FIELD OF THE INVENTION

The present invention relates to anti-B7-H4 antibodies and immunoconjugates and methods of using the same.

BACKGROUND

B7-H4 is a Type I transmembrane protein and is a member of the B7 superfamily of proteins that provides co-signal in conjunction with a T-cell receptor antigenic signal. B7-H4 is a negative regulator of T-cell function and ligation of T-cells inhibits their growth, cytokine secretion and cytotoxicity. Elimination of B7-H4 in mice does not affect immune cell homeostasis and no signs of autoimmunity. Zhu et al., Blood, 113(8): 1759-1767 (2009); Suh et al., Molecular and Cellular Biology, 26(17): 6403-6411 (2006). The receptor for B7-H4 is unknown and unidentified.

Human B7-H4 is a 282 amino acid protein (including the amino-terminal signal sequence), of which ˜227 amino acids are predicted to be in the extracellular space following cleavage of the amino-terminal signal sequence. B7-H4 comprises an Ig-like V-domain, an Ig-like C domain, a transmembrane domain and a short cytoplasmic tail.

There is a need in the art for agents that target B7-H4 for the diagnosis and treatment of B7-H4-associated conditions, such as cancer. The invention fulfills that need and provides other benefits.

SUMMARY

The invention provides anti-B7-H4 antibodies and immunoconjugates and methods of using the same.

In some embodiments, an isolated antibody that binds to B7-H4 is provided. In some embodiments, the antibody binds to B7-H4 and has at least one of the following characteristics: (a) binds to an epitope within all or a portion of the B7-H4 Ig-V containing domain (amino acids 29-157 of SEQ ID NO: 73); or binds to an epitope within all or a portion of the B7-H4 Ig-C containing domain (amino acids 158-250 of SEQ ID NO: 73); or binds to an epitope within all or a portion of the B7-H4 Ig-V and Ig-C domains (amino acids 29-250 of SEQ ID NO:73); or binds to an epitope within all or a portion of SEQ ID NO: 74 (mature human B7-H4) or binds to an epitope within all or a portion of SEQ ID NO: 73 (precursor human B7-H4). In some embodiments, the antibody binds to B7-H4 with an affinity of ≤50 nM. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is an IgG1, IgG2a or IgG2b antibody. In some embodiments, the antibody is an antibody fragment that binds B7-H4. In some embodiments, B7-H4 is human B7-H4 has the sequence of SEQ ID NO: 73.

In some embodiments, the antibody comprises (a) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 6; or (b) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; or (c) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; or (d) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30; or (e) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 40; or (f) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59.

In some embodiments, the antibody comprises (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 5, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7; or (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15; or (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; or (d) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31; or (e) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 39, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 40, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41; or (f) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60.

In some embodiments, the antibody further comprising a heavy chain framework FR3 sequence of SEQ ID NO: 51, 52 or 53.

In some embodiments, the antibody further comprises: (a) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10; or (b) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or (c) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; or (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34; or (e) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 42, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 44; or (f) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In some embodiments, the antibody further comprises (a) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10; or (b) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or (c) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; or (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34; or (e) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 42, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 44; or (f) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In some embodiments, the antibody further comprises a light chain framework FR2 sequence of SEQ ID NO: 65 or 66 or a light chain framework FR3 sequence of SEQ ID NO: 47.

In some embodiments, an isolated antibody (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4; or (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3; or (c) a VH sequence as in (a) and a VL sequence as in (b); or (d) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12; or (e) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11; or (f) a VH sequence as in (d) and a VL sequence as in (e); or (g) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20; or (h) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19; or (i) a VH sequence as in (g) and a VL sequence as in (h); or (j) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 28; or (k) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 27; or (l) a VH sequence as in (j) and a VL sequence as in (k); or (m) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 36; or (n) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 35; or (o) a VH sequence as in (m) and a VL sequence as in (n); or (p) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 37; or (q) a VH sequence as in (p) and a VL sequence as in (n); or (r) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38; or (s) a VH sequence as in (r) and a VL sequence as in (n); or (t) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 56; or (u) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 55; or (v) a VH sequence as in (t) and a VL sequence as in (v); or (w) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 57; or (x) a VH sequence as in (t) and a VL sequence as in (w).

