Antibodies and immunoconjugates and uses therefor

1. A humanized monoclonal antibody that binds to STEAP-1,

wherein the antibody comprises (a) heavy chain variable domain comprising:

(1) an HC-FR1 comprising the amino acid sequence of SEQ ID NO:25;

(2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:14;

(3) an HC-FR2 comprising the amino acid sequence of SEQ ID NO:22;

(4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:15;

(5) an HC-FR3 comprising the amino acid sequence of SEQ ID NO:138;

(6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:16; and

(7) an HC-FR4 comprising the amino acid sequence of SEQ ID NO:24,

and (b) a light chain variable domain comprising:

(1) an HC-FR1 comprising the amino acid sequence of SEQ ID NO:17;

(2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:11;

(3) an HC-FR2 comprising the amino acid sequence of SEQ ID NO:18;

(4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:12;

(5) an HC-FR3 comprising the amino acid sequence of SEQ ID NO:19;

(6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:13; and

(7) an HC-FR4 comprising the amino acid sequence of SEQ ID NO:20,

wherein the binding affinity to STEAP- 1 of said humanized antibody is higher than the binding affinity to STEAP-1 of a humanized antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:6.

2. The antibody of claim 1 , wherein the antibody is an antibody fragment selected from a Fab, Fab′-SH, Fv, scFv, or (Fab′) 2 fragment.

3. The antibody of claim 1 covalently attached to a cytotoxic agent.

4. The antibody covalently attached to a cytotoxic agent of claim 3 , wherein the cytotoxic agent is selected from a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioactive isotope, and a nucleolytic enzyme.

5. An immunoconjugate, wherein the immunoconjugate has the formula Ab-(L-D)p, and wherein: (a) Ab is the antibody of claim 1 (b) L is a linker; (c) D is a drug moiety; and (d) p is 1 to 20.

6. The immunoconjugate of claim 5 , wherein L is selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1carboxylate (SMCC), N-Succinimidyl (4-iodo-acetyl) aminobenzoate (SLAB), and 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

7. The immunoconjugate of claim 5 , wherein D is selected from an auristatin and dolastatin.

8. The immunoconjugate of claim 7 , wherein D is a drug moiety of formula D E or D F :

and wherein R 2 and R 6 are each methyl, R 3 and R 4 are each isopropyl; R 5 is H; R 7 is sec-butyl; each R 8 is independently selected from CH 3 , O—CH 3 , OH, and H; R 9 is H; R 10 is aryl; Z is —O— or —NH—; R 11 is H, C 1 -C 8 alkyl, or —(CH 2 ) 2 —O—(CH 2 ) 2 —OCH 2 ) 2 —O—CH 3 ; and R 18 is —C(R 8 ) 2 —C(R 8 ) 2 -aryl; and

p ranges from about 1 to 8.

9. The immunoconjugate of claim 5 , having in vitro or in vivo cell killing activity.

10. The immunoconjugate of claim 5 , wherein the immunoconjugate has the formula Ab-(L-MMAE)p, wherein L is a linker and p ranges from 2 to 5.

11. The immunoconjugate of claim 10 , wherein L comprises val-cit.

12. The immunoconjugate of claim 10 , wherein L comprises MC.

13. The immunoconjugate of claim 10 , wherein L comprises PAB.

14. The immunoconjugate of claim 10 , wherein L comprises MC-vc-PAB.

15. The immunoconjugate of claim 5 , wherein D is a maytansinoid.

16. The immunoconjugate of claim 15 , wherein D is selected from DM1, DM3, and DM4.

17. The immunoconjugate of claim 15 , wherein the linker is attached to the antibody through a thiol group on the antibody.

18. The immunoconjugate of claim 15 , wherein the linker, L, is selected from N-Succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate (SLAB).

19. The immunoconjugate of claim 18 , wherein D is DM4.

20. The immunoconjugate of claim 19 , wherein L comprises SPP.

21. The immunoconjugate of claim 19 , wherein L comprises SMCC.

22. The immunoconjugate of claim 19 , wherein p is from 2-6.

23. A pharmaceutical composition comprising the immunoconjugate of claim 5 and a pharmaceutically acceptable carrier.

24. The antibody of claim 1 comprising a heavy chain constant domain and/or a light chain constant domain in which an amino acid residue naturally occurring in said heavy chain constant domain or said light chain constant domain is replaced with a free cysteine amino acid having a thiol reactivity value in the range of 0.6 to 1.0.

