HK1211597B - Her3 antigen binding proteins binding to the beta-hairpin of her3 - Google Patents

Description

HER3 antigen binding protein that binds to HER3 β-hairpin

The present invention relates to anti-HER3 antigen binding proteins (eg, anti-HER3 antibodies) that bind to the HER3 β-hairpin, methods for selecting these antigen binding proteins, their preparation, and use as pharmaceuticals.

Background of the Invention

The HER protein family consists of four members: HER1, also known as epidermal growth factor receptor (EGFR) or ErbB-1; HER2, also known as ErbB-2; ErbB-3, also known as HER3; and ErbB-4, also known as HER4. ErbB family proteins are receptor tyrosine kinases and represent important mediators of cell growth, differentiation, and survival. HER family proteins represent receptors for different ligands, such as the neuregulin (NRG) family, amphiregulin, EGF, and TGF-a. Heregulin (also known as HRG or neuregulin NRG-1) is a ligand for, for example, HER3 and HER4.

Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine protein kinase erbB-3, SEQ ID NO: 3) encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4 (Kraus, M.H. et al., PNAS 86: 9193-9197 (1989); Plowman, G.D. et al., PNAS 87: 4905-4909 (1990); Kraus, M.H. et al., PNAS 90: 2900-2904 (1993)). Like the prototypical EGFR, the transmembrane receptor HER3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD), and a C-terminal phosphorylation domain. This membrane-bound protein has a heregulin (HRG) binding domain within its intracellular domain, but lacks an active kinase domain. Therefore, it can bind this ligand but does not transmit the signal into the cell via protein phosphorylation. However, it does form heterodimers with other HER family members that do have kinase activity. Heterodimerization leads to activation of receptor-mediated signaling pathways and transphosphorylation of its intracellular domain. Dimer formation between HER family members expands the signaling potential of HER3 and serves not only as a means of signal diversification but also as a means of signal amplification. For example, HER2/HER3 heterodimers induce one of the most important mitogenic signals among HER family members through the PI3K and AKT pathways (Sliwkowski, M.X. et al., J.Biol.Chem. 269:14661-14665 (1994); Alimandi, M. et al., Oncogene. 10:1813-1821 (1995); Hellyer, N.J., J.Biol.Chem. 276:42153-4261 (2001); Singer, E., J.Biol.Chem. 276:44266-44274 (2001); Schaefer, K.L., Neoplasia 8:613-622 (2006)). For an overview of HER3 and its various interactions within the HER receptor family and the NGR ligand family, see, eg, Sithanandam, G. et al., Cancer Gene Therapy 15:413-448 (2008).

Amplification of this gene and/or overexpression of its protein have been reported in numerous cancers, including prostate, bladder, and breast tumors. Alternative transcript splice variants encoding different isoforms have been characterized. One isoform lacks the intermembrane region and is secreted extracellularly. This form functions to regulate the activity of the membrane-bound form. Additional splice variants have also been reported, but they have not yet been thoroughly characterized.

Interestingly, in its equilibrium state, the HER3 receptor exists in its "closed conformation," which indeed means that the heterodimerization of the HER3 β-hairpin motif is tethered to the HER3 ECD domain IV via non-covalent interactions (see Figures 1c and 1d). It is hypothesized that the "closed" HER3 conformation can be opened by the binding of the ligand heregulin at a specific HER3 heregulin binding site. This occurs at the HER3 interface formed by HER3 ECD domains I and III. Through this interaction, the HER3 receptor is believed to be activated and converted to its "open conformation" (see Figures 1e and 1b and, for example, Baselga, J. et al., Nat. Rev. Cancer 9:463-475 (2009) and Desbois-Mouthon, C. et al., Gastroenterol Clin Biol 34:255-259 (2010)). In this open conformation, heterodimerization with HER2 and transsignaling induction are possible (see Figure 1b).

WO 2003/013602 relates to inhibitors of HER activity, including HER antibodies. WO 2007/077028 and WO 2008/100624 also relate to HER3 antibodies. WO 97/35885 and WO 2010/127181 relate to HER3 antibodies.

WO 2012/22814 relates to HER3 antibodies that freeze in a "closed or inactive conformation", meaning that they freeze the equilibrium state of HER3 (see Figure 1 ), rendering the HER3 β-hairpin inaccessible when in this equilibrium state. The HER3 antibodies of WO 2012/22814 do not bind to the HER3 β-hairpin presented in an active three-dimensional orientation, such as within a SlyD scaffold (see, e.g., Figures 17 and 2 , and, e.g., the polypeptide of SEQ ID NO: 18).

Human HER4 (also known as ErbB-4, ERBB4, v-erb-a, erythroblastic leukemia virus oncogene homolog 4, p180erbB4, avian erythroblastic leukemia virus (v-erb-b2) oncogene homolog 4; SEQ ID NO: 5) is a type I single-pass transmembrane protein with multiple furin-like cysteine-rich domains, a tyrosine kinase domain, a phosphatidylinositol-3-kinase binding site, and a PDZ domain binding motif (Plowman, G.D. et al., PNAS 90: 1746-50 (1993); Zimonjic, D.B. et al., Oncogene 10: 1235-7 (1995); Culouscou, J.M. et al., J. Biol. Chem. 268: 18407-10 (1993)). This protein binds to and is activated by neuregulin-2 and -3, heparin-binding EGF-like growth factor, and betacellulin. Ligand binding induces a variety of cellular responses, including mitosis and differentiation. Multiple proteolytic events allow the release of cytoplasmic and extracellular fragments. Mutations in this gene have been associated with cancer. Alternative splicing variants encoding different protein isoforms have been described; however, not all variants have been fully characterized.

Anti-HER4 antibodies for use in anticancer therapy are known, for example from US Pat. No. 5,811,098, US Pat. No. 7,332,579 or Hollmén, M. et al., Oncogene. 28: 1309-19 (2009) (anti-ErbB-4 antibody mAb 1479).

It has been impossible so far to select antigen binding proteins (in particular antibodies) that specifically bind to the HER3 β-hairpin because this HER3 β-hairpin presents a cryptic epitope that is inaccessible when HER3 is in the equilibrium state (see Figure 1).

SUMMARY OF THE INVENTION

We have now found a way to obtain such antibodies using HER3 (and HER4 for counter-selection) β-hairpins functionally presented in a three-dimensional orientation within a SlyD scaffold (see e.g. FIG2 , and polypeptides of SEQ ID NOs: 13 and 17 to 24).

The present invention provides a method for selecting an antigen binding protein (particularly an antibody) that binds to human HER3 (and does not cross-react with human HER4), wherein the antigen binding protein (particularly an antibody) binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3,

Among them use

a) at least one polypeptide comprising the amino acid sequence SEQ ID NO: 1 selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3

and

b) at least one polypeptide comprising the amino acid sequence SEQ ID NO: 2 selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4

to select an antigen binding protein (in particular an antibody) that shows binding to at least one polypeptide under a) and does not show binding to at least one polypeptide under b),

Thus, antigen binding proteins (particularly antibodies) are selected that bind within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3 and do not cross-react with human HER4.

The present invention provides antigen-binding proteins (particularly antibodies) obtained by such selection methods.

The present invention provides an isolated antigen-binding protein (particularly an antibody) that binds to human HER3 (and does not cross-react with human HER4), wherein the antigen-binding protein (particularly an antibody) binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3.

The present invention further provides an isolated antigen binding protein that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antigen binding protein binds to the polypeptide SEQ ID NO: 18TtSlyDcys-Her3, and

b) wherein the antigen binding protein does not cross-react with the polypeptide SEQ ID NO: 22TtSlyDcys-Her4.

The present invention further provides an isolated antigen binding protein that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antigen binding protein binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in the polypeptide SEQ ID NO: 18 (TtSlyDcas-Her3), and

b) wherein the antigen binding protein does not cross-react with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) contained in the polypeptide SEQ ID NO: 22 (TtSlyDcas-Her4).

The present invention further provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antibody binds to the polypeptide SEQ ID NO: 18TtSlyDcys-Her3, and

b) wherein the antibody does not cross-react with the polypeptide SEQ ID NO: 22TtSlyDcys-Her4.

The present invention further provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antibody binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in the polypeptide SEQ ID NO: 18 (TtSlyDcas-Her3), and

b) wherein the antibody does not cross-react with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) contained in the polypeptide SEQ ID NO: 22 (TtSlyDcas-Her4).

The present invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1 in activated HER3.

The present invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody

binds to the amino acid sequence SEQ ID NO: 1 in activated HER3; and

Inhibits the heterodimerization of HER3/HER2 heterodimers.

The present invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody

a) binds to the amino acid sequence SEQ ID NO: 1; and/or

b) binds to the amino acid sequence SEQ ID NO: 1 in activated HER3; and/or

c) binding within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3; and/or

d) binds to the β-hairpin region of HER3; and/or

e) inhibiting heterodimerization of HER3/HER2 heterodimers; and/or

f) has a ratio of the binding constant (Ka) for binding to Thermus thermophilus SlyD FKBP-HER3 (SEQ ID NO: 13) to the binding constant (Ka) for binding to HER3-ECD (SEQ ID NO: 4) (Ka(Thermus thermophilus SlyD FKBP-Her3)/Ka(HER3-ECD)) of 1.5 or greater, when measured in a surface plasmon resonance assay; and/or

g) has a ratio of the molar ratio MR for binding to Thermus thermophilus SlyDFKBP-HER3 (SEQ ID NO: 13) to the molar ratio MR for binding to HER3-ECD (SEQ ID NO: 4) (MR(Thermus thermophilus SlyDFKBP-Her3)/MR(HER3-ECD)) of 2.0 or higher when measured in a surface plasmon resonance assay; and/or

h) has no cross-reactivity to the amino acid sequence of SEQ ID NO: 2; and/or

i) has no cross-reactivity to the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4; and/or

j) has no cross-reactivity to the β-hairpin region of HER4; and/or

k) does not compete with heregulin for binding to HER3; and/or

1) inducing heregulin to bind to HER3; and/or

m) binds to HER3-ECD with an affinity of a KD value ≤ 1 x ^10-8 M (in one embodiment, a KD value of 1 x ^10-8 M to 1 x ^10-13 M; in one embodiment, a KD value of 1 x ^10-9 M to 1 x ^10-13 M); and/or

n) binds to a polypeptide consisting of PLVYNKLTFQLE (SEQ ID NO: 48); and/or

o) binds to a polypeptide consisting of PLVYNKLTFQLE (SEQ ID NO: 48) and does not cross-react with a polypeptide consisting of PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2); and/or

p) exhibits at least two-fold higher binding in the presence of heregulin compared to the level of binding in the absence of heregulin, as detected at 0 minutes after incubation with the antibody in a FACS assay with T47D cells expressing HER3; and/or

q) shows nearly complete HER3 internalization in the presence of heregulin after 4 h of incubation with antibodies in a FACS assay performed with HER3-expressing T47D cells.

In one embodiment, such anti-HER3 antibodies are monoclonal antibodies.

In one embodiment, such anti-HER3 antibodies are human antibodies, humanized antibodies, or chimeric antibodies.

In one embodiment, such anti-HER3 antibodies are antibody fragments that bind human HER3.

In one embodiment, the present invention provides an anti-HER3 antibody comprising:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In one embodiment, the present invention provides an anti-HER3 antibody comprising:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In one embodiment, the present invention provides an anti-HER3 antibody comprising:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:44;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:46;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In one embodiment, the present invention provides an anti-HER3 antibody comprising:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In one embodiment, such anti-HER3 antibodies comprise:

i) VH sequence SEQ ID NO: 31 and VL sequence SEQ ID NO: 32; or

ii) Humanized variants of the antibody VH and VL under i).

In one embodiment, such anti-HER3 antibodies comprise

i) VH sequence SEQ ID NO: 39 and VL sequence SEQ ID NO: 40; or

ii) Humanized variants of the antibody VH and VL under i).

In one embodiment, such anti-HER3 antibodies comprise

i) VH sequence SEQ ID NO: 47 and VL sequence SEQ ID NO: 48; or

ii) Humanized variants of the antibody VH and VL under i).

In one embodiment, such anti-HER3 antibodies comprise

i) VH sequence SEQ ID NO: 56 and VL sequence SEQ ID NO: 57; or

ii) Humanized variants of the antibody VH and VL under i).

In one embodiment, such anti-HER3 antibodies are full-length IgG1 antibodies or IgG4 antibodies.

In one embodiment, such anti-HER3 antibodies are Fab fragments.

The present invention further provides isolated nucleic acids encoding such anti-HER3 antibodies.

The present invention further provides a host cell comprising such a nucleic acid.

The present invention further provides a method of producing an antibody, comprising culturing such a host cell such that the antibody is produced.

In one embodiment, such methods further comprise recovering the antibody from the host cell.

The present invention further provides an immunoconjugate comprising such an anti-HER3 antibody and a cytotoxic agent.

The present invention further provides a pharmaceutical formulation comprising such an anti-HER3 antibody and a pharmaceutically acceptable carrier.

The present invention further provides an anti-HER3 antibody as described herein for use as a medicament. The present invention further provides an anti-HER3 antibody as described herein or an immunoconjugate comprising the anti-HER3 antibody and a cytotoxic agent for use in treating cancer. The present invention further provides an anti-HER3 antibody as described herein for use in inhibiting HER3/HER2 dimerization.

The present invention further provides the use of such an anti-HER3 antibody or an immunoconjugate comprising the anti-HER3 antibody and a cytotoxic agent in the manufacture of a medicament. The present invention further provides such a use, wherein the medicament is for treating cancer. The present invention further provides such a use, wherein the medicament is for inhibiting HER3/HER2 dimerization.

The present invention further provides a method of treating an individual having cancer, comprising administering to the individual an effective amount of an anti-HER3 antibody described herein or an immunoconjugate comprising the anti-HER3 antibody and a cytotoxic agent.

The present invention further provides a method of inducing apoptosis of cancer cells in an individual suffering from cancer, comprising administering to the individual an effective amount of an immunoconjugate comprising an anti-HER3 antibody described herein and a cytotoxic agent, thereby inducing apoptosis of cancer cells in the individual.

One embodiment of the present invention is a polypeptide selected from the group consisting of:

i) SEQ ID NO:12TtSlyD-FKBP-Her3,

ii) SEQ ID NO:16TtSlyDcas-Her3,

iii) SEQ ID NO:17TtSlyDcys-Her3,

iv) SEQ ID NO: 18 TgSlyDser-Her3, and

v) SEQ ID NO:19TgSlyDcys-Her3,

The polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

The present invention further provides the use of one of such polypeptides selected from the group consisting of:

i) SEQ ID NO:12TtSlyD-FKBP-Her3,

ii) SEQ ID NO:16TtSlyDcas-Her3,

iii) SEQ ID NO:17TtSlyDcys-Her3,

iv) SEQ ID NO: 18 TgSlyDser-Her3, and

v) SEQ ID NO: 19TgSlyDcys-Her3.

The present invention further provides a method for producing an antibody that specifically binds to the HER3 β-hairpin having the amino acid sequence of SEQ ID NO: 1, comprising the following steps:

a) administering to the experimental animal at least once a polypeptide selected from the group consisting of:

i) SEQ ID NO:12TtSlyD-FKBP-Her3,

ii) SEQ ID NO:16TtSlyDcas-Her3,

iii) SEQ ID NO:17TtSlyDcys-Her3,

iv) SEQ ID NO: 18 TgSlyDser-Her3, and

v) SEQ ID NO:19TgSlyDcys-Her3,

wherein the polypeptide comprises a HER3 β-hairpin having an amino acid sequence of SEQ ID NO: 1,

b) recovering B cells that produce antibodies that specifically bind to HER3 β-hairpin having the amino acid sequence of SEQ ID NO: 1 from the experimental animal 3 to 10 days after the last administration of the polypeptide, and

c) culturing cells containing a nucleic acid encoding an antibody that specifically binds to HER3 β-hairpin having the amino acid sequence of SEQ ID NO: 1 and recovering the antibody from the cells or the culture medium, thereby producing an antibody that specifically binds to the target antigen.

The present invention further provides use of a polypeptide selected from the group consisting of:

i) SEQ ID NO:12TtSlyD-FKBP-Her3,

ii) SEQ ID NO:16TtSlyDcas-Her3,

iii) SEQ ID NO:17TtSlyDcys-Her3,

iv) SEQ ID NO: 18 TgSlyDser-Her3, and

v) SEQ ID NO:19TgSlyDcys-Her3,

The polypeptide comprises an epitope in the HER3 β-hairpin having the amino acid sequence of SEQ ID NO: 1.

The use of a HER3 β-hairpin functionally presented in a three-dimensional orientation within the SlyD scaffold (see, e.g., FIG2 , and polypeptides of SEQ ID NOs: 13 and 17 to 24) enables the selection of anti-HER3 antigen-binding proteins (particularly antibodies) that bind to this β-hairpin as described herein. Antigen-binding proteins (particularly antibodies) according to the present invention have been found to have highly valuable properties, such as internalization of HER3 in HER3-expressing cancer cells, or very specific pharmacokinetic properties (such as faster binding rates and higher molar ratios for binding to the Thermus thermophilus SlyD FKBP-Her3 scaffold (which presents the HER3 β-hairpin in a three-dimensional functional structure and mimics the open HER conformation of the HER3 β-hairpin) compared to binding to the HER3-ECD in its closed conformation (i.e., in the absence of heregulin).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Schematic overview of the "closed" and "open" HER3 conformations and the effect of neuregulin family ligands (such as, for example, heregulin, abbreviated HR) on the conformational changes.

Figure 2 3D structure of the HER3 β-hairpin functionally presented in a three-dimensional orientation within the SlyD scaffold of Thermus thermophilus.

Figure 3 SDS-PAGE analysis of Ni-NTA purification of TtSlyD-FKBP-Her3. E1 and E2 show purified fractions 12 and 13. SN: E. coli lysate supernatant before purification.

FIG4 SEC elution profile of Ni-NTA purified fractions of Thermus thermophilus SlyD-FKBP-Her3.

FIG5 A) Testing of the specificity and reactivity of selected clones in IHC. As shown, all clones were specific for HER3 detection and showed no cross-reactivity with other members of the HER family (HER1, HER2, and HER4). - Antibodies M-08-11, 17-02, and 17-07.

B) Testing of specificity and reactivity in IHC of selected clones. As shown, all clones were specific for HER3 detection and showed no cross-reactivity with other members of the HER family (HER1, HER2, and HER4). - Antibodies M-08-11, 17-02, M-43-01, and M-46-01.

FIG6 FACS analysis of time-dependent HER3 internalization induced by M-08-11 antibody in T47D cells.

Figure 7 Two-state analysis of anti-HER3 antibodies:

In Figure 7a) and b) a two-state analysis of the anti-HER3 antibody M-08-11 is shown.

Figure 7a: Anti-HER3 antibody M-08-11: Only at very high Her3 ECD concentrations is the "closed" Her3 ECD bound according to a 1:1 Langmuir interaction (since the dissociation curves can be superimposed to form coincident dissociation curves).

Figure 7b: Anti-HER3 antibody M-08-11: Dissociation curves of heregulin/Her3 ECD interaction are not superimposable.

Figure 7c: Interaction diagram of two-state kinetic analysis of anti-HER3 antibodies M-08-11, M-43-01 and M-46-01.

Figure 7d: Interaction diagram of two-state kinetic analysis of anti-HER3 antibody M-43-01.

Figure 7e: Interaction diagram of two-state kinetic analysis of anti-HER3 antibody M-46-01.

Figure 8. Overlay of Biacore sensorgrams. 1: 100 nM M-05-74* heregulin/Her3 ECD interaction. 2: 100 nM M-08-11* heregulin/Her3 ECD interaction. 3 and 4: 100 nM M-05-74 and 100 nM M-08-11* Her3 ECD interaction. 5: Buffer reference.

Figure 9 Overlay of sensorgrams from a Biacore epitope binding experiment. Primary antibody M-05-74 (M-074 in the figure) presents Her3 ECD to secondary antibodies M-208, GT (=8B8), M-05-74, and M-08-11 (M-011 in Figure 9) (M-. The measured noise is 5 RU.

Figure 10 Overlay of Biacore sensorgrams. 1: 90 nM heregulin*Her3 ECD complex on M-05-74. 2: 90 nM heregulin*Her3 ECD complex on M-08-11. 3: 90 nM heregulin*Her3 ECD complex on 8B8 antibody.

Figure 11 Schematic representation of the effects identified by Biacore functional assays. 1: M-08-11 binds to heregulin-activated Her3 ECD and induces delayed heregulin dissociation, with M-08-11 remaining in the Her3 ECD receptor complex. 2: M-05-74 binds to heregulin-activated Her3 ECD. Heregulin is trapped in the complex, and the antibody remains in the complex. 3: 8B8 binds to heregulin-activated Her3 ECD. The entire complex dissociates from the antibody.

