below 10 percent from unconjugated proteinaceous carrier, cytotoxic drug derivative, and aggregated conjugates. 
 
     
     
         2 . The method of  claim 1 , wherein the proteinaceous carrier is selected from a group consisting of hormones, growth factors and their genetically or enzymatically engineered counterparts. 
     
     
         3 . The method of  claim 1 , wherein the proteinaceous carrier is an antibody. 
     
     
         4 . The method of  claim 3 , wherein the antibody is selected from a group consisting of a monoclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, a Fab fragment and a F(ab) 2  fragment. 
     
     
         5 . The method of  claim 4 , wherein the humanized antibody is directed against the cell surface antigen CD22. 
     
     
         6 .- 9 . (canceled) 
     
     
         10 . The method of  claim 5 , wherein the humanized anti-CD22 antibody is a CDR-grafted antibody that is a variant antibody obtained by an affinity maturation protocol and has increased specificity for human CD22. 
     
     
         11 .- 22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the chromatographic separation process of step (3) is size exclusion chromatography (SEC). 
     
     
         24 . The method of  claim 1 , wherein the chromatographic separation process of step (3) is HPLC, FPLC or SEPHACRYL™ S-200 chromatography. 
     
     
         25 . The method of  claim 1 , wherein the chromatographic separation process of step (3) is hydrophobic interaction chromatography (HIC). 
     
     
         26 . The method of  claim 25 , wherein the hydrophobic interaction chromatography (HIC) is carried out using Phenyl SEPHAROSE™ 6 Fast Flow chromatographic medium, Butyl SEPHAROSE™ 4 Fast Flow chromatographic medium, Octyl SEPHAROSE™ 4 Fast Flow chromatographic medium, TOYOPEARL® Ether-650M chromatographic medium, MACRO-PREP® methyl HIC medium or MACRO-PREP® t-Butyl HIC medium. 
     
     
         27 . The method of  claim 25 , wherein the hydrophobic interaction chromatography (HIC) is carried out using Butyl SEPHAROSE™ 4 Fast Flow chromatographic medium. 
     
     
         28 .- 42 . (canceled) 
     
     
         43 . A monomeric calicheamicin derivative/anti-CD22 antibody conjugate having the formula, Pr(—X—S—S—W)m, wherein:
 Pr is an anti-CD22 antibody; 
 X is a hydrolyzable linker that comprises a product of any reactive group that can react with an antibody; 
 W is a calicheamicin radical; 
 m is the average loading for a purified conjugation product such that the calicheamicin constitutes 4-10% of the conjugate by weight; and 
 (—X—S—S—W)m is a calicheamicin derivative. 
 
     
     
         44 . The monomeric calicheamicin derivative/anti-CD22 antibody conjugate of  claim 43 , wherein the antibody is selected from a group consisting of a monoclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, a Fab fragment and a F(ab) 2  fragment. 
     
     
         45 .- 47 . (canceled) 
     
     
         48 . The monomeric calicheamicin derivative/anti-CD22 antibody conjugate of  claim 44 , wherein the humanized antibody is a CDR-grafted anti-CD22 antibody. 
     
     
         49 .- 57 . (canceled) 
     
     
         58 . The monomeric calicheamicin derivative/anti-CD22 antibody conjugate of  claim 48 , wherein the CDR-grafted antibody is a variant antibody obtained by an affinity maturation protocol and has increased specificity for human CD22. 
     
     
         59 . (canceled) 
     
     
         60 . The monomeric calicheamicin derivative/anti-CD22 antibody conjugate of  claim 44 , wherein the anti-CD22 antibody comprises a hybrid CDR comprising a truncated donor CDR sequence wherein the missing portion of the donor CDR is replaced by a different sequence and forms a functional CDR. 
     
