Immunoconjugates and methods

1. An immunoconjugate having Formula (I),

Ab-[S-L 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n   (I)

wherein:

Ab is an antibody or an antigen-binding fragment thereof;

L 1 is

L 2 is absent,

Z 1 and Z 2 are each individually hydrogen, halogen, NO 2 , —O—(C 1 -C 6 alkyl), or C 1 -C 6 alkyl;

L 3 is —(CH 2 )n 1 -C(═O)— or —(CH 2 CH 2 O)n 1 -(CH 2 )n 1 C(═O)—;

each n 1 is independently an integer of 0 to 12;

L 4 is a tetrapeptide residue;

L 5 is absent or —[NH(CH 2 )n 2 ]n 3 -;

n 2 is an integer of 0 to 6;

n 3 is an integer of 0 to 2;

L 6 is absent or

L 7 is absent,

D is a drug moiety; and

n is an integer from 1 to 10, wherein said antibody or binding fragment thereof is selected from:

an antibody or binding fragment thereof comprising:

a heavy chain comprising:

VHCDR 1 comprising an amino acid sequence of SEQ ID NO:29;

VHCDR 2 comprising an amino acid sequence of SEQ ID NO:30; and

VHCDR 3 comprising an amino acid sequence of SEQ ID NO:31; and

a light chain comprising:

VLCDR 1 comprising an amino acid sequence of SEQ ID NO:36;

VLCDR 2 comprising an amino acid sequence of DAS; and

VLCDR 3 comprising an amino acid sequence of SEQ ID NO:38;

wherein the antibody or antigen-binding fragment specifically binds to the extracellular domain of human receptor tyrosine kinase like orphan receptor 1 (ROR1).

2. The immunoconjugate of claim 1 , wherein D is a drug moiety of Formula (II) having the structure:

wherein:

R 1 and R 2 are each individually selected from the group consisting of hydrogen, halogen, —CN, —OR 5 , —NR 5 R 6 , a substituted or an unsubstituted C 1 -C 6 alkyl, a substituted or an unsubstituted C 1 -C 6 haloalkyl, a substituted or an unsubstituted —O—(C 1 -C 6 alkyl), a substituted or an unsubstituted —O—(C 1 -C 6 haloalkyl), and —[(CY 2 ) p O(CY 2 ) q ] t CY 3 , or a substituted or an unsubstituted —O—(CR 5 R 6 ) m —O— such that R 1 and R 2 taken together form a ring;

R 3 is hydrogen or a substituted or an unsubstituted C 1 -C 6 alkyl, a substituted or an unsubstituted C 1 -C 6 haloalkyl, or —[(CY 2 ) p O(CY 2 ) q ] t CY 3 ;

R 4 is hydrogen, a substituted or an unsubstituted —(C 1 -C 6 alkyl)-X 2 , a substituted or an unsubstituted —(C 1 -C 6 haloalkyl)-X 2 , a substituted or an unsubstituted —(C 1 -C 6 alkenyl)-X 2 , a substituted or an unsubstituted —(C 1 -C 6 haloalkenyl)-X 2 , a substituted or an unsubstituted —(C 1 -C 6 alkynyl)-X 2 , or a substituted or an unsubstituted —(C 1 -C 6 haloalkynyl)-X 2 ;

X 1 is —O—, —S(O n6 )—, —NH—, —O—(C═O)—, —NH—(C═O)—, —NH—(C═O)—O—, —NH—(C═O)—NH—, or —NH—S(O n6 )—;

X 2 is —OR 9 , —SR 9 , or —NHR 9 ;

R 5 and R 6 are each individually hydrogen, halogen, a substituted or an unsubstituted C 1 -C 6 alkyl, a substituted or an unsubstituted C 1 -C 6 haloalkyl, or —[(CY 2 ) p O(CY 2 ) q ] t CY 3 ;

m is 1 or 2;

n 4 and n 5 are each individually 0, 1 or 2, with the proviso that n 4 and n 5 are not both 0;

n 6 is 0, 1 or 2;

each Y is individually H or halogen;

each p is individually 1, 2, 3, 4, 5, or 6;

each q is individually 0, 1, 2, 3, 4, 5, or 6;

each t is individually 1, 2, 3, 4, 5, or 6;

R 7 is H, —COR 8 , —CO 2 R 8 , —(C 0 )—NHR 8 , L 4 , L 5 , L 6 , or L 7 ;

R 8 is a substituted or an unsubstituted C 1 -C 6 alkyl-X 3 , a substituted or an unsubstituted C 1 -C 6 haloalkyl-X 3 , or —[(CY 2 ) p O(CY 2 ) q ] t CY 2 —X 3 ;

R 9 is H, —COR 8 , —CO 2 R 8 , —(C 0 )—NHR 8 , L 4 , L 5 , L 6 , or L 7 , with the proviso that exactly one of R 7 and R 9 is L 4 , L 5 , L 6 , or L 7 ; and

each X 3 is individually —H, —OH, —SH, or —NH 2 .

