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 - 206 . (canceled) 
     
     
         207 . A composition with reduced low conjugated fraction (LCF) below 10% comprising antibody-calicheamicin conjugates having the formula, Pr(—X—W)m, wherein:
 Pr is an antibody, 
 X is a linker having the formula (CO-Alk 1 -Sp 1 -Ar-Sp 2 -Alk 2 -C(Z 1 )=Q-Sp), wherein 
 Alk 1  and Alk 2  are independently a bond or branched or unbranched (C 1 -C 10 ) alkylene chain; 
 Sp 1  is a bond, —S—, —O—, —CONH—, —NHCO—, —NR′—, —N(CH 2 CH 2 ) 2 N—, or —X—Ar′—Y—(CH 2 ) n —Z wherein X, Y, and Z are independently a bond, —NR′—, —S—, or —O—, with the proviso that when n=0, then at least one of Y and Z must be a bond and Ar′ is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C 1 -C 5 ) alkyl, (C 1 -C 4 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen, nitro, —COOR′, —CONHR′, —(CH 2 ) n COOR′, —S(CH 2 ) n COOR′, —O(CH 2 ) n CONHR′, or —S(CH 2 ) n CONHR′, with the proviso that when Alk 1  is a bond, Sp 1  is a bond, n is an integer from 0 to 5, and R′ is a branched or unbranched (C 1 -C 5 ) chain optionally substituted by one or two groups of —OH, (C 1 -C 4 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen, nitro, (C 1 -C 3 ) dialkylamino, or (C 1 -C 3 ) trialkylammonium-A −  where A −  is a pharmaceutically acceptable anion completing a salt; 
 Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C 1 -C 6 ) alkyl, (C 1 -C 5 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen, nitro, —COOR′, —CONHR′, —O(CH 2 ) n COOR′, —S(CH 2 ) n COOR′, —O(CH 2 ) n CONHR′, or —S(CH 2 ) n CONHR′ wherein n and R′ are as hereinbefore defined or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene or 
 
       
         
           
           
               
               
           
         
       
       with each naphthylidene or phenothiazine optionally substituted with one, two, three, or four groups of (C 1 -C 6 ) alkyl, (C 1 -C 5 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen, nitro, —COOR′, —CONHR′, —O(CH 2 ) n COOR′, —S(CH 2 ) n COOR′, or —S(CH 2 ) n CONHR′ wherein n and R′ are as defined above, with the proviso that when Ar is phenothiazine, Sp 1  is a bond only connected to nitrogen;
 Sp 2  is a bond, —S—, or —O—, with the proviso that when Alk 2  is a bond, Sp 2  is a bond; 
 Z 1  is H, (C 1 -C 5 ) alkyl, or phenyl optionally substituted with one, two, or three groups of (C 1 -C 5 ) alkyl, (C 1 -C 5 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen, nitro, —COOR′, —ONHR′, —O(CH 2 ) n COOR′, —S(CH 2 ) n COOR′, —O(CH 2 ) n CONHR′, or —S(CH 2 ) n CONHR′ wherein n and R′ are as defined above; 
 Sp is a straight or branched-chain divalent or trivalent (C 1 -C 18 ) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-aryl (C 1 -C 18 ) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radical or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radical, and wherein if Sp is a trivalent radical, Sp can be additionally substituted by lower (C 1 -C 5 ) dialkylamino, lower (C 1 -C 5 ) alkoxy, hydroxy, or lower (C 1 -C 5 ) alkylthio groups; and 
 Q is ═NHNCO—, ═NHNCS—, ═NHNCONH—, ═NHNCSNH—, or ═NHO—; 
 W is a calicheamicin; 
 m is the average loading for a purified conjugation product such that the calicheamicin constitutes 4-10% of the conjugate by weight; and 
 (—X—W)m is a calicheamicin derivative. 
 
     
     
         208 . The composition of  claim 207 , wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, and a biologically active antibody fragment wherein the biologically active fragment is a Fab, a modified Fab, Fab′, F(ab′) 2  or Fv, or a heavy chain monomer or dimer. 
     
     
         209 . The composition of  claim 207 , wherein the antibody is a humanized antibody. 
     
     
         210 . The composition of  claim 207 , wherein the antibody is a CDR-grafted antibody. 
     
     
         211 . The composition of  claim 207 , wherein the calicheamicin constitutes 7-9% of the conjugate by weight. 
     
     
         212 . The composition of  claim 207 , wherein the antibody is an anti-CD22 antibody. 
     
     
         213 . The composition of  claim 212 , wherein the anti-CD22 antibody comprises 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 to 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. 
     
     
         214 . The composition of  claim 212 , wherein the anti-CD22 antibody comprises SEQ ID NO: 1 for CDR-H1, residues 50 to 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. 
     
     
         215 . The composition of  claim 214 , wherein the antibody comprises a variable domain comprising human acceptor framework regions and non-human donor CDRs. 
     
     
         216 . The composition of  claim 215 , 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 set forth as SEQ ID NOs: 21 or 22. 
     
     
         217 . The composition of  claim 215 , 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 set forth as SEQ ID NOs: 17 or 18. 
     
     
         218 . The composition of  claim 215 , wherein the anti-CD22 antibody comprises
 (a) 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 set forth as SEQ ID NOs: 21 or 22; and   (b) 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 set forth as SEQ ID NOs: 17 or 18.   
     
     
         219 . The composition of  claim 215 , wherein the anti-CD22 antibody comprises a heavy chain variable domain comprising SEQ ID NO: 27. 
     
     
         220 . The composition of  claim 215 , wherein the anti-CD22 antibody comprises a light chain variable domain comprising SEQ ID NO: 19. 
     
     
         221 . The composition of  claim 215 , 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. 
     
     
         222 . The composition of  claim 215 , wherein the anti-CD22 antibody is expressed in a mammalian cell and comprises a heavy chain consisting of residues 20-466 of SEQ ID NO: 30. 
     
     
         223 . The composition of  claim 215 , wherein the anti-CD22 antibody is expressed in a mammalian cell and comprises a light chain consisting of residues 21-239 of SEQ ID NO: 28. 
     
     
         224 . The composition of  claim 215 , wherein the anti-CD22 antibody is expressed in a mammalian cell and comprises a heavy chain consisting of residues 20-466 of SEQ ID NO: 30 and a light chain consisting of residues 21-239 of SEQ ID NO: 28. 
     
     
         225 . The composition of  claim 224 , wherein the anti-CD22 antibody results from the expression of SEQ ID NO: 29 and SEQ ID NO: 31 in the mammalian cell. 
     
     
         226 . The composition of  claim 207 , wherein the calicheamicin is selected from the group consisting of β-calicheamicin, γ-calicheamicin and N-acetyl γ-calicheamicin.