Conjugates of cysteine engineered antibodies

Abstract

The invention relates to novel cell-binding agent-cytotoxic agent conjugates, wherein the cell-binding agent (CBA) is covalently linked to the cytotoxic agent through an engineered Cys, such as an engineered Cys in the heavy chain CH3 domain, at a position corresponds to the EU/OU numbering position 442 (or C442) on an antibody CBA. The invention also provides methods of preparing the conjugates of the present invention. The invention further provides composition and methods useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal using the conjugates of the invention.

1 . An antibody-cytotoxic agent conjugate represented by the following formula: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 Ab is an antibody having a cysteine residue at the EU/OU numbering position 442 of a heavy chain of the antibody, and is covalently linked to a linking moiety J CB ′ through the thiol group S 442  of the cysteine residue; 
 D is a cytotoxic agent covalently linked to a linker L that is covalently linked to J CB ′; and, 
 w is 1 or 2. 
 
       
     
     
         2 . The conjugate of  claim 1 , wherein the cysteine residue at position 442 is recombinantly introduced into said Ab. 
     
     
         3 . The conjugate of  claim 1 , wherein:
 J CB ′ is   
       
         
           
           
               
               
           
         
         s1 is the site covalently linked to the cysteine residue, and s2 is the site covalently linked to the group L; and, 
         R a , R b , R c , and R e , for each occurrence, are independently H or an optionally substituted alkyl. 
       
     
     
         4 - 6 . (canceled) 
     
     
         7 . The conjugate of  claim 1 , wherein -L- is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         wherein:
 s3 is the site covalently linked to J CB ′, and s4 is the site covalently linked to D; 
 E is —(CR 10 R 11 ) q —, cycloalkyl, or cycloalkylalkyl; 
 Z is absent, —SO 2 NR 9 —, —NR 9 SO 2 —, —C(═O)—NR 9 —, —NR 9 —C(═O)—, —C(═O)—O—, —O—C(═O)—, —C(═)—NR 9 —(CH 2 CH 2 O) p —, —NR 9 —C(═O)—(CH 2 CH 2 O) p —, —(OCH 2 CH 2 ) p —C(═O)NR 9 —, or —(OCH 2 CH 2 ) p —NR 9 —C(═O)—; 
 p is an integer from 1 to 24; 
 Q is H, a charged substituent, or an ionizable group; 
 R 9 , R 10 , R 11 , R 12 , and R 13 , for each occurrence, are independently H or an optionally substituted alkyl; and, 
 q and r, for each occurrence, are independently an integer between 0 and 10. 
 
       
     
     
         8 - 11 . (canceled) 
     
     
         12 . The conjugate of  claim 7 , wherein Q is:
 i) H;   ii) —SO 3 H, —Z′—SO 3 H, —OPO 3 H 2 , —Z′—OPO 3 H 2 , —PO 3 H 2 , —Z′—PO 3 H 2 , —CO 2 H, —Z′—CO 2 H, —NR 11 R 12 , or —Z′—NR 14 R 15 , or a pharmaceutically acceptable salt thereof; or,   iii) —N + R 14 R 15 R 16 X −  or —Z′—N + R 14 R 15 R 16 X − ;
 Z′ is an optionally substituted alkylene, an optionally substituted cycloalkylene, or an optionally substituted phenylene; 
 R 14 , R 15  and R 16  are each independently an optionally substituted alkyl; and, 
 X −  is a pharmaceutically acceptable anion. 
   
     
     
         13 - 16 . (canceled) 
     
     
         17 . The conjugate of  claim 1 , wherein -L- is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         wherein:
 s3 is the site covalently linked to J CB ′, and s4 is the site covalently linked to D; 
 R 19  to R 22 , for each occurrence, are independently H or an optionally substituted alkyl; 
 m and n are each independently an integer between 0 and 10; 
 R h  is H or an optionally substituted alkyl; 
 P is an optionally substituted alkylene, —(CH 2 —CH 2 —O) j — (wherein the oxygen atom is connected to the —(C═O)— group connected to P), an amino acid residue or a peptide containing 2 to 20 amino acid residues; and 
 j is an integer from 1 to 24. 
 