3. The immunoconjugate of claim 2 , wherein D is a drug moiety of Formula (II) having the structure:

wherein:

R 1 and R 2 are each individually selected from the group consisting of hydrogen, halogen, —OR 5 , an unsubstituted C 1 -C 6 alkyl, an unsubstituted C 1 -C 6 haloalkyl and an unsubstituted —O—(C 1 -C 6 alkyl), or an unsubstituted —O—(CR 5 R 6 ) m —O— such that R 1 and R 2 taken together form a ring;

R 3 is hydrogen or an unsubstituted C 1 -C 6 alkyl;

R 4 is (C 1 -C 6 alkyl)-X 2 ;

X 1 is —O—;

X 2 is —OR 9 ;

R 5 and R 6 are each individually hydrogen;

m is 1;

n 4 is 1 or 2;

n 5 is 0;

R 7 is H; and

R 9 is L 4 , L 5 , L 6 , or L 7 .

4. The immunoconjugate of claim 3 , wherein n 4 is 1.

5. The immunoconjugate of claim 3 , wherein n 4 is 2.

6. The immunoconjugate of claim 1 , wherein the immunoconjugate is:

wherein Z 1 and Z 2 are each individually hydrogen.

7. The immunoconjugate of claim 1 , wherein the immunoconjugate is:

8. The immunoconjugate of claim 1 , wherein the immunoconjugate is:

9. The immunoconjugate of claim 2 , wherein D is a compound of Formula (II) having the structure:

10. The immunoconjugate of claim 9 , wherein D is a compound of Formula (II) having the structure:

11. The immunoconjugate of claim 9 , wherein D is a compound of Formula (II) having the structure:

12. A compound of Formula (II), or a pharmaceutically acceptable salt thereof, having the structure:

wherein:

R 1 and R 2 are each individually selected from the group consisting of hydrogen, halogen, —OR 5 , an unsubstituted C 1 -C 6 alkyl, an unsubstituted C 1 -C 6 haloalkyl, and an unsubstituted —O—(C 1 -C 6 alkyl), or an unsubstituted —O—(CR 5 R 6 ) m —O— such that R 1 and R 2 taken together form a ring;

R 3 is hydrogen or an unsubstituted C 1 -C 6 alkyl;

R 4 is hydrogen or an unsubstituted —(C 1 -C 6 alkyl)-X 2 ;

X 1 is —O—;

X 2 is —OH;

R 5 and R 6 are each individually hydrogen;

R 7 is H;

m is 1;

n 4 is 1 or 2; and

n 5 is 0;

with the proviso that Formula (II) does not represent exatecan.

13. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein at least one of R 1 and R 2 is hydrogen.

14. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein at least one of R 1 and R 2 is halogen.

15. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein at least one of R 1 and R 2 is an unsubstituted C 1 -C 6 alkyl.

16. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are each individually selected from the group consisting of hydrogen, fluoro, methoxy, methyl, and difluoromethyl, or —O—(CH 2 )—O— such that R 1 and R 2 taken together form a ring.

17. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein R 3 is hydrogen.

18. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein R 3 is an unsubstituted C 1 -C 6 alkyl.

19. The compound of claim 18 , or a pharmaceutically acceptable salt thereof, wherein R 3 is methyl.

20. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein R 4 is hydrogen.

21. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein R 4 is an unsubstituted —(C 1 -C 6 alkyl)-X 2 .

22. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein the compound of Formula (II) is selected from the group consisting of:

23. The compound of claim 12 , wherein n 4 is 1.

24. The compound of claim 12 , wherein n 4 is 2.

25. The compound of claim 12 , or a pharmaceutically acceptable salt thereof, wherein the compound of Formula (II) is

26. The compound of claim 25 , or a pharmaceutically acceptable salt thereof, wherein the compound of Formula (II) is

27. The compound of claim 25 , or a pharmaceutically acceptable salt thereof, wherein the compound of Formula (II) is

28. A compound, or a pharmaceutically acceptable salt thereof, represented by Formula (IVa):

wherein:

R 1 and R 2 are each individually selected from the group consisting of hydrogen, halogen, —OR 5 , an unsubstituted C 1 -C 6 alkyl, an unsubstituted C 1 -C 6 haloalkyl and an unsubstituted —O—(C 1 -C 6 alkyl), or an unsubstituted —O—(CR 5 R 6 ) m —O— such that R 1 and R 2 taken together form a ring;

R 3 is hydrogen or an unsubstituted C 1 -C 6 alkyl;

R 4 is hydrogen or an unsubstituted —(C 1 -C 6 alkyl)-X 2 ;

X 1 is —O—;

X 2 is —OH;

R 5 and R 6 are each individually hydrogen;

R 7 is H; and

m is 1.