Figure 12 Strategies for epitope mapping and alanine scanning approaches. Peptide hairpin sequences of EGFR, Her2 ECD, Her3 ECD, and Her4 ECD were investigated (peptide hairpins), including their structural burial (structural). Cysteine was replaced with serine.

Figure 13. CelluSpots ^™ synthesis and epitope mapping of the epitope of antibody M-08-11 on HER3. Anti-HER3 antibody M-08-11 binds to the HER3 ECD binding epitope PLVYNKLTFQLE (SEQ ID NO: 48) and does not bind to the β-hairpin of HER4.

Figure 14 Results from CelluSpots ^™ alanine scanning of the anti-HER3/HER4 antibody M-05-74 (referred to as M-074 in the figure) and the anti-HER3 antibody M-08-11 (referred to as M-011) without HER4 cross-reactivity - the amino acids that contribute most to binding of the anti-HER3 antibody M-08-11 to its HER3 ECD binding epitope PLVYNKLTFQLE (SEQ ID NO: 48) are underlined/bold.

Figure 15 A) Binding of M-08-11 (M-011) induces/promotes H(HRG) binding to HER3 ECD. Thus, M-08-11 does not compete with heregulin (HRG) for binding to HER3 ECD.

B) Binding of M-08-11, M-43-01, and M-46-01 induces/promotes binding of heregulin (HRG) to HER3 ECD. Thus, M-08-11, M-43-01, and M-46-01 do not compete with heregulin (HRG) for binding to HER3 ECD.

Figure 16 Inhibition of HER2/HER3 heterodimerization (immunoprecipitation and Western blot) by the anti-HER3 antibody M-08-11 (referred to as M-011) in MCF7 cells (HER3-IP=immunoprecipitation with HER3 antibody/HER2-IP=immunoprecipitation with HER3 antibody).

Figure 17 Biacore sensorgram overlay: Binding of the antibody M-08-11 (1) of the present invention to TtSlyDcys-HER3 (SEQ ID NO: 18) compared to the anti-HER3 antibody MOR09823 (2) described in WO2012/22814. The antibody M-08-11 (1) of the present invention shows a clear signal for binding to TtSlyDcys-HER3 (SEQ ID NO: 18), while the anti-HER3 antibody MOR09823 (2) shows no binding to TtSlyDcys-HER3 (SEQ ID NO: 18) at all. Control measurements without any antibody also do not show any binding to TtSlyDcys-HER3 (SEQ ID NO: 18).

Detailed description of embodiments of the present invention

I. Definition

For purposes herein, " acceptor human framework " refers to a framework comprising a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a shared framework as defined below. The acceptor human framework "derived" from a human immunoglobulin framework or a shared framework of people can comprise the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the shared framework sequence of people.

An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody which does not possess such alterations, such alterations resulting in improvements in the affinity of the antibody for antigen.

As used herein, the term "antigen binding protein" refers to an antibody or scaffold antigen binding protein as described herein. In a preferred embodiment, the antigen binding protein is an antibody as described herein. Scaffold antigen binding proteins are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13: 245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discov Today 13: 695-701 (2008), both of which are incorporated herein by reference in their entirety. B. Criteria for selecting parent variable domains and receptors for the antigen binding proteins of the present invention. In one embodiment, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (Evibody); lipocalin; protein A derived molecules such as the Z domain (Affibody, SpA), A domain (Avimer/Maxibody) of protein A; heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamers; C-type lectin domain (Tetranectin); human γ-crystallin and human ubiquitin (affilin); PDZ domain; scorpion toxin Kunitz-type domain of human protease inhibitors; and fibronectin (adnectin); which has been protein engineered to obtain binding to ligands other than the natural ligand.

CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a CD28 family receptor primarily expressed on CD4+ T cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to antibody CDRs can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also called Evibodies. For more details, see Journal of Immunological Methods 248(1-2), 31-45 (2001).

Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bile pigments, retinoids, and lipids. They have a rigid β-sheet secondary structure with loops at the open ends of the canonical structure that can be engineered to bind different target antigens. Anticalins range in size from 160 to 180 amino acids and are derived from lipocalins. For more details, see Biochim Biophys Acta 1482:337-350 (2000); US Pat. No. 7,250,297 B1 and US Pat. No. 2007,0224,633.

Affibody is a scaffold derived from Staphylococcus aureus protein A that can be engineered to bind antigens. The domain consists of a three-helix bundle of approximately 58 amino acids. Libraries have been generated by randomizing surface residues. For more details, see Protein Eng. Des. Sel. 17: 455-462 (2004) and EP1641818A1. Avimers are multidomain proteins derived from the A domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide bond structure. Diversity is generated by shuffling natural variations exhibited by the A domain family. For more details, see Nature Biotechnology 23 (12): 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16 (6): 909-917 (June 2007).

Transferrin is a monomeric serum transport glycoprotein. By inserting peptide sequences into permissive surface loops, transferrin can be engineered to bind to different target antigens. Examples of engineered transferrin scaffolds include Transbody. For more details, see J. Biol. Chem 274, 24066-24073 (1999).

Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, a family of proteins that mediate the attachment of membrane integral proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. By randomizing the residues in the first α-helix and β-turn of each repeat, they can be engineered to bind to different target antigens. Their binding interface can be expanded by increasing the number of modules (an affinity maturation approach). For more details, see J. Mol. Biol. 332: 489-503 (2003); PNAS 100 (4): 1700-1705 (2003) and J. Mol. Biol. 369: 1015-1028 (2007) and US20040132028A1.

Fibronectin is a scaffold that can be engineered to bind antigens. Adnectins consist of the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3) as their backbone. Three loops at one end of the β-sandwich can be engineered to enable adnectins to specifically recognize a desired therapeutic target. For more details, see Protein Eng. Des. Sel. 18:435-444 (2005); US20080139791; WO2005056764; and US6818418B1.

Peptide aptamers are combinatorial recognition molecules composed of a constant scaffold protein, usually thioredoxin (TrxA), containing a constrained variable peptide loop inserted at the active site. For more details, see Expert Opin. Biol. Ther. 5, 783-797 (2005).

Microbodies are derived from naturally occurring microproteins, 25-50 amino acids long, that contain three to four cysteine bridges. Examples of microproteins include KalataBI, piscitoxin, and knottin. Microproteins have a loop that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For more details on engineered knottin domains, see WO2008098796.

Other antigen-binding proteins include proteins that have been used as scaffolds to engineer different target antigen binding properties, including human γ-crystallin and human ubiquitin (affilin), the Kunitz-type domain of human protease inhibitors, the PDZ domain of Ras binding protein AF-6, scorpion toxins (charybdo scorpion toxins), and C-type lectin domains (tetranectin), reviewed in Chapter 7-Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15: 14-27 (2006). The epitope binding domains of the present invention can be derived from any of these alternative protein domains.

The terms "anti-HER3 antigen binding protein," "antigen binding protein that binds to (human) HER3," and "antigen binding protein that specifically binds to human HER3" refer to antigen binding proteins that are capable of binding to HER3 with sufficient affinity such that the antigen binding protein can be used as a diagnostic and/or therapeutic agent in targeting HER3.

The terms "anti-HER3 antibody,""antibody that binds to (human) HER3," and "antibody that specifically binds to human HER3" refer to antibodies that are capable of binding to HER3 with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent in targeting HER3. In one embodiment, the extent of binding of the anti-HER3 antibody to unrelated, non-HER3 proteins is less than about 10% of the binding of the antibody to HER3, as measured, for example, by a surface plasmon resonance assay (such as, for example, BIACORE). In certain embodiments, the antibody that binds to human HER3 has a KD value for binding to human HER3 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M). In a preferred embodiment, with respect to HER3 binding affinity, the corresponding KD value for binding affinity is determined in a surface plasmon resonance assay using the wild-type extracellular domain (ECD) of human HER3 (HER3-ECD).

As used herein, the term "anti-HER3 antigen-binding protein or anti-HER3 antibody that binds to the amino acid sequence SEQ ID NO: 1 in activated HER3" refers to an anti-HER3 antigen-binding protein or anti-HER3 antibody that binds to the amino acid sequence SEQ ID NO: 1 contained in human HER3-ECD in the presence of heregulin (HRG). In a preferred embodiment, the term "anti-HER3 antigen-binding protein or anti-HER3 antibody that binds to the amino acid sequence SEQ ID NO: 1 in activated HER3" refers to an anti-HER3 antigen-binding protein or anti-HER3 antibody that binds to the amino acid sequence SEQ ID NO: 1 contained in the polypeptide SEQ ID NO: 18 (TtSlyDcys-Her3).

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

"Antibody fragments" refer to molecules other than intact antibodies that contain a portion of an intact antibody that binds to the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope as a reference antibody" is one that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

The term "antibody (or antigen-binding protein) having/showing cross-reactivity to (human) HER4, HER4 β-hairpin, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or HER4 ECD" refers to an antibody (or antigen-binding protein) that binds to (human) HER4, HER4 β-hairpin, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (or cross-reacts with) in, for example, a polypeptide of SEQ ID NO: 22 (TtSlyDcas-Her4), a polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4) or the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) contained in HER4 ECD, similar to the above definition of antibodies (or antigen-binding proteins) that bind to human HER3. In this context, the term “an antibody (or antigen binding protein) that does not have/show cross-reactivity to (or does not cross-react with) the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) comprised in, for example, a polypeptide of SEQ ID NO: 22 (TtSlyDcas-Her4), a polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4) or HER4 ECD” refers to an antibody (or antigen binding protein) that does not bind to (human) HER4, HER4 β-hairpin, a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, respectively, within, for example, a polypeptide of SEQ ID NO: 22 (TtSlyDcas-Her4), a polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4) or HER4 ECD, respectively. In a preferred embodiment, the antibody does not cross-react with the extracellular domain (ECD) of human HER4 (human HER4-ECD) SEQ ID NO: 6, i.e., when the binding signal (in relative units (RU)) measured in a surface plasmon assay is three times lower than the background signal (noise) (e.g., an immobilized (e.g., captured) antibody with human HER4-ECD as the antigen is injected at 25°C as a soluble analyte). In a preferred embodiment, the antibody of the present invention does not cross-react with human HER4 when it does not cross-react with the polypeptide SEQ ID NO: 22 (TtSlyDcys-Her4). The definitions of cross-reactivity and non-cross-reactivity for other antigens (e.g., for HER2, HER1, etc.) are similar.

As used herein, the term "cancer" can be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethra cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary cancer, Cancer, central nervous system (CNS) neoplasms, spinal axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, lymphomas, lymphocytic leukemias, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers. In a preferred embodiment, such cancers are breast cancer, ovarian cancer, cervical cancer, lung cancer, or prostate cancer. In a preferred embodiment, such cancers are further characterized by HER3 expression or overexpression. Yet another embodiment of the invention is an anti-HER3 antibody of the invention for use in the simultaneous treatment of a primary tumor and new metastases.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamicin, vinca alkaloids); [00145] The present invention also includes but is not limited to alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below. In a preferred embodiment, the "cytotoxic agent" is Pseudomonas exotoxin A or a variant thereof. In a preferred embodiment, the "cytotoxic agent" is Amanita toxin or a variant thereof.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) NIH Publication 91-3242.

"Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. Generally, the FR of a variable domain is composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences in VH (or VL) generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard for the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initial transformed cell are included herein.

A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

"Human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

"Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, typically two, substantially entire variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all FRs correspond to those of human antibodies. Optionally, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized variant" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. In a preferred embodiment, mouse HVRs are grafted into the framework regions of human antibodies to prepare "humanized antibodies." See, for example, Riechmann, L. et al., Nature 332 (1988) 323-327; and Neuberger, M.S. et al., Nature 314 (1985) 268-270. The mouse variable region amino acid sequences are aligned with a human germline antibody V gene set and sorted according to sequence identity and homology. Acceptor sequences are selected based on high overall sequence homology and, optionally, the presence of the correct canonical residues already present in the acceptor sequence (see Poul, M-A. and Lefranc, M-P., in "Ingenieries des anticorps banques combinatores", ed. by Lefranc, M-P. and Lefranc, G., Les Editions INSERM, 1997). Germline V genes only encode regions up to the beginning of HVR3 for the heavy chain and up to the middle of HVR3 for the light chain. Therefore, the genes of the germline V genes are not aligned with the complete V domain. The humanized constructs contain human frameworks 1 to 3, mouse HVRs, and human framework 4 sequences derived from human JK4 and JH4 sequences (for the light and heavy chains, respectively). Before selecting a specific acceptor sequence, the so-called canonical loop structure of the donor antibody can be determined (see Morea, V. et al., Methods, Vol 20, Issue 3 (2000) 267-279). These canonical loop structures are determined by the type of residues present at the so-called canonical positions. These positions (parts) are located outside the HVR region and should remain functionally equivalent in the final construct, thereby retaining the HVR conformation of the parent (donor) antibody. In WO2004/006955, a method for humanizing antibodies is reported, which comprises the following steps: identifying the canonical HVR structural type of the HVR in a non-human mature antibody; obtaining a peptide sequence library of a human antibody variable region; determining the canonical HVR structural type of the variable region in the library; and selecting a human sequence in which the canonical HVR structure is identical to the canonical HVR structural type of the non-human antibody at the corresponding position in the non-human and human variable regions. In short, potential recipient sequences are selected based on the presence of high overall homology and the correct (right) canonical residues that already exist in the optional recipient sequence. In some cases, simple HVR grafting only results in partial retention of the binding specificity of non-human antibodies. It has been found that at least some specific non-human framework residues are required to reconstruct binding specificity and also have to be grafted into the human framework, that is, so-called "back mutations" have to be performed in addition to the introduction of non-human HVRs (see, for example, Queen et al., Proc. Natl. Acad. Sci. USA 86: 10,029-10,033 (1989); Co et al., Nature 351: 501-502 (1991)). These specific framework amino acid residues are involved in FR-HVR interactions and stabilize the conformation (loop) of the HVRs (see, for example, Kabat et al., J. Immunol. 147: 1709 (1991)). In some cases, forward mutations are also introduced to more closely adopt the human germline sequence. Thus, a “humanized variant of an antibody according to the invention (e.g., of mouse origin)” refers to an antibody that is based on a mouse antibody sequence, wherein the VH and VL are humanized by standard techniques as described above (including HVR grafting and optionally mutagenesis of the subsequent framework regions and certain amino acids in HVR-H1, HVR-H2, HVR-L1 or HVR-L2, while HVR-H3 and HVR-L3 remain unmodified).

As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain antigen contact residues ("antigen contacts"). Generally, antibodies comprise six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops, which occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs, which occur at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigenic contacts, which occur at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

(d) a combination of (a), (b), and/or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

"Individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject refers to a human.

An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chromatogr. B 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-HER3 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical and/or bind to the same epitope, except for example containing naturally occurring mutations or possible variant antibodies occurring during the generation of monoclonal antibody preparations, such variants are generally present in very small amounts. Unlike polyclonal antibody preparations that typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a group of substantially homogeneous antibodies, and should not be interpreted as requiring the generation of antibodies by any particular method. For example, the monoclonal antibody to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and such methods and other exemplary methods for generating monoclonal antibodies are described herein.

The term "Mab" refers to a monoclonal antibody, while the term "hMab" refers to a humanized version of such a monoclonal antibody.

A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations (including immunoconjugates if available in the prior art).

"Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded together by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Depending on the amino acid sequence of its constant domain, antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ).

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences. However, for purposes of the present invention, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted, along with user documentation, to the U.S. Copyright Office (Washington D.C., 20559), where it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is available to the public from Genentech (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

In the case of using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or which can be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

Multiply the fraction X/Y by 100

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that would be unacceptably toxic to a subject to which the formulation would be administered.

"Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the term "HER3" refers to any native HER3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed HER3 as well as any form of HER3 derived from processing in cells. The term also includes naturally occurring HER3 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human HER3 is shown in SEQ ID NO: 3. "Human HER3" (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine protein kinase erbB-3, SEQ ID NO: 3) encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4 (Kraus, M.H. et al., PNAS 86:9193-9197 (1989); Plowman, G.D. et al., PNAS 87:4905-4909 (1990); Kraus, M.H. et al., PNAS 90:2900-2904 (1993)). Like the prototypical EGFR, the transmembrane receptor HER3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD), and a C-terminal phosphorylation domain. This membrane-bound protein has a heregulin (HRG) binding domain that is located in the extracellular domain rather than the active kinase domain. Therefore, it can bind this ligand but does not transmit the signal into the cell via protein phosphorylation. However, it does form dimers with other HER family members that do not have kinase activity. Heterodimerization leads to activation of receptor-mediated signaling pathways and transphosphorylation of its intracellular domain. Dimer formation between HER family members expands the signaling potential of HER3 and serves not only as a means of signal diversification but also as a means of signal amplification. For example, HER2/HER3 heterodimers induce one of the most important mitogenic signals among HER family members, through the PI3K and AKT pathways (Sliwkowski M.X. et al., J. Biol. Chem. 269:14661-14665 (1994); Alimandi M et al., Oncogene. 10:1813-1821 (1995); Hellyer, N.J., J. Biol. Chem. 276:42153-4261 (2001); Singer, E., J. Biol. Chem. 276:44266-44274 (2001); Schaefer, K.L., Neoplasia 8:613-622 (2006)). For an overview of HER3 and its various interactions within the HER receptor family and the NGR ligand family, see, eg, Sithanandam, G. et al Cancer Gene Therapy 15:413-448 (2008).

Interestingly, in its equilibrium state, the HER3 receptor exists in its "closed conformation," which indeed means that the heterodimeric HER3 β-hairpin motif is tethered to the HER3 ECD domain IV via non-covalent interactions (see Figure 1c). It is hypothesized that the "closed" HER3 conformation can be opened by the binding of the ligand heregulin at a specific HER3 heregulin binding site. This occurs at the HER3 interface formed by HER3 ECD domains I and III. Through this interaction, the HER3 receptor is believed to be activated and converted to its "open conformation" (see Figure 1b and, for example, Baselga, J. et al., Nat. Rev. Cancer 9:463-475 (2009) and Desbois-Mouthon, C., at al., Gastroenterol Clin Biol 34:255-259 (2010)). In this open conformation, heterodimerization with HER2 and transsignaling induction are possible (see Figure 1b).

As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of disease or slow the progression of disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, a library of complementary VL or VH domains can be screened using a VH or VL domain from an antibody that binds to a specific antigen to isolate antibodies that bind to that antigen. See, e.g., Portolano, S. et al., J. Immunol. 150:880-887 (1993); Clarkson, T. et al., Nature 352:624-628 (1991)).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

II. Compositions and Methods

In one aspect, the present invention is based in part on the discovery that using HER3 and HER4 β-hairpins functionally presented in a three-dimensional orientation within a SlyD scaffold (see, e.g., FIG. 2 , and polypeptides of SEQ ID NOs. 13 and 17 to 24) it is possible to select antibodies that are specific for the HER3 β-hairpin and do not cross-react with the HER4 β-hairpin.

In certain embodiments, the invention provides antibodies that bind to human HER3 (and do not cross-react with human HER4), wherein the antibody binds within the amino acid sequence of human HER3: PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1).

The antibodies of the present invention are useful for, for example, diagnosis or treatment of cancer.

A. Exemplary Anti-HER3 Antigen Binding Proteins and Antibodies

The present invention provides an isolated antigen binding protein that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antigen binding protein binds to a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3,

and

b) wherein the antigen binding protein does not cross-react with a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4.

The present invention further provides an isolated antigen binding protein that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antigen binding protein binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3;

and

b) wherein the antigen binding protein does not cross-react with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4.

The present invention further provides an isolated antigen binding protein that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antigen binding protein binds to the polypeptide SEQ ID NO: 18TtSlyDcys-Her3, and

b) wherein the antigen binding protein does not cross-react with the polypeptide SEQ ID NO: 22TtSlyDcys-Her4.

The present invention further provides an isolated antigen binding protein that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antigen binding protein binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in the polypeptide SEQ ID NO: 18 (TtSlyDcas-Her3), and

b) wherein the antigen binding protein does not cross-react with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) contained in the polypeptide SEQ ID NO: 22 (TtSlyDcas-Her4).

The present invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antibody binds to a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3,

and

b) wherein the antibody does not cross-react with a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4.

The present invention further provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antibody binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3;

and

b) wherein the antibody does not cross-react with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4.

The present invention further provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antibody binds to the polypeptide SEQ ID NO: 18TtSlyDcys-Her3, and

b) wherein the antibody does not cross-react with the polypeptide SEQ ID NO: 22TtSlyDcys-Her4.

The present invention further provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4),

a) wherein the antibody binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in the polypeptide SEQ ID NO: 18 (TtSlyDcas-Her3), and

b) wherein the antibody does not cross-react with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) contained in the polypeptide SEQ ID NO: 22 (TtSlyDcas-Her4).