       
     
     
         18 - 27 . (canceled) 
     
     
         28 . The conjugate of  claim 1 , wherein -L- is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         wherein:
 s3 is the site covalently linked to J CB ′, and s4 is the site covalently linked to D; 
 R 23  and R 24 , for each occurrence, are independently H or an optionally substituted alkyl; 
 m′ is an integer between 0 and 10; and 
 R h′  is H or an optionally substituted alkyl. 
 
       
     
     
         29 - 31 . (canceled) 
     
     
         32 . The conjugate of  claim 1 , wherein D is a benzodiazepine compound. 
     
     
         33 . (canceled) 
     
     
         34 . The conjugate of  claim 1 , wherein D is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 the double line   between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is —H, and when it is a single bond, X is selected from —H, or an amine protecting group; and Y is selected from —OR, —OCOR′, —SR, —NR′R″, —SO 3 M, —SO 2 M or —OSO 3 M, wherein M is —H or a cation; 
 R is —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group —(CH 2 CH 2 O) n —R c , wherein n is an integer from 1 to 24, and R c  is a linear or branched alkyl having 1 to 4 carbon atoms; 
 R′ and R″ are each independently selected from —H, —OH, —OR, —NRR g′ , —COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted aryl having from 6 to 18 carbon atoms, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P, a PEG group —(CH 2 CH 2 O) n —R c , and R g′  is —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group —(CH 2 CH 2 O) n —R c ; 
 X′ is selected from the group consisting of —H, —OH, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl, and an amine-protecting group; 
 Y′ is selected from the group consisting of —H, an oxo group, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; 
 A and A′ are selected from —O— and —S—; 
 W′ is absent, or selected from —O—, —N(R e )—, —N(R e )—C(═O)—, —N(C(═O)R e )—, —S— or —CH 2 —S—, —CH 2 NR e —; 
 R x  is absent or selected from a linear, branched or cyclic alkyl having 1 to 10 carbon atoms; 
 R e  is —H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or —(CH 2 —CH 2 —O) n —R k , wherein R k  is a —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g., —NHR 101 ) or tertiary amino (—NR 101 R 102 ) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R 101  and R 102  are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms; 
 G is selected from —CH— or —N—; and 
 R 6  is —H, —R, —OR, —SR, —NR′R″, —NO 2 , or halogen. 
 
       
     
     
         35 - 38 . (canceled) 
     
     
         39 . The conjugate of  claim 28 , wherein D is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 one of L′, L″, and L′″ is represented by the following formula:
   —Z 1 —P 1 —Z 2 —R x1 —C(═O)—  (A′), or
 
   —N(R e )—R x1 —C(═O)—  (D′);
 
