Anti-LGR5 antibodies and immunoconjugates

1. An immunoconjugate comprising an isolated antibody that binds to LgR5, wherein the antibody comprises:

a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 30, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 27, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 28, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or

b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 61, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 62, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 58, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 59.

2. The immunoconjugate of claim 1 , wherein the antibody is a monoclonal antibody.

3. The immunoconjugate of claim 1 , wherein the antibody is a human, humanized, or chimeric antibody.

4. The immunoconjugate of claim 1 , wherein the antibody is an antibody fragment that binds LgR5.

5. The immunoconjugate of claim 1 , wherein LgR5 is human LgR5 of SEQ ID NO: 67.

6. The immunoconjugate of claim 1 , wherein the antibody comprises:

a) heavy chain framework FR3 sequence of SEQ ID NO: 40;

b) heavy chain framework FR3 sequence of SEQ ID NO: 41;

c) heavy chain framework FR3 sequence of SEQ ID NO: 42; or

d) heavy chain framework FR3 sequence of SEQ ID NO: 43.

7. The immunoconjugate of claim 1 , wherein the antibody comprises a light chain framework FR3 sequence of SEQ ID NO: 35.

8. The immunoconjugate of claim 1 , wherein the antibody comprises:

a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6;

b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5;

c) a VH sequence as in (a) and a VL sequence as in (b);

d) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8;

e) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7;

f) a VH sequence as in (d) and a VL sequence as in (e);

g) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10;

h) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9;

i) a VH sequence as in (g) and a VL sequence as in (h);

j) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12;

k) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11;

l) a VH sequence as in (j) and a VL sequence as in (k);

m) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14;

n) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13;

o) a VH sequence as in (m) and a VL sequence as in (n);

p) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16;

q) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15;

r) a VH sequence as in (p) and a VL sequence as in (q);

s) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 18;

t) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17;

u) a VH sequence as in (s) and a VL sequence as in (t);

v) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20;

w) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19;

x) a VH sequence as in (v) and a VL sequence as in (w);

y) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26;

z) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25; or

aa) a VH sequence as in (y) and a VL sequence as in (z).

9. The immunoconjugate of claim 8 , comprising a VH sequence selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 26.

10. The immunoconjugate of claim 8 , comprising a VL sequence selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, and 25.

11. An immunoconjugate comprising:

a) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 5;

b) a VH sequence of SEQ ID NO: 8 and a VL sequence of SEQ ID NO: 7;

c) a VH sequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO: 9;

d) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 11;

e) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 13;

f) a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 15;

g) a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 17;

h) a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 19; or

i) a VH sequence of SEQ ID NO: 26 and a VL sequence of SEQ ID NO: 25;

and a cytotoxic agent.

12. The immunoconjugate of claim 1 , which is an IgG1, IgG2a or IgG2b antibody.

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

(a) Ab is the antibody;

(b) L is a linker;

(c) D is a drug selected from a maytansinoid, an auristatin, a calicheamicin, a pyrrolobenzodiazepine, and a nemorubicin derivative; and

(d) p ranges from 1-8.

14. The immunoconjugate of claim 13 , wherein D is an auristatin.

15. The immunoconjugate of claim 14 , 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.

16. The immunoconjugate of claim 13 , wherein the drug is MMAE.

17. The immunoconjugate of claim 13 , wherein D 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).

18. The immunoconjugate of claim 17 , wherein D has the structure:

wherein n is 0 or 1.

19. The immunoconjugate of claim 17 , wherein D has a structure selected from:

wherein R E and R E″ are each independently selected from H or R D , wherein R D is independently selected from R, CO 2 R, COR, CHO, CO 2 H, and halo;

wherein Ar 1 and Ar 2 are each independently optionally substituted C 5-20 aryl; and

wherein n is 0 or 1.

20. The immunoconjugate of claim 13 , wherein D is a pyrrolobenzodiazepine of Formula B:

wherein the horizontal wavy line indicates the covalent attachment site to the linker;

R V1 and R V2 are independently selected from H, methyl, ethyl, phenyl, fluoro-substituted phenyl, and C 5-6 heterocyclyl; and

n is 0 or 1.

21. The immunoconjugate of claim 13 , wherein D is a nemorubicin derivative.

22. The immunoconjugate of claim 21 , wherein D has a structure selected from:

23. The immunoconjugate of claim 13 , wherein the linker is cleavable by a protease.

24. The immunoconjugate of claim 23 , wherein the linker comprises a val-cit dipeptide or a Phe-Lys dipeptide.

25. The immunoconjugate of claim 13 , wherein the linker is acid-labile.

26. The immunoconjugate of claim 25 , wherein the linker comprises hydrazone.

27. The immunoconjugate of claim 15 having the formula:

wherein S is a sulfur atom.

28. The immunoconjugate of claim 18 having the formula:

29. The immunoconjugate of claim 22 having a formula selected from:

wherein R 1 and R 2 are independently selected from H and C 1 -C 6 alkyl.

30. The immunoconjugate of claim 13 , wherein p ranges from 2-5.

31. A pharmaceutical formulation comprising the immunoconjugate of claim 13 and a pharmaceutically acceptable carrier.