29. The compound of claim 28 , or pharmaceutically acceptable salt thereof, wherein the compound of Formula (IVa) is:

or a pharmaceutically acceptable salt thereof.

30. A conjugate having Formula (III),

Mi-L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D  (III)

wherein:

Mi is

L 2 is absent,

Z 1 and Z 2 are each individually hydrogen, halogen, NO 2 , —O—(C 1 -C 6 alkyl), or C 1 -C 6 alkyl;

L 3 is —(CH 2 )n 1 -C(═O)—;

n 1 is an integer of 0 to 12;

L 4 is a tetrapeptide residue;

L 5 is absent or —[NH(CH 2 )n 21 ]n 3 -;

n 2 is an integer of 0 to 6;

n 3 is an integer of 0 to 2;

L 6 is absent;

L 7 is absent,

and

D is a drug moiety, wherein D is a compound of Formula (II) having the structure:

wherein:

R 1 and R 2 are each individually selected from the group consisting of hydrogen, halogen, —OR 5 , an unsubstituted C 1 -C 6 alkyl, an unsubstituted C 1 -C 6 haloalkyl and an unsubstituted —O—(C 1 -C 6 alkyl), or an unsubstituted —O—(CR 5 R 6 ) m —O— such that R 1 and R 2 taken together form a ring;

R 3 is hydrogen or an unsubstituted C 1 -C 6 alkyl;

R 4 is (C 1 -C 6 alkyl)-X 2 ;

X 1 is —O—;

X 2 is —OR 9 ;

R 5 and R 6 are each individually hydrogen;

m is 1;

n 4 is 1 or 2;

n 5 is 0;

R 7 is H; and

R 9 is L 4 , L 5 or L 7 .

31. The conjugate of claim 30 , wherein D is a compound of Formula (II) having the structure:

wherein:

R 1 is methyl;

R 2 is fluoro;

R 3 is hydrogen;

R 4 is (C 2 alkyl)-X 2 ; and

n 4 is 2.

32. The conjugate of claim 30 , wherein:

L 2 is

Z 1 and Z 2 are each individually hydrogen;

n 1 is the integer 2;

L 4 is gly-gly-phe-gly;

L 5 is —[NH(CH 2 )n 2 ]n 3 -;

n 2 is the integer of 1;

n 3 is the integer of 1; and

L 7 is absent.

33. The conjugate of claim 30 , wherein Formula (III) is:

34. The conjugate of claim 30 , wherein Formula (III) is:

35. The conjugate claim 30 , wherein Formula ( 111 ) is:

36. The conjugate of claim 30 , wherein D is a compound of Formula (II) having the structure:

wherein:

R 1 is methyl;

R 2 is fluoro;

R 3 is hydrogen;

R 4 is an unsubstituted —(C 2 alkyl)-X 2 ; and

n 4 is 2.

37. The conjugate of claim 32 , wherein D is a compound of Formula (II) having the structure:

wherein:

R 1 is methyl;

R 2 is fluoro;

R 3 is hydrogen;

R 4 is an unsubstituted —(C 2 alkyl)-X 2 ; and

n 4 is 2.

38. The conjugate of claim 30 , wherein:

L 2 is absent;

n 1 is the integer 5;

L 4 is gly-gly-phe-gly;

L 5 is —[NH(CH 2 )n 2 ]n 3 -;

n 2 is the integer of 1;

n 3 is the integer of 1; and

L 7 is absent.

39. The conjugate of claim 38 , wherein D is a compound of Formula (II) having the structure:

wherein:

R 1 is methyl;

R 2 is fluoro;

R 3 is hydrogen;

R 4 is an unsubstituted —(C 2 alkyl)-X 2 ; and

n 4 is 2.

40. The conjugate of claim 30 , wherein Formula (III) is:

41. A method for treating a cancer or a tumor comprising administering an effective amount of a compound of claim 12 , or a pharmaceutically active salt thereof, to a subject having the cancer or the tumor.

42. The method of claim 41 , wherein the cancer or the tumor is selected from lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer, penis cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.