In some embodiments, the antibody comprises a VH sequence of SEQ ID NO: 4, a VH sequence of SEQ ID NO: 12, a VH sequence of SEQ ID NO: 20, a VH sequence of SEQ ID NO: 4, 28, a VH sequence of SEQ ID NO: 36, a VH sequence of SEQ ID NO: 37, a VH sequence of SEQ ID NO: 38, or a VH sequence of SEQ ID NO: 56.

In some embodiments, the antibody comprises a VL sequence of SEQ ID NO: 3, a VL sequence of SEQ ID NO: 11, a VL sequence of SEQ ID NO: 19, a VL sequence of SEQ ID NO: 27, a VL sequence of SEQ ID NO: 35, a VL sequence of SEQ ID NO: 55, or a VL sequence of SEQ ID NO: 57.

In some embodiments, an isolated antibody comprises (a) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 3; or (b) a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 19; or (c) a VH sequence of SEQ ID NO: 28 and a VL sequence of SEQ ID NO: 27; or (d) a VH sequence of SEQ ID NO: 36 and a VL sequence of SEQ ID NO: 35; or (e) a VH sequence of SEQ ID NO: 37 and a VL sequence of SEQ ID NO: 35; or (f) a VH sequence of SEQ ID NO: 38 and a VL sequence of SEQ ID NO: 35; or (g) a VH sequence of SEQ ID NO: 56 and a VL sequence of SEQ ID NO: 55; or (h) a VH sequence of SEQ ID NO: 56 and a VL sequence of SEQ ID NO: 57.

In some embodiments, an isolated nucleic acid that encodes an antibody described herein is provided. In some embodiments, a host cell comprising the nucleic acid is provided. In some embodiments, a method of producing an antibody described herein is provided. In some embodiments, the method comprises culturing the host cell comprising the nucleic acid that encodes an antibody.

In some embodiments, immunoconjugates are provided. In some embodiments, an immunoconjugate comprises an anti-B7-H4 antibody and a cytotoxic agent. In some embodiments, an immunoconjugate has the formula Ab-(L-D)p, wherein: (a) Ab is the an antibody described herein; (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. In some embodiments, D is an auristatin. In some such embodiments, D has formula DE


wherein R2and R6are each methyl, R3and R4are each isopropyl, R5is H, R7is sec-butyl, each R8is independently selected from CH3, O—CH3, OH, and H; R9is H; and R18is —C(R8)2—C(R8)2-aryl. In some embodiments, D is MMAE having the structure:



In some embodiments, 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; R2is independently selected from H, OH, ═O, ═CH2, CN, R, OR, ═CH—RD, ═C(RD)2, O—SO2—R, CO2R and COR, and optionally further selected from halo or dihalo, wherein RDis independently selected from R, CO2R, COR, CHO, CO2H, and halo; R6and R9are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, NO2, Me3Sn and halo; R7is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, NO2, Me3Sn and halo; Q is independently selected from O, S and NH; R11is either H, or R or, where Q is O, SO3M, where M is a metal cation; R and R′ are each independently selected from optionally substituted C1-8alkyl, C1-12alkyl, C3-8heterocyclyl, C3-20heterocyclyl, and C5-20aryl 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; R12, R16, R19and R17are as defined for R2, R6, R9and R7respectively; R″ is a C3-12alkylene 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). In some such embodiments, D is


wherein n is 0 or 1.

In some embodiments, D is a pyrrolobenzodiazepine having a structure selected from:


wherein REand RE″are each independently selected from H or RD, wherein RDis independently selected from R, CO2R, COR, CHO, CO2H, and halo; wherein Ar1and Ar2are each independently optionally substituted C5-20aryl; and wherein n is 0 or 1.

In some embodiments, D is a pyrrolobenzodiazepine of Formula B:


wherein the horizontal wavy line indicates the covalent attachment site to the linker; RV1and RV2are independently selected from H, methyl, ethyl, phenyl, fluoro-substituted phenyl, and C5-6heterocyclyl; and n is 0 or 1.

In some embodiments, D is a nemorubicin derivative. In some embodiments, D has a structure selected from:



In some embodiments, an immunoconjugate comprises a linker that is cleavable by a protease. In some embodiments, the linker comprises a val-cit dipeptide, a Phe-Lys dipeptide, or a Phe-homoLys dipeptide. In some embodiments, an immunoconjugate comprises a linker that is acid-labile. In some such embodiments, the linker comprises hydrazone.

