Anti-HER2 antibody-maytansine conjugates and methods of use thereof

1. A method of treating breast cancer in a subject, the method comprising:

administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate, wherein the administering is effective to treat breast cancer in the subject, and

wherein the conjugate is of formula (I):

wherein

Q 1 is C or N, wherein if Q 1 is N, then Y 1 is absent;

Y 1 is selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl;

L is a linker comprising -(T 1 -Z 1 ) a -(T 2 -Z 2 ) b -(T 3 -Z 3 ) c -(T 4 -Z 4 ) a , wherein a, b, c, and d are each independently 0 or 1, where the sum of a, b, c, and dis 1 to 4;

T 1 , T 2 , T 3 , and T 4 are each independently selected from (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, (EDA) w , (PEG) n , (AA) p , —(CR 13 OH) h —, piperidin-4-amino (4AP), an acetal group, a hydrazine, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol or a modified polyethylene glycol, and AA is an amino acid residue, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12;

Z 1 , Z 2 , Z 3 , and Z 4 are each independently selected from the group consisting of a covalent bond, —CO—, —NR 15 —, —NR 15 (CH 2 ) q —, —NR 15 (C 6 H 4 )—, —CONR 15 —, —NR 15 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 15 —, —NR 15 SO 2 — and —P(O)OH—, wherein q is an integer from 1 to 6;

each R 13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl;

each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

W 1 is a maytansinoid; and

W 2 is an anti-HER2 antibody comprising a sulfatase motif comprising a formylglycine residue, wherein the formylglycine residue is coupled to the maytansinoid through the linker.

2. The method of claim 1 , wherein the administering is effective to deliver a therapeutically effective amount of maytansinoid to a target site in the subject.

3. The method of claim 1 , wherein the breast cancer is a HER2-positive breast carcinoma.

4. The method of claim 1 , wherein:

T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl;

T 2 , T 3 , and T 4 are each independently selected from (EDA) w , (PEG) n , (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, (AA) p , —(CR 13 OH) h —, piperidin-4-amino (4AP), an acetal group, a hydrazine, and an ester; and

Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from the group consisting of a covalent bond, —CO—, —NR 15 —, —NR 15 (CH 2 ) q —, —NR 15 (C 6 H 4 )—, —CONR 15 —, —NR 15 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 15 —, —NR 15 SO 2 —, and —P(O)OH—;

wherein:

(PEG) n is

 where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where y is an integer from 1 to 6 and r is 0 or 1;

piperidin-4-amino is

and

each R 12 and R 15 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring.

5. The method of claim 1 , wherein T 1 , T 2 , T 3 , and T 4 and Z 1 , Z 2 , Z 3 , and Z 4 are selected from the following table:

T 1

Z 1

T 2

Z 2

T 3

Z 3

T 4

Z 4

(C 1 -C 12 )alkyl

—CONR 15 —

(PEG) n

—CO—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(PEG) n

—CO—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

(PEG) n

—NR 15 —

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(PEG) n

—NR 15 —

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

(C 1 -C 12 )alkyl

—NR 15 —

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

(PEG) n

—CO—

(EDA) w

—

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

(CR 13 OH) h

—CONR 15 —

(C 1 -C 12 )alkyl

—CO—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(C 1 -C 12 )alkyl

—CO—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

(PEG) n

—CO—

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

(CR 13 OH) h

—CO—

(AA) p

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(PEG) n

—CO—

(AA) p

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(PEG) n

—SO 2 —

(AA) p

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

(CR 13 OH) h

—CONR 15 —

(PEG) n

—CO—

(C 1 -C 12 )alkyl

—CO—

(CR 13 OH) h

—CO—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

substituted

—NR 15 —

(PEG) n

—CO—

—

—

(C 1 -C 12 )alkyl

(C 1 -C 12 )alkyl

—SO 2 —

(C 1 -C 12 )alkyl

—CO—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

(C 1 -C 12 )alkyl

—

(CR 13 OH) h

—CONR 15 —

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(PEG) n

—CO—

(AA) p

—NR 15 —

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 15 —

(PEG) n

—P(O)OH—

(AA) p

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

(C 1 -C 12 )alkyl

—NR 15 —

—

—CO—

—

—

(C 1 -C 12 )alkyl

—CONR 15 —

(C 1 -C 12 )alkyl

—NR 15 —

—

—CO—

(C 1 -C 12 )alkyl

—NR 15 —

(C 1 -C 12 )alkyl

—CO—

4AP

—CO—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

(C 1 -C 12 )alkyl

—CO—

4AP

—CO—

(C 1 -C 12 )alkyl

—CO—

—

 —.

6. The method of claim 1 , wherein L is selected from one of the following structures:

wherein

each f is independently 0 or an integer from 1 to 12;

each y is independently 0 or an integer from 1 to 20;

each n is independently 0 or an integer from 1 to 30;

each p is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each R is independently hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

each R′ is independently H, a sidechain group of an amino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

7. The method of claim 1 , wherein the maytansinoid is of the formula:

where indicates the point of attachment between the maytansinoid and L.

8. The method of claim 1 , wherein the anti-HER2 antibody binds an epitope within Domain I, Domain II, Domain III, or Domain IV of HER2.