25. The antibody of claim 24 wherein the free cysteine amino acid is located in a light chain constant domain.

26. The antibody of claim 24 wherein the free cysteine amino acid is located in a heavy chain constant domain.

27. An antibody of claim 24 covalently attached to a cytotoxic agent.

28. The antibody covalently attached to a cytotoxic agent of claim 27 , wherein the cytotoxic agent is selected from a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioactive isotope, and a nucleolytic enzyme.

29. The antibody of claim 24 wherein the antibody is covalently attached to a capture label, a detection label, or a solid support.

30. The antibody of claim 24 covalently attached to an auristatin or maytansinoid drug moiety.

31. An antibody-drug conjugate, wherein the antibody-drug conjugate has the formula Ab-(L-D)p, and wherein: (a) Ab is the antibody of claim 24 ; (b) L is a linker moiety attached to said free cysteine amino acid; (c) D is a drug moiety; and (d) p ranges from 1 to 4.

32. The antibody-drug conjugate compound of claim 31 wherein p is 2.

33. The antibody-drug conjugate compound of claim 31 wherein L has the formula:

-A a —W w —Y y —

where:

A is a Stretcher unit covalently attached to a cysteine thiol of the cysteine engineered antibody (Ab);

a is 0 or 1;

each W is independently an Amino Acid unit;

w is an integer ranging from 0 to 12;

Y is a Spacer unit covalently attached to the drug moiety; and

y is 0, 1 or 2.

34. The antibody-drug conjugate compound of claim 33 having the formula:

where PAB is para-aminobenzylcarbamoyl, and R 17 is a divalent radical selected from (CH 2 ) r , C 3 -C 8 carbocyclyl, O—(CH 2 ) r , arylene, (CH 2 ) r -arylene, -arylene-(CH 2 ) r —, (CH 2 ) r —(C 3 -C 8 carbocyclyl), (C 3 -C 8 carbocyclyl)-(CH 2 ) r , C 3 -C 8 heterocyclyl, (CH 2 ) r —C 3 -C 8 heterocyclyl), —(C 3 -C 8 heterocyclyl)-(CH 2 ) r —, —(CH 2 ) r C(O)NR b (CH 2 ) r —, —(CH 2 CH 2 O) r —, —(CH 2 CH 2 O) r —CH 2 —, —(CH 2 ) r C(O)NR b (CH 2 CH 2 O) r —, (CH 2 ) r C(O)NR b (CH 2 CH 2 O) r —CH 2 —, —(CH 2 CH 2 O) r C(O)NR b (CH 2 CH 2 O) r —, —(CH 2 CH 2 O) r C(O)NR b (CH 2 CH 2 O) r —CH 2 —, and CH 2 CH 2 O) r C(O)NR b (CH 2 ) r ; where R b is H, C 1 -C 6 alkyl, phenyl, or benzyl; and r is independently an integer ranging from 1 to 10.

35. The antibody-drug conjugate compound of claim 33 wherein W w is valine-citrulline.

36. The antibody-drug conjugate compound of claim 34 wherein R17 is (CH 2 ) 5 or (CH 2 ) 2 .

37. The antibody-drug conjugate compound of claim 33 having the formula:

38. The antibody-drug conjugate compound of claim 31 wherein D is MMAE, having the structure:

where the wavy line indicates the attachment site to the linker L.

39. A pharmaceutical formulation comprising the antibody covalently attached to an auristatin or maytansinoid drug moiety of claim 30 , and a pharmaceutically acceptable diluent, carrier or excipient.

40. The antibody of claim 24 in which an amino acid residue naturally occurring in said heavy chain constant domain or said light chain constant domain is replaced with a cysteine at a position selected from 110, 144, 168 and 205 of the light chain constant domain according to Kabat numbering convention or a position selected from 115, 118, 120, 171,172, 282, 375, and 400 of the heavy chain constant domain according to EU numbering convention.

41. The antibody of claim 40 , wherein a cysteine is at position 205 of the light chain constant domain.

42. The antibody of claim 40 , wherein a cysteine is at position 118 of the heavy chain constant domain.

43. The antibody-drug conjugate compound of claim 31 wherein D is DM4, having the structure:

wherein the wavy lite indicates the attachment site to the linker L.

44. An antibody-drug conjugate compound selected from the structures:

wherein Val is valine; Cit is citrulline; p ranges from 1 to 4; and Ab is an anti-STEAP-1antibody of claim 24 .