Anti-her2 binding molecules

Abstract

The present invention relates to binding proteins of the extracellular domain (ECD) of HER2 / ErbB2. More specifically, binding proteins bind to conformational epitopes exposed in cells that respond to HER2 expansion or activation.

Description

[0001] All documents cited or referenced in this application, as well as all documents cited or referenced in the documents cited in this application, together with the instructions, descriptions, product specifications, and product sheets of any manufacturer of any product mentioned in any document cited herein or incorporated herein by reference, are incorporated herein by reference in their entirety.

[0002] The entire content of the electronic submission of the sequence listing is incorporated herein by reference for all purposes.

[0003] This application is a divisional application of Chinese Patent Application No. 202080034298.1 and claims priority to Australian Provisional Patent Application AU 2019900973 filed on March 22, 2019. The entire content of this document is incorporated herein by reference. Technical Field

[0004] The present invention relates to a binding protein for the extracellular domain (ECD) of HER2 / ErbB2. More specifically, the binding protein binds to a conformational epitope that is exposed in cells responsive to HER2 amplification or activation. Background Art

[0005] The ErbB family of receptors includes four homologous proteins that reside on the cell surface: epidermal growth factor receptor (EGFR; also known as ERBB1; HER1); ERBB2 (also known as HER2; Neu); ERBB3 (or HER3); and ERBB4 (or HER4).

[0006] Overexpression and amplification of HER2 have been identified in many cancer types, including breast cancer, biliary tract cancer, colon cancer, endometrial cancer, gastric cancer and gastroesophageal junction cancer, glioblastoma multiforme, head and neck cancer, ovarian cancer, pancreatic cancer, and urothelial cancer. HER2 overexpression and amplification have been shown to be associated with poor outcomes in breast cancer and gastric / gastroesophageal junction (GEJ) cancers (Nagaraja et al., (2016) Eur J Surg Oncol 42(1):140-8); however, its role in other tumor types is not well defined.

[0007] The market for HER2 antibodies exceeded $10 billion in 2017, and increased sales of HER2 antibodies are expected in the future, with new clinical indications and markets emerging. The response to HER2-targeted therapy in patients with HER2 overexpression / amplification is affected by several clinicopathological features, including tumor histology, degree of dysplasia, disease stage, and the presence of potential risk factors. Amplification or overexpression of HER2 is also used as a predictive biomarker for anti-HER therapy in multiple tumor types, including breast cancer, gastric cancer, and gynecologic cancers (Slamon DJ et al., (1987) Science 235{4785):177-82; Santin AD et al., (2005) Cancer 104(7):1391-7; Morrison C et al., (2006) J Clin Oncol 24(15):2376-85; Liu et al., (2010) J Thoracic Oncol 5(12):1922-32). For a review, see Parakh S et al., (2017) Cancer Treatment Reviews 59:1-21.

[0008] HER2 function

[0009] The extracellular domain of HER2 cannot bind any known natural ligand (Klapper LN et al., (1999) PNAS 96(9):4995-5000). Different from other members of the ErbB family, HER2 adopts a conformation that favors oligomerization and activation of the HER2 kinase in the absence of ligand (for other ErbB family members, ligand activation is required) (Garrett TP et al., (2002) Cell 110(6):763-73). The disclosure of HER2 (Cho H-S et al., (2003) Nature 421(6924):756-60) renders the dimerization arms permanently available for homodimer or heterodimer interactions between monomer family members, as well as conformational changes and oligomerization of pre-existing inactive dimers (Maruyama IN et al., (2014) Cells 3(2):304-30), leading to autophosphorylation and signal transduction of the intracellular kinase domain. HER2 overexpression increases the affinity of EGF and neuregulin for their receptors and decreases the rate of ligand dissociation from the active dimer. HER2 overexpression has also been shown to affect the recycling and degradation rates of HER2-containing heterodimers: the EGFR-HER2 heterodimer undergoes endocytic recycling rather than degradation, which results in prolonged EGFR signal transduction (Huang G et al., (1999) J Cell Biochem 74(1):23-30). Among the heterodimers formed upon ligand activation, the HER2-HER3 heterodimer appears to be the most efficient signal transduction complex in HER2-amplified tumors (Tzahar E et al., (1996) Mol Cell Biol 16(10):5276-87). Signal transduction through the HER2-HER3 complex is mediated by HER3-dependent phosphorylation and subsequent activation of the PI3K / Akt signaling pathway (Pinkas-Kramarski R et al., (1996) EMBO J 15(10):2452). In vitro studies have demonstrated that HER2 overexpression leads to malignant transformation, development of anti-apoptotic properties, increased invasiveness, and drug resistance. Receptor activation through HER2-heterooligomerization may be the key mechanism driving cell proliferation observed in HER2-overexpressing cells (Wolf-Yadlin A et al., (2006) Mol Syst Biol 2(1):54).

[0010] Anti-HER2 directed therapies

[0011] Many different methods of inhibiting HER2 have been attempted and many of these have entered clinical practice.

[0012] (i) Monoclonal antibodies against domain IV of HER2: Earlier HER2 inhibition methods used monoclonal antibodies against the extracellular domain, and the main example is Trastuzumab (Albanell J et al., (1999) Drugs Today (Barc) 35(12):931-46). Other anti-HER2 antibodies have also been described, for example, in Ko B-K et al., (2015) Mol Oncol 9(2):398-408; Mahdavi M et al., (2015) Monoclon Antib Immunodiagn Immunother 34(3):213-21; Ceran C et al., (2012) Cancer Cell 6(2):117-27).

[0013] (ii) Antibodies against domain II of HER2: Pertuzumab, a class of drugs known as HER dimerization inhibitors (Adams CW et al., (2006) 55(6):717-27), binds to the extracellular dimerization domain II of HER2 (an epitope different from the epitope of Trastuzumab), inhibiting dimerization between HER receptors (Adams CW, ibid.).

[0014] (iii) Small molecule inhibitors of HER2: Small molecule inhibitors targeting HER2 have been developed. Many target multiple receptors, including other members of the ErbB family, which may be advantageous as collateral signal transduction can be a mechanism of resistance to HER2 inhibitors (Ritter CA et al., (2007) Clin Cancer Res 13(16):4909-19). There are two approved tyrosine kinase inhibitors; lapatinib is an oral small molecule reversible inhibitor that inhibits both EGFR and HER2 kinases (Tevaarwerk AJ et al., (2009) Clin Ther 31:2332-48), and afatinib is an irreversible inhibitor of EGFR, HER2, and HER4 tyrosine kinase activities as well as dimers containing EGFR and HER2 (Li D et al., (2008) Oncogene 27(34):4702-11). Neratinib is an irreversible pan-tyrosine kinase inhibitor of HER1, HER2, and HER4. It appears to affect downstream signal transduction in cells overexpressing HER2 and cells with amplified EGFR, and results in apoptosis and reduced tumor growth (Rabindran SK et al., (2004) Can Res 64(11):3958-65). Tucatinib is a selective oral HER2 inhibitor (Moulder-Thompson S et al., (2017) Clin Cancer Res clincanres 1496.2016).