 
 and the other two are each independently selected from —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , halogen, guanidinium [—NH(C═NH)NH 2 ], —OR, —NR′R″, —NO 2 , —NR′COR″, —SR, —SOR′, —SO 2 R′, —SO 3 H, —OSO 3 H, —SO 2 NR′R″, cyano, an azido, —COR′, —OCOR′, and —OCONR′R″; 
 one of the Z 1  and Z 2  is —C(═O)—, and the other is —NR 5 —; 
 P 1  is an amino acid residue or a peptide containing between 2 to 20 amino acid residues; 
 R x1  is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; 
 R e  is —H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or —(CH 2 —CH 2 —O) n —R k , wherein R k  is a —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g., —NHR 101 ) or tertiary amino (—NR 101 R 102 ) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R 101  and R 102  are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms; 
 the double line   between N and C represents a single bond or a double bond, provided that when it is a double bond X 1  is absent and Y 1  is —H, or a linear or branched alkyl having 1 to 4 carbon atoms, and when it is a single bond, X 1  is —H or an amine protecting moiety; and Y 1  is a leaving group selected from —OR, —OCOR′, —OCOOR′, —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionally substituted 5- or 6-membered nitrogen-containing heterocycle, a guanidinium represented by —NR′(C═NH)NR′R″, an amino acid, or a peptide represented by —NRCOP′, —SR, —SOR′, halogen, cyano, azido, —OSO 3 H (or a salt thereof), sulfite (—SO 3 H or —SO 2 H or a salt thereof), metabisulfite (H 2 S 2 O 5  or a salt thereof), mono-, di-, tri-, and tetra-thiophosphate (PO 3 SH 3 , PO 2 S 2 H 2 , POS 3 H 2 , PS 4 H 2  or a salt thereof), thio phosphate ester (R i O) 2 PS(OR i ), R i S—, R i SO, R i SO 2 , R i SO 3 , thiosulfate (HS 2 O 3  or a salt thereof), dithionite (HS 2 O 4  or salt thereof), phosphorodithioate (P(═S)(OR k′ )(S)(OH) or a salt thereof), hydroxamic acid (R k′ C(═O)NOH or a salt thereof), and formaldehyde sulfoxylate (HOCH 2 SO 2   −  or a salt thereof) or a mixture thereof, wherein R i  is a linear or branched alkyl having 1 to 10 carbon atoms and is substituted with at least one substituent selected from —N(R j ) 2 , —CO 2 H, —SO 3 H, and —PO 3 H; R i  can be further optionally substituted with a substituent for an alkyl described herein; R j  is a linear or branched alkyl having 1 to 6 carbon atoms; R k′  is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl; 
 P′ is an amino acid residue or a peptide containing between 2 to 20 amino acid residues, 
 R, for each occurrence, is independently selected from the group consisting of —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P; 
 R′ and R″ are each independently selected from —H, —OH, —OR, —NHR, —NR 2 , —COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , and an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N and P; 
 R c  is —H or an optionally substituted linear or branched alkyl having 1 to 4 carbon atoms; 
 n is an integer from 1 to 24; 
 X 1 ′ is selected from —H, an amine-protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P; 
 Y 1 ′ is selected from —H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted 6- to 18-membered aryl, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms; 
 R 1 , R 2 , R 3 , R 4 , R 1 ′, R 2 ′, R 3 ′ and R 4 ′ are each independently selected from the group consisting of —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2C H 2 O) n —R c , halogen, guanidinium [—NH(C═NH)NH 2 ], —OR, —NR′R″, —NO 2 , —NCO, —NR′COR″, —SR, —SOR′, —SO 2 R′, —SO 3   − —H, —OSO 3 H, —SO 2 NR′R″, cyano, an azido, —COR′, —OCOR′, and —OCONR′R″; 
 R 6  is —H, —R, —OR, —SR, —NR′R″, —NO 2 , or halogen; 
 G is —CH— or —N—; 
 A and A′ are the same or different, and are independently selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—, —NR 5  and —CRR′N(R 5 )—; and 
 R 5  for each occurrence is independently —H or an optionally substituted linear or branched alkyl having 1 to 10 carbon atoms. 
 
       
     
     
         40 - 73 . (canceled) 
     
     
         74 . The conjugate of  claim 39 , wherein D is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein M is H +  or a pharmaceutically acceptable cation. 
       
     
     
         75 . The conjugate of  claim 1 , wherein the conjugate is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody; and M is H +  or a pharmaceutically acceptable cation. 
       
     
     
         76 . The conjugate of  claim 1 , wherein M, when present, is H + , Na +  or K + . 
     
     
         77 . The conjugate of  claim 1 , wherein w is 2. 
     