In one aspect, the invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody binds within the amino acid sequence of human HER3: PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1).

In certain embodiments, the invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody has one or more of the following properties (and every combination of each single property is contemplated herein):

a) the antibody binds to the amino acid sequence SEQ ID NO: 1; and/or

b) the antibody binds to the amino acid sequence SEQ ID NO: 1 in activated HER3; and/or

c) the antibody binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3; and/or

d) the antibody binds to the β-hairpin region of HER3; and/or

e) the antibody inhibits heterodimerization of HER3/HER2 heterodimers (see Example 6); and/or

f) the antibody has a ratio of the binding constant (Ka) for binding to Thermus thermophilus SlyD FKBP-HER3 (SEQ ID NO: 13) to the binding constant (Ka) for binding to HER3-ECD (SEQ ID NO: 4) (Ka(Thermus thermophilus SlyD FKBP-Her3)/Ka(HER3-ECD)) of 1.5 or greater when measured in a surface plasmon resonance assay; and/or

g) the antibody has a ratio of the molar ratio MR for binding to Thermus thermophilus SlyD FKBP-HER3 (SEQ ID NO: 13) to the molar ratio MR for binding to HER3-ECD (SEQ ID NO: 4) (MR(Thermus thermophilus SlyD FKBP-Her3)/MR(HER3-ECD)) of 2.0 or higher when measured in a surface plasmon resonance assay; and/or

h) the antibody has no cross-reactivity to the amino acid sequence of SEQ ID NO: 2; and/or

i) the antibody has no cross-reactivity to the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4; and/or

j) the antibody has no cross-reactivity to the β-hairpin region of HER4; and/or

k) the antibody does not compete with heregulin for binding to HER3 (see Example 5); and/or

1) the antibody induces heregulin to bind to HER3 (see Example 5); and/or

m) the antibody binds to HER3-ECD with an affinity of a KD value ≤ 1 x ^10-8 M (in one embodiment, a KD value of 1 x ^10-8 M to 1 x ^10-13 M; in one embodiment, a KD value of 1 x ^10-9 M to 1 x ^10-13 M); and/or

n) the antibody binds to a polypeptide consisting of PLVYNKLTFQLE (SEQ ID NO: 48) (see Examples 2 and 3); and/or

o) the antibody binds to a polypeptide consisting of PLVYNKLTFQLE (SEQ ID NO: 48) and does not cross-react with a polypeptide consisting of PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) (see Examples 2 and 3); and/or

p) exhibits at least two-fold higher binding in the presence of heregulin compared to the level of binding in the absence of heregulin, as detected at 0 minutes after incubation with the antibody in a FACS assay with HER3-expressing T47D cells (see Example 2b); and/or

q) shows nearly complete HER3 internalization in the presence of heregulin after 4 hours of incubation with the antibody in a FACS assay performed with HER3-expressing T47D cells (see Example 2b).

In certain embodiments, the invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody has one or more of the following properties (and every combination of each single property is contemplated herein):

a) the antibody binds to the amino acid sequence SEQ ID NO: 1; and

The antibody does not cross-react with the amino acid sequence SEQ ID NO: 2;

and/or

b) the antibody binds to the amino acid sequence SEQ ID NO: 1 in activated HER3; and

The antibody does not cross-react with the amino acid sequence SEQ ID NO: 2 in activated HER4;

and/or

c) the antibody binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3; and

The antibody does not bind within the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) contained in a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4;

and/or

d) the antibody binds to a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3; and

The antibody does not cross-react with a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4;

and/or

e) the antibody binds to the β-hairpin region of HER3; and

This antibody does not cross-react with the β-hairpin region of HER4;

and/or

f) the antibody binds to a polypeptide having a length of 15 amino acids and comprising the amino acid sequence PVYNKLTFQLE (SEQ ID NO: 48) and

It does not cross-react with the polypeptide consisting of the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2);

and/or

g) The antibody binds to a 15-amino acid polypeptide comprising the amino acid sequence PVYNKLTFQLE (SEQ ID NO: 48).

In certain embodiments, the present invention provides an isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody binds to a 15 amino acid polypeptide comprising the amino acid sequence PVYNKLTFQLE (SEQ ID NO: 48) and does not cross-react with a polypeptide consisting of the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2).

In certain embodiments, the present invention provides an isolated antibody that binds to human HER3 and does not cross-react with human HER4, wherein the antibody binds to a 15 amino acid polypeptide comprising the amino acid sequence PVYNKLTFQLE (SEQ ID NO: 48).

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In yet another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25. In yet another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:26; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27.

In another aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In another aspect, the present invention provides an anti-HER3 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:26; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:28; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

In one embodiment, such anti-HER3 antibodies comprise:

i) VH sequence SEQ ID NO: 31 and VL sequence SEQ ID NO: 32; or

ii) Humanized variants of the antibody VH and VL under i).

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38. In yet another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38, and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33. In yet another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:34; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:35.

In another aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.

In another aspect, the present invention provides an anti-HER3 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:34; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:35; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:36; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:37; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:38.

In one embodiment, such anti-HER3 antibodies comprise:

i) VH sequence SEQ ID NO: 39 and VL sequence SEQ ID NO: 40; or

ii) Humanized variants of the antibody VH and VL under i).

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:44; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:45; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:46.

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 41; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 42; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 46. In yet another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 46, and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 41. In yet another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43.

In another aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 44; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 46. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 44; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 46.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 41, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 42, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 44, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 46.

In another aspect, the present invention provides an anti-HER3 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:44; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:45; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:46.

In one embodiment, such anti-HER3 antibodies comprise:

i) VH sequence SEQ ID NO: 47 and VL sequence SEQ ID NO: 48; or

ii) Humanized variants of the antibody VH and VL under i).

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:50; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:51; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:52; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:53; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:54.

In one aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 49; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54. In yet another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54, and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 49. In yet another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:50; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:51.

In another aspect, the present invention provides an anti-HER3 antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 49, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

In another aspect, the present invention provides an anti-HER3 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:50; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:51; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:52; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:53; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:54.

In one embodiment, such anti-HER3 antibodies comprise:

i) VH sequence SEQ ID NO: 56 and VL sequence SEQ ID NO: 57; or

ii) Humanized variants of the antibody VH and VL under i).

In another aspect, the present invention provides an anti-HER3 antibody comprising:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In another aspect, the present invention provides an anti-HER3 antibody comprising: i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In another aspect, the present invention provides an anti-HER3 antibody comprising:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:44;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:46;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In another aspect, the present invention provides an anti-HER3 antibody comprising:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

In any of the above embodiments, the anti-HER3 antibody is humanized. In one embodiment, the anti-HER3 antibody comprises the HVRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-HER3 antibody comprises the HVRs of any of the above embodiments and further comprises a VH comprising human germline IMGT_hVH_1_46 or IMGT_hVH_3_15 framework regions and a VL comprising human germline IMGT_hVK_1_33 framework regions. The sequences and framework regions of human germlines are described in Poul, M-A. and Lefranc, M-P., in "Ingénierie des anticorps banques combinatores" ed. by Lefranc, M-P. and Lefranc, G., Les Editions INSERM, 1997. Human heavy and light chain variable framework regions of all human germline lines are listed, for example, in Lefranc, M.P., Current Protocols in Immunology (2000) - Appendix 1 P A.1 P.1-A.1 P.37 and are available via IMGT (the international ImMunoGeneTics information http://imgt.cines.fr) or via http://vbase.mrc-cpe.cam.ac.uk.

In another aspect, the present invention provides an anti-HER3 antibody comprising:

A)

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30;

or

ii) a humanized variant of an antibody HVR under i)(a), (b), (d) and/or (e); or

B)

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38;

or

ii) a humanized variant of an antibody HVR under i)(a), (b), (d) and/or (e); or

C)

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:44;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:46;

or

ii) a humanized variant of an antibody HVR under i)(a), (b), (d) and/or (e); or

D)

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54;

or

ii) humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e);

The antibody has one or more of the following characteristics:

a) the antibody binds to the amino acid sequence SEQ ID NO: 1; and/or

b) the antibody does not cross-react with human HER4; and/or

c) the antibody binds to the amino acid sequence SEQ ID NO: 1 in activated HER3; and/or

d) the antibody binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3; and/or

e) the antibody binds to the β-hairpin region of HER3; and/or

f) the antibody inhibits heterodimerization of HER3/HER2 heterodimers; and/or

g) the antibody has a ratio of the binding constant (Ka) for binding to Thermus thermophilus SlyD FKBP-HER3 (SEQ ID NO: 13) to the binding constant (Ka) for binding to HER3-ECD (SEQ ID NO: 4) (Ka(Thermus thermophilus SlyD FKBP-Her3)/Ka(HER3-ECD)) of 1.5 or greater when measured in a surface plasmon resonance assay; and/or

h) the antibody has a ratio of the molar ratio MR for binding to Thermus thermophilus SlyD FKBP-HER3 (SEQ ID NO: 13) to the molar ratio MR for binding to HER3-ECD (SEQ ID NO: 4) (MR(Thermus thermophilus SlyD FKBP-Her3)/MR(HER3-ECD)) of 2.0 or higher when measured in a surface plasmon resonance assay; and/or

i) the antibody has no cross-reactivity to the amino acid sequence of SEQ ID NO: 2; and/or

j) the antibody shows no cross-reactivity to the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4; and/or

k) the antibody has no cross-reactivity to the β-hairpin region of HER4; and/or

1) the antibody does not compete with heregulin for binding to HER3; and/or

m) the antibody induces heregulin binding to HER3; and/or

n) the antibody binds to HER3-ECD with an affinity of a KD value ≤ 1 x ^10-8 M (in one embodiment, a KD value of 1 x ^10-8 M to 1 x ^10-13 M; in one embodiment, a KD value of 1 x ^10-9 M to 1 x ^10-13 M); and/or

o) the antibody binds to a polypeptide consisting of PLVYNKLTFQLEP (SEQ ID NO: 48); and/or

p) the antibody binds to a polypeptide consisting of PLVYNKLTFQLEP (SEQ ID NO:48) and does not cross-react with a polypeptide consisting of PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2); and/or

q) the antibody exhibits at least a two-fold higher level of binding in the presence of heregulin compared to the level of binding in the absence of heregulin, as detected at 0 minutes after incubation with the antibody in a FACS assay with T47D cells expressing HER3; and/or

r) The antibody showed nearly complete HER3 internalization in the presence of heregulin after 4 hours of incubation with the antibody in a FACS assay performed with HER3-expressing T47D cells.

In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-HER3 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as an anti-HER3 antibody comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32. In certain embodiments, antibodies are provided that bind to an epitope within a human HER3 fragment consisting of the amino acid sequence PLVYNKLTFQLE (SEQ ID NO: 48). In certain embodiments, antibodies are provided that bind to an epitope within a human HER3 fragment consisting of the amino acid sequence PLVYNKLTFQLE (SEQ ID NO: 48) and do not cross-react with a human HER4 fragment consisting of the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2).

1. Antibody affinity

In certain embodiments, the antibodies provided herein have a dissociation constant KD of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M).

In a preferred embodiment, KD is measured using a surface plasmon resonance assay at approximately 10 response units (RU) using an immobilized antigen CM5 chip at 25°C. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl/min to obtain approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) were injected at 25° C. at a flow rate of approximately 25 μl/min. Association rates (k _on or ka) and dissociation rates (k _off or kd) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (Evaluation software version 3.2). The equilibrium dissociation constant (KD) was calculated as the ratio kd/ka (k _off /k _on ). See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10 ⁶ M ^-1 s ^-1 according to the surface plasmon resonance assay above, the on-rate can be determined using a fluorescence quenching technique, i.e., measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer such as a spectrophotometer equipped with a cut-off device (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette.

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Plueckthun, A., In: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-lives, see US Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 0 404 097; WO 1993/01161; Hudson, P.J. et al., Nat. Med. 9:129-134 (2003); and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson, P.J. et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production in recombinant host cells (eg, E. coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described in, for example, U.S. Patent No. 4,816,567; and Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or part thereof) are derived from non-human antibodies, and FR (or part thereof) are derived from human antibody sequences. Optionally, humanized antibodies also include at least a portion of human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (such as antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their generation are reviewed in, for example, Almagro, J.C. and Fransson, J., Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann, I. et al., Nature 332:323-329 (1988); Queen, C. et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri, S.V. et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, E.A., Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua, W.F. et al., Methods 36:43-60 (2005) (describing “FR shuffling”); Osbourn, J. et al., Methods 36:61-68 (2005) and Klimka, A. et al., Br. J. Cancer 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling); Morea, V. et al., Methods, Vol 20, Issue 3 (2000) 267-279) and WO 2004/006955 (the canonical structure approach).

4. Human Antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Generally, human antibodies are described in van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to generate complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or it exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE ^™ technology; U.S. Patent No. 5,770,429, which describes technology; U.S. Patent No. 7,041,870, which describes KM technology, and U.S. Patent Application Publication No. US 2007/0061900, which describes technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with a different human constant region.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for generating human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133: 3001-3005 (1984); Brodeur, B.R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147: 86-95 (1991)). Human antibodies generated via human B cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103: 3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26: 265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, H. P. and Brandlein, S., Histology and Histopathology 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27: 185-91 (2005).

It is also possible to generate human antibodies as follows, that is, to separate the Fv clone variable domain sequence selected from the phage display library derived from humans.Then, this type of variable domain sequence can be combined with the desired human constant domain.The technology of selecting human antibodies from antibody libraries is described below.

5. Library-derived Antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired one or more activities. For example, a variety of methods for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom, H.R. et al., Methods in Molecular Biology 178:1-37 (2001), and further described, for example, in McCafferty, J. et al., Nature 348:552-554 (1990); Clackson, T. et al., Nature 352:624-628 (1991); Marks, J.D. et al., J. Mol. Biol. 222:581-597 (1992); Marks, J.D. and Bradbury, A., Methods in Molecular Biology 248:161-175 (2003); Sidhu, S.S. et al., J. Mol. Biol. 338(2):299-310 (2004); Lee, C.V. et al., J. Mol. Biol. 339(3):411-412 (2005). al., J. Mol. Biol. 340: 1073-1093 (2004); Fellouse, F. A., Proc. Natl. Acad. Sci. USA 101: 12467-12472 (2004); and Lee, C. V. et al., J. Immunol. Methods 284:119-132(2004).

In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage, as described in Winter, G. et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, non-immunized repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens in the absence of any immunization, as described by Griffiths, A.D. et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences encoding highly variable CDR3 regions and achieving rearrangement in vitro, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for HER3, while the other is for any other antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing HER3. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature, 305:537-540 (1983); WO 93/08829; and Traunecker, A. et al., EMBO J. 10:3655-3659 (1991)), and “knob-into-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). It can also be achieved by engineering electrostatic manipulation effects for generating antibody Fc-heterodimer molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan, M. et al., Science 229:81-83 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148:1547-1553 (1992)); using "diabody" technology for generating bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 148:1547-1553 (1992)). et al., J. Immunol. 152:5368-5374 (1994)); and by preparing trispecific antibodies as described, for example, in Tutt, A. et al., J. Immunol. 147:60-69 (1991) to generate multispecific antibodies.

Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576).

The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs" that comprise antigen binding sites that bind to both HER3/HER4 and another, different antigen (see, eg, US 2008/0069820).

The antibodies or fragments herein also include the multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793.

7. Antibody variants

In certain embodiments, the amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Suitable modifications may be introduced into the nucleotide sequence encoding the antibody, or the amino acid sequence variants of the antibody may be prepared by peptide synthesis. Such modifications include, for example, deletion and/or insertion and/or substitution of the residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution may be performed to obtain the final construct, as long as the final construct has the desired characteristics, for example, antigen binding.

a) Substitution, insertion, and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 1

According to common side chain properties, amino acids can be grouped as follows:

(1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

A class of substitution variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated, and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots," i.e., residues encoded by codons that undergo mutations at high frequency during the somatic maturation process (see, e.g., Chowdhury, P.S., Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described in, e.g., Hoogenboom, H.R. et al., in Methods in Molecular Biology 178: 1-37 (2002)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-guided mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made in HVRs that do not substantially reduce binding affinity. Such changes may be outside of HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

A method for identifying residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham, B.C. and Wells, J.A., Science, 244: 1081-1085 (1989). In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively or in addition, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. As candidates for substitution, such contact residues and neighboring residues can be targeted or eliminated. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N or C-terminus of an antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

In the case where the antibody comprises an Fc region, the carbohydrate attached to the Fc region can be changed. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region via an N-link. See, for example, Wright, A. and Morrison, S.L., TIBTECH 15: 26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) in the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants can have improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A. et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87: 614-622 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94:680-688 (2006); and WO 2003/085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; U.S. Patent No. 6,602,684; and US 2005/0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can be comprised of a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention encompasses antibody variants that possess some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/reduction of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, JVand Kinet, JP, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA; and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes, R. et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and, therefore, lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202: 163-171 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and M. J. Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18: 1759-1769 (2006)).

Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Pat. No. 6,737,056 ). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine ( U.S. Pat. No. 7,332,581 ).

Certain antibody variants with improved or decreased binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056; WO 2004/056312; and Shields, R.L. et al., J. Biol. Chem. 276:6591-6604 (2001)).

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, eg, substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and/or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551; WO 99/51642; and Idusogie, E.E. et al., J. Immunol. 164:4178-4184 (2000).

Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005/0014934, which is responsible for the transfer of maternal IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117:587-593 (1976) and Kim, J.K. et al., J. Immunol. 24:2429-2434 (1994)). These antibodies comprise an Fc region with one or more substitutions that improve Fc region binding to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

See also Duncan, A. R. and Winter, G., Nature 322:738-740 (1988); US 5,648,260; US 5,624,821; and WO 94/29351, which concern other examples of Fc region variants.

d) Cysteine-engineered antibody variants

In certain embodiments, it may be desirable to create an antibody engineered with cysteine, such as a "thioMAb," in which one or more residues of the antibody are replaced with cysteine residues. In a specific embodiment, the substituted residue is present in an accessible site of the antibody. By replacing those residues with cysteine, reactive thiol groups are thus located at accessible sites of the antibody and can be used to conjugate the antibody to other modules, such as drug modules or linker-drug modules, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Antibodies engineered with cysteine can be generated as described in, for example, U.S. Patent No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous modules known in the art and readily available. Modules suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in production. Polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be identical or different molecules. Generally, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically for a given condition, etc.

In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are harmless to normal cells but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Recombinant methods and compositions can be used to generate antibodies, for example as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-HER3 antibody as described herein is provided. Such nucleic acid can encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH (e.g., the light and/or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector and a second vector, wherein the first vector comprises a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and the second vector comprises a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-HER3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture fluid).

For the recombinant generation of anti-HER3 antibodies, nucleic acids encoding the antibodies are isolated (e.g., as described above) and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237; US 5,789,199; and US 5,840,523. See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell slurry in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, T.U., Nat. Biotech. 22: 1409-1414 (2004); and Li, H. et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429 (which describe PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, for example, in Graham, FL et al., J. Gen Virol. 36:59-74 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, JP, Biol. Reprod. 23:243-252 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, JP et al., Annals of NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

C. Assays and Antibody (or Antigen Binding Protein) Selection Methods

The anti-HER3 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

One aspect of the invention is a method for selecting an antibody (or antigen binding protein) that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody (or antigen binding protein) binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3,

Among them use

a) at least one polypeptide comprising the amino acid sequence SEQ ID NO: 1 selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3

and

b) at least one polypeptide comprising the amino acid sequence SEQ ID NO: 2 selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4

to select (in a binding assay) antibodies (or antigen binding proteins) that show binding to at least one polypeptide under a) and show no binding to at least one polypeptide under b),

Thus, antibodies (or antigen binding proteins) are selected that bind within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3 and do not cross-react with human HER4.

One aspect of the invention is a method for selecting an antibody (or antigen binding protein) that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody (or antigen binding protein) binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3,

Among them use

a) at least one polypeptide comprising the amino acid sequence SEQ ID NO: 1 selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3

and

b) a human HER4 ECD polypeptide having the amino acid sequence of SEQ ID NO: 6 to select (in a binding assay) antibodies (or antigen binding proteins) that show binding to at least one polypeptide under a) and do not bind to the human HER4 ECD polypeptide under b), and thereby select antibodies (or antigen binding proteins) that bind within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3 and do not cross-react with human HER4.

In one embodiment, such selection methods further comprise a step wherein the selected antibodies are counter-screened with a polypeptide (for which binding is tested) selected from the group consisting of:

SEQ ID NO:14TtSlyD-wild type

SEQ ID NO:15TtSlyDcas

SEQ ID NO:16TgSlyDΔIF

To confirm that the selected antibodies do not bind to polypeptide scaffolds that do not contain the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) or the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2).