[0015] (iv) Anti-HER2 antibody-drug conjugates: High expression on HER2-positive cancer cells and low expression on normal tissues make it a target for antibody-drug conjugates (ADCs). The trastuzumab antibody-drug conjugate (ado-Trastuzumab emtansine, T-DM1) is the first anti-HER2 ADC approved in solid tumors. It consists of trastuzumab linked to the potent cytotoxic drug DM1, a microtubule dimerization inhibitor. The antitumor effect of T-DM1 is related to trastuzumab and DM1 metabolites (Juntilla T et al., (2011) Breast Cancer Res Treat 128(2):347-56). DM1 metabolites disrupt the microtubule network, leading to cell cycle arrest and apoptotic cell death. Although T-DM1 shows the greatest benefit in tumors with high HER2 expression, it shows efficacy in different HER2 expression subgroups (Baselga J et al., (2016) Clin Cancer Res clincanres.2499.015). The ADC MM-302 consists of a HER2-targeting antibody linked to liposomal doxorubicin. SYD985 is another HER2-targeting trastuzumab-based ADC that is linked to the toxic alkylating antibiotic duocarmycin (Dokter W et al., (2014) Mol Cancer Ther 13(11):2618-29). DS-8201a is a HER2-targeting ADC that includes a humanized anti-HER2 antibody linked to a topoisomerase I inhibitor (Ogitani Y et al., (2016) Clin Cancer Res 22(20):5097-108). XMT-1522 is an anti-HER2 ADC that includes an anti-HER2 antibody with HT-19 linked to an auristatin-based cytotoxic payload (AF-HPA) (Bergstrom D et al., (2016) Can Res 76(4 Suppl) P4-14-28).

[0016] (v) Monoclonal antibodies: Many newer anti-HER2 monoclonal antibodies are in clinical development, including 10H8 and 8H11 (Kim AY et al., (2013) Proceedings of the ASCO Annual Meeting), MGAH22 (margetuximab), which is an Fc-optimized chimeric anti-HER2 monoclonal antibody, ertumaxomab, which targets HER2, the T cell-specific CD3 antigen, and FcγI / III type receptors; and CMAB302 (cipterbin), which is a biosimilar of trastuzumab.

[0017] (vi) Bispecific antibodies: The bispecific antibody MM-111 targeting the HER2 / HER3 heterodimer has been developed (McDonagh CF et al., (2012) Mol Cancer Ther 11(3):582-93). Many of the first in human early studies were to evaluate MM-111 in HER2-positive solid tumors as a single agent and in combination with trastuzumab and various chemotherapy regimens or lapatinib. MM-111 is also being studied in gastrointestinal malignancies. MCLA-128 is a humanized bispecific antibody targeting HER2 and Her3 with enhanced ADCC activity (Calvo E et al., Abstract CT050 AACR 2016). MCLA-128 blocks downstream signal transduction through the HER2:Her3 heterodimer even at high protein concentrations. GBR 1302 is another bispecific antibody targeting CD3ε and HER2, which was evaluated in early clinical trials after showing effective anti-tumor activity in tumors overexpressing HER2 and non-overexpressing HER2 (Moretti P et al., (2016) BEAT GBR 1302). ZW25 is a bispecific antibody targeting two different epitopes on the extracellular domain of the HER2 receptor and is currently being evaluated in HER2-expressing cancers in a Phase 1 clinical trial. Another bispecific immunotoxin has been developed, which comprises an anti-HER2 single-chain variable fragment (scFv) fused to diphtheria toxin-anti-EpCAM.

[0018] Despite the various approaches, challenges remain. Primary resistance to the single agent trastuzumab occurs in 70% of breast cancers overexpressing HER2 (Vogel CL et al., (2002) 20(3):719-26), and most patients develop resistance during treatment. Many mechanisms have been proposed (see Parakh S et al., ibid). The resistance mechanisms to pertuzumab are poorly understood compared to trastuzumab. Similar to other anti-HER2 therapies, primary and acquired resistance to T-DM1 also occurs (Tan X et al., (2013) Can Res 73(8 Suppl):4629). While the resistance mechanisms to T-DM1 seem to depend on the size of the tumor and the duration of treatment; resistance is observed even after a long latency (Barok M et al., (2011) BreastCancer Res 13(2):R46).

[0019] Despite the success of trastuzumab (Herceptin), pertuzumab, and the T-DM1 conjugate, the toxicity of current HER2 antibodies is dose-limiting, and resistance always develops as described above. Clearly, new strategies are needed to target HER2 without the associated toxicological profiles. Summary of the Invention

[0020] The present invention provides an isolated specific binding protein that binds to the extracellular domain (ECD) of HER2 in a conformationally flexible region of domain II. In particular, the binding protein recognizes a HER2 epitope that does not exhibit any amino acid sequence alterations or substitutions from the wild-type HER2 sequence and is exposed in cells in response to HER2 amplification or activation. The conformationally exposed epitope is found only in neoplastic, hyperproliferative, or abnormal cells and is undetectable in normal or wild-type cells. "Wild-type" means a cell that expresses endogenous HER2, but specifically excludes cells that overexpress the HER2 gene; the term "wild-type" refers to the genotype, phenotype, or other characteristics present in normal cells and not in abnormal or neoplastic cells.

[0021] Interestingly, the binding proteins of the present invention do not block the binding of pertuzumab or trastuzumab / Herceptin to HER2 on cancer cells, indicating that this epitope region of domain II allows the binding of this binding molecule without blocking the binding of these antibodies when conformationally exposed, potentially allowing a dual therapy approach. More specifically, the inventors have found that although the binding molecules of the present invention bind to a smaller proportion of HER2 on the cancer cell surface (e.g., when compared to pertuzumab or trastuzumab), they are equally effective in vivo at concentrations equivalent to those of pertuzumab or trastuzumab, despite binding to fewer receptors. Since the binding molecules of the present invention are internalized and are tumor cell-specific, they are ideally suited as drug conjugates or agents in a dual therapy approach with other HER2 antibodies.

[0022] The specific binding protein of the present invention can be an antibody or a fragment thereof, such as an immunogenic fragment thereof, which does not bind to or recognize normal or wild-type cells containing normal or wild-type HER2 epitopes in the absence of abnormal expression and in the presence of normal HER2 post-translational modifications. More specifically, the specific binding protein of the present invention can be an antibody or a fragment thereof that recognizes a HER2 epitope that is present in cells overexpressing HER2 (e.g., the HER2 gene is amplified), particularly in the presence of abnormal post-translational modifications, and is undetectable in cells expressing HER2 under normal conditions, particularly in the presence of normal post-translational modifications.