     
         78 . A compound represented by the following structural formula:
   D-L-J CB ,   or a pharmaceutically acceptable salt thereof, wherein:
 D is represented by the following structural formula: 
   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 the double line   between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is —H, and when it is a single bond, X is selected from —H, or an amine protecting group; and Y is selected from —OR, —OCOR′, —SR, —NR′R″, —SO 3 M, —SO 2 M or —OSO 3 M, wherein M is —H or a cation; 
 R is —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group —(CH 2 CH 2 O) n —R c , wherein n is an integer from 1 to 24, and R c  is a linear or branched alkyl having 1 to 4 carbon atoms; 
 R′ and R″ are each independently selected from —H, —OH, —OR, —NRR g′ , —COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted aryl having from 6 to 18 carbon atoms, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P, a PEG group —(CH 2 CH 2 O) n —R c , and R g′  is —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group —(CH 2 CH 2 O) n —R c ; 
 X′ is selected from the group consisting of —H, —OH, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl, and an amine-protecting group; 
 Y′ is selected from the group consisting of —H, an oxo group, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; 
 A and A′ are selected from —O— and —S—; 
 W′ is absent, or selected from —O—, —N(R e )—, —N(R e )—C(═O)—, —N(C(═O)R e )—, —S— or —CH 2 —S—, —CH 2 NR e —; 
 R x  is absent or selected from a linear, branched or cyclic alkyl having 1 to 10 carbon atoms; 
 R e  is —H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or —(CH 2 —CH 2 —O) n —R k , wherein R k  is a —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g., —NHR 101 ) or tertiary amino (—NR 101 R 102 ) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R 101  and R 102  are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms; 
 G is selected from —CH— or —N—; 
 R 6  is —H, —R, —OR, —SR, —NR′R″, —NO 2 , or halogen; 
 -L- is represented by the following structural formula: 
 
       
       
         
           
           
               
               
           
         
         
           wherein:
 s3 is the site covalently linked to J CB , and s4 is the site covalently linked to D; 
 E is —(CR 10 R 11 ) q —, cycloalkyl or cycloalkylalkyl; 
 Z is absent, —SO 2 NR 9 —, —NR 9 SO 2 —, —C(═O)—NR 9 —, —NR 9 —C(═O)—, —C(═O)—O—, —O—C(═O)—, —C(═O)—NR 9 —(CH 2 CH 2 O) p —, —NR 9 —C(═O)—(CH 2 CH 2 O) p —, —(OCH 2 CH 2 ) p —C(═O)NR 9 —, or —(OCH 2 CH 2 ) p —NR 9 —C(═O)—; 
 p is an integer from 1 to 1000; 
 Q is H, a charged substituent or an ionizable group; 
 R 9 , R 10 , R 11 , R 12 , and R 13 , for each occurrence, are independently H or an optionally substituted alkyl; 
 q and r, for each occurrence, are independently an integer between 0 and 10; and 
 J CB  is 
 
         
       
       
         
           
           
               
               
           
         
       
       X′—CR b R c —C(═)—, X′—CR b R c —C(═O)—NR e —, 
       
         
           
           
               
               
           
         
       
     
     
         79 - 95 . (canceled) 
     
     
         96 . A compound represented by the following structural formula:
   D-L-J CB ,   or a pharmaceutically acceptable salt thereof, wherein:
 D is represented by the following structural formula: 
   
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 one of L′, L″, and L′″ is represented by the following formula:
   —Z 1 —P 1 —Z 2 —R x1 —C(═O)—  (A′),or
 
   —N(R e )—R x1 —C(═O)—  (D′);
 