The present invention provides antibodies (or antigen-binding proteins) obtained by such selection methods.

A method for selecting an antibody (or antigen-binding protein) that specifically binds to human HER3 (and does not cross-react with human HER4), comprising the following steps:

a) determining the binding affinity of a plurality of antibodies (or antigen binding proteins) to a HER3 β-hairpin having the amino acid sequence of SEQ ID NO: 1, wherein the HER3 β-hairpin is presented as a polypeptide comprising a HER3 β-hairpin having the amino acid sequence of SEQ ID NO: 1 selected from the group consisting of:

i) SEQ ID NO:12TtSlyD-FKBP-Her3,

ii) SEQ ID NO:16TtSlyDcas-Her3,

iii) SEQ ID NO:17TtSlyDcys-Her3,

iv) SEQ ID NO: 18 TgSlyDser-Her3, and

v) SEQ ID NO:19TgSlyDcys-Her3,

b) selecting an antibody (or antigen binding protein) having an apparent complex stability above a predetermined threshold level;

c) determining the binding affinity of the antibody (or antigen binding protein) selected under step b) to the HER4 β-hairpin having the amino acid sequence of SEQ ID NO: 2, wherein the HER4 β-hairpin is presented as a polypeptide comprising the HER4 β-hairpin having the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of:

i) SEQ ID NO:20TtSlyDcas-Her4,

ii) SEQ ID NO:21TtSlyDcys-Her4,

iii) SEQ ID NO: 22 TgSlyDser-Her4, and

iv) SEQ ID NO:23TgSlyDcys-Her4,

d) selecting antibodies (or antigen binding proteins) that do not have apparent complex stability above a predetermined threshold level.

1. Binding assays and other assays

In one aspect, the antibodies (or antigen binding proteins) of the invention are tested for their antigen binding activity, e.g., by known methods such as ELISA, Western blot, including surface plasmon resonance (e.g., BIACORE), and the like.

On the other hand, competition assays can be used to identify antibodies that compete with M-08-11 for binding to HER3. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the epitope bound by M-08-11. Detailed exemplary methods for locating epitopes bound by antibodies are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). Other methods are described in detail in Example 4, using CelluSpot ^™ technology.

In an exemplary competitive assay, immobilized HER3 is incubated in a solution comprising a first labeled antibody (which binds to HER3, such as M-08-11) and a second unlabeled antibody (which is to be tested for its ability to compete with the first antibody for binding to HER3). The second antibody may be present in the hybridoma supernatant. As a control, immobilized HER3 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to HER3, excess unbound antibody is removed, and the amount of the label associated with immobilized HER3 is measured. If the amount of the label associated with immobilized HER3 in the test sample is substantially reduced relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to HER3. See Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988).

2. Activity Assay

On the one hand, an assay for identifying an anti-HER3 antibody (or antigen-binding proteins) with biological activity is provided. Biological activity can include, for example, inhibition of HER3 phosphorylation, inhibition of cancer cell proliferation of cancer cells expressing or overexpressing HER3, inhibition of HER3/HER2 heterodimerization, (time-dependent) internalization through FACS assay, inhibition of tumor growth in vivo in xenograft animals (e.g., mice or rats) models of xenograft cancer cells expressing or overexpressing HER3. Also provided are antibodies with such biological activity in vivo and/or in vitro, either alone or as immunoconjugates with cytotoxic agents.

In certain embodiments, the antibodies of the invention are tested for such biological activities. Exemplary in vitro or in vivo assays for specified biological activities are described in Example 2e and Examples 5, 6 and 8.

D. Immunoconjugates

The present invention also provides immunoconjugates comprising an anti-HER3 antibody (or antigen binding protein) described herein coupled to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see US 5,208,020, US 5,416,064 and EP 0 425 235 B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see US 5,635,483, US 5,780,588, and US 7,498,298); dolastatin; calicheamicin or its derivatives (see US 5,712,374, US 5,714,586, US 5,739,116, US 5,767,285, US 5,770,701, US 5,770,710, US 5,773,001, and US 5,877,296; Hinman, L.M. et al., Cancer Res. 53:3336-3342 (1993); and Lode, H.N. et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz, F. et al., Current Med. Chem. 13:477-523 (2006); Jeffrey, S.C. et al., Bioorg Med. Chem. Letters 16:358-362 (2006); Torgov, M.Y. et al., Bioconjug. Chem. 16:717-721 (2005); Nagy, A. et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik, G.M. et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King, H.D. et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis toxin, scutellaria serrata ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to Pseudomonas exotoxin A or a variant thereof. Pseudomonas exotoxin A or a variant thereof is described in, for example, WO 2011/32022, WO 2009/32954, WO 2007/031741, WO 2007/016150, WO 2005/052006, and Liu, W. et al., PNAS 109: 11782-11787 (2012).

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to generate radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When the radioconjugate is used for detection, it can contain a radioactive atom for scintigraphic studies, such as TC ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of the antibody and cytotoxic agent can be prepared using a) recombinant expression techniques (e.g., for expression in, for example, E. coli of an amino acid sequence-based toxin fused to a Fab or Fv antibody fragment) or b) polypeptide coupling techniques (like enzyme sortase-based coupling of an amino acid sequence-based toxin to a Fab or Fv antibody fragment) or c) using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane Bifunctional derivatives of alkane-1-carboxylates (SMCC), iminothiolane (IT), imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta, E.S. et al., Science 238:1098-1104 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari, R.V. et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

The immunoconjugates or ADCs herein specifically encompass, but are not limited to, such conjugates prepared with the following cross-linkers, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology Inc., Rockford, IL, U.S.A.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any anti-HER3 antibody (or antigen binding protein) provided herein is useful for detecting the presence of HER3 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues, such as tumor tissue.

In one embodiment, an anti-HER3 antibody for use in a diagnostic or detection method is provided. In yet another aspect, a method for detecting the presence of HER3 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-HER3 antibody as described herein under conditions that allow the anti-HER3 antibody to bind to HER3, and detecting whether a complex is formed between the anti-HER3 antibody and HER3. Such methods can be in vitro or in vivo methods. In one embodiment, an anti-HER3 antibody is used to select a subject suitable for treatment with an anti-HER3 antibody, for example, where HER3 is a biomarker for selecting a patient.

Exemplary disorders that can be diagnosed using the antibodies of the invention include cancer.

In certain embodiments, labeled anti-HER3 antibodies are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and indirectly detected moieties, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of the anti-HER3 antibodies (or antigen binding proteins) described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)), in the form of a lyophilized formulation or an aqueous solution. Generally, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polyols. Peptides；Proteins such as serum albumin, gelatin, or immunoglobulins；Hydrophilic polymers such as polyvinylpyrrolidone；Amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine；Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins；Chelating agents such as EDTA；Sugars such as sucrose, mannitol, trehalose, or sorbitol；Salt-forming counterions such as sodium；Metal complexes (e.g., Zn-protein complexes)；And/or nonionic surfactants such as polyethylene glycol (PEG).Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated.

The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-HER3 antibodies (or antigen binding proteins) or immunoconjugates of an anti-HER3 antibody (or antigen binding protein) conjugated to a cytotoxic agent provided herein can be used in therapeutic methods.

On the one hand, there is provided an immunoconjugate of an anti-HER3 antibody or an anti-HER3 antibody conjugated to a cytotoxic agent, which is used as a medicine. In some other aspects, there is provided an immunoconjugate of an anti-HER3 antibody or an anti-HER3 antibody conjugated to a cytotoxic agent, which is used to treat cancer. In certain embodiments, there is provided an immunoconjugate of an anti-HER3 antibody or an anti-HER3 antibody conjugated to a cytotoxic agent, which is used for a method of treating a subject having cancer, comprising administering an effective amount of an anti-HER3 antibody or an anti-HER3 antibody conjugated to a cytotoxic agent to the subject. In some other embodiments, the present invention provides an immunoconjugate of an anti-HER3 antibody or an anti-HER3 antibody conjugated to a cytotoxic agent, which is used to induce apoptosis and/or inhibit cancer cell proliferation in cancer cells. In certain embodiments, the present invention provides an anti-HER3 antibody or an immunoconjugate of an anti-HER3 antibody conjugated to a cytotoxic agent, for use in a method of inducing apoptosis of cancer cells and/or inhibiting the proliferation of cancer cells in an individual, comprising administering to the individual an effective amount of the anti-HER3 antibody or the immunoconjugate of the anti-HER3 antibody conjugated to a cytotoxic agent to induce apoptosis of cancer cells and/or inhibit the proliferation of cancer cells. According to any of the above embodiments, the "individual" is preferably a human.

In another aspect, the present invention provides the use of an anti-HER3 antibody or an immunoconjugate of an anti-HER3 antibody conjugated to a cytotoxic agent in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat cancer. In another embodiment, the medicament is used in a method for treating cancer, comprising administering an effective amount of the medicament to an individual having cancer. In another embodiment, the medicament is used to induce apoptosis of cancer cells and/or inhibit the proliferation of cancer cells. In another embodiment, the medicament is used in a method for inducing apoptosis of cancer cells and/or inhibiting the proliferation of cancer cells in an individual suffering from cancer, comprising administering an effective amount of the medicament to the individual to induce apoptosis of cancer cells and/or inhibit the proliferation of cancer cells. According to any of the above embodiments, the "individual" may be a "human".

In another aspect, the present invention provides a method for treating cancer. In one embodiment, the method comprises administering an effective amount of an anti-HER3 antibody to an individual with cancer. According to any of the above embodiments, the "individual" can be a human.

In another aspect, the present invention provides a method for inducing apoptosis and/or inhibiting cancer cell proliferation in an individual suffering from cancer. In one embodiment, the method comprises administering to the individual an effective amount of an anti-HER3 antibody or an immunoconjugate of an anti-HER3 antibody conjugated to a cytotoxic compound to induce apoptosis and/or inhibit cancer cell proliferation in the individual suffering from cancer. In one embodiment, the "individual" is a human.

In another aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-HER3 antibodies provided herein, for use in, for example, any of the above-described methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-HER3 antibodies provided herein and a pharmaceutically acceptable carrier.

The antibodies of the present invention (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosage can be by any suitable route (e.g., by injection, such as intravenous or subcutaneous injection), depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

The antibodies of the present invention should be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this regard include the specific condition being treated, the specific mammal being treated, the clinical status of the individual patient, the cause of the condition, the agent delivery site, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibodies need not be formulated with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other agents depends on the amount of the antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These are typically used in the same dosage and route of administration as described herein, or in about 1-99% of the dosage described herein, or in any dosage and any route determined to be suitable empirically/clinically.

For the prevention or treatment of a disease, the appropriate dosage of the antibodies of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapies, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg/kg to 15 mg/kg (e.g., 0.5 mg/kg to 10 mg/kg) can be used as an initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion. Depending on the factors described above, a typical daily dose can range from about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the situation, treatment generally continues until the desired containment of disease symptoms occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about 2 to about 20 doses, or, for example, about 6 doses of the antibody). A higher initial loading dose can be administered, followed by one or more lower doses. An exemplary dosing regimen includes administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the antibody. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

It will be understood that any of the above-described formulations or therapeutic methods may be practiced using the immunoconjugates of the invention, either as an alternative to or in addition to anti-HER3 antibodies.

III. Products

In another aspect of the present invention, an article of manufacture is provided that includes materials useful for treating, preventing, and/or diagnosing the conditions described above. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container contains a composition that is effective for treating, preventing, and/or diagnosing the condition, either alone or in combination with another composition, and may have a sterile access port (e.g., the container may be a vial or intravenous solution bag with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may include (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising another cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be understood that any of the above-described preparations may include an immunoconjugate of the invention, either instead of or in addition to an anti-HER3 antibody.

Description of amino acid sequences

SEQ ID NO: 1 β-hairpin of human HER3

SEQ ID NO: 2 Human HER4 β-hairpin

SEQ ID NO: 3 people HER3

SEQ ID NO:4 Human HER3 extracellular domain (ECD)

SEQ ID NO:5 people HER4

SEQ ID NO:6 Human HER4 extracellular domain (ECD)

SEQ ID NO: 7 people HER1

SEQ ID NO:8 Human HER1 extracellular domain (ECD)

SEQ ID NO:9 human HER2

SEQ ID NO: 10 Human HER2 extracellular domain (ECD)

SEQ ID NO: 11 Human heregulin fragment (HRG)

SEQ ID NO: 12 Human heregulin β-1 fragment (provided by Preprotech)

SEQ ID NO:13TtSlyD-FKBP-Her3

SEQ ID NO:14TtSlyD-wild type

SEQ ID NO:15TtSlyDcas

SEQ ID NO:16TgSlyDΔIF

SEQ ID NO:17TtSlyDcas-Her3

SEQ ID NO:18TtSlyDcys-Her3

SEQ ID NO:19TgSlyDser-Her3

SEQ ID NO:20TgSlyDcys-Her3

SEQ ID NO:21TtSlyDcas-Her4

SEQ ID NO:22TtSlyDcys-Her4

SEQ ID NO:23TgSlyDser-Her4

SEQ ID NO:24TgSlyDcys-Her4

SEQ ID NO:25 Heavy chain HVR-H1, M-08-11

SEQ ID NO:26 Heavy chain HVR-H2, M-08-11

SEQ ID NO:27 Heavy chain HVR-H3, M-08-11

SEQ ID NO:28 Light chain HVR-L1, M-08-11

SEQ ID NO:29 Light chain HVR-L2, M-08-11

SEQ ID NO:30 Light chain HVR-L3, M-08-11

SEQ ID NO:31 Heavy chain variable domain VH, M-08-11

SEQ ID NO:32 Light chain variable domain VL, M-08-11

SEQ ID NO:33 Heavy chain HVR-H1, M-17-02

SEQ ID NO:34 Heavy chain HVR-H2, M-17-02

SEQ ID NO:35 Heavy chain HVR-H3, M-17-02

SEQ ID NO:36 Light chain HVR-L1, M-17-02

SEQ ID NO:37 Light chain HVR-L2, M-17-02

SEQ ID NO:38 Light chain HVR-L3, M-17-02

SEQ ID NO:39 Heavy chain variable domain VH, M-17-02

SEQ ID NO:40 Light chain variable domain VL, M-17-02

SEQ ID NO:41 Heavy chain HVR-H1, M-43-01

SEQ ID NO:42 Heavy chain HVR-H2, M-43-01

SEQ ID NO:43 Heavy chain HVR-H3, M-43-01

SEQ ID NO:44 Light chain HVR-L1, M-43-01

SEQ ID NO:45 Light chain HVR-L2, M-43-01

SEQ ID NO:46 Light chain HVR-L3, M-43-01

SEQ ID NO:47 Heavy chain variable domain VH, M-43-01

SEQ ID NO:48 Light chain variable domain VL, M-43-01

SEQ ID NO:49 Heavy chain HVR-H1, M-46-01

SEQ ID NO:50 Heavy chain HVR-H2, M-46-01

SEQ ID NO:51 Heavy chain HVR-H3, M-46-01

SEQ ID NO:52 Light chain HVR-L1, M-46-01

SEQ ID NO:53 Light chain HVR-L2, M-46-01

SEQ ID NO:54 Light chain HVR-L3, M-46-01

SEQ ID NO:55 Heavy chain variable domain VH, M-46-01

SEQ ID NO:56 Light chain variable domain VL, M-46-01

SEQ ID NO:57 Binding epitope within human HER3 β-hairpin

SEQ ID NO: 58 Pseudomonas exotoxin variant PE24LR8M_3G (including GGG linker)

SEQ ID NO:59M-08-11 light chain; M-08-11_LC

SEQ ID NO:60 M-08-11 heavy chain HC with sortase tag; M-08-11_HC

SEQ ID NO: 61 M-08-11 heavy chain HC conjugated to Pseudomonas exotoxin variant PE24LR8M (Fab-011-PE heavy chain 1)

SEQ ID NO:62 M-08-11 heavy chain HC conjugated to Pseudomonas exotoxin variant PE24LR8M (Fab-011-PE heavy chain 2) as a direct PE24LR8M fusion

SEQ ID NO:63 Soluble Staphylococcus aureus sortase A

SEQ ID NO:64 Heavy chain variable domain VH, M-05-74

SEQ ID NO:65 Light chain variable domain VL, M-05-74

SEQ ID NO:66 Human kappa light chain constant region

SEQ ID NO:67 Human lambda light chain constant region

SEQ ID NO: 68 Human heavy chain constant region derived from IgG1

SEQ ID NO: 69 Human heavy chain constant region derived from IgG1, mutated at L234A and L235A SEQ ID NO: 70 Human heavy chain constant region derived from IgG1, mutated at L234A, L235A and P329G

SEQ ID NO: 71 Human heavy chain constant region derived from IgG4

The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Several embodiments of the present invention are listed below:

1. A method for selecting an antigen binding protein that binds to human HER3 (and does not cross-react with human HER4), wherein the antigen binding protein binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3, wherein the antigen binding protein is selected using

a) at least one polypeptide comprising the amino acid sequence SEQ ID NO: 1 selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3

and

b) at least one polypeptide comprising the amino acid sequence SEQ ID NO: 2 selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4

to select an antigen binding protein that shows binding to at least one polypeptide under a) and shows no binding to at least one polypeptide under b),

Thus, an antigen binding protein was selected that binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3 and does not cross-react with human HER4.

2. The antigen-binding protein obtained by the selection method of embodiment 1.

3. The method of embodiment 1 or the antigen binding protein of embodiment 2, wherein the antigen binding protein is an antibody.

4. An isolated antigen binding protein that binds to human HER3,

a) wherein the antigen binding protein binds to the polypeptide SEQ ID NO: 18TtSlyDcys-Her3, and

b) wherein the antigen binding protein does not cross-react with the polypeptide SEQ ID NO: 22TtSlyDcys-Her4.

5. An isolated antigen binding protein that binds to human HER3,

a) wherein the antigen binding protein binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) contained in the polypeptide SEQ ID NO: 18 (TtSlyDcas-Her3), and

b) wherein the antigen binding protein does not cross-react with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) contained in the polypeptide SEQ ID NO: 22 (TtSlyDcas-Her4).

6. The antigen binding protein of embodiment 4 or 5, wherein the antigen binding protein is an antibody.

7. An isolated antibody that binds to human HER3, wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1 in activated HER3.

8. The antibody of embodiment 6 or 7, wherein the antibody exhibits at least two-fold higher binding level in the presence of heregulin compared to the binding level in the absence of heregulin, as detected at 0 minutes after incubation with the antibody in a FACS assay performed with T47D cells expressing HER3.

9. An isolated antibody that binds to human HER3 (and does not cross-react with human HER4), wherein the antibody:

a) binds to the amino acid sequence SEQ ID NO: 1; and/or

b) binds to the amino acid sequence SEQ ID NO: 1 in activated HER3; and/or

c) binding within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:13TtSlyD-FKBP-Her3,

SEQ ID NO:17TtSlyDcas-Her3,

SEQ ID NO:18TtSlyDcys-Her3,

SEQ ID NO: 19 TgSlyDser-Her3, and

SEQ ID NO:20TgSlyDcys-Her3; and/or

d) binds to the β-hairpin region of HER3; and/or

e) inhibiting heterodimerization of HER3/HER2 heterodimers; and/or

f) has a ratio of the binding constant (Ka) for binding to Thermus thermophilus SlyDFKBP-HER3 (SEQ ID NO: 13) to the binding constant (Ka) for binding to HER3-ECD (SEQ ID NO: 4) (Ka(Thermus thermophilus SlyDFKBP-Her3)/Ka(HER3-ECD)) of 1.5 or greater when measured in a surface plasmon resonance assay; and/or

g) has a ratio of the molar ratio MR for binding to Thermus thermophilus SlyDFKBP-HER3 (SEQ ID NO: 13) to the molar ratio MR for binding to HER3-ECD (SEQ ID NO: 4) (MR(Thermus thermophilus SlyDFKBP-Her3)/MR(HER3-ECD)) of 2.0 or higher when measured in a surface plasmon resonance assay; and/or

h) has no cross-reactivity to the amino acid sequence of SEQ ID NO: 2; and/or

i) shows no cross-reactivity to the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2) comprised in a polypeptide selected from the group consisting of:

SEQ ID NO:21TtSlyDcas-Her4,

SEQ ID NO:22TtSlyDcys-Her4,

SEQ ID NO:23 TgSlyDser-Her4, and

SEQ ID NO:24TgSlyDcys-Her4; and/or

j) has no cross-reactivity to the β-hairpin region of HER4; and/or

k) does not compete with heregulin for binding to HER3; and/or

1) inducing heregulin to bind to HER3; and/or

m) binds to HER3-ECD with an affinity of a KD value ≤ 1 x ^10-8 M (in one embodiment, a KD value of 1 x ^10-8 M to 1 x ^10-13 M; in one embodiment, a KD value of 1 x ^10-9 M to 1 x ^10-13 M); and/or

n) binds to a polypeptide consisting of PLVYNKLTFQLE (SEQ ID NO: 48); and/or

o) binds to a polypeptide consisting of PLVYNKLTFQLE (SEQ ID NO: 48) and does not cross-react with a polypeptide consisting of PQTFVYNPTTFQLEHNFNA (SEQ ID NO: 2); and/or

p) exhibits at least two-fold higher binding in the presence of heregulin compared to the level of binding in the absence of heregulin, as detected at 0 minutes after incubation with the antibody in a FACS assay with T47D cells expressing HER3; and/or

q) shows nearly complete HER3 internalization in the presence of heregulin after 4 h of incubation with the antibody in a FACS assay performed with HER3-expressing T47D cells.