[0023] The inventors have discovered new monoclonal antibodies, exemplified herein by the antibody designated mAb104, which specifically recognize abnormally expressed HER2. In particular, the antibodies of the present invention recognize HER2 epitopes that are found in neoplastic, hyperproliferative, or abnormal cells and are undetectable in normal or wild-type cells. The antibodies of the present invention are further exemplified by the antibodies mAb105, mAb106, and mAb107 described herein.

[0024] The present invention provides a HER2 / ErbB binding protein comprising an antigen-binding domain, wherein the antigen-binding domain specifically binds to an epitope within domain II of HER2 that is exposed in response to HER2 amplification or activation. In one instance, the HER2 binding protein binds to a region of HER2 that is conformationally exposed in neoplastic, hyperproliferative, or abnormal cells, but not in normal or wild-type cells.

[0025] In another instance, the binding of the binding protein to its epitope does not block the binding of pertuzumab or trastuzumab / Herceptin. In one instance, the binding protein is not pertuzumab or trastuzumab. In one instance, the binding protein binds to a region comprising residues 293 to 309 of the mature normal or wild-type human HER2 sequence as shown in Figure 1 (SEQ ID NO: 27). This region forms part of domain II in the extracellular domain (ECD) of HER2. In a specific instance, the epitope comprises the amino acid sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1). The epitope is shown in Figure 1 as the bold and underlined sequence. Although this epitope includes P294, L295, and H296 that are also present in the epitope bound by pertuzumab, the binding protein described herein is non-pertuzumab-blocking and permits pertuzumab to bind to HER2 simultaneously. The epitope can be determined by any conventional epitope mapping technique known to those skilled in the art.

[0026] In one instance, the binding protein does not bind or binds substantially not to human EGFR (HER1) or HER3 or HER4.

[0027] In one instance, the binding protein is a binding protein having the characteristics of an antibody that the inventors have identified and characterized, particularly a binding protein that recognizes aberrantly expressed HER2, such as that found in amplified HER2. In another instance, the binding protein binds to tumor cell lines that express high levels of HER2. In one instance, HER2 overexpression is determined using immunohistochemical analysis. In a specific instance, the staining pattern is evaluated and scored using the recommendations of the American Society of Clinical Oncology and the College of American Pathologists (ASCO / CAP) for HER2 testing in breast cancer (Wolff AC et al. (2013) Journal of Clinical Oncology 31(31):3997-4013).

[0028] In another example, the binding protein binds to cancer cells selected from the group consisting of breast cancer, gastric cancer, squamous cell carcinoma, and colon cancer. In another example, the binding protein binds to cell lines selected from the group consisting of breast (e.g., BT 474, SK-BR3, SUM 159PT, MDA-MB-453), stomach (e.g., NCI-N87, MK N7), squamous cell carcinoma (e.g., A431), and colon cancer (COLO205, LIM1215).

[0029] In another example, compared to pertuzumab and / or trastuzumab, the binding protein binds to a smaller proportion of HER2 / ErbB on the cell surface of cancer cells. In another example, when evaluated by flow cytometry, the binding protein binds to HER2+-expressing cells at an order of magnitude of at least 1 log, at least 2 logs, or at least 3 logs lower than the binding of trastuzumab or pertuzumab. In certain examples, the binding protein binds to less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the total HER2 / ErB2 expressed on the surface of cancer cells. In one example, the proportion of the binding protein that binds to HER2 / Erb2 is about 0.35 - 0.5% of the total HER2 / Erb2 expressed on the surface of cancer cells.

[0030] In one example, the binding protein does not bind to normal gastric mucosa. In another example, the binding protein does not bind to normal breast cells.

[0031] In another example, the binding protein is capable of internalizing into tumor cells. In certain examples, the binding protein has an anti-proliferative effect on tumor cells (e.g., gastric cells) in vivo. In other examples, the binding protein has an anti-tumor effect in vivo comparable to that of pertuzumab or trastuzumab. In another example, the binding protein causes necrosis of tumor cells (e.g., breast tumor cells).

[0032] Exemplary HER2 binding proteins having such binding characteristics as described herein include the variable regions and / or CDRs of antibodies designated as mAb104 or mAb106.

[0033] In one example, the binding protein binds to a peptide comprising the sequence shown in SEQ ID NO: 1 or consisting of the sequence shown in SEQ ID NO: 1, or binds to a sequence in human HER2ECD, at a level similar or substantially the same as, or with a similar or substantially the same affinity as, the antibody designated as mAb104 or mAb106. In a particular example, the binding protein binds to the residues 293 to 309 or a continuous amino acid sequence consisting thereof of the mature normal or wild-type human HER2 sequence as shown in Figure 1 shown.

[0034] In another instance, the HER2-binding protein competitively inhibits the binding of antibodies designated as mAb104 or mAb106 to human HER2. In another instance, the protein competitively inhibits the binding of antibodies designated as mAb104 or mAb106 to a peptide consisting of the sequence shown in SEQ ID NO:1.

[0035] In one instance, the HER2-binding protein binds a peptide comprising the sequence shown in SEQ ID NO:1 or consisting of the sequence shown in SEQ ID NO:1 in an amount within 75% of the antibody-binding amount, the antibody comprising a VH comprising the sequence shown in SEQ ID NO:2 or SEQ ID NO:4 and a VL comprising the sequence shown in SEQ ID NO:3 or SEQ ID NO:5.

[0036] In one instance, the amount of the bound protein or antibody is evaluated by contacting the HER2-binding protein with a peptide consisting of the sequence shown in SEQ ID NO:1 and an amount of the HER2-binding protein contacting the peptide (e.g., 10 μg / ml). Then the amount of the HER2-binding protein bound to the peptide is determined and compared with the amount of an antibody comprising a VH comprising the sequence shown in SEQ ID NO:2 or SEQ ID NO:4 and a VL comprising the sequence shown in SEQ ID NO:3 or SEQ ID NO:5 that binds to the peptide, respectively. In one instance, the amount of the HER2-binding protein bound to the peptide is within about 80%, or 70%, or 60%, or 40% of the antibody-binding amount.

[0037] The present invention also provides a HER2-binding protein that competitively inhibits the binding of antibodies designated as follows:

[0038] (i) mAb104, the antibody comprising a VH and a VL, the VH comprising the sequence shown in SEQ ID NO:2 and the VL comprising the sequence shown in SEQ ID NO:3; or

[0039] (ii) mAb106, which comprises a VH and a VL, the VH comprising the sequence shown in SEQ ID NO:4 and the VL comprising the sequence shown in SEQ ID NO:5, to a peptide comprising the sequence shown in SEQ ID NO:1 or consisting of the sequence shown in SEQ ID NO:1 or the ECD of human HER2 (e.g., Figure 1 ).