In some embodiments, an immunoconjugate has a formula selected from:



wherein S is a sulfur atom;


In some embodiments, p ranges from 2-5.

In some embodiments, an immunoconjugate comprises an antibody that comprises (a) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 3; or (b) a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 19; or (c) a VH sequence of SEQ ID NO: 28 and a VL sequence of SEQ ID NO: 27; or (d) a VH sequence of SEQ ID NO: 36 and a VL sequence of SEQ ID NO: 35; or (e) a VH sequence of SEQ ID NO: 37 and a VL sequence of SEQ ID NO: 35; or (f) a VH sequence of SEQ ID NO: 38 and a VL sequence of SEQ ID NO: 35; or (g) a VH sequence of SEQ ID NO: 56 and a VL sequence of SEQ ID NO: 55; or (h) a VH sequence of SEQ ID NO: 56 and a VL sequence of SEQ ID NO: 57.

In some embodiments, an immunoconjugate comprises an antibody that comprises (a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 5, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7; (b) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10; or (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15; (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or (e) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (f) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; or (g) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31; (h) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34; or (i) (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, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41; (j) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 42, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 44; or (k) (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, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (l) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In some embodiments, pharmaceutical formulations are provided. In some such embodiments, a pharmaceutical formulation comprises an immunoconjugate comprising an antibody that binds B7-H4, e.g., as described herein. In some embodiments, a pharmaceutical formulation further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is Avastin® (bevacizumab).

In some embodiments, methods of treating individuals having B7-H4 positive cancers are provided. In some such embodiments, a method comprises administering a pharmaceutical formulation comprising an immunoconjugate comprising an antibody that binds B7-H4, e.g., as described herein. In some such embodiments, the method comprises administering a pharmaceutical formulation comprising an immunoconjugate comprising an antibody that bind to the extracellular domain of B7-H4. In some embodiments, the B7-H4-positive cancer is selected from breast cancer, ovarian cancer, and endometrial cancer. In some embodiments, a method comprises administering an additional therapeutic agent to the individual. In some such embodiments, the additional therapeutic agent is Avastin® (bevacizumab).

In some embodiments, methods of inhibiting proliferation of a B7-H4-positive cell are provided. In some embodiments, the method comprising exposing the cell to an immunoconjugate comprising an antibody that binds B7-H4 under conditions permissive for binding of the immunoconjugate to B7-H4 on the surface of the cell. In some embodiments, an antibody that binds B7-H4 is an antibody described herein. In some embodiments, proliferation of the cell is thereby inhibited. In some embodiments, the cell is a breast, ovarian, or endometrial cancer cell.

In some embodiments, an antibody that binds B7-H4 is conjugated to a label. In some embodiments, an antibody that binds B-H4 is an antibody described herein. In some embodiments, the label is a positron emitter. In some embodiments, the positron emitter is89Zr.

In some embodiments, a method of detecting human B7-H4 in a biological sample is provided. In some embodiments, a method comprises contacting the biological sample with an anti-B7-H4 antibody under conditions permissive for binding of the anti-B7-H4 antibody to a naturally occurring human B7-H4, and detecting whether a complex is formed between the anti-B7-H4 antibody and a naturally occurring human B7-H4 in the biological sample. In some embodiments, an anti-B7-H4 antibody is an antibody described herein. In some embodiments, the biological sample is a breast cancer sample, ovarian cancer sample, or endometrial cancer sample.

In some embodiments, a method for detecting an B7-H4-positive cancer is provided. In some such embodiments, a method comprises (i) administering a labeled anti-B7-H4 antibody to a subject having or suspected of having an B7-H4-positive cancer, and (ii) detecting the labeled anti-B7-H4 antibody in the subject, wherein detection of the labeled anti-B7-H4 antibody indicates a B7-H4-positive cancer in the subject. In some embodiments, an anti-B7-H4 antibody is an antibody described herein. In some such embodiments, the labeled anti-B7-H4 antibody comprises an anti-B7-H4 antibody conjugated to a positron emitter. In some embodiments, the positron emitter is89Zr.