     
         61 .- 64 . (canceled) 
     
     
         65 . A method for the preparation of a stable lyophilized composition of a monomeric cytotoxic drug derivative/carrier conjugate produced, the method comprising:
 (a) dissolving the monomeric cytotoxic drug derivative/carrier conjugate to a final concentration of 0.5 to 2 mg/mL in a solution comprising a cryoprotectant at a concentration of 1.5%-5% by weight, a polymeric bulking agent at a concentration of 0.5-1.5% by weight, electrolytes at a concentration 0.01M to 0.1 M, a solubility facilitating agent at a concentration of 0.005-0.05% by weight, buffering agent at a concentration of 5-50 mM such that the final pH of the solution is 7.8-8.2, and water;   (b) dispensing the above solution into vials at a temperature of +5° C. to +10° C.;   (c) freezing the solution at a freezing temperature of −35° C. to −50° C.;   (d) subjecting the frozen solution to an initial freeze drying step at a primary drying pressure of 20 to 80 microns at a shelf-temperature at −10° C. to −40° C. for 24 to 78 hours; and   (e) subjecting the freeze-dried product of step (d) to a secondary drying step at a drying pressure of 20 to 80 microns at a shelf temperature of +10° C. to +35° C. for 15 to 30 hours.   
     
     
         66 .- 72 . (canceled) 
     
     
         73 . The method of  claim 65 , further optionally comprising a bioactive agent at a therapeutically effective concentration. 
     
     
         74 . (canceled) 
     
     
         75 . The method of  claim 73 , wherein the bioactive agent is a growth factor. 
     
     
         76 . The method of  claim 73 , wherein the bioactive agent is a hormone. 
     
     
         77 .- 144 . (canceled) 
     
     
         145 . A method for preparing monomeric cytotoxic drug/carrier conjugates with reduced low conjugated fraction (LCF) having the formula, Pr(—X—W)m, wherein:
 Pr is a CDR-grafted humanized anti-CD22 antibody that is a variant antibody obtained by an affinity maturation protocol and has increased specificity for human CD22, 
 X is a linker that comprises a product of any reactive group that can react with a proteinaceous carrier, 
 W is a cytotoxic drug; 
 m is the average loading for a purified conjugation product such that the cytotoxic drug constitutes 7-9% of the conjugate by weight; and 
 (—X—W)m is a cytotoxic drug derivative, 
 said method comprising the steps of:
 (1) adding the cytotoxic drug derivative to the CDR-grafted humanized anti-CD22 antibody wherein the cytotoxic drug derivative is 4.5-11% by weight of the CDR-grafted humanized anti-CD22 antibody; 
 (2) incubating the cytotoxic drug derivative and the CDR-grafted humanized anti-CD22 antibody in a non-nucleophilic, protein-compatible, buffered solution having a pH in the range from about 7 to 9 to produce a monomeric cytotoxic drug/carrier conjugate, wherein solution further comprises (a) a suitable organic cosolvent, and (b) an additive comprising at least one C 6 -C 18  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 conjugate produced in step (2) to a chromatographic separation process to separate monomeric cytotoxic drug derivative/CDR-grafted humanized anti-CD22 antibody conjugates with a loading in the range of 4-10% by weight cytotoxic drug and with low conjugated fraction (LCF) below 10 percent from unconjugated CDR-grafted humanized anti-CD22 antibody, cytotoxic drug derivative, and aggregated conjugates. 
 
 
     
     
         146 . A monomeric calicheamicin derivative/anti-CD22 antibody conjugate having the formula, Pr(—X—S—S—W)m, wherein:
 Pr is a CDR-grafted anti-CD22 antibody obtained by an affinity maturation protocol that has increased specificity for human CD22; 
 X is a hydrolyzable linker that comprises a product of any reactive group that can react with an antibody; 
 W is a calicheamicin radical; 
 m is the average loading for a purified conjugation product such that the calicheamicin constitutes 4-10% of the conjugate by weight; and 
 (—X—S—S—W)m is a calicheamicin derivative.