[0040] In one instance, the HER2-binding protein binds the ECD of HER2 with an affinity dissociation constant (KD) of 2.90 - 3.20 nM, e.g., having Figure 1 the sequence shown. In another instance, the KD is between about 2.90 and about 3 nM. In another instance, the KD is about 3 nM.

[0041] In one instance, the KD is evaluated by utilizing surface plasmon resonance (SPR) in a biosensor equipped with a streptavidin (SA) chip, capturing a biotinylated human HER2 peptide (e.g., the peptide according to SEQ ID NO: 1) on the chip surface, and passing a HER2 binding protein over it.

[0042] When evaluated by SA chip biotinylated peptide SPR, the exemplary HER2 binding proteins of the present invention have a KD of about 3 nM (e.g., + / - 0.2 nM). In one instance, the HER2 binding protein has the KD shown in Table 7 for mAb104 or mAb106.

[0043] In one instance, the HER2 binding protein of the present invention specifically binds to human HER2. In one instance, the binding of the protein is evaluated by ELISA.

[0044] The HER2 binding protein of the present invention can be an anti-HER2 recombinant or synthetic or monoclonal antibody or an antigen-binding fragment thereof.

[0045] In one instance, the HER2 binding protein is a chimeric antibody comprising human heavy and light chain constant region sequences. In another instance, the HER2 binding protein is a humanized or fully human antibody.

[0046] In one instance, the HER2 binding protein comprises a heavy chain variable region sequence (VH) having at least 55% identity to the heavy chain variable region sequence of mAb104 (SEQ ID NO: 2).

[0047] In one instance, the HER2 binding protein comprises a light chain variable region sequence (VL) having at least 50% identity to the light chain variable region sequence of mAb104 (SEQ ID NO: 3).

[0048] In one instance, the binding protein comprises;

[0049] (i) A VH CDR1 having the following sequence:

[0050] GYX 7 FTX 8 YX 9 MX 10 (SEQ ID NO: 6)

[0051] wherein X 7 is S or T; X 8 is G or D; X 9 is F or G; X 10 is H or N;

[0052] (ii) A VH CDR2 having the following sequence:

[0053] X 19 INX 20 YX 21 GX 22 X 23 X 24 YX 25 X 26 X 27 FKX 28 (SEQ ID NO: 7)

[0054] wherein X 19 is R or W; X 20 is P or T; X 21 is N or T; X 22 is D or K; X 23 is I or P; X 24 is R or T; X 25 is N or D; X 26 is Q or D; X 27 is N or D; and X 28 is D or G;

[0055] (iii) having a VH CDR3 with the following sequence:

[0056] X 50 X 51 X 52 X 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 X 61 FX 62 Y(SEQ ID NO: 8)

[0057] wherein X 50 is absent or is R; X 51 is absent or is F; X 52 is absent or is L; X 53 is absent or is N; X 54 is absent or is T; X 55 is absent or is V; X 56 is absent or is A; X 57 is absent or is G; X 58 is absent or is R; X 59 is absent or is S; X 60 is L or V; X 61 is N or Y; and X 62 is A or D;

[0058] and / or

[0059] (iv) A VL CDR1 having the following sequence:

[0060] X 14 X 15 SX 16 SX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 (SEQ ID NO: 9)

[0061] where X 14 is K or S; X 15 is S or V; X 16 is Q or S; X 17 is L or absent; X 18 is L or absent; X 19 is D or absent; X 20 is S or absent; X 21 is D or absent; X 22 is G or absent; X 23 is K or V; X 24 is T or G; X 25 is F or S; X 26 is L or M; and X 27 is N or Y;

[0062] (v) A VL CDR2 having the following sequence:

[0063] LX 35 SX 36 LX 37 S(SEQ ID NO: 10)

[0064] X 35 is D or E; X 36 is K or T; X 37 is S or A; and

[0065] (vi) A VL CDR3 having the following sequence:

[0066] X 49 QX 50 X 51 X 52 X 53 PX 54 T(SEQ ID NO: 11)

[0067] Wherein X 49 is W or Q; X 50 is G or W; X 51 is T or S; X 52 is H or S; X 53 is F or N; and X 54 is W or P.

[0068] In one example, the HER2 binding protein comprises the heavy chain variable region sequence (VH) listed below:

[0069] X 1 X 2 QLX 3 QSGPELX 4 KPGX 5 X 6 VKISCKAS GYX 7 FTX 8 YX 9 MX 10 WVX 11 QX 12 X 13

[0070] X 14 X 15 X 16 LX 17 WX 18 G X 19 INX 20 YX 21 GX 22 X 23 X 24 YX 25 X 26 X 27 FKX 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 SX 37 STAYX 38 X 39 X 40 X 41 X 42 LX 43 X 44 EDX 45 AX 46 X 47 X 48 CAX 49 X 50 X 51

[0071] X 52 X 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 X 61 FX 62 Y WGQGTX 63 X 64 TVSX 65 (SEQ ID NO: 12)

[0072] Wherein

[0073] X 1 is E or Q; X 2 is V or I; X 3is Q or V; X 4 is V or K; X 5 is A or E; X 6 is S or T; X 7 is S or T; X 8 is G or D; X 9 is F or G; X 10 is H or N; X 11 is R or K; X 12 is S or A; X 13 is H or P; X 14 is V or G; X 15 is R or K; X 16 is S or G; X 17 is E or K; X 18 is I or M; X 19 is R or W; X 20 is P or T; X 21 is N or T; X 22 is D or K; X 23 is I or P; X 24 is R or T; X 25 is N or D; X 26 is Q or D; X 27 is N or D; X 28 is D or G; X 29 is K or R; X 30 is A or F; X 31 is S or A; X 32 is L or F; X 33 is T or S; X 34 is V or L; X 35 is D or E; X 36 is K or T; X 37 is S or A; X 38 is M or L; X 39 is E or Q; X 40 is L or I; X 41 is H or N; X 42 is R or N; X 43 is T or K; X 44 is S or N; X 45 is S or M; X 46 is V or T; X 47 is F or Y; X 48 is Y or F; X 49 is S or R; X 50 does not exist or is R; X 51 does not exist or is F; X 52 does not exist or is L; X 53 does not exist or is N; X 54 does not exist or is T; X 55Absent or V; X 56 Absent or A; X 57 Absent or G; X 58 Absent or R; X 59 Absent or S; X 60 Is L or V; X 61 Is N or Y; X 62 Is A or D; X 63 Is P or T; X 64 Is V or L; and X 65 Is A or S.

