SITE-SPECIFIC ANTIBODY CONJUGATION METHODS AND COMPOSITIONS

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.