10. An isolated antibody that binds to human HER3 and does not cross-react with human HER4, wherein the antibody binds to a 15 amino acid polypeptide comprising the amino acid sequence PLVYNKLTFQLE (SEQ ID NO: 48).

11. The antibody of any one of embodiments 6 to 10, which is a human antibody, a humanized antibody, or a chimeric antibody.

12. The antibody of any one of embodiments 6 to 10, which is an antibody fragment that binds to human HER3 (and does not cross-react with human HER4).

13. The antibody of any one of embodiments 6 to 12, wherein the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27.

14. The antibody of any one of embodiments 6 to 12 or 13, comprising: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

15. An isolated antibody that binds to human HER3, wherein the antibody comprises:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

16. The antibody of any one of embodiments 6 to 12, wherein the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35.

17. The antibody of any one of embodiments 6 to 12 or 16, comprising: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.

18. An isolated antibody that binds to human HER3, wherein the antibody comprises:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

19. The antibody of any one of embodiments 6 to 12, wherein the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43.

20. The antibody of any one of embodiments 6 to 12 or 19, comprising: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 44; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 46.

21. An isolated antibody that binds to human HER3, wherein the antibody comprises:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:41;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:42;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:44;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:46;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

22. The antibody of any one of embodiments 6 to 12, wherein the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:50; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:51.

23. The antibody of any one of embodiments 6 to 12 or 22, comprising: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

24. An isolated antibody that binds to human HER3, wherein the antibody comprises:

i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:49;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 53; and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54;

or

ii) Humanized variants of the antibody HVRs under i)(a), (b), (d) and/or (e).

25. The antibody according to any one of embodiments 6 to 24, which is a full-length IgG1 antibody or an IgG4 antibody.

26. The antibody according to any one of embodiments 6 to 24, which is a Fab fragment.

27. An immunoconjugate comprising the antibody of any one of embodiments 6 to 24 and a cytotoxic agent.

28. A pharmaceutical formulation comprising the antibody of any one of embodiments 6 to 24 or the immunoconjugate of embodiment 27, and a pharmaceutically acceptable carrier.

29. The antibody according to any one of embodiments 6 to 24 or the immunoconjugate according to embodiment 27 for use as a medicament.

30. The antibody of any one of embodiments 6 to 24 or the immunoconjugate of embodiment 27, for use in treating cancer.

31. The antibody according to any one of embodiments 6 to 24, for use in inhibiting HER3/HER2 dimerization.

32. Use of the antibody according to any one of embodiments 6 to 24 or the immunoconjugate according to embodiment 27 in the manufacture of a medicament.

33. The use according to embodiment 32, wherein the medicament is for treating cancer.

34. Use of the antibody according to any one of embodiments 6 to 24 in the manufacture of a medicament, wherein the medicament is for inhibiting HER3/HER2 dimerization.

35. A method of treating an individual having cancer comprising administering to the individual an effective amount of the antibody of any one of the preceding embodiments or an immunoconjugate comprising the antibody of any one of the preceding embodiments and a cytotoxic agent.

36. A method of inducing apoptosis of cancer cells in an individual suffering from cancer, comprising administering to the individual an effective amount of an immunoconjugate comprising the antibody of any one of the preceding embodiments and a cytotoxic agent, thereby inducing apoptosis of cancer cells in the individual.

37. An isolated nucleic acid encoding the antibody of any one of embodiments 6 to 24.

38. A host cell comprising the nucleic acid of embodiment 37.

39. A method of producing an antibody, comprising culturing the host cell of embodiment 38 such that the antibody is produced.

40. A polypeptide selected from the group consisting of:

i) SEQ ID NO:12TtSlyD-FKBP-Her3,

ii) SEQ ID NO:16TtSlyDcas-Her3,

iii) SEQ ID NO:17TtSlyDcys-Her3,

iv) SEQ ID NO: 18 TgSlyDser-Her3, and

v) SEQ ID NO:19TgSlyDcys-Her3,

The polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

Example

Materials and general methods

Recombinant DNA technology

DNA was manipulated using standard methods, such as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions.

gene synthesis

The desired gene segments were prepared from oligonucleotides generated by chemical synthesis. 400-1600 bp long gene segments flanked by single restriction endonuclease cleavage sites were assembled by annealing and ligating oligonucleotides, including PCR amplification, and subsequently cloned into the expression vectors described below via designated restriction sites, such as EcoRI/BlpI or BsmI/XhoI. The DNA sequences of the subcloned gene segments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to the instructions provided by Geneart (Regensburg, Germany).

DNA sequencing

DNA sequences were determined by double-strand sequencing performed at Sequiserve GmbH (Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data management

Sequence creation, positioning, analysis, annotation, and interpretation were performed using the Vector NT1Advance suite version 11.5.0 from Infomax.

Example 1

Preparation of Antigens and Screening Proteins - Generation of Functional β-Hairpin HER3 and β-Hairpin HER4 Constructs for Selection of Antibodies Binding to HER3 β-Hairpin and HER4 β-Hairpin

To generate functional β-hairpin HER3 and HER4 constructs, the amino acid sequences of the β-hairpins of HER3 (SEQ ID NO: 1) and HER4 (SEQ ID NO: 2) were grafted into the SlyD polypeptide framework containing the FKBP domain. In such constructs, the grafted β-hairpins are freely accessible, in contrast to the hidden structure in the native inactive conformation of HER3 or HER4 (in the absence of a ligand such as HRG) (see Figures 1c and 1d, where the β-hairpin of HER3 is hidden).

All fusion SlyD polypeptides can be purified and refolded using almost the same protocol. The E. coli BL21 (DE3) cells transformed with specific expression plasmids were grown at 37°C in LB medium containing the corresponding antibiotics for selective growth (kanamycin 30 μg/ml, or ampicillin 100 μg/ml) to an OD600 of 1.5, and cytosol overexpression was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). 3 hours after induction, cells were harvested by centrifugation (5,000 g, 20 minutes), frozen and stored at -20°C. For cell lysis, the frozen pellet was resuspended in a cooling 50 mM sodium phosphate buffer (pH 8.0) supplemented with 7 M GdmCl and 5 mM imidazole. Thereafter, the suspension was stirred on ice for 2-10 hours until the cells were completely lysed. After centrifugation (25,000g, 1h) and filtration (nitrocellulose membrane, 8.0 μm, 1.2 μm, 0.2 μm), lysate was applied to a Ni-NTA column balanced with lysis buffer. In subsequent cleaning steps, imidazole concentration was increased to 10mM (in 50mM sodium phosphate buffer (pH 8.0) comprising 7M GdmCl), and 5mM TCEP was added to keep the thiol module in reduced form and to prevent premature disulfide bridge formation. The reducing cleaning buffer of at least 15-20 volumes was used. Thereafter, GdmCl solution was replaced with a 50mM sodium phosphate buffer (pH 8.0) comprising 100mM NaCl, 10mM imidazoles, and 5mM TCEP to induce the conformational refolding of the SlyD fusion polypeptide bound by the matrix. In order to avoid the reactivation of the co-purified protease, a protease inhibitor cocktail (no EDTA, Roche) was added to the refolding buffer. In the overnight process, a total of 15-20 column volumes of refolding buffer are used. Thereafter, TCEP and EDTA-free inhibitor mixture are removed by cleaning with 10 column volumes of 50mM sodium phosphate buffer (pH 8.0) containing 100mM NaCl and 10mM imidazoles. In the final cleaning step, the imidazole concentration is increased to 30mM (10 column volumes) to remove adhered pollutants. The refolded polypeptide is then eluted by using 250mM imidazoles in the same buffer. The purity of the protein fraction is assessed by Tricine-SDS-PAGE (Schaegger, H. and von Jagow, G., Anal. Biochem. 166 (1987) 368-379). Subsequently, the protein was subjected to size exclusion chromatography (Superdex ^™ HiLoad, Amersham Pharmacia) using potassium phosphate as a buffer system (50 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, 0.5 mM EDTA). Finally, the protein-containing fractions were combined and concentrated to a concentration of approximately 5 mg/ml in an Amicon cell (YM10). An exemplary SDS-PAGE analysis of the Ni-NTA purification of TtSlyD-FKBP-Her3 is shown in FIG3 , while the SEC elution profile of the Ni-NTA purified fractions of Thermus thermophilus SlyD-FKBP-Her3 is shown in FIG4 . The Thermus thermophilus SlyD (TtSlyD)-Her3 fusion polypeptide was successfully purified as a soluble and stable polypeptide in monomeric form. The final yield was quantified as 16.4 mg of purified protein from fractions 12 and 13.

Table 2: Summary of amino acid sequences of developed SlyD-based epitope scaffolds carrying either a HER3 dimerization domain fragment (HER3 β-hairpin (SEQ ID NO: 1)) or a HER4 dimerization domain fragment (HER4 β-hairpin (SEQ ID NO: 2)) as insert.

TtSlyD-FKBP-Her3, TtSlyDcas-Her3, TtSlyDcys-Her3, Thermococcus gammatolerans TgSlyDser-Her3 and TgSlyDcys-Her3 carry a HER3 dimerization domain fragment (HER3 β-hairpin (SEQ ID NO: 1)) as an insert and are used as immunogens and positive controls in ELISA screening.

TtSlyD-wild type, TtSlyDcas, and TgSlyDΔIF were used as negative controls in ELISA screening (without HER3 dimerization domain fragment (HER3 β-hairpin (SEQ ID NO: 1)) or Her4 dimerization domain fragment (HER4 β-hairpin (SEQ ID NO: 2)) as inserts).

The cross-reactivity of the developed clones to HER4 was examined in ELISA screening using TtSlyDcas-Her4, TtSlyDcys-Her4, TgSlyDser-Her4 and TgSlyDcys-Her4, which carry a fragment of the Her4 dimerization domain (HER4 β-hairpin (SEQ ID NO: 2)) as an insert.

When expressing epitope scaffolds in E. coli, the N-terminal methionine residue can be present or absent. (Nt = N-terminus; Ct = C-terminus)

Table 2

Example 2

a) Immunization and selection of HER3 antibodies

To generate antibodies against the HER3 β-hairpin, Balb/C, NMRI, or SJL mice were immunized with different antigens. The following proteins were used as antigens: full-length HER3 ECD, or the epitope scaffold proteins TtSlyD-FKBP12-Her3, TtSlyDcys-Her3, TtSlyDcas-Her3, TgSlyDcys-Her3, and TgSlyDser-Her3. The TtSlyD-FKBP12-Her3 variants represent the first generation of epitope scaffolds for generating antibodies specific for the HER3 dimerization domain. Although the general principle of using SlyD variants as epitope scaffolds has long been demonstrated using the first generation SlyD-FKBP12 scaffold, improved scaffold variants with higher stability were developed. These SlyD variants were derived from Thermus thermophilus and Thermococcus gamma-resistant.

All mice were immunized three times at 0, 6, and 10 weeks after the start of the immunization campaign. At each time point, each mouse was immunized with 100 μg of endotoxin-free immunogen dissolved in 100 μl PBS. For the first immunization, the immunogen was mixed with 100 μl CFA. For the second and third immunizations, the immunogen was mixed with IFA. The first and third immunizations were applied via the intraperitoneal route, while the second immunization was applied subcutaneously. 2 and 3 days before preparing spleen cells for antibody development using hybridoma technology, mice were given an intravenous booster immunization with 12.5 μg of immunogen in 100 μl PBS without adjuvant.

Titer analysis

To determine serum titers against the corresponding immunogens and screening proteins, a small amount of serum was collected from each mouse 11 weeks after the start of the immunization campaign. For ELISA, the immunogen or screening scaffold protein was immobilized on the plate surface. HER3 ECD was immobilized at a concentration of 1 μg/ml, and the scaffold proteins TtSlyD-FKBP12-Her3, TtSlyD-FKBP12, TtSlyDcys-Her3, TtSlyDcas-Her3, TtSlyDcas, TgSlyDcys-Her3, TgSlyDser-Her3, and TgSlyDΔIF were used at a concentration of 0.5 μg/ml. The scaffold proteins TtSlyDcas and TgSlyDΔIF were used as negative controls. Serum from each mouse was diluted in PBS containing 1% BSA and the dilutions were added to the plates. Sera were tested at dilutions of 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700, and 1:656100. Bound antibodies were detected with HRP-labeled F(ab') ₂ goat anti-mouse Fcγ (Dianova) using ABTS (Roche) as substrate.

Even at the level of serum titration, it is already apparent that mice immunized produce antibodies against the HER3 dimerization β-hairpin domain. In mice immunized with HER3 ECD, this can be shown by titration against one of the scaffold proteins comprising the dimerization β-hairpin loop. The strong reduction signal can be explained by the fact that most of the antibodies generated by immunization with HER3 ECD target other parts within the ECD, with only a small portion binding the dimerization β-hairpin domain. In mice immunized with a scaffold comprising the HER3 dimerization loop, the portion of the antibodies targeting the loop can be shown by titration against HER3 ECD (positive control) and titration against a control scaffold without HER3 insertion (negative control).

b) Antibody development and ELISA screening/selection

The epitope scaffold technology described herein provides two main strategies for developing antibodies targeting the HER3 dimerization domain (HER3 β-hairpin (SEQ ID NO: 1)). One strategy is to immunize the full-length HER3 ECD and use the scaffold to screen for dimerization domain β-hairpin-specific antibodies. Another strategy is to directly use the scaffold for immunization and counter-screen using HER3 ECD, a scaffold with another main chain, or a scaffold without an insert. Antibodies are produced by fusing primary B cells with P3X63Ag8.653 myeloma cells using hybridoma technology. Two days after the final booster immunization, the immunized mice were sacrificed and spleen cell populations were prepared. Splenocytes were fused with P3X63Ag8.653 using PEG fusion technology. The cell batch culture from the fusion was incubated overnight at 37°C under 5% _CO2 . The next day, the cell batch containing the fused cells was centrifuged at 400g for 10 minutes. Afterwards, the cells were suspended in hybridoma selection medium supplemented with 0.1x azaserine-hypoxanthine (Sigma) and seeded in 96-well plates at a concentration of 2.5 x 10 ⁴ cells per well. The plates were incubated at 37°C in 5% CO ₂ for at least 1 week. The selection medium was replaced 3 days before ELISA analysis.

Primary culture supernatants were tested in ELISAs against HER3ECD and various scaffold proteins. Tests against scaffold proteins were performed to demonstrate that the selected clones bound to the dimerization domain of the native HER3ECD. Tests against control scaffolds TtSlyDcas and TgSlyDΔIF were performed to demonstrate that the selected clones bound to inserted HER3-derived sequences rather than the scaffold backbone. In order to examine cross-reactivity, clones were generated against the full-length ECDs of other members of the Her family (i.e., HER1, HER2, and HER4). As shown, all selected clones were highly specific to HER3, and no cross-reactivity was detected for other members of the Her family. For ELISA screening, an antigen down format was used. HER3ECD was immobilized at a concentration of 1 μg/ml, and scaffold proteins TtSlyD-FKBP12-Her3, TtSlyD-FKBP12, TtSlyDcys-Her3, TtSlyDcas-Her3, TtSlyDcas, TgSlyDcys-Her3, TgSlyDser-Her3, and TgSlyDΔIF were immobilized at a concentration of 0.5 μg/ml. Hybridoma supernatants were added to the plates and incubated at room temperature for 1 hour. Bound antibodies were detected with HRP-labeled F(ab') ₂ goat anti-mouse Fcγ (Dianova), and ABTS (Roche) was used as an HRP substrate.

Table 3: Evaluation of selected clones by ELISA. Clones were tested against the scaffold proteins TtSlyDcas-Her3, TtSlyDcys-Her3, TgSlyDser-Her3 and TgSlyDcys-Her3 and the full-length Her3 ECD to verify their Her3 dimerization domain insert (HER3 β-hairpin (SEQ ID NO: 1)) specificity. Scaffold proteins TtSlyDcas and TgSlyDΔIF were used as negative controls. In addition, clones were tested against the full-length ECDs of HER1, HER2, HER3 and HER4 to verify potential cross-reactivity. Clones showed binding to the full-length HER3 ECD and showed no cross-reactivity against the full-length HER1, HER2, and HER4 ECDs. Numbers are OD values measured at 405 nm.

c) Immunohistochemistry

All selected clones were tested for reactivity and specificity in IHC. HEK293 cells were transiently transfected with plasmids encoding full-length HER1, HER2, HER3, or HER4, respectively. Two days after transfection, the various cell lines, now expressing HER1, HER2, HER3, or HER4, were harvested, fixed in formalin, and embedded in agarose to generate IHC controls. Additionally, agarose blocks were embedded in paraffin after overnight fixation in formalin. Untransfected HEK293 cells served as negative controls and were processed as transfected cells. After paraffin embedding, 3 μm thin sections were prepared using a microtome. Sections were mounted on glass microscope slides and dried for 2 hours. All further steps of the immunohistochemical staining protocol were performed using the Ventana Benchmark XT. Slides were dewaxed, and antigen retrieval was performed by heating for 1 hour. For antigen retrieval, Ventana Buffer CC1 was used. Antibodies were used at a concentration of 1 μg/ml. Bound antibodies were detected using the Ventana UltraView Detection Kit. The results are shown in Figure 5. As shown, all clones were specific for HER3 detection and showed no cross-reactivity with other members of the HER family (HER1, HER2, and HER4).

d) DNA sequencing of selected anti-Her3 hybridomas

In order to obtain the DNA sequence dna of selected hybridoma clone, 5 ' Race PCR is carried out.For RT-PCR, by using total RNA purification kit (Qiagen) from 5x10 ⁶ individual cells prepare total RNA.Use 5 ' to trigger RACE PCR kit (Roche) to carry out reverse transcription and PCR.By gel electrophoresis and subsequent gel purification, purify the PCR fragment produced from heavy chain and light chain.Use Topo Zero-Blunt cloning kit (Invitrogen) to clone the PCR fragment, and transform into competent cells.To order-checking is carried out to obtain the consensus sequence for selected clone from several clones of each hybridoma.To M-08-11, M-17-02, M-17-07 and M-17-12, M-43-01 and M-46-01 are ordered.For M-17-02, M-17-07 and M-17-12, identify same VH and VL sequence.Similarly, M-17-01 and M-17-11 are ordered.

e) Time-dependent internalization analysis of M-08-11 via FACS

M-08-11 binding to HER3 and internalization of M-08-11 were analyzed by FACS using the HER3-expressing tumor cell line T47D. ⁵ x 105 cells were treated with 50 ng of recombinant human heregulin fragment (HRG) (SEQ ID NO: 11). The fragment containing the amino acid sequence of SEQ ID NO: 11 was cloned into the pCDNA.1 vector (Invitrogen). The HRG fragment was expressed in FreeStyle ^™ 293-F cells according to the protocol described by Invitrogen (FreeStyle ^™ 293 Expression System, catalog number K9000-01). The purified HRG fragment was dissolved in 20 mM histidine, 140 mM NaCl, pH 6.0, and stored at -80°C.

Untreated (-) cells were used as negative controls. 1 μg of M-08-11 was added to the cells shortly after heregulin-induced activation. The cells were incubated at 37°C for 0, 5, 15, 30, 45, 60, 75, 90, 105, 120, 180, or 240 minutes. After incubation, the cells were immediately placed on ice. The cells were washed once with 3 ml of FACS buffer and then stained with 1 μg of R-phycoerythrin goat anti-mouse IgG (H+L) secondary antibody for 30 minutes. Flow cytometry was performed using a FACSCanto ^™ flow cytometer (BD Biosciences). Results of FACS analysis of time-dependent HER3 receptor internalization induced by M-08-11 in T47D cells: M-08-11 showed binding to expressed HER3 ECD in the presence or absence of supplementation with recombinant human heregulin fragment (HRG). M-08-11 caused internalization of the HER3 receptor over a 4-hour period. The results are shown in Figure 6. Isotype controls are indicated as constant horizontal black bars.

