Anti-B7-H4 antibodies and immunoconjugates

1. An immunoconjugate comprising a bi-epitopic antibody and a cytotoxic agent, wherein the bi-epitopic antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that binds a first epitope of B7-H4, and wherein the second half antibody comprises a second VH/VL unit that binds a second epitope of B7-H4, wherein the first half antibody comprises:

(a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 39, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 40, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 42, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 129;

(b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 39, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 40, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 42, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 129;

(c) a VH sequence of SEQ ID NO: 38 and a VL sequence of SEQ ID NO: 126; or

(d) a VH sequence of SEQ ID NO: 127 and a VL sequence of SEQ ID NO: 126.

2. The immunoconjugate of claim 1 , wherein the second half antibody comprises:

(a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63; or

(b) a VH sequence of SEQ ID NO: 56 and a VL sequence of SEQ ID NO: 55.

3. The immunoconjugate of claim 1 , wherein the antibody is an IgG1 or IgG4 antibody.

4. The immunoconjugate of claim 1 , wherein the first half antibody comprises a first heavy chain constant region comprising a knob mutation and the second heavy chain comprises a second heavy chain constant region comprising a hole mutation; or wherein the first half antibody comprises a first heavy chain constant region comprising a hole mutation and the second heavy chain comprises a second heavy chain constant region comprising a knob mutation.

5. The immunoconjugate of claim 4 , wherein the antibody is an IgG1 or IgG4 antibody and wherein the knob mutation comprises a T366W mutation.

6. The immunoconjugate of claim 5 , wherein the antibody is an IgG1 or IgG4 antibody and wherein the hole mutation comprises at least one, at least two, or three mutations selected from T366S, L368A, and Y407V.

7. The immunoconjugate of claim 1 , wherein:

a) the first half antibody comprises a heavy chain sequence of SEQ ID NO: 159 or 163 and a light chain sequence of SEQ ID NO: 145 or 146;

b) the first half antibody comprises a heavy chain sequence of SEQ ID NO: 160 or 164 and a light chain sequence of SEQ ID NO: 145 or 146;

c) the second half antibody comprises a heavy chain sequence of SEQ ID NO: 161 or 165 and a light chain sequence of SEQ ID NO: 147 or 148; or

d) the second half antibody comprises a heavy chain sequence of SEQ ID NO: 162 or 166 and a light chain sequence of SEQ ID NO: 147 or 148.

8. The immunoconjugate of claim 1 , wherein:

the first half antibody comprises a heavy chain sequence of SEQ ID NO: 159 or 163 and a light chain sequence of SEQ ID NO: 145 or 146, and the second half antibody comprises a heavy chain sequence of SEQ ID NO: 162 or 166 and a light chain sequence of SEQ ID NO: 147 or 148.

9. The immunoconjugate of claim 1 , wherein B7-H4 is human B7-H4 of SEQ ID NO: 73.

10. The immunoconjugate of claim 1 , wherein the cytotoxic agent is conjugated to the antibody through an engineered cysteine in the antibody sequence.

11. The immunoconjugate of claim 1 having the formula Ab-(L-D)p, wherein:

(a) Ab is the bi-epitopic antibody;

(b) L is a linker;

(c) D is the cytotoxic agent; and

(d) p ranges from 1-8.

12. The immunoconjugate of claim 1 , wherein the cytotoxic agent is selected from a maytansinoid, an auristatin, a calicheamicin, a pyrrolobenzodiazepine, a nemorubicin derivative, and a 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI).

13. The immunoconjugate of claim 12 , wherein the cytotoxic agent is an auristatin.

14. The immunoconjugate of claim 11 , wherein D has formula D E

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; and R 18 is —C(R 8 ) 2 —C(R 8 ) 2 -aryl.