[0074] In one example, the HER2 binding protein further comprises the following light chain variable region sequences (VL):

[0075] X 1 IVX 2 TQSPX 3 X 4 X 5 SVX 6 X 7 GX 8 X 9 X 10 X 11 X 12 X 13 C X 14 X 15 SX 16 SX 17 X 18 X 19 X 20 X 21

[0076] X 22 X 23 X 24 X 25 X 26 X 27 WX 28 X 29 QX 30 PX 31 X 32 SPKX 33 X 34 IY LX 35 SX 36 LX 37 S GVPX 38 RFX 39 GSGSGTX 4 0 X 41 X 42 LX 43 ISX 44 X 45 EAEDX 46 X 47 X 48 YY C X 49 QX 50 X 51 X 52 X 53 PX 54 T FGX 55 GTKLEX 56 KR (SEQ ID NO: 13)

[0077] Wherein

[0078] X 1 Is D or Q; X 2is I or L; X 3 is L or A; X 4 is T or L; X 5 is L or M; X 6 is T or S; X 7 is F or P; X 8 is Q or E; X 9 is P or K; X 10 is A or V; X 11 is S or T; X 12 is I or M; X 13 is S or T; X 14 is K or S; X 15 is S or V; X 16 is Q or S; X 17 is L or absent; X 18 is L or absent; X 19 is D or absent; X 20 is S or absent; X 21 is D or absent; X 22 is G or absent; X 23 is K or V; X 24 is T or G; X 25 is F or S; X 26 is L or M; X 27 is N or Y; X 28 is L or Y; X 29 is L or Q; X 30 is R or K; X 31 is G or R; X 32 is Q or S; X 33 is R or P; X 34 is L or W; X 35 is V or T; X 36 is K or N; X 37 is D or A; X 38 is D or P; X 39是 T or S; X 40 is D or S; X 41 is F or Y; X 42 is T or S; X 43 is K or T; X 44 is R or S; X 45 is V or M; X 46 is L or A; X 47 is G or A; X 48 is V or T; X 49 is W or Q; X 50 is G or W; X 51 is T or S; X 52 is H or S; X 53 is F or N; X 54is W or P; X 55 is G or A; and X 56 is I or L.

[0079] In one instance, VH comprises or consists of a CDR1 sequence selected from GYSFTGYFMH (SEQ ID NO: 14) or GYTFTDYGMN (SEQ ID NO: 15).

[0080] In one instance, VH comprises or consists of a CDR2 sequence selected from RINPYNGDIRYNQNFKD (SEQ ID NO: 16) or WINTYTGKPTYDDDFKG (SEQ ID NO: 17).

[0081] In one instance, VH comprises or consists of a CDR3 sequence selected from LNFAY (SEQ ID NO: 18) or RFLNTVAGRSVYFDY (SEQ ID NO: 19).

[0082] In one instance, VL comprises or consists of a CDR1 sequence selected from KSSQSLLDSDGKTFLN (SEQ ID NO: 20) or SVSSSVGSMY (SEQ ID NO: 21).

[0083] In one instance, VL comprises or consists of a CDR2 sequence selected from LVSKLDS (SEQ ID NO: 22) or LTSNLAS (SEQ ID NO: 23).

[0084] In one instance, VL comprises or consists of a CDR3 sequence selected from WQGTHFPWT (SEQ ID NO: 24) or QQWSSNPPT (SEQ ID NO: 25).

[0085] The present invention also provides a HER2-binding protein comprising a heavy chain variable region sequence (VH), the heavy chain variable region sequence having CDR1, CDR2, and CDR3 sequences that respectively comprise or consist of the following:

[0086] (i) SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18; or

[0087] (ii) SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0088] The present invention also provides a HER2-binding protein comprising a light chain variable region sequence (VL), the light chain variable region sequence having CDR1, CDR2, and CDR3 sequences that respectively comprise or consist of the following:

[0089] (i) SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24; or

[0090] (ii) SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25;

[0091] In one instance, the HER2-binding protein comprises CDRs having a sequence comprising or consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and / or SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24.

[0092] In one instance, the HER2-binding protein comprises CDRs having a sequence comprising or consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and / or SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25.

[0093] In one instance, the HER2-binding protein comprises a VH and / or a VL, the VH comprises a sequence having at least 55% identity to the sequence shown in SEQ ID NO: 2, the VL comprises a sequence having at least 50% identity to the sequence shown in SEQ ID NO: 3, or a humanized, chimeric, or deimmunized form thereof.

[0094] In one instance, the HER2-binding protein comprises a VH and / or a VL, the VH comprises a sequence having at least 55% identity to the sequence shown in SEQ ID NO: 4, the VL comprises a sequence having at least 50% identity to the sequence shown in SEQ ID NO: 5, or a humanized, chimeric, or deimmunized form thereof.

[0095] In one instance, the VH comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 2 or SEQ ID NO: 4.

[0096] In one instance, the VL comprises a sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 3 or SEQ ID NO: 5.

[0097] The present invention also provides a HER2-binding protein comprising or consisting of the following:

[0098] (i)VH shown in SEQ ID NO: 2 and VL shown in SEQ ID NO: 3; or

[0099] (ii)VH shown in SEQ ID NO: 4 and VL shown in SEQ ID NO: 5.

[0100] In one example, the HER2-binding protein is an antigen-binding fragment selected from the following:

[0101] (i) Single-chain Fv fragment (scFv);

[0102] (ii) Dimeric scFv (di-scFv);

[0103] (iii) At least one of (i) and / or (ii) linked to a heavy-chain constant region or Fc or heavy-chain constant domain (CH)2 and / or CH3; or

[0104] (iv) At least one of (i) and / or (ii) linked to a protein that enhances the antibody half-life (e.g., human serum albumin (HSA)).

[0105] In another example of the present invention, VL and VH are in separate polypeptide chains. For example, the HER2-binding protein is:

[0106] (i) Bispecific antibody;

[0107] (ii) Trispecific antibody;

[0108] (iii) Tetraspecific antibody;

[0109] (iv) Fab;

[0110] (v) F(ab′)2;

[0111] (vi) Fv; or

[0112] (vii) At least one of (i) to (vi) linked to a heavy-chain constant region or Fc or heavy-chain constant domain (CH)2 and / or CH3; or

[0113] (viii) At least one of (i) to (vi) linked to a protein that enhances the antibody half-life (e.g., HSA).

[0114] The present invention also provides a chimeric antibody comprising VH and VL as described herein, wherein VH is linked to a heavy-chain constant region and VL is linked to a light-chain constant region.

[0115] The present invention also provides a chimeric antibody comprising VH and VL as described herein, wherein VH is linked to a human heavy-chain constant region and VL is linked to a human light-chain constant region.

[0116] Based on the disclosure herein, it will be apparent to those skilled in the art that the HER2-binding proteins of the present invention include human, humanized, human-like, chimeric, and primatized proteins.