9. The method of claim 1 , wherein the anti-HER2 antibody binds an epitope within amino acids 561-625 or within amino acids 529-625 of SEQ ID NO:1, and wherein the anti-HER2 antibody comprises:

(a) a VH region comprising:

a VH CDR1 comprising the amino acid sequence DTYIH (SEQ ID NO:20);

a VH CDR2 comprising the amino acid sequence RIYPTNGYTRYADSVKG (SEQ ID NO:21); and

a VH CDR3 comprising the amino acid sequence WGGDGFYAMDV (SEQ ID NO:22); and

(b) a VL region comprising:

a VL CDR1 comprising the amino acid sequence RASQDVNTAVA (SEQ ID NO:23);

a VL CDR2 comprising the amino acid sequence SASFLES (SEQ ID NO:24); and

a VL CDR3 comprising the amino acid sequence QQHYTTPPT (SEQ ID NO:25).

10. The method of claim 1 , wherein the the anti-HER2 antibody is huMAb4D5-8.

11. The method of claim 1 , wherein the anti-HER2 antibody comprises a sequence of the formula (II):

X 1 (FGly′)X 2 Z 20 X 3 Z 30   (II)

wherein

FGly′ is the formylglycine residue coupled to the maytansinoid through the linker;

Z 20 is either a proline or alanine residue;

Z 30 is a basic amino acid or an aliphatic amino acid;

X 1 may be present or absent and, when present, can be any amino acid, with the proviso that when the sequence is at the N-terminus of the conjugate, X 1 is present; and

X 2 and X 3 are each independently any amino acid.

12. The method of claim 11 , wherein the sequence is L(FGly′)TPSR.

13. The method of claim 11 , wherein

Z 30 is selected from R, K, H, A, G, L, V, I, and P;

X 1 is selected from L, M, S, and V; and

X 2 and X 3 are each independently selected from S, T, A, V, G, and C.

14. The method of claim 1 , wherein the formylglycine residue is positioned at a C-terminus of a heavy chain constant region of the anti-HER2 antibody.

15. The method of claim 14 , wherein the heavy chain constant region comprises a sequence of the formula (II):

X 1 (FGly′)X 2 Z 20 X 3 Z 30   (II)

wherein

FGly′ is the formylglycine residue coupled to the maytansinoid through the linker;

Z 20 is either a proline or alanine residue;

Z 30 is a basic amino acid or an aliphatic amino acid;

X 1 may be present or absent and, when present, can be any amino acid, with the proviso that when the sequence is at the N-terminus of the conjugate, X 1 is present; and

X 2 and X 3 are each independently any amino acid, and

wherein the sequence is C-terminal to the amino acid sequence SLSLSPG (SEQ ID NO:223).

16. The method of claim 15 , wherein the heavy chain constant region comprises the sequence SPGSL(FGly′)TPSRGS (SEQ ID NO:224).

17. The method of claim 15 , wherein

Z 30 is selected from R, K, H, A, G, L, V, I, and P;

X 1 is selected from L, M, S, and V; and

X 2 and X 3 are each independently selected from S, T, A, V, G, and C.

18. The method of claim 1 , wherein the formylglycine residue is positioned in a light chain constant region of the anti-HER2 antibody.

19. The method of claim 18 , wherein the light chain constant region comprises a sequence of the formula (II):

X 1 (FGly′)X 2 Z 20 X 3 Z 30   (II)

wherein

FGly′ is the formylglycine residue coupled to the maytansinoid through the linker;

Z 20 is either a proline or alanine residue;

Z 30 is a basic amino acid or an aliphatic amino acid;

X 1 may be present or absent and, when present, can be any amino acid, with the proviso that when the sequence is at the N-terminus of the conjugate, X 1 is present; and

X 2 and X 3 are each independently any amino acid, and

wherein the sequence C-terminal to the sequence KVDNAL (SEQ ID NO:123), and/or is N-terminal to the sequence QSGNSQ (SEQ ID NO:124).

20. The method of claim 19 , wherein the light chain constant region comprises the sequence KVDNAL(FGly′)TPSRQSGNSQ (SEQ ID NO:125).

21. The method of claim 19 , wherein

Z 30 is selected from R, K, H, A, G, L, V, I, and P;

X 1 is selected from L, M, S, and V; and

X 2 and X 3 are each independently selected from S, T, A, V, G, and C.

22. The method of claim 1 , wherein the formylglycine residue is positioned in a heavy chain CH1 region of the anti-HER2 antibody.

23. The method of claim 22 , wherein the heavy chain CH1 region comprises a sequence of the formula (II):

X 1 (FGly′)X 2 Z 20 X 3 Z 30   (II)

wherein

FGly′ is the formylglycine residue coupled to the maytansinoid through the linker;

Z 20 is either a proline or alanine residue;

Z 30 is a basic amino acid or an aliphatic amino acid;

X 1 may be present or absent and, when present, can be any amino acid, with the proviso that when the sequence is at the N-terminus of the conjugate, X 1 is present; and

X 2 and X 3 are each independently any amino acid, and

wherein the sequence is C-terminal to the amino acid sequence SWNSGA (SEQ ID NO:126) and/or is N-terminal to the amino acid sequence GVHTFP (SEQ ID NO:127).

24. The method of claim 23 , wherein the heavy chain CH1 region comprises the sequence SWNSGAL(FGly′)TPSRGVHTFP (SEQ ID NO:128).

25. The method of claim 23 , wherein

Z 30 is selected from R, K, H, A, G, L, V, I, and P;

X 1 is selected from L, M, S, and V; and

X 2 and X 3 are each independently selected from S, T, A, V, G, and C.

26. The method of claim 1 , wherein the formylglycine residue is positioned in a heavy chain CH2 region of the anti-HER2 antibody.

27. The method of claim 1 , wherein the formylglycine residue is positioned in a heavy chain CH3 region of the anti-HER2 antibody.