15. The immunoconjugate of claim 14 , wherein the cytotoxic agent is MMAE.

16. The immunoconjugate of claim 1 , wherein the cytotoxic agent is a pyrrolobenzodiazepine of Formula A:

wherein the dotted lines indicate the optional presence of a double bond between C1 and C2 or C2 and C3;

R 2 is independently selected from H, OH, ═O, ═CH 2 , CN, R, OR, ═CH—R D , ═C(R D ) 2 , O—SO 2 —R, CO 2 R and COR, and optionally further selected from halo or dihalo, wherein

R D is independently selected from R, CO 2 R, COR, CHO, CO 2 H, and halo;

R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, NO 2 , Me 3 Sn and halo;

R 7 is independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, NO 2 , Me 3 Sn and halo;

Q is independently selected from O, S and NH;

R 11 is either H, or R or, where Q is O, SO 3 M, where M is a metal cation;

R and R′ are each independently selected from optionally substituted C 1-8 alkyl, C 3-8 heterocyclyl and C 5-20 aryl groups, and optionally in relation to the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

R 12 , R 16 , R 19 and R 17 are as defined for R 2 , R 6 , R 9 and R 7 respectively;

R″ is a C 3-12 alkylene group, which chain may be interrupted by one or more heteroatoms and/or aromatic rings that are optionally substituted; and

X and X′ are independently selected from O, S and N(H).

17. The immunoconjugate of claim 16 , wherein the cytotoxic agent has the structure:

wherein n is 0 or 1.

18. The immunoconjugate of claim 1 , wherein the cytotoxic agent is a nemorubicin derivative.

19. The immunoconjugate of claim 18 , wherein the cytotoxic agent has a structure selected from:

20. The immunoconjugate of claim 1 , wherein the cytotoxic agent comprises a 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI).

21. The immunoconjugate of claim 20 , wherein the cytotoxic agent has the formula:

where

R 1 is selected from H, P(O) 3 H 2 , C(O)NR a R b , or a bond to L;

R 2 is selected from H, P(O) 3 H 2 , C(O)NR a R b , or a bond to L;

R a and R b are independently selected from H and C 1 -C 6 alkyl optionally substituted with one or more F,

or R a and R b form a five or six membered heterocyclyl group;

T is a tether group selected from C 3 -C 12 alkylene, Y, (C 1 -C 6 alkylene)-Y—(C 1 -C 6 alkylene), (C 1 -C 6 alkylene)-Y—(C 1 -C 6 alkylene)-Y—(C 1 -C 6 alkylene), (C 2 -C 6 alkenylene)-Y—(C 2 -C 6 alkenylene), and (C 2 -C 6 alkynylene)-Y—(C 2 -C 6 alkynylene);

where Y is independently selected from O, S, NR 1 , aryl, and heteroaryl;

where alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with F, OH, O(C 1 -C 6 alkyl), NH 2 , NHCH 3 , N(CH 3 ) 2 , OP(O) 3 H 2 , and C 1 -C 6 alkyl, where alkyl is optionally substituted with one or more F;

or alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with a bond to L;

D′ is a drug moiety selected from:

where the wavy line indicates the site of attachment to T;

X 1 and X 2 are independently selected from O and NR 3 , where R 3 is selected from H and C 1 -C 6 alkyl optionally substituted with one or more F;

R 4 is H, CO 2 R, or a bond to a linker (L), where R is C 1 -C 6 alkyl or benzyl; and

R 5 is H or C 1 -C 6 alkyl.

22. The immunoconjugate of claim 20 , wherein the cytotoxic agent has a structure selected from:

23. The immunoconjugate of claim 20 , wherein the cytotoxic agent comprises the structure:

24. The immunoconjugate of claim 11 , wherein the linker is cleavable by a protease.

25. The immunoconjugate of claim 24 , wherein the linker comprises a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

26. The immunoconjugate of claim 11 , wherein the linker is acid-labile.

27. The immunoconjugate of claim 26 , wherein the linker comprises hydrazone.

28. The immunoconjugate of claim 11 having the formula:

wherein S is a sulfur atom.

29. The immunoconjugate of claim 11 having the formula:

30. The immunoconjugate of claim 11 having a formula selected from:

31. The immunoconjugate of claim 11 , having a structure selected from:

32. The immunoconjugate of claim 11 , wherein p ranges from 2-5.

33. A pharmaceutical formulation comprising the immunoconjugate of claim 1 and a pharmaceutically acceptable carrier.

34. The pharmaceutical formulation of claim 33 , further comprising an additional therapeutic agent.

35. The pharmaceutical formulation of claim 34 , wherein the additional therapeutic agent is bevacizumab.

36. The immunoconjugate of claim 11 , wherein p ranges from 1.4-2.