Site-specific antibody conjugation methods and compositions

Abstract

Provided are novel antibody drug conjugates (ADCs), and methods of using such ADCs to treat proliferative disorders.

1 .- 17 . (canceled) 
     
     
         18 . A method of treating cancer in a subject comprising administering to said subject a pharmaceutical composition comprising an engineered antibody comprising one or more unpaired cysteine residues, wherein the engineered antibody immunospecifically reacts with SEZ6. 
     
     
         19 .- 20 . (canceled) 
     
     
         21 . The method of  claim 18 , wherein the engineered antibody binds to a human SEZ6 protein and is conjugated to a cytotoxic agent via the one or more unpaired cysteine residues. 
     
     
         22 . The method of  claim 21 , wherein the antibody is an internalizing antibody. 
     
     
         23 . The method of  claim 21 , wherein the engineered antibody is an IgG1 monoclonal antibody that binds SEZ6;
 wherein the engineered antibody comprises (i) a cysteine residue at heavy chain position 220 and a deletion of a cysteine residue at light chain position 214, (ii) a cysteine residue at heavy chain position 220 and substitution of a cysteine residue at a light chain position 214, (iii) a cysteine residue at light chain position 214 and a deletion of a cysteine residue at heavy chain position 220, or (iv) a cysteine residue at light chain position 214 and a substitution of a cysteine residue heavy chain position 220; and   wherein the engineered antibody comprises native cysteine residues at heavy chain positions 226 and 229.   
     
     
         24 . The method of  claim 23 , wherein the engineered antibody comprises (a) three complementarity determining regions of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 190; and three complementarity determining regions of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 191; or (b) three complementarity determining regions of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 172; and three complementarity determining regions of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 173. 
     
     
         25 . The method of  claim 24 , wherein the engineered antibody comprises a humanized antibody or a CDR grafted antibody. 
     
     
         26 . The method of  claim 24 , wherein the engineered antibody comprises two light chains, two heavy chains and two unpaired cysteine residues. 
     
     
         27 . The method of  claim 26 , wherein each of the two light chains comprises an unpaired cysteine residue at position 214. 
     
     
         28 . The method of  claim 26 , wherein each of the two heavy chains comprises an unpaired cysteine residue at position C220. 
     
     
         29 . The method of  claim 24 , wherein the engineered antibody comprises residues 24-34 of SEQ ID NO: 190 for CDR-L1, residues 50-56 of SEQ ID NO: 190 for CDR-L2, residues 89-97 of SEQ ID NO: 190 for CDR-L3, residues 26-32 of SEQ ID NO: 191 for CDR-H1, residues 52-56 of SEQ ID NO: 191 for CDR-H2, and residues 95-102 of SEQ ID NO: 191 for CDR-H3, wherein the residues are numbered according to Chothia. 
     
     
         30 . The method of  claim 24 , wherein the engineered antibody comprises residues 30-36 of SEQ ID NO: 190 for CDR-L1, residues 46-55 of SEQ ID NO: 190 for CDR-L2, residues 89-96 of SEQ ID NO: 190 for CDR-L3, residues 30-35 of SEQ ID NO: 191 for CDR-H1, residues 47-58 of SEQ ID NO: 191 for CDR-H2, and residues 93-101 of SEQ ID NO: 191 for CDR-H3, wherein the residues are numbered according to MacCallum. 
     
     
         31 . The method of  claim 24 , wherein the engineered antibody comprises residues 24-34 of SEQ ID NO: 190 for CDR-L1, residues 50-56 of SEQ ID NO: 190 for CDR-L2, residues 89-97 of SEQ ID NO: 190 for CDR-L3, residues 31-35 of SEQ ID NO: 191 for CDR-H1, residues 50-65 of SEQ ID NO: 191 for CDR-H2, and residues 95-102 of SEQ ID NO: 191 for CDR-H3, wherein the residues are numbered according to Kabat. 
     
     
         32 . The method of  claim 24 , wherein the engineered antibody comprises
 (a) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 403 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 500;   (b) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 403 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 501;   (c) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 502 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 404;   (d) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 503 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 404;   (e) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 504 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 500;   (f) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 504 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 501;   (g) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 505 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 404; or   (h) a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 506 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 404.   
     
     
         33 . The method of  claim 24 , wherein the engineered antibody comprises a light chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 403 and a heavy chain constant region comprising an amino acid sequence set forth as SEQ ID NO: 500. 
     
     
         34 . The method of  claim 21 , wherein the cytotoxic agent is an auristatin, a maytansinoid, a calicheamicin or a radioisotope. 
     
     
         35 . The method of  claim 21 , wherein the pharmaceutical composition comprises a drug to antibody ratio (DAR) of 1, 2, 3, 4, 5, 6, 7, or 8, each +/−0.4. 
     
     
         36 . The method of  claim 21 , wherein the pharmaceutical composition comprises a drug to antibody ratio (DAR) of 2+/−0.4. 
     
     
         37 . The method of  claim 21 , wherein the pharmaceutical composition comprises a predominant antibody drug conjugate species present at a concentration of greater than 70%. 
     
     
         38 . The method of  claim 21 , wherein the engineered antibody is conjugated to the cytotoxic agent via a linker. 
     
     
         39 . The method of  claim 38 , wherein the linker comprises a non-cleavable linker. 
     
     
         40 . The method of  claim 21 , wherein the cancer is small cell lung cancer or medullary thyroid cancer. 
     
     
         41 . The method of  claim 40 , wherein the small cell lung cancer is resistant to a platinum based agent. 
     
     
         42 . The method of  claim 41 , wherein the platinum based agent is cisplatin. 
     
     
         43 . A method of treating small cell lung cancer comprising administering to a subject in need thereof a therapeutically effective amount of an antibody drug conjugate comprising an antibody conjugated to a cytotoxic agent;
 wherein the antibody binds to a human SEZ6 protein and comprises (i) a cysteine residue at heavy chain position 220 and a deletion of a cysteine residue at light chain position 214, (ii) a cysteine residue at heavy chain position 220 and substitution of a cysteine residue at a light chain position 214, (iii) a cysteine residue at light chain position 214 and a deletion of a cysteine residue at heavy chain position 220, or (iv) a cysteine residue at light chain position 214 and a substitution of a cysteine residue heavy chain position 220;   wherein the antibody comprises native cysteine residues at heavy chain positions 226 and 229;   wherein the antibody comprises three complementarity determining regions of a light chain variable region set forth as SEQ ID NO: 190 and three complementarity determining regions of a heavy chain variable region set forth as SEQ ID NO: 191; and   wherein the cytotoxic agent is a calicheamicin.   
     
     
         44 . A method of treating medullary thyroid cancer comprising administering to a subject in need thereof a therapeutically effective amount of an antibody drug conjugate comprising an antibody conjugated to a cytotoxic agent;
 wherein the antibody binds to a human SEZ6 protein and comprises (i) a cysteine residue at heavy chain position 220 and a deletion of a cysteine residue at light chain position 214, (ii) a cysteine residue at heavy chain position 220 and substitution of a cysteine residue at a light chain position 214, (iii) a cysteine residue at light chain position 214 and a deletion of a cysteine residue at heavy chain position 220, or (iv) a cysteine residue at light chain position 214 and a substitution of a cysteine residue heavy chain position 220;   wherein the antibody comprises native cysteine residues at heavy chain positions 226 and 229;   wherein the antibody comprises three complementarity determining regions of a light chain variable region set forth as SEQ ID NO: 190 and three complementarity determining regions of a heavy chain variable region set forth as SEQ ID NO: 191; and   wherein the cytotoxic agent is a calicheamicin.