[0117] The antibodies of the present invention can belong to any class, including IgM, IgG, IgE, IgA, IgD, or subclasses. Exemplary subclasses of IgG are IgG1, IgG2, IgG3, and IgG4.

[0118] In one instance, the HER2-binding protein is recombinant. In one instance, the HER2-binding protein is synthetic.

[0119] The present invention also provides anti-idiotypic antibodies or antigen-binding fragments thereof that are capable of binding to mAb104 or mAb106.

[0120] In one instance, the HER2-binding protein or antibody of the present invention is conjugated to another moiety. The moiety can be a detectable or functional moiety. For example, the moiety is selected from the group consisting of radioisotopes, detectable labels, therapeutic compounds, colloids, toxins, nucleic acids, peptides, proteins, drugs, compounds that increase the half-life of the HER2-binding protein in a subject, and mixtures thereof. In certain instances, the moiety can be selected from immunoglobulins or fragments or portions of immunoglobulins, therapeutic compounds (e.g., chemotherapy), drugs or bioactive agents, toxins, or radionuclides. Alternatively, the moiety can include siRNA, DNAzyme, or ribozyme. Combinations of any of the foregoing moieties are also included in the present invention. In one instance, the HER2-binding protein is an antibody-drug conjugate. In another instance, the antibody-drug conjugate includes the HER2-binding protein of the present invention linked to monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), pyrrolobenzodiazepine (PBD), or emtansine (DM1). In certain instances, the linkage is achieved through a linking chemistry. In one instance, the drug is conjugated to the HER2-binding protein via a cysteine or lysine residue present in the HER2-binding protein. In other instances, the linkage is via a linker known in the art (e.g., a G-S linker). In another instance, the antibody-drug conjugate is capable of internalizing upon binding to the HER2 receptor on tumor cells. The present invention also extends to compositions comprising such conjugates as described herein.

[0121] The serum half-life of a binding protein or antibody can be increased, for example, by incorporating a salvage receptor binding epitope into the antibody as described in US 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule. In another example, the half-life is increased by pegylation.

[0122] The invention also provides an isolated nucleic acid encoding the HER2 binding protein or antibody of the invention.

[0123] The invention also provides an expression construct comprising the nucleic acid of the invention operably linked to a promoter. Such an expression construct can be in a vector such as a plasmid.

[0124] In an example of the disclosure involving a single polypeptide HER2 binding protein, the expression construct can comprise a promoter linked to the nucleic acid encoding the polypeptide chain.

[0125] In an example involving multiple polypeptides forming a HER2 binding protein, the expression construct of the invention comprises a nucleic acid encoding one of the polypeptides (e.g., comprising VH) operably linked to a promoter and a nucleic acid encoding another of the polypeptides (e.g., comprising VL) operably linked to another promoter.

[0126] In another example, the expression construct is a bicistronic expression construct, for example comprising the following components operably linked in a 5′ to 3′ order:

[0127] (i) a promoter

[0128] (ii) a nucleic acid encoding a first polypeptide;

[0129] (iii) an internal ribosome entry site; and

[0130] (iv) a nucleic acid encoding a second polypeptide.

[0131] For example, the first polypeptide comprises VH and the second polypeptide comprises VL, or the first polypeptide comprises VL and the second polypeptide comprises VH.

[0132] The invention also contemplates separate expression constructs, one encoding a first polypeptide (e.g., comprising VH and optionally a heavy chain constant region or a portion thereof) and the other encoding a second polypeptide (e.g., comprising VL and optionally a light chain constant region). For example, the invention also provides a composition comprising:

[0133] (i) a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising VH operably linked to a promoter); and

[0134] (ii) A second expression construct, which comprises a nucleic acid encoding a polypeptide (e.g., comprising a VL operably linked to a promoter),

[0135] wherein the first and second polypeptides associate to form the HER2-binding protein of the invention.

[0136] The invention further provides an isolated cell expressing the HER2-binding protein or antibody of the invention or a recombinant cell genetically modified to express the HER2-binding protein or antibody of the invention. In one example, the cell is an isolated hybridoma cell. In another example, the cell comprises the nucleic acid or expression construct of the invention or:

[0137] (i) A first expression construct, which comprises a nucleic acid encoding a polypeptide (e.g., comprising a VH) operably linked to a promoter; and

[0138] (ii) A second expression construct, which comprises a nucleic acid encoding a polypeptide (e.g., comprising a VL) operably linked to a promoter,

[0139] wherein the first and second polypeptides associate to form the HER2-binding protein or antibody of the invention.

[0140] The invention also provides a composition comprising the HER2-binding protein or nucleic acid or expression construct or cell of the invention and a suitable carrier. In one example, the composition comprises the HER2-binding protein of the invention.

[0141] In one example, the carrier is pharmaceutically acceptable.

[0142] The composition of the invention can be administered alone or in combination with other treatments, therapeutic agents or medicaments, simultaneously / parallelly or sequentially. In one example, the HER2-binding protein or composition of the invention is administered in combination with pertuzumab or trastuzumab. In another example, the HER2-binding protein or composition of the invention is administered in combination with a tyrosine kinase inhibitor (e.g., lapatinib). It is also contemplated that the HER2-binding protein or composition herein is administered simultaneously or sequentially with an anti-cancer therapy (e.g., chemotherapy or radiotherapy). In another example, the HER2-binding protein or composition described herein is administered simultaneously or successively with an immunotherapeutic agent or immunomodulator.

[0143] The HER2-binding protein of the invention can be used for treatment, diagnosis or detection. In some examples, the HER2-binding protein is linked to a chemotherapeutic agent used for theranostics.

[0144] The invention also provides a diagnostic agent comprising the HER2-binding protein described herein conjugated to a detectable label. In one example, the diagnostic agent is used for in vivo or in vitro detection of tumor cells expressing HER2.

[0145] In one instance, a diagnostic agent can be used to detect the presence of tumor cells expressing HER2 in a subject or in a biological sample obtained from a subject having or suspected of having a HER2-positive tumor. Examples of detectable labels include various enzymes, cofactors, fluorescent materials, luminescent materials, electron-dense labels, MRI labels, and radioactive materials.

[0146] The present invention also provides a HER2-binding protein or diagnostic agent as described herein for histological examination of a biological sample. Methods for preparing histological samples are well known to those skilled in the art.

[0147] The present invention also provides a method for treating or preventing cancer expressing HER2 in a subject, the method comprising administering to the subject a HER2-binding protein or nucleic acid or expression construct or cell or composition of the present invention. In one instance, the subject is a subject having cancer such as breast cancer.

[0148] In one instance, the method comprises administering to the subject an antibody or a humanized or deimmunized form thereof, the antibody comprising a VH containing the sequence shown in SEQ ID NO: 2 and / or a VL containing the sequence shown in SEQ ID NO: 3.