 
 and the other two are each independently selected from —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , halogen, guanidinium [—NH(C═NH)NH 2 ], —OR, —NR′R″, —NO 2 , —NR′COR″, —SR, —SOR′, —SO 2 R′, —SO 3 H, —OSO 3 H, —SO 2 NR′R″, cyano, an azido, —COR′, —OCOR′, and —OCONR′R″; 
 one of the Z 1  and Z 2  is —C(═O)—, and the other is —NR 5 —; 
 P 1  is an amino acid residue or a peptide containing between 2 to 20 amino acid residues; 
 R x1  is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; 
 R e  is —H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or —(CH 2 —CH 2 —O) n —R k , wherein R k  is a —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g., —NHR 101 ) or tertiary amino (—NR 101 R 102 ) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R 101  and R 102  are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms; 
 the double line   between N and C represents a single bond or a double bond, provided that when it is a double bond X 1  is absent and Y 1  is —H, or a linear or branched alkyl having 1 to 4 carbon atoms, and when it is a single bond, X 1  is —H or an amine protecting moiety; and Y 1  is a leaving group selected from —OR, —OCOR′, —OCOOR′, —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionally substituted 5- or 6-membered nitrogen-containing heterocycle, a guanidinium represented by —NR′(C═NH)NR′R″, an amino acid, or a peptide represented by —NRCOP′, —SR, —SOR′, halogen, cyano, azido, —OSO 3 H (or a salt thereof), sulfite (—SO 3 H or —SO 2 H or a salt thereof), metabisulfite (H 2 S 2 O 5  or a salt thereof), mono-, di-, tri-, and tetra-thiophosphate (P 3 SH 3 , PO 2 S 2 H 2 , POS 3 H 2 , PS 4 H 2  or a salt thereof), thio phosphate ester (R i O) 2 PS(OR i ), R i S—, R i SO, R i SO 2 , R i SO 3 , thiosulfate (HS 2 O 3  or a salt thereof), dithionite (HS 2 O 4  or salt thereof), phosphorodithioate (P(═S)(OR k′ )(S)(OH) or a salt thereof), hydroxamic acid (R k′ C(═O)NOH or a salt thereof), and formaldehyde sulfoxylate (HOCH 2 SO 2   −  or a salt thereof) or a mixture thereof, wherein R i  is a linear or branched alkyl having 1 to 10 carbon atoms and is substituted with at least one substituent selected from —N(R j ) 2 , —CO 2 H, —SO 3 H, and —PO 3 H; R i  can be further optionally substituted with a substituent for an alkyl described herein; R j  is a linear or branched alkyl having 1 to 6 carbon atoms; R k′  is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl; 
 P′ is an amino acid residue or a peptide containing between 2 to 20 amino acid residues, 
 R, for each occurrence, is independently selected from the group consisting of —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P; 
 R′ and R″ are each independently selected from —H, —OH, —OR, —NHR, —NR 2 , —COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , and an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N and P; 
 R c  is —H or an optionally substituted linear or branched alkyl having 1 to 4 carbon atoms; 
 n is an integer from 1 to 24; 
 X 1 ′ is selected from —H, an amine-protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P; 
 Y 1 ′ is selected from —H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted 6- to 18-membered aryl, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms; 
 R 1 , R 2 , R 3 , R 4 , R 1 ′, R 2 ′, R 3 ′ and R 4 ′ are each independently selected from the group consisting of —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , halogen, guanidinium [—NH(C═NH)NH 2 ], —OR, —NR′R″, —NO 2 , —NCO, —NR′COR″, —SR, —SOR′, —SO 2 R′, —SO 3   − H, —OSO 3 H, —SO 2 NR′R″, cyano, an azido, —COR′, —OCOR′, and —OCONR′R″; 
 R 6  is —H, —R, —OR, —SR, —NR′R″, —NO 2 , or halogen; 
 G is —CH— or —N—; 
 A and A′ are the same or different, and are independently selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—, —NR 5  and —CRR′N(R 5 )—; and 
 R 5  for each occurrence is independently —H or an optionally substituted linear or branched alkyl having 1 to 10 carbon atoms; 
 -L- is represented by the following structural formula: 
 
       
       
         
           
           
               
               
           
         
         wherein:
 s3 is the site covalently linked to J CB ′, and s4 is the site covalently linked to D; 
 R 23  and R 24 , for each occurrence, are independently H or an optionally substituted alkyl; 
 m′ is an integer between 0 and 10; 
 R h′  is H or an optionally substituted alkyl; and 
 J CB  is 
 
       
       
         
           
           
               
               
           
         
       
       X′—CR b R c —C(═O)—, X′—CR b R c —C(═O)—NR e —, 
       
         
           
           
               
               
           
         
       
     
     
         97 - 140 . (canceled) 
     
     
         141 . A pharmaceutical composition comprising the conjugate of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         142 . A method of inhibiting abnormal cell growth or treating a proliferative disorder in a mammal, comprising administering to said mammal a therapeutically effective amount of the conjugate of  claim 1 , and, optionally, a second therapeutic agent. 
     
     
         143 . The method of  claim 142 , wherein the proliferative disorder is cancer. 
     
     
         144 . The method of  claim 143 , wherein the cancer is a hematological cancer or a solid tumor. 
     
     
         145 - 146 . (canceled)