In the absence of HRG (-), M-08-11 showed weak binding to the expressed HER3 ECD. In contrast, in the presence of the human heregulin fragment (+HRG), stronger binding was detected. Binding in FACS assays performed with the HER3-expressing tumor cell line T47D showed that the level of binding in the presence of HRG was at least 2-fold higher than that in the absence of HRG, as detected immediately (0 minutes) after antibody exposure (0 minute incubation). M-08-11 caused HER3 receptor internalization over a 4-hour period. Isotype controls are indicated as constant horizontal black bars.

Example 3

a) Kinetic screening/binding properties of HER3 antibodies

Kinetic screening was performed on a BIAcore 4000 instrument equipped with a Biacore CM5 sensor according to Schraeml et al. (Schraeml, M. and M. Biehl, Methods Mol Biol 901 (2012) 171-181). Test antibodies were captured in all assays. The system was controlled by software version V1.1. The instrument buffer was HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). The system was operated at 25°C. A 30 μg/ml rabbit polyclonal antibody (RAM IgG (rabbit anti-mouse IgG with Fcγ specificity) from GE Healthcare) in 10 mM sodium acetate buffer (pH 4.5) was immobilized on spots 1, 2, 4, and 5 in flow cells 1, 2, 3, and 4 using EDC/NHS chemistry according to the manufacturer's instructions. The sensor was saturated using 1 M ethanolamine. In each flow cell, points 1-2 and 5-4 were used to calculate the reference signal, and point 3 served as a blank control. Antigens (human recombinant HER3 ECD (68 kDa) and recombinant Thermus thermophilus SlyD FKBP-HER3 (15 kDa) containing HER3 β-hairpin peptide (SEQ ID NO: 1)) were diluted to 150 nM in instrument buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran, Sigma) to inhibit nonspecific binding. Before applying them, the hybridoma culture supernatant was diluted 1:5 in instrument buffer. The diluted mixture was injected at a flow rate of 30 μl / min for 2 minutes. The capture level (CL) of the antibody in response units was monitored. Immediately thereafter, the corresponding antigen was injected at a flow rate of 30 μl / min for 3 minutes of binding time. Afterwards, the antibody-antigen complex dissociation signal was recorded for 5 minutes. The sensor was regenerated by injecting 10 mM glycine-HCl solution (pH 1.7) at a flow rate of 30 μl / min for 2 minutes. The signal recorded shortly before the end of the antigen injection represents the late binding phase (BL) in response units. The signal recorded shortly before the end of the dissociation recording represents the late stabilization phase (SL) in response units. Determine and calculate the dissociation rate constant. The stability of the antibody-antigen complex in minutes is calculated using the following formula: ln(2)/60*kd. The molar ratio is calculated using the following formula: MW(antibody)/MW(antigen)*BL(antigen)/CL(antibody).

The late binding time (BL) represents the response units at the end of the analyte injection. The amount of antibody captured as ligand on the sensor surface is measured as the capture level (CL) in response units. Together with the molecular weight information of the analyte being tested, the molar ratio of antibody to analyte in solution can be calculated. Assuming the sensor is configured with the appropriate amount of antibody-ligand capture level, each antibody should be able to functionally bind to at least one analyte in solution, which is represented by a molar ratio MR = 1.0. The molar ratio is then also an indicator of the potency pattern of analyte binding. For an antibody binding two analytes, the maximum potency can be MR = 2, one for each Fab valency. In the case of sterically restricted or functionally impaired analyte binding, the molar ratio can indicate stoichiometric binding, similar to the case where the HER3 ECD is bound in its "closed" conformation by the anti-HER3 β-hairpin antibody of the present invention (because this β-hairpin is hidden in the closed conformation). The maximum measurement deviation in the molar ratio determination is MR = 0.2.

Screening/Selection of Anti-HER3 Antibody M-08-11:

In one experiment, kinetic screening was driven by primary hybridoma cultures from different fusions obtained from mice immunized with human recombinant Her3 ECD. The goal was to select cultures with binding specificity for the HER3 heterodimerization domain β-hairpin peptide (SEQ ID NO: 1). As antigens/analytes in solution, human recombinant HER3 ECD (68 kDa) and recombinant Thermus thermophilus SlyD FKBP-HER3 (15 kDa) ((SEQ ID NO: 13), abbreviated as thermo SlyD-Her3 in the tables below) containing the HER3 β-hairpin peptide (SEQ ID NO: 1) were used. Positive hits were classified as primary culture supernatants with binding activity for both antigen/analyte. Table 4 exemplifies primary culture supernatants from which M-08-11 met these requirements, demonstrating epitope specificity for the HER3 β-hairpin. This is therefore a suitable method for screening anti-HER3 antibodies that bind to the HER3 hairpin of SEQ ID NO: 1.

Table 4: Exemplary results obtained from a kinetic screening experiment performed with a set of hybridoma primary cultures from a fusion in which antibodies M-08-11, M-43-01 and M-46-01 were identified as binding to both the HER3 ECD and the HER3 β-hairpin (SEQ ID NO: 1) within the thermo SlyD-Her3 construct.

It was found that M-08-11, M-17-01, M-17-02, M-43-01 and M-46-01 showed reduced molar ratios in their binding to human HER3 ECD (MR = 0.4, 0.04, 0.1, 0.1 and 0.2, respectively) (= closed conformation of HER3-ECD in the absence of heregulin), whereas M-08-11, M-17-01, M-17-02, M-43-01 and M-46-01 showed increased molar ratios in their binding to Thermus thermophilus SlyDFKBP-Her3 containing the HER3 β-hairpin (SEQ ID NO: 1) (MR = 1.3, 1.5, 1.7, 1.7 and 1.8, respectively), indicating functional stoichiometric 1:1 binding and improved epitope accessibility (compared to human Her3 ECD, where HER3 The β-hairpin presents a hidden epitope in the absence of heregulin (in its inactive state/closed conformation). Looking at pure antigen binding affinity independently, M-08-11, M-17-01, M-17-02, M-43-01, and M-46-01 showed slightly stronger binding affinity to the HER3 ECD compared to the HER3-HERG complex (see below).

b) Kinetics of HER3 antibodies M-08-11, M-17-01, M-17-02, M-43-01 and M-46-01 to investigate the mode of action of these antibodies in the absence and presence of heregulin (HRG)

In its equilibrium state, the Her3 ECD is in its "closed conformation", which indeed means that the heterodimeric Her3 β-hairpin motif is tethered to the Her3 ECD domain IV via non-covalent interactions (see Figures 1c and d). It has been suggested that the "closed" Her3 conformation can be opened by the binding of the ligand heregulin at a specific Her3 heregulin binding site. This occurs at the Her3 interface formed by Her3 ECD domain I and domain III. It is believed that through this interaction, the Her3 receptor is activated and converted to its "open conformation" (see Figures 1b and e). When this occurs, the Her3 β-hairpin is accessible to the antibody. This mode of action can be simulated in vitro by Biacore experiments.

Monoclonal antibodies were kinetically evaluated for their behavior against the heregulin-activated Her3 extracellular domain (Her3-ECD) using a Biacore T100 instrument (GE Healthcare). CM5 series sensors were installed into the system according to the manufacturer's instructions and standardized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20). Sample buffer was system buffer supplemented with 1 mg/ml CMD (carboxymethyl dextran, Sigma #86524). The system was operated at 25°C. 6500 RU RAM-Fcγ (relative units of Fcγ fragment RamIgG, GE Healthcare) were immobilized on all four flow cells using EDC/NHS chemistry according to the manufacturer's instructions. The sensors were inactivated using 1 M ethanolamine.

The binding activity of the corresponding antibodies against the analyte was tested kinetically. Antibodies were captured by injecting at 5 μl/min for 1 minute at a concentration of 35 nM. The flow rate was set at 100 μl/min.

The analytes in the solution tested were human heregulin fragment (HRG) (SEQ ID NO: 11), a 44 kDa homodimeric protein (prepared according to Example 2e, recombinant Thermus thermophilus SlyD FKBP-Her3 (15 kDa) (SEQ ID NO: 13), abbreviated as T.T. SlyD-Her3 in the table below), human recombinant HER3 ECD (SEQ ID NO: 4) (68 kDa), human recombinant HER4 ECD (SEQ ID NO: 6), and 100 nM Her4 ECD (incubated with a 5-fold molar excess of heregulin at room temperature for 60 minutes to generate a HER4 ECD-HRG complex (increased molecular weight of the complex)).

The analyte was injected in solution at different concentrations of 0 nM, 1.1 nM, 3.7 nM, 11.1 nM, 33.1 nM, and 90 nM for 3.5 minutes. Dissociation was monitored for 15 minutes. Where possible, kinetic characteristics were evaluated according to Langmuir fitting.

Table 5a: SPR-analyzed kinetic data of M-08-11

MR = molar ratio, BL = late binding phase, CL = capture level; n.d. = no detectable binding signal

In another experiment, HER1 ECD, T.T.SlyD-cysHer3 and T.T.SlyD-cas without the HER3 β-hairpin were also included in the measurements - the results are shown in Table 5b, which reveal essentially the same binding properties of M-05-74.

A Biacore T200 instrument (GE Healthcare) was equipped with a CM5 series sensor. The sensor was standardized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% w/v Tween 20) according to the manufacturer's instructions. The sample buffer was system buffer supplemented with 1 mg/ml CMD (carboxymethyl dextran, Sigma #86524). The system was operated at 25°C. 6500 RU RAM-Fcγ (relative units of Fcγ fragment RamIgG, GE Healthcare) were immobilized on all four flow cells using amine-coupled EDC/NHS chemistry according to the manufacturer's instructions. The sensor was inactivated using 1 M ethanolamine. Monoclonal antibodies (CL, capture level) were captured on the sensor surface by injecting at 10 μl/min for 1 minute. Concentration-dependent kinetics were measured. A concentration series of each of the analytes HER1-ECD, HER2-ECD, HER3-ECD, HER4-ECD, T.T.SlyD-cysHer3 and T.T.SlyD-cas was injected at 0 nM, 1.1 nM, 3.3 nM, 2 x 10 nM, 30 nM and 90 nM. Heregulin β (HRG) was injected at 0 nM, 17 nM, 2 x 50 nM, 150 nM and 450 nM, 90 nM HER3 ECD and 90 nM HER4 ECD were pre-incubated for 2 hours with a 5-fold molar excess of HRG β and injected at 0 nM, 1.1 nM, 3.3 nM, 2 x 10 nM, 30 nM and 90 nM HER concentration steps. All analytes were injected at a flow rate of 100 μl/min for 5 minutes of association time and 10 minutes of dissociation time. The sensor capture system was regenerated by injecting 10 mM glycine, pH 1.7, for 3 minutes at 10 μl/min. Kinetic data were evaluated using Biacore T200 evaluation software where possible. HER3-ECD, HER4-ECD, and T.T.SlyD-cysHer3 kinetics were evaluated using a Langmuir fit model whenever possible, or using interaction map two-state kinetics.

Table 5b: SPR analysis kinetic data of M-08-11, M-43-01 and M-46-01

MR = molar ratio, BL = late binding phase, CL = capture level; n.d. = no detectable binding signal, IM = interaction map two-state kinetics – see Table 6b and Figures 7c, d, e.

The molar ratio was calculated using the following formula: MW(antibody)/MW(antigen)*BL(antigen)/CL(antibody).

Anti-HER3 antibodies M-08-11, M-43-01, and M-46-01 bound to the "open" Her3 ECD conformation presented in recombinant Thermus thermophilus SlyD-HER3 (15 kDa) with nanomolar affinity (see Tables 5a-c) and functional stoichiometry of MR = 1.4, 1.5, 1.7, 1.7, and 1.8, respectively. The "closed" HER ECD was bound with reduced functional MR of 0.5, 0.04, 0.1, 0.1, and 0.2, respectively, indicating steric hindrance in the proximity of the HER3 hairpin domain. No binding was detected for human heregulin beta (HRG), wild-type Thermus thermophilus SlyD protein, Her4 ECD, and Her4 ECD in complex with heregulin. Thus, the anti-HER3 antibodies M-08-11, M-43-01, and M-46-01 specifically bind to the Her3 ECD and the HER3 β-hairpin, but not to the HER4-ECD.

The ratio of the binding constant (Ka) of M-08-11 binding to Thermus thermophilus SlyD FKBP-HER3 (SEQ ID NO: 13) to the binding constant (Ka) of M-08-11 binding to HER3-ECD (SEQ ID NO: 4) was 1.5 or higher (Ka(Thermus thermophilus SlyD FKBP-Her3)/Ka(HER3-ECD)).

The ratio of the molar ratio MR of M-08-11 binding to Thermus thermophilus SlyD FKBP-HER3 (SEQ ID NO: 13) to the molar ratio MR of M-08-11 binding to HER3-ECD (SEQ ID NO: 4) was 2.0 or higher (MR(Thermus thermophilus SlyD FKBP-Her3)/MR(HER3-ECD)).

Thus, M-08-11, M-43-01, and M-46-01 together belong to a class of epitopes that differ from the M-5-74 epitope in that the antibodies specifically interact with HER3-ECD and HER3-ECD-HRG, but not with HER4-ECD. No measurable interactions were observed with HER1-ECD, HER2-ECD, HER4-ECD, and HRG. M-08-11, M-43-01, and M-46-01 bind to T.T.SlyD-cysHer3 with a 1:2 stoichiometry and do not interact with T.T.SlyD-cas. Compared to the inactive HER3-ECD, M-08-11, M-43-01, and M-46-01 bind to HER3-ECD-HRG with increased stoichiometry. M-46-01 exhibits the highest stoichiometry for HER3-ECD-HRG binding.

M-08-11, M-43-01, and M-46-01 belong to the same class of antibodies and interact with HER3-ECD-HRG via a two-state mechanism. Therefore, the complex interaction at 25°C was resolved by interaction mapping (Figure 7c, d, e).

Kinetic analysis of clone M-08-11

Surprisingly, it was found that the anti-HER3 antibodies M-08-11, M-17-01, M-17-02, M-43-01, and M-46-01 bind to Her3 ECD in complex with heregulin in a different interaction mode than M-05-74. Anti-HER/HER antibody M-05-74 is another antibody that not only binds to the β-hairpin of HER3, but also shows specific binding to HER4-ECD and HER4 β-hairpin (M-05-74 has VH and VL of SEQ ID NO: 64 and SEQ ID NO: 65). M-08-11 binds to heregulin-activated Her3 ECD in a non-Langmuir, non-1:1 interaction, but rather in a complex kinetic mode. The 1:1 kinetic direct interaction results in a binary complex [A] + [B] → [AB], and the complex shows a constant dissociation rate. The half-life of complex dissociation can be described by the formula t1/2 dissociation = ln(2)/ _kd . In contrast, multi-state kinetics revealed a complex, nonlinear curve in the dissociation phase. The reactants can interact in such a way that intermediates can form, which then react further to form the stable product [A]+[B]→[AB]*→[AB]. To elucidate the binding mode, Biacore experiments were performed.

The binding mode of clone culture M-08-11 to heregulin-activated Her3 ECD was kinetically evaluated using a Biacore B3000 instrument (GE Healthcare). CM5 series sensors were installed into the system according to the manufacturer's instructions and normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20). Sample buffer was system buffer supplemented with 1 mg/ml CMD (carboxymethyl dextran). The system was operated at 25°C. 10,000 RURAM-Fcγ (Relative Units of Fcγ fragment rabbit anti-mouse IgG, Jackson Laboratories) was immobilized on all flow cells using EDC/NHS chemistry according to the manufacturer's instructions. Sensors were inactivated using 1 M ethanolamine.

Since the interaction of M-08-11 with Her3 ECD in complex with heregulin did not reveal a monophasic Langmuir 1:1 interaction but rather a complex interaction, the binding mode of the antibody was investigated by two-state analysis (Jenkins, J.L., M.K. Lee, et al., J Biol Chem 275 (2000) 14423-14431).

The anti-HER3 antibody M-08-11 was captured from its crude hybridoma supernatant on the sensor surface by injecting at 10 μl/min for 2 minutes. The flow rate was set to 100 μl/min. In each cycle, the analyte complex (5:1) heregulin/Her3-ECD was injected at a concentration of 100 nM. In another embodiment, the analyte Her3 ECD was injected at 1 μM. In each consecutive cycle, the analyte injection time was extended from 30 seconds, 90 seconds, 180 seconds and 300 seconds. Binding was monitored with a sensorgram. Regeneration of the sensor surface was achieved using three consecutive injections of 10 mM glycine pH 1.7 for 60 seconds at 30 μl/min. Finally, the obtained sensorgrams were plotted as an overlay and standardized. The standardized data were superimposed at the reporting point at the beginning of the analyte dissociation phase. 1:1 Langmuir interactions typically produce overlapping dissociation characteristics, while complex interactions show non-overlapping, sometimes biphasic dissociation phases.

Figures 7a and b show a two-state analysis of the anti-HER3 antibody M-08-11. The "closed" Her3 ECD is bound according to a 1:1 Langmuir interaction only at very high Her3 ECD concentrations (Figure 7a), as the dissociation curves are superimposable, forming overlapping dissociation curves. The dissociation curves for the heregulin/Her3 ECD interaction are not superimposable (Figure 7b). Therefore, the complex is bound via a non-Langmuir mechanism. When the M-08-11 (M-011) interaction was calculated using a four-parameter two-state model, the following data were obtained.

Binding of M-08-11 to heregulin/Her3 ECD is functional (MR = 1.1). The two-state model delivers four kinetic parameters. The apparent dissociation constant (affinity) KD _ap = 18 nM can be calculated by forming a quotient from the rate-determining steps _kal and _kd2 .

Table 6a: Two-state fitting calculations of M-08-11 calculated using the Biacore two-state model

In another experiment, two-state kinetic analysis was performed using the anti-HER3 antibodies M-08-11, M-43-01, and M-46-01 based on the interaction map (Altschuh, D., et al., Biochem Biophys Res Commun. 428(1)(2012)74-79). The data were calculated using TraceDrawer software (version 1.5.) (Ridgeview Diagnostics) according to the manufacturer's recommendations. The results are shown in Table 6b and Figures 7c, d, and e.

Overall, the interaction maps (Figure 7c, d, e) show that the interaction of M-08-11, M-43-01 and M-46-01 with HER3-ECD-HRG is composed of two different kinetic ratio contributions. Two-state kinetic number: number of subcomponents identified in the apparent kinetics; ka 1/Ms: association rate constant; kd 1/s: dissociation rate constant; KD (M): dissociation rate constant; Weight (%): contribution to the total interaction. Table 6b: Two-state fit calculations of the antibody/heroin/Her3 ECD interaction of M-08-11, M-43-01 and M-46-01 based on the interaction map (Altschuh, D., et al., Biochem Biophys Res Commun. 428 (1) (2012) 74-79) calculated from the data using TraceDrawer software (version 1.5.) (Ridgeview Diagnostics) according to the manufacturer's recommendations:

Interaction mapping software identified two kinetic components in the interactions of M-08-11, M-43-01, and M-46-01 with heregulin/Her3 ECD. The apparent kinetics consisted of two interactions: a low-affinity binding event and a second, higher-affinity component in the same region, similar to the apparent affinities calculated using the Biacore two-state model.

The interpretation of the two-state interaction pattern of the exemplary anti-HER3 antibody M-08-11 becomes apparent from the data in the overlay graph of Figure 8. Figure 8 shows two different modes of binding of anti-HER3 antibody M-08-11 and anti-HER3/HER4 antibody M-05-74 to the Her3 ECD complex activated by heregulin (HER3 ECD HRG). M-08-11 binds to the activated complex with an accelerated association rate constant ka because the accessibility of the Her3 hairpin is improved in the Her3 ECD "open conformation." In this manner, heregulin acts as a catalyst for the interaction. During the interaction, heregulin dissociates from the M-08-11*Her3 ECD complex. In the dissociation phase of this trimeric complex, the binding signal level asymptotically shifts to the binding level of the pure Her3 ECD interaction signal. This is also true for antibodies M-43-01 and M-46-01. Anti-HER3/HER4 antibody M-05-74 captures and prevents heregulin from dissociating from the complex. M-05-74 is a trap for heregulin ("heluin pool").

Example 4

Epitope mapping of the anti-HER3 antibody M-08-11 and analysis of its mode of action with a unique epitope, such as the HER3 β-hairpin within TtSlyDcys-HER3 (SEQ ID NO: 18) and no cross-reactivity with the HER4 β-hairpin

Binding specificity was assessed using culture supernatants of accessible epitope clones on a Biacore 2000 (GE Healthcare) instrument. CM5 sensors were installed in the system according to the manufacturer's instructions and normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20). Sample buffer was system buffer supplemented with 1 mg/ml CMD (carboxymethyl dextran, Sigma). The system was operated at 37°C. 10,000 RU of RAM-Fcγ (Relative Units of Fcγ fragment rabbit anti-mouse IgG, Jackson Laboratories) was immobilized on all four flow cells using EDC/NHS chemistry according to the manufacturer's instructions. The sensors were inactivated with 1 M ethanolamine.