[0149] In one instance, the method comprises administering to the subject an antibody or a humanized or deimmunized form thereof, the antibody comprising a VH containing the sequence shown in SEQ ID NO: 4 and / or a VL containing the sequence shown in SEQ ID NO: 5.

[0150] The present invention also provides a HER2-binding protein or nucleic acid or expression construct or cell or composition of the present invention for use in a medicament.

[0151] The present invention further provides a HER2-binding protein or nucleic acid or expression construct or cell or composition of the present invention for treating a cell proliferative disorder expressing HER2.

[0152] In one instance, the present invention provides a method for treating a cell proliferative disorder expressing HER2, the method comprising administering to a subject in need thereof a HER2-binding protein or nucleic acid or expression construct or cell or composition of the present invention. In one instance, the cancer is selected from the group consisting of breast cancer, gastric cancer, gastroesophageal cancer, colon cancer, and squamous cell carcinoma.

[0153] In one instance, the HER2-binding protein is administered to the subject in a therapeutically effective amount.

[0154] Preferably, the subject is a human.

[0155] The present invention also provides the use of the HER2-binding protein or nucleic acid or expression construct or cell of the present invention in the preparation of a medicament for treating cancers expressing HER2.

[0156] The present invention further provides a method for detecting HER2 in a biological sample, the method comprising contacting the sample with the HER2-binding protein or antibody of the present invention to form an antigen-protein complex and detecting the complex, wherein detection of the complex indicates HER2 expression in the sample.

[0157] The present invention also provides a vaccine antigen comprising the sequence according to SEQ ID NO: 1 or consisting thereof, and a pharmaceutically acceptable carrier for generating human HER2 antibodies.

[0158] The present invention also provides a method for generating a HER2 / ErbB2-binding protein, which comprises immunizing a rodent with a cyclic peptide comprising the sequence H-GCPLHNQEVTAEDGTQRC-NH 2 (SEQ ID NO: 26); obtaining a hybridoma cell line from the B cells of the immunized rodent and purifying the antibody from the hybridoma cell line. In one example, the peptide is cyclized by a disulfide bond. In one example, the peptide is cyclized by a disulfide bond between the side chains of Cys2 and Cys18. In another example, the peptide is linked to keyhole limpet hemocyanin (KLH) protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] Figure 1 : The full-length protein sequence of human receptor tyrosine-protein kinase HER2 comprising a 22-amino acid leader sequence. The peptide epitope bound by mAb104 within the cysteine-rich domain II is shown in underlined bold text.

[0160] Figure 2 : Comparison by ELISA of the binding of 10 μg / mL of (A) mAb104, (B) mAb105, (C) mAb106, and (D) mAb107 to the extracellular domain of HER2, to cyclic and linear polypeptide immunogens linked to keyhole limpet hemocyanin (KLH) that generated the antibodies, or to a control irrelevant peptide linked to KLH. The binding activity of triplicate samples was measured by absorbance readings of the optical density at 405 nm using a Versamax microplate reader (Molecular Devices), and determined using Softmax Pro 4.8 software and mean ± SD. mAb104 (A) and mAb106 (C) showed the strongest binding activity to all immobilized HER2 forms, while mAb105 (B) showed the weakest binding. Specificity was confirmed by lack of binding to the control peptide. Results represent two independent experiments.

[0161] Figure 3: Antibodies that bind to cell lysates by western blot. SK-BR-3, BT-474, MDA-MB-453, and NCI-N87 cells were washed, lysed, and immunoblotted for endogenous HER2 (commercial positive control antibody 2242, Cell Signaling Technology, Beverly, MA), mAb104, mAb105, mAb106, mAb107. Results represent two independent experiments.

[0162] Figure 4 : CDRs (shown in bold and underlined) of the heavy and light chains of mAb104 (A and B) and mAb106 (C and D) are designated respectively. CDRs are named by Kabat and Chothia numbering.

[0163] Figure 5 : Comparison of the binding of HER2 antibodies (A) trastuzumab (B) mAb106 and (C) mAb104 to HER2 ECD was detected by surface plasmon resonance using a BIAcore T200 biosensor at antibody concentrations from 320 μg / mL to 10 μg / mL (2133 to 66 nM). Traces represent the binding and dissociation of the antibody in solution to immobilized recombinant HER2 ECD. Results represent two or more experiments

[0164] Figure 6 : ELISA-based HER2-ECD binding competition assay (A) Trastuzumab and pertuzumab do not affect mAb104 binding (B) mAb104 does not affect trastuzumab binding (C) mAb104 partially affects pertuzumab binding to recombinant HER2 ECD immobilized on the microtiter plate. (Data; mean ± SE; n = 3) Results represent two experiments.

[0165] Figures 7-1 to 7-4 : FACS-based competition assay. Pre-incubation with 10-fold excess of mAb104 (100 μg / mL) does not affect the binding of 10 μg / mL trastuzumab or pertuzumab to HER2 on the surface of cancer cells in BT-474 cells (A and B), SK-BR-3 cells (C and D), NCI-N87 cells (E and F), or OE-19 cells (G and H). Results represent two or more experiments.

[0166] Figure 8-1 : Lysates of cancer cell lines were separated on 4% SDS-PAGE and blotted with (A) mAb104, (B) anti-HER2, and (C) anti-HER3. GAPDH was used as a loading control for protein normalization. Lane 1: molecular weight marker. Results represent three experiments.

[0167] Figure 8-2. ELISA analysis. Specificity of mAb104 (3 - 10,000 ng / mL) coated on ELISA plates was bound with recombinant sEGFR extracellular domain or ECD of HER2, HER3 or HER4. Controls included separate pNPP substrate and secondary anti - mouse antibody - alkaline phosphatase conjugate. (Data; mean ± SE; n = 3).

[0168] Figures 9-1 to 9-3 : Effects of mAb104 (A and B) alone or in combination with trastuzumab (C and D) or pertuzumab (E and F) on the in vitro growth of SK - BR - 3 (A, C and E) or BT - 474 cells (B, D and F), as measured by MTS assay. Cells were incubated with mAb104, trastuzumab, pertuzumab, isotype control as single therapies or in combination with trastuzumab + pertuzumab, trastuzumab + mAb104 or pertuzumab + mAb104 in serum - free medium for 5 - 7 days. Viable cell numbers were determined at baseline and at the end of the experiment. Results are expressed as mean ± SD, n = 3. Data represent two or more independent experiments. * mAb014, pertuzumab and isotype control antibody had no anti - proliferative effects and overlapped with each other.