The primary antibody, 50 nM anti-HER3 M-05-74, was captured on all flow cells at a flow rate of 10 μl/min for 1 minute. The flow rate was set to 30 μl/min, and IgG blocking solution (50 μg/ml IgG (20:2:1 IgG1-Fcγ, IgG2a-Fcγ, IgG2b), Roche) was injected for 5 minutes. The antigen Her3 ECD was injected at 1.5 μM for 3 minutes.

Afterwards, 100 nM of anti-HER3 secondary antibodies (a) M-05-74, b) 8B8 from WO 97/35885 (referred to as GT in the figure), c) M-208, which binds to HER3 domain IV, and d) M-08-11, another HER3 β-hairpin binder without HER4 ECD and HER4 β-hairpin cross-reactivity, were injected at 30 μl/min for 3 minutes. Acidic regeneration of the sensor surface was achieved using three consecutive injections of 10 mM glycine, pH 1.7, for 60 seconds at 30 μl/min.

The measurement noise is defined by the rebinding of the secondary M-05-74 injection (which resaturates the dissociated primary M-05-74). The experiment shows (see Figure 9) that M-208 and M-05-74 occupy different epitopes on Her3 ECD because, in the presence of M-05-74, the secondary M-208 signal completely saturates Her3 ECD. The M-08-11 binding is completely blocked by the presence of M-05-74. The M-08-11 secondary signal is even lower than the noise. Nevertheless, M-08-11 binds to an epitope different from M-05-74 because M-08-11 does not bind to human HER4 ECD and HER4 β-hairpin. (See also the precise epitope mapping data for the β-hairpin of HER3 and HER4 below). In the presence of M-05-74, the 8B8 (=GT) secondary antibody produces a significant signal that is higher than the noise. Thus, the 8B8 (=GT) antibody binds to a different epitope than M-05-74 and M-08-11.

Figure 10 is an overlay of Biacore sensorgrams of anti-HER3 antibody M-08-11, anti-HER3/HER4 antibody M-05-74, and anti-HER3 antibody 8B8 (from WO97/35885), showing different modes of binding. Anti-HER3 antibody M-08-11HER3 (a β-hairpin binder without HER4 ECD and HER4 β-hairpin cross-reactivity) delays heregulin dissociation (2) and produces complex two-state kinetics. Anti-HER3/HER4 antibody M-05-74 traps heregulin-activated Her3 ECD (1) (t1/2 dissociation = 18 minutes) and acts as a heregulin sink. 8B8 antibody (3) does not trap heregulin and does not delay heregulin dissociation from the Her3 ECD/heterologous complex. Since this is a perfect Langmuir interaction, the heregulin/Her3 ECD complex dissociates rapidly and completely from the 8B8 antibody as an intact complex.

A schematic diagram of these binding modes of action is shown in Figure 11: 1: M-08-11 binds to heregulin-activated Her3 ECD and induces delayed heregulin dissociation, with M-08-11 remaining in the Her3 ECD receptor complex. 2: M-05-74 binds to heregulin-activated Her3 ECD. Heregulin is trapped in the complex and the antibody remains in the complex. 3: 8B8 binds to heregulin-activated Her3 ECD. The entire complex dissociates from the antibody.

Peptide-based two-dimensional epitope mapping

In another embodiment, a peptide-based epitope mapping experiment was performed using CelluSpots ^™ synthesis and epitope mapping technology to characterize the HER3 ECD epitope. Epitope mapping was performed using a library of overlapping, immobilized peptide fragments (length: 15 amino acids) corresponding to the sequences of human HER1 ECD, HER2 ECD, HER3 ECD, and HER4 ECD peptide hairpins. The strategy for epitope mapping and the alanine scanning approach are shown in Figure 12. The peptide hairpin sequences (β-hairpins) of HER1 (EGFR) ECD, HER2 ECD, HER3 ECD, and HER4 ECD were investigated, including their structural burial (structurally). Cysteine was replaced with serine. For antibody selection that binds to the HER3 β-hairpin and does not bind to the HER4 β-hairpin, the β-hairpins of HER3 and HER4 were defined by SEQ ID NO: 1 and SEQ ID NO: 2.

Each synthesized peptide is shifted by one amino acid, i.e., it overlaps with the preceding and following peptides by 14 amino acids. To prepare the peptide arrays, Intavis CelluSpots ^™ technology is used. In this approach, peptides are synthesized on modified cellulose discs using an automated synthesizer (Intavis MultiPep RS) and dissolved after synthesis. Solutions of individual peptides covalently linked to high-molecular-weight cellulose are then spotted onto coated microscope slides. CelluSpots ^™ synthesis is performed stepwise using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on amino-modified cellulose discs in a 384-well synthesis plate. In each coupling cycle, the corresponding amino acid is activated with a DIC/HOBt solution in DMF. Between coupling steps, unreacted amino groups are capped with a mixture of acetic anhydride, diisopropylethylamine, and 1-hydroxybenzotriazole. After synthesis, the cellulose discs were transferred to a 96-well plate and treated with a mixture of trichloroacetic acid (TFA), dichloromethane, triisopropylsilane (TIS), and water for side chain deprotection. After removing the cleavage solution, the cellulose-bound peptides were dissolved with a mixture of TFA, TFMSA, TIS, and water, precipitated with diisopropyl ether, and resuspended in DMSO. The peptide solutions were then spotted onto Intavis CelluSpots ^™ slides using an Intavis slide spotting robot.

For epitope analysis, slides prepared as described above were washed with ethanol and then with Tris-buffered saline (TBS; 50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 8) before being blocked with 5 mL of 10x Western blocking reagent (Roche Applied Science), 2.5 g of sucrose in TBS, 0.1% Tween 20 at 4°C for 16 hours. Slides were washed with TBS and 0.1% Tween 20 and then incubated with 1 μg/mL of the corresponding IGF1 antibody in TBS and 0.1% Tween 20 at ambient temperature for 2 hours, followed by washing with TBS + 0.1% Tween 20. For detection, slides were incubated with anti-rabbit/anti-mouse HRP-secondary antibodies (1:20,000 in TBS-T), followed by incubation with the chemiluminescent substrate luminol and visualization using a LumiImager (Roche Applied Science). ELISA-positive SPOTs were quantified and the antibody-binding epitopes were identified by assignment of corresponding peptide sequences.

As depicted in Figure 13, M-08-11 shows a HER3 ECD-specific epitope having the amino acid sequence PLVYNKLTFQLE (SEQ ID NO: 48), with no detectable cross-reactivity to other hairpin sequences of the Her family (particularly, no binding to the HER4 β-hairpin was detected). M-05-74 shows cross-reactivity to a HER3 ECD epitope having the amino acid sequence VYNKLTFQLEP and a HER4 ECD epitope having the amino acid sequence VYNPTTFQLE, with no detectable signal for hairpin motifs in EGFR and HER2 ECD. There was no detectable signal at all with the 8B8 antibody, so the 8B8 antibody targets an epitope that is different from the anti-HER3 antibody M-08-11 and the anti-HER3/HER4 antibody M-05-74 and from the β-hairpin peptide motif in general.

In FIG14 , the amino acids identified by alanine scanning as contributing most to the binding of the anti-HER3 antibody M-08-11 to the HER3 ECD binding epitope are underlined/bold.

Example 5

Binding of HRG to HER3-ECD in the presence of HER3 antibodies (ELISA)

The streptavidin-coated 96-well plates were incubated at 4°C with cell culture supernatants containing HER3-ECD with SBP tags. The next day, the wells were washed 3 times with wash buffer (PBS+0.05% Tween-20) and blocked with PBS containing 1% BSA for 1 hour. After washing 3 more times with wash buffer, 40 μl of antibody solution (in Delfia binding buffer) was added to each well as a 2x stock solution of the expected final concentration ( ^10-3 to ¹⁰³ nM, or ^10-4 to ¹⁰² nM). Immediately add 40 μl of 20nM europium-labeled heregulin-β (PeproTech, catalog number 100-03) to achieve a final concentration of 10nM. The plate was incubated on a shaker at room temperature for 2 hours. After washing 3 times with Delfia wash buffer, Delfia enhancement solution was added and incubated on a shaker for 15 minutes (protected from light). Finally, the plate was measured on a Tecan Infinite F200 reader using a time-resolved fluorescence measurement protocol. The binding of anti-HER3 antibodies M-08-11, M-43-01, and M-46-01 promoted/induced HRG binding to HER3-ECD until the signal reached a plateau at 200% (for M-08-11 and M-43-01) and 330% (for M-46-01). M-33 served as an isotype control and showed a concentration-independent value of approximately 100%. The results are shown in Figures 15A and B.

Example 6

Inhibition of HER2/HER3 heterodimerization/heterodimerization in MCF7 cells (immunoprecipitation and Western blotting)

MCF-7 cells are seeded into 6-well plates (2ml RPMI, 10% FCS, 8x10 ⁵ cells per well) and grown overnight. The next day, the culture medium is replaced with 2ml of starvation culture medium containing 0.5% FCS. On the third day, antibodies are added to a final concentration of 10 μg/ml and the plates are incubated at 37°C. After 50 minutes, add β-adjusted protein (PeproTech, catalog number 100-03) to a final concentration of 500ng/ml and the plates are incubated at 37°C for another 10 minutes. The cells are washed with PBS and lysed in 250μl of Triton lysis buffer containing 1% digitonin. 60μl of the collected lysate is transferred to a reaction tube and incubated with 40μl of Sepharose (Herceptin or HER3 antibody #208) coupled to the antibody and 500μl of a buffer containing 0.3% digitonin. The reaction mixture is incubated overnight at 4°C on a wheel rotator. The next day, 500 μ l of the buffer solution containing 0.3% digitonin was used to clean the reaction mixture 3 times. After the last cleaning, supernatant was discarded, and 10 μ l 4x loading buffer was added. Pipe was incubated at 70 ° C for 10 minutes, and supernatant was subsequently loaded onto the gel and carried out SDS-PAGE. After subsequent semi-dry Western blotting, the film containing the sample immunoprecipitated with HER2 antibody was incubated with anti-HER3 antibodies M-08-11 (M-011 in Figure 16), and vice versa. The film was then incubated with the second antibody that was conjugated with HRP, and the ECL signal was transferred to the X-ray film. The result is shown in Figure 16, which shows that anti-HER3 antibodies M-08-11 inhibits HER2/HER heterodimer formation (HER2/HER heterodimerization).

Example 7

Production of M-08-11-Fab-Pseudomonas exotoxin conjugate (designated M-08-11-PE or M-08-11-Fab-PE)

Expression, purification and renaturation of Fab fragments of M-08-11, PE24 variants, and M-08-11 Fab fragments based on the sequences of SEQ ID NO: 49, 50, 51, 52 (or 53) conjugated to Pseudomonas exotoxin variant PE24LR8M.

Fab expression (e.g. for sortase coupling) – expression vector

For expression of the described Fab fragments, variants of the expression plasmids based on cDNA organization with or without CMV-Intron A promoter or based on genomic organization with CMV promoter were applied to transient expression (eg HEK293-F) cells.

In addition to the antibody expression cassette, the vector also contains:

- an origin of replication, which allows this plasmid to replicate in E. coli, and

-β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

The transcription unit of the antibody gene is composed of the following elements:

- one or more unique restriction sites at the 5' end,

- immediate early enhancer and promoter from human cytomegalovirus, |

- followed by, in the case of cDNA tissue, the intron A sequence,

- 5' untranslated region of human antibody gene,

- immunoglobulin heavy chain signal sequence,

- Human antibody chains with immunoglobulin exon-intron organization as cDNA or as genomic organization

- a 3' untranslated region having a polyadenylation signal sequence, and

- One or more unique restriction sites at the 3' end.

The fusion gene that comprises antibody chain as described below is produced by PCR and/or gene synthesis, and is assembled by connecting (for example, using the unique restriction site in the corresponding vector) nucleic acid segment by known recombination method and technology.Verify the nucleotide sequence of subclone by DNA sequencing.For transient transfection, by preparing a large amount of plasmid from the plasmid preparation (Nucleobond AX, Macherey-Nagel) of the Escherichia coli culture through transformation.

Cell culture technology

Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

Fab fragments were expressed by transient co-transfection of heavy and light chain expression plasmids into HEK29-F cells cultured in suspension as described below.

Transient transfection in the HEK293-F system

Fab fragments were produced by transient transfection with the corresponding plasmids (e.g., encoding heavy and modified heavy chains, and corresponding light and modified light chains) using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle ^™ 293 expression medium (Invitrogen) in shake flasks or stirred fermenters were transfected with a mixture of the four expression plasmids and 293-Free ^™ (Novagen) or Fectin (Invitrogen). For 2 L shake flasks (Corning), HEK293-F cells were seeded in 600 mL at a density of 1.0E*6 cells/mL and incubated at 120 rpm, 8% CO ₂ . The next day, cells were transfected at a cell density of approximately 1.5E*6 cells/mL with a mixture of approximately 42 mL of the following solutions: A) 20 mL of Opti-MEM (Invitrogen) containing 600 μg of total plasmid DNA (1 μg/mL) encoding the heavy chain or modified heavy chain and the corresponding light chain (equimolar ratio), and B) 20 ml of Opti-MEM + 1.2 mL of 293-Free (Novagen) or Fectin (2 μl/mL). Glucose solution was added during the fermentation process depending on glucose consumption. After 5-10 days, the supernatant containing the secreted antibody was harvested and the antibody was purified directly from the supernatant, or the supernatant was frozen and stored.

Expression of the sortase-coupled expression vector, Pseudomonas exotoxin variant PE24-LR8M

To express PE24-LR8M, an E. coli expression plasmid was used.

In addition to the expression cassette for Pseudomonas exotoxin A domain III, the vector also contains:

- an origin of replication from the vector pBR322 for replication in E. coli (according to Sutcliffe, G., et al., Quant. Biol. 43 (1979) 77-90),

- lacI repressor gene from Escherichia coli (Farabaugh, P.J., Nature 274 (1978) 765-769),

- The Saccharomyces cerevisiae URA3 gene encoding orotidine 5'-phosphate decarboxylase (Rose, M. et al., Gene 29 (1984) 113-124), which allows plasmid selection by complementation of an Escherichia coli pyrF deletion strain (uracil auxotroph).

The transcription unit of the toxin gene consists of the following elements:

- one or more unique restriction sites at the 5' end,

- T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according to Bujard, H., et al., Methods. Enzymol. 155 (1987) 416-433 and Stueber, D., et al., Immunol. Methods IV (1990) 121-152), including a synthetic ribosome binding site according to Stueber, D., et al. (supra), - Pseudomonas exotoxin A domain III with an N-terminal coupling tag followed by a furin site (SEQ ID NO: 45, Pseudomonas exotoxin variant PE24LR8M_3G, including a GGG linker for sortase coupling),

- two phage-derived transcription terminators, the λ-T0 terminator (Schwarz, E., et al., Nature 272 (1978) 410-414) and the fd terminator (Beck E. and Zink, B., Gene 1-3 (1981) 35-58), | - one or more unique restriction sites at the 3' end.

Cultivation and expression of Pseudomonas exotoxin A construct variant PE24-LR8M_3G in a fed-batch process in Escherichia coli on chemically defined medium

For expression of PE24-LR8M_3G_Ecoli (25 kDa), an E. coli host/vector system was used with antibiotic-free plasmid selection by complementation of an E. coli auxotroph (PyrF) (EP 0 972 838 and US 6,291 ,245).

E. coli K12 strain was transformed by electroporation with the expression plasmid. The transformed E. coli cells were first grown on agar plates at 37°C. One colony from this plate was transferred to a 3 mL roller bottle and grown at 37°C to an optical density of 1-2 (measured at 578 nm). 1000 μl of this culture was then mixed with 1000 μl of sterile 86% glycerol and immediately frozen at -80°C for long-term storage. Correct product expression from this clone was first verified in a small-scale shake flask experiment and analyzed by SDS-PAGE before being transferred to a 10 L fermentor.

Pre-culture:

For pre-fermentation, chemically defined medium was used. For pre-fermentation, 220 ml of medium in a 1000 ml Erlenmeyer shake flask with 4 baffles was inoculated with 1.0 ml from the original seed bank ampoule. Culture was carried out on a rotary shaker at 32 ° C and 170 rpm for 8 hours until an optical density (578 nm) of 2.9 was obtained. 100 ml of pre-culture was used to inoculate the batch culture medium of a 10 L bioreactor.

Fermentation:

For fermentation in a 10 L Biostat C, DCU3 fermentor (Sartorius, Melsungen, Germany), a chemically defined batch medium was used. All components were dissolved in deionized water. The base solution used for pH adjustment was a 12.5% (w/v) _NH3 solution in water supplemented with 11.25 g/l L-methionine.

Batch fermentation was started with 4.2 L sterile batch medium plus 100 ml inoculum from a pre-culture at 31° C., pH 6.9 ± 0.2, 800 mbar back pressure and an initial aeration rate of 10 L/min. Relative dissolved oxygen values (pO ₂ ) were maintained at 50% by increasing the agitator speed to 1500 rpm throughout the fermentation. After the initial glucose supplement was exhausted, the temperature was changed to 25° C., as indicated by a sharp increase in dissolved oxygen values, and after 15 minutes the fermentation entered fed-batch mode starting with two feeds (60 and 14 g/h, respectively). The rate of feed 2 remained constant, while the rate of feed 1 gradually increased from 60 to a final 160 g/h in 7 hours, with a predetermined feed profile. When the carbon dioxide waste gas concentration level was above 2%, the aeration rate was increased from 10 to 20 L/min in 5 hours. The expression of recombinant PE24-LR8M_3G_Ecoli protein was induced by adding 2.4 g IPTG at an optical density of approximately 120. The target protein was expressed soluble in the cytoplasm.

After 24 hours of cultivation, the optical density reached 209, and the entire culture was cooled to 4-8°C. The bacteria were harvested by centrifugation using a flow-through centrifuge (13,000 rpm, 13 L/h), and the resulting biomass was stored at -20°C until further processing (cell disruption). The yield was 67.5 g of dry cells per liter.

Analysis of product formation:

The samples taken from the fermentation tank were analyzed by SDS-polyacrylamide gel electrophoresis, one before induction and the other at a specific time point after induction of protein expression. The same amount of cells from each sample (OD _target = 10) were suspended in 5 mL PBS buffer and broken on ice through ultrasonic treatment. Then 100 μL of each suspension was centrifuged (15,000 rpm, 5 minutes), each supernatant was taken out and transferred to a separate vial. This is to distinguish between soluble and insoluble expressed target proteins. 100 μL and 200 μL SDS sample buffer (Laemmli, UK, Nature 227 (1970) 680-685) were added to each supernatant (= soluble protein fraction) and each pellet (= insoluble protein fraction) respectively. The samples were heated at 95°C for 15 minutes under vigorous mixing to dissolve and reduce all proteins in the sample. After cooling to room temperature, 5 μL of each sample was transferred to a 4-20% TGX Criterion dye-free polyacrylamide gel (Bio-Rad). Additionally, 5 μL of a molecular weight standard (Precision Plus Protein Standard, Bio-Rad) was applied.

Electrophoresis was run at 200 V for 60 minutes, after which the gel was transferred to a GelDOC EZ imager (Bio-Rad) and processed with UV irradiation for 5 minutes. Gel images were analyzed using Image Lab analysis software (Bio-Rad). Relative quantification of protein expression was performed by comparing the volume of the product band with the volume of the 25 kD band of the molecular weight standard.

Cultivation and expression of antibody fragment light chain constructs (VL) and antibody fragment heavy chain Pseudomonas exotoxin A variant fusions (Fab-PE24) in Escherichia coli fed-batch process on chemically defined medium

For expression of Fab-light chain (23.4 kDa) and Fab-heavy chain PE24 fusions (48.7 kDa), an E. coli host/vector system with antibiotic-free plasmid selection by complementation of an E. coli auxotroph (PyrF) was employed (EP 0972 838 and US 6,291,245).

E. coli K12 strains were transformed by electroporation with the corresponding expression plasmids. The transformed E. coli cells were first grown on agar plates at 37°C. For each transformation, one colony picked from this plate was transferred to a 3 mL roller bottle culture and grown at 37°C to an optical density of 1-2 (measured at 578 nm). 1000 μl of culture was then mixed with 1000 μl of sterile 86% glycerol and immediately frozen at -80°C for long-term storage. These clones were first verified for correct product expression in small-scale shake flask experiments and analyzed by SDS-PAGE before being transferred to 10 L fermentors.

Pre-culture:

For pre-fermentation, chemically defined medium was used. For pre-fermentation, 220 ml of medium in a 1000 ml Erlenmeyer shake flask with 4 baffles was inoculated with 1.0 ml from the original seed bank ampoule. Culture was carried out on a rotary shaker at 37°C and 170 rpm for 9 hours until an optical density (578 nm) of 7-8 was obtained. 100 ml of pre-culture was used to inoculate the batch culture medium of a 10 L bioreactor.

Fermentation (RC52#003):

For fermentation in 10 L Biostat C, DCU3 fermenters (Sartorius, Melsungen, Germany), a chemically defined batch medium was used. The base solution used for pH adjustment was a 12.5% (w/v) NH ₃ aqueous solution supplemented with 11.25 g/l L-methionine.

Batch fermentation was started with 4.2 L sterile batch medium plus 100 ml inoculum from a pre-culture at 31° C., pH 6.9 ± 0.2, 800 mbar back pressure and an initial aeration rate of 10 L/min. Relative dissolved oxygen values (pO ₂ ) were maintained at 50% by increasing the agitator speed to 1500 rpm throughout the fermentation. After the initial glucose supplement was exhausted, the temperature was changed to 37° C., as indicated by a sharp increase in dissolved oxygen values, and after 15 minutes the fermentation entered fed-batch mode starting with two feeds (60 and 14 g/h, respectively). The rate of feed 2 remained constant, while the rate of feed 1 gradually increased from 60 to a final 160 g/h in 7 hours, with a predetermined feed profile. When the carbon dioxide waste gas concentration level was above 2%, the aeration rate was increased from 10 to 20 L/min in 5 hours. The expression of the recombinant target protein as insoluble inclusion bodies located in the cytoplasm was induced by adding 2.4 g IPTG at an optical density of approximately 40.

After 24 hours of cultivation, the optical density reached 185, and the entire culture was cooled to 4-8°C. The bacteria were harvested by centrifugation using a flow-through centrifuge (13,000 rpm, 13 L/h), and the resulting biomass was stored at -20°C until further processing (cell disruption). The yield was 40-60 g dry cells per liter.

Analysis of product formation:

The samples taken from the fermentation tank were analyzed by SDS-polyacrylamide gel electrophoresis, one before induction and the other at a specific time point after induction of protein expression. The same amount of cells from each sample (OD _target = 10) were suspended in 5 mL PBS buffer and broken on ice through ultrasonic treatment. Then 100 μL of each suspension was centrifuged (15,000 rpm, 5 minutes), each supernatant was taken out and transferred to a separate vial. This is to distinguish between soluble and insoluble expressed target proteins. 100 μL and 200 μL SDS sample buffer (Laemmli, UK, Nature 227 (1970) 680-685) were added to each supernatant (= soluble protein fraction) and each pellet (= insoluble protein fraction), respectively. The samples were heated at 95°C for 15 minutes under vigorous mixing to dissolve and reduce all proteins in the sample. After cooling to room temperature, 5 μL of each sample was transferred to a 4-20% TGX Criterion dye-free polyacrylamide gel (Bio-Rad). In addition, 5 μL of a molecular weight standard (Precision Plus Protein Standard, Bio-Rad) and three amounts (0.3 μL, 0.6 μL, and 0.9 μL) of a quantitative standard with a known target protein concentration (0.1 μg/μL) were applied.

Electrophoresis was run at 200 V for 60 minutes, after which the gel was transferred to a GelDOC EZ imager (Bio-Rad) and processed with UV irradiation for 5 minutes. Gel images were analyzed using Image Lab analysis software (Bio-Rad). Using three standards, a linear regression curve was calculated with a coefficient > 0.99, and the concentration of the target protein in the original sample was calculated.

Purification, sortase coupling, and renaturation of the Fab fragment of M-08-11, the PE24 variant, and the M-08-11 Fab fragment conjugated to the Pseudomonas exotoxin variant PE24LR8M

Fab fragment

The Fab fragments were purified by affinity chromatography (Ni Sepharose ^™ High-Efficiency HisTrap ^™ ) according to the manufacturer's description. Briefly, the supernatant was loaded onto a column balanced in 50 mM sodium phosphate pH 8.0, 300 mM NaCl. Protein elution was performed using the same buffer at pH 7.0 containing 4 mM imidazole, followed by a wash step with a gradient rising to 100 mM imidazole. Fractions containing the desired Fab fragment were combined and dialyzed against 20 mM His, 140 mM NaCl, pH 6.0.

PE24 for sortase conjugation

E. coli cells expressing PE24 were lysed by high pressure homogenization in 20mM Tris, 2mM EDTA, pH 8.0 + complete protease inhibitor cocktail tablets (Roche) (for details: Christian Schantz). The lysate was filtered and loaded onto a Q sepharose FF (GE Healthcare) equilibrated in 20mM Tris, pH 7.4. The protein was eluted with a gradient up to 500mM NaCl in the same buffer. Fractions containing PE24 were identified by SDS-PAGE. The pool was concentrated and applied to a HiLoad ^™ Superdex ^™ 75 (GE Healthcare) equilibrated in 20mM Tris, 150mM NaCl, pH 7.4. Fractions containing PE24 were combined according to SDS-PAGE and frozen at -80°C.

Sortase coupling of Fab fragments to PE24

The Fab fragment and PE24 were diafiltered separately into 50mM Tris, 150mM NaCl, 5mM CaCl2 pH _7.5 using an Ultra 4 centrifugal filter device (Merck Millipore) and concentrated to 5-10mg/ml. The two proteins and the sortase were combined in a 1:1:0.8 molar ratio. After incubation at 37°C for 1 hour, the mixture was loaded onto a Ni Sepharose ^™ high-efficiency HisTrap ^™ equilibrated in 50mM sodium phosphate, pH 8.0, 300mM NaCl. Elution was performed using a gradient increasing to 100mM imidazole in the same buffer pH 7.0. The flow-through fraction containing the final product Fab-PE24 was concentrated and loaded onto a HiLoad ^™ Superdex ^™ 200 (GE Healthcare) equilibrated in 20mM Tris, 150mM NaCl, pH 7.4. The fractions containing the desired coupled protein were combined and stored at -80°C. Soluble Staphylococcus aureus sortase A was used as the sortase (SEQ ID NO: 54). Soluble Staphylococcus aureus sortase A was expressed and purified using the following expression plasmid: The sortase gene encodes an N-terminally truncated Staphylococcus aureus sortase A (60-206) molecule. The expression plasmid used for transient expression of soluble sortase in HEK293 cells contained, in addition to the soluble sortase expression cassette, an origin of replication from the vector pUC18 (which allows this plasmid to replicate in E. coli) and a β-lactamase gene (which confers ampicillin resistance in E. coli). The transcription unit of the soluble sortase contained the following functional elements:

- the immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A, - the human heavy chain immunoglobulin 5' untranslated region (5'UTR),

- mouse immunoglobulin heavy chain signal sequence,

- an N-terminally truncated Staphylococcus aureus sortase A encoding nucleic acid, and

- Bovine growth hormone polyadenylation sequence (BGH pA).

Renaturation of Fab-PE24 derived from E. coli inclusion bodies

Inclusion bodies of VH-PE24 and VL-C _kappa were dissolved separately in 8M guanidine hydrochloride, 100mM Tris-HCl, 1mM EDTA, pH 8.0+100mM dithiothreitol (DTT). After 12-16 hours at room temperature, the pH of the solubilized material was adjusted to 3.0 and the centrifuged solution was dialyzed against 8M guanidine hydrochloride, 10mM EDTA, pH 3.0. Protein concentration was determined by biuret reaction and the purity of the inclusion body preparation was assessed by SDS-PAGE. Equimolar amounts of the two chains were diluted in two steps into 0.5M arginine, 2mM EDTA, pH 10+1mM GSH/1mM GSSG to a final concentration of 0.2-0.3mg/ml. After 12-16 hours at 4-10°C, the renatured protein was diluted with _H2O to <3 mS/cm and loaded onto a Q sepharose FF (GE Healthcare) equilibrated in 20 mM Tris/HCl, pH 7.4. Elution was performed using a gradient increasing to 400 mM NaCl in the same buffer. Fractions containing the correct product were identified by SDS-PAGE and analytical size exclusion chromatography (SEC). Pooled fractions were concentrated and loaded onto a HiLoad ^™ Superdex ^™ 200 (GE Healthcare) equilibrated in 20 mM Tris, 150 mM NaCl, pH 7.4 or in 20 mM histidine, 140 mM NaCl, pH 6.0. Fractions were analyzed and pooled by analytical SEC and stored at -80°C.

Based on SEQ ID NO: 50 and SEQ ID NO: 53, the immunoconjugate of the Fab fragment of M-08-11 and the Pseudomonas exotoxin variant PE24LR8M (M-08-11-PE) can also be recombinantly expressed, purified and renatured as a direct PE24LR8M fusion protein.

Example 8

Cytotoxicity of M-08-11-Fab-Pseudomonas exotoxin conjugate (M-08-11-PE) against different tumor cell lines

A549 cells overexpressing HER3 were seeded into white 96-well plates (transparent flat bottom, 1x10 ⁴ cells per well) and grown overnight in RPMI (10% FCS). The next day, the culture medium was replaced with 50 μl starvation medium (RPMI, 0.5% FCS). After at least 4 hours, 5 μl of heregulin-β (PeproTech, catalog number 100-03) (HRGβ) was added to a final concentration of 500 ng/ml. 50 μl of M-08-11-Fab-Pseudomonas exotoxin conjugate (M-08-11-Fab-PE) solution was added to a final concentration of 10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.005 and 0.002 μg/ml. The plates were incubated for 72 hours. After 24 and 48 hours, 5 μl of heregulin-β was added again to a final concentration of 500 ng/ml. After 72 hours, luminescence was measured using Promega's CellTiter-Glo luminescent cell viability assay (Cat. No. G7571) in a Tecan Infinite F200 reader. EC50 values of M-08-11-Fab-Pseudomonas exotoxin conjugate (M-08-11-Fab-PE) in the absence and presence of HRGβ were calculated.

Example 9

Humanized variants of antibodies according to the present invention

The binding specificity of the mouse antibody is transferred to a human acceptor framework to eliminate potential immunogenicity issues derived from sequence segments that the human body would recognize as foreign. This is done by grafting the entire HVR of the mouse (donor) antibody onto a human (acceptor) antibody framework, a process known as HVR (or CDR) grafting or antibody humanization.

The mouse variable region amino acid sequences were aligned with the human germline antibody V gene collection and sorted based on sequence identity and homology. Acceptor sequences were selected based on high overall sequence homology and, optionally, the presence of the correct canonical residues already present in the acceptor sequence (see Poul, M-A. and Lefranc, M-P., in "Ingénierie desanticorps banques combinatores" ed. by Lefranc, M-P. and Lefranc, G., Les Editions INSERM, 1997).

The germline V genes only encode regions up to the beginning of HVR3 of the heavy chain and to the middle of HVR3 of the light chain. Therefore, the germline V genes are not aligned across the entire V domain. The humanized constructs contain human frameworks 1 to 3, mouse HVRs, and human framework 4 sequences derived from human JK4 and JH4 sequences for the light and heavy chains, respectively.

Before selecting a specific acceptor sequence, the so-called canonical loop structures of the donor antibody can be determined (see Morea, V., et al., Methods, Vol 20, Issue 3 (2000) 267-279). These canonical loop structures are determined by the types of residues present at the so-called canonical positions. These positions are (partially) located outside the HVR regions and should remain functionally equivalent in the final construct in order to maintain the HVR conformation of the parent (donor) antibody.

In WO 2004/006955 a method for humanizing antibodies is reported, comprising the steps of identifying a canonical HVR structure type for HVRs in a non-human mature antibody; obtaining a peptide sequence library for human antibody variable regions; determining the type of canonical HVR structure for the variable regions in the library; and selecting human sequences in which the canonical HVR structure is identical to the canonical HVR structure type of the non-human antibody at corresponding positions within the non-human and human variable regions.

In general, potential receptor sequences are selected based on high overall homology and the presence of correct canonical residues already present in the additional, optional receptor sequence.

In some cases, simple HVR grafting results in only partial retention of the binding specificity of the non-human antibody. It has been found that at least some specific non-human framework residues are required for reconstitution of binding specificity and have to be grafted into the human framework, i.e., in addition to introducing the non-human HVRs, so-called "back mutations" have to be performed (see, e.g., Queen et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10, 029-10, 033; Co et al., Nature 351 (1991) 501-502). These specific framework amino acid residues participate in FR-HVR interactions and stabilize the conformation (loops) of the HVRs (see, e.g., Kabat et al., J. Immunol. 147 (1991) 1709).

In some cases, forward mutations were also introduced in order to adopt closer human germline sequences.

Genes for the designed antibody sequences were generated using conventional PCR techniques. The heavy chain variable region was fused to the human IgG1 heavy chain constant region (if effector function is desired) or human IgG1/IgG4 heavy chain constant region variants (if effector function is not desired; IgG1 L234A L235A P329G; IgG4 S228P L235E P329G). The light chain variable domain was fused to the light chain kappa or lambda constant domain for the construction of expression plasmids. Thus, humanized mouse anti-HER3 antibodies M-08-11, M-17-02, M-43-01, and M-46-01 were generated. Antibodies were expressed in mammalian cell culture systems such as HEK or CHO and purified by protein A and size exclusion chromatography. Humanized antibodies were expressed as full-length antibodies or antibody fragments, or included in immunotoxin conjugates (similar to Example 7).

Example 11

Binding of antibody M-08-11 (1) to TtSlyDcys-HER3 (SEQ ID NO: 18) compared to the anti-HER3 antibody MOR09823 (2) described in WO 2012/22814

A Biacore T200 instrument (GE Healthcare) was equipped with a CM5 series sensor according to the manufacturer's instructions and standardized in HBS-ET+ buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% w/v Tween 20). The sample buffer was system buffer supplemented with 1 mg/ml CMD (carboxymethyl dextran). The system was operated at 37°C. A dual antibody capture system was established on the sensor surface. 6500 RU of monoclonal antibody <M-IgG>R was immobilized on all flow cells using EDC/NHS chemistry according to the manufacturer's instructions. The sensor was inactivated using 1 M ethanolamine. Flow cell 1 served as a reference and captured anti-TSH IgG1 antibody at 10 μl/min for 1 minute. M-08-11 was captured at 10 μl/min for 1 minute on flow cell 2. Mouse anti-human Fc pan antibody was captured on flow cell 3 at 10 μl/min for 1 minute, followed by injection of anti-HER3 antibody M-08-11 (1) (90 nM) or anti-HER3 antibody MOR09823 (150 nM) antibody at 10 μl/min for 1 minute. The flow rate was set to 60 μl/min. The analyte TtSlyDcys-HER3 (SEQ ID NO: 18) in solution was injected at concentrations of 0 nM and 150 nM for 5 minutes, and dissociation was monitored for 600 seconds. The sensor was fully regenerated by injecting 10 mM glycine pH 1.7 buffer once at 10 μl/min for 3 minutes.

Figure 17 depicts a sensorgram overlay showing the binding signals of 150 nM TtSlyDcys-Her3 and buffer. The overlay shows that antibody M-08-11 binds to 150 nM TtSlyDcys-HER3 (1). The MOR09823 antibody does not bind to TtSlyDcas-HER3 (2). Control measurements without any antibody at all also do not show any binding to TtSlyDcys-HER3 (SEQ ID NO: 18). Thus, the anti-HER3 antibody MOR09823 (2) described in WO 2012/22814 does not show any binding interaction with TtSlyDcys-Her3. The positive control antibody M-08-11 (1) shows significant binding to TtSlyDcas-Her3. No binding interaction could be detected for either antibody when 150 nM TtSlyDcys (no HER3 β-hairpin insertion) was injected (data not shown). The anti-HER3 antibody MOR09823 described in WO 2012/22814 was also tested for its binding affinity to HER3-ECD as an analyte. In this setting, using HER3-ECD instead of TtSlyDcys-Her3, MOR09823 showed a clear binding signal to HER3 ECD with a KD (affinity) of 0.3 nM. Thus, MOR09823 binds to human HER3, but not to the β-hairpin of human HER3 (when it is contained within TtSlyDcys-HER3 (SEQ ID NO: 18)).

Claims (17)

1. A method for selectively binding an antigen-binding protein to human HER3, wherein the antigen-binding protein binds within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) of human HER3. Among them, the use of a) A polypeptide containing the amino acid sequence SEQ ID NO:1: SEQ ID NO:18TtSlyDcys-Her3 and b) A polypeptide containing the amino acid sequence SEQ ID NO:2: SEQ ID NO:22TtSlyDcys-Her4 To select antigen-binding proteins that show binding to the peptide in a) and not binding to the peptide in b), and thereby select antigen-binding proteins that bind to the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) of human HER3 and do not cross-react with human HER4. The antigen-binding protein is an antibody. 2. An isolated antibody binding to human HER3, wherein the antibody comprises: (a) The amino acid sequence of HVR-H1 shown in SEQ ID NO:25; (b) The amino acid sequence HVR-H2 shown in SEQ ID NO:26; (c) The amino acid sequence HVR-H3 shown in SEQ ID NO:27; (d) HVR-L1 with the amino acid sequence shown in SEQ ID NO:28; (e) The amino acid sequence of HVR-L2 as shown in SEQ ID NO:29; and (f) HVR-L3, whose amino acid sequence is shown in SEQ ID NO:30. 3. An isolated antibody binding to human HER3, wherein the antibody comprises: (a) HVR-H1, as shown in SEQ ID NO:33, amino acid sequence; (b) The amino acid sequence HVR-H2 shown in SEQ ID NO:34; (c) The amino acid sequence HVR-H3 shown in SEQ ID NO:35; (d) HVR-L1 with the amino acid sequence shown in SEQ ID NO:36; (e) The amino acid sequence of HVR-L2 as shown in SEQ ID NO:37; and (f) HVR-L3, whose amino acid sequence is shown in SEQ ID NO:38. 4. An isolated antibody binding to human HER3, wherein the antibody comprises: (a) HVR-H1, as shown in SEQ ID NO:41; (b) The amino acid sequence HVR-H2 shown in SEQ ID NO:42; (c) The amino acid sequence HVR-H3 shown in SEQ ID NO:43; (d) HVR-L1 with the amino acid sequence shown in SEQ ID NO:44; (e) The amino acid sequence of HVR-L2 as shown in SEQ ID NO:45; and (f) HVR-L3, whose amino acid sequence is shown in SEQ ID NO:46. 5. An isolated antibody binding to human HER3, wherein the antibody comprises: (a) HVR-H1, as shown in SEQ ID NO:49; (b) The amino acid sequence HVR-H2 shown in SEQ ID NO:50; (c) HVR-H3, as shown in SEQ ID NO:51; (d) HVR-L1 with the amino acid sequence shown in SEQ ID NO:52; (e) The amino acid sequence of HVR-L2 as shown in SEQ ID NO:53; and (f) HVR-L3, whose amino acid sequence is shown in SEQ ID NO:54. 6. The antibody of any one of claims 2 to 5, wherein the antibody has one or more of the following characteristics: a) The antibody binds to the amino acid sequence SEQ ID NO:1 in activated HER3; and/or b) This antibody inhibits the heterodimerization of the HER3/HER2 heterodimer; and/or c) The antibody has no cross-reactivity with the amino acid sequence SEQ ID NO:2; and/or d) This antibody shows no cross-reactivity with the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) contained in a polypeptide selected from the group consisting of: SEQ ID NO:22TtSlyDcys-Her4; and/or e) This antibody does not compete with motrin for binding to HER3; and/or f) The antibody induces the binding of HER3 to the modulator protein. 7. The antibody according to any one of claims 2 to 5, wherein the antibody a) Binding peptide SEQ ID NO:18TtSlyDcys-Her3, and b) Does not cross-react with peptide SEQ ID NO:22TtSlyDcys-Her4. 8. The antibody of any one of claims 2 to 7, wherein it is a humanized antibody or a chimeric antibody. 9. The antibody of any one of claims 2 to 8, wherein it is a full-length IgG1 antibody or an IgG4 antibody. 10. The Fab fragment of the antibody according to any one of claims 2 to 8. 11. An immunoconjugate comprising (1) an antibody of any one of claims 2 to 9 or a Fab fragment of claim 10, and (2) a cytotoxic agent. 12. A pharmaceutical formulation comprising (1) an antibody of any one of claims 2 to 9, or a Fab fragment of claim 10, or an immunoconjugate of claim 11, and (2) a pharmaceutically acceptable carrier. 13. Use of the antibody of any one of claims 2 to 9, or the Fab fragment of claim 10, or the immunoconjugate of claim 11, in the manufacture of a medicament for treating cancer. 14. Use of the antibody of any one of claims 2 to 9, or the Fab fragment of claim 10, or the immunoconjugate of claim 11, in the manufacture of a medicament for inhibiting HER3/HER2 dimerization. 15. An isolated nucleic acid encoding an antibody of any one of claims 2 to 9 or a Fab fragment of claim 10. 16. A host cell comprising the nucleic acid of claim 15. 17. A method for generating an antibody or Fab fragment, comprising culturing a host cell of claim 16 to generate the antibody or Fab fragment, and recovering the antibody or Fab fragment from the cell culture or cell culture supernatant.