[0169] Figure 10-1 and 10-2 : mAb104 did not affect downstream signal transduction of the MAPK pathway and Akt in (A) SK - BR - 3 and (B) BT474 cells in vitro when used as single therapy (A and B) or in combination with trastuzumab or pertuzumab (C and D). Cells were incubated in serum - free medium and treated with 100 μg / mL of mAb104, trastuzumab, pertuzumab alone or in combination for 24 h before whole - cell lysis. Equal amounts of lysates were then loaded and separated on 4 - 12% gels and then transferred to nitrocellulose membranes. Membranes were immunoblotted as shown. Results represent two experiments.

[0170] Figure 11-1 and 11-2 : mAb104 did not affect ligand - dependent phosphorylation of the MAPK pathway and Akt in (A) SK - BR - 3 and (B) BT474 cells in vitro when used as single therapy (A and B) or in combination with trastuzumab or pertuzumab (C and D). Cells were incubated in serum - free medium, treated with 100 μg / mL of mAb104, trastuzumab and pertuzumab alone or in combination for 24 h, and then 100 ng EGF was added for 10 min. After whole - cell lysis, equal amounts of lysates were loaded and separated on 4 - 12% gels and then transferred to nitrocellulose membranes. Membranes were immunoblotted as shown. Results represent two experiments.

[0171] Figures 12-1 to 12-4 : After treating BT474 cells (A to H) and SK-BR-3 cells (I to P) with trastuzumab, pertuzumab, and mAb104 as single therapies and in combination (total antibody in all groups was 0.1 mg / mL) for 4 h, the effects of the treatments on breast cancer cell viability and apoptosis were evaluated by flow cytometry analysis with annexin-V and propidium iodide (PI) staining.

[0172] Figure 13 : Antitumor effect of mAb104 in BT-474 breast cancer xenografts. Mice (n = 5) were treated with 1 mg mAb104, trastuzumab, pertuzumab, and isotype control. The tumor volume at the start of treatment was 100 - 120 mm 3 . Data shown in the growth curves represent mean tumor volume ± S.E. Tumors in each treatment group were significantly smaller than the control group * p < 0.001 for control vs. mAb104; ** p < 0.0001 for control vs. trastuzumab and pertuzumab.

[0173] Figure 14 : Antitumor effect of mAb104 in BT-474 xenografts. Mice (n = 5 / group) were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, and isotype control. The tumor volume at the start of treatment was 120 - 150 mm 3 . Data shown in the growth curves represent mean tumor volume ± S.E. * p < 0.01, control vs. mAb104 treatment group.

[0174] Figures 15-1 to 15-3 : (A) Antitumor effect of mAb104 in HER2-positive breast PDX models. Mice (n = 5 / group) were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, and isotype control. The tumor volume at the start of treatment was 100 - 120 mm 3 . (B) Antitumor effect of the combination of mAb104 and trastuzumab in BT-474 xenografts. Mice (n = 5) were treated with a total dose of 0.5 mg of mAb104 + trastuzumab, trastuzumab + pertuzumab, or isotype control. The tumor volume at the start of treatment was 120 - 150 mm 3 . * p < 0.0001 for control vs. trastuzumab / mAb104. (c) Antitumor effect of the combination of mAb104 and trastuzumab in HER2-positive breast PDX models. Mice (n = 5) were treated with a total dose of 0.5 mg of mAb104 + trastuzumab, trastuzumab + pertuzumab, or isotype control. The tumor volume at the start of treatment was 120 - 150 mm3 . * p < 0.0001, control group vs. mAb104; ** p < 0.001, trastuzumab vs. trastuzumab / mAb104. Data shown in the growth curves of groups A - C represent mean tumor volume ± S.E.

[0175] Figure 16-1 and 16-2 : The following effects on BT - 474 xenograft tumors were evaluated by immunohistochemistry: anti - HER2 monotherapy (0.5 mg dose) on (A) proliferation by Ki67, (B) downstream signal transduction by staining phosphorylated - Akt, (C) effect on vasculature by staining podoplanin; or the combination of mAb104 with trastuzumab (0.5 mg total protein dose) on (D) proliferation by Ki67, (E) downstream signal transduction by staining phosphorylated - Akt, (F) effect on vasculature by staining podoplanin. * p < 0.001 control group vs. trastuzumab.

[0176] Figure 17 : As determined by MTS assay, mAb104 did not inhibit the growth of (A) NCI - N87 and (B) OE19 gastric cancer cells in vitro. Cells were incubated with mAb104, trastuzumab, pertuzumab, isotype control as monotherapy (A and B) in serum - free medium for 5 - 7 days. Viable cell numbers were determined at baseline and at the end of the experiment. Results are expressed as mean ± SD, n = 3. Data represent two or more independent experiments. * p < 0.0001, control vs. trastuzumab.

[0177] Figure 18 : As determined by MTS assay, mAb104 did not inhibit the growth of (A) NCI - N87 and (B) OE19 gastric cancer cells in vitro. Cells were incubated with the combination of mAb104 with trastuzumab + pertuzumab, trastuzumab + mAb104, or pertuzumab + mAb104 in serum - free medium for 5 - 7 days. Viable cell numbers were determined at baseline and at the end of the experiment. Results are expressed as mean ± SD, n = 3. Data represent two or more independent experiments. * p ≤ 0.005, compared with control

[0178] Figure 19-1 and 19-2: When used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), mAb104 does not affect downstream signal transduction of the MAPK pathway and Akt in (A) NCI-N87 and (B) OE-19 cells in vitro. Cells were incubated in serum-free medium and treated with 100 μg / mL of mAb104, trastuzumab, pertuzumab alone or in combination with trastuzumab or pertuzumab for 24 h before whole cell lysis. Equal amounts of lysates were then loaded and separated on a 4-12% gel and then transferred to a nitrocellulose membrane. The membrane was immunoblotted as shown. Results represent two experiments.

[0179] Figure 20-1 and 20-2 : When used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), mAb104 does not affect ligand-dependent phosphorylation of the MAPK pathway and Akt in (A) NC-N87 and (B) OE-19 cells in vitro. Cells were incubated in serum-free medium, treated with 100 μg / mL of mAb104, trastuzumab, and pertuzumab alone or in combination for 24 h, and then 100 ng EGF was added for 10 min. After whole cell lysis, equal amounts of lysates were loaded and separated on a 4-12% gel and then transferred to a nitrocellulose membrane. The membrane was immunoblotted as shown. Results represent two experiments.

[0180] Figure 21-1 and 21-2 : After treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy and in combination (A and B) in NCI-N87 gastric cancer and (C) OE-19 esophageal cancer cells for 4 h, the effects of the treatment on cancer cell viability and apoptosis were evaluated by propidium iodide (PI) staining.

[0181] Figure 22 : Confluent OE-19 cells were used for migration assays. Images were collected at 0 h and 90 h of incubation with 0 or 100 μg / mL antibody. The antibody did not delay the migration of OE-19 cells at 90 h post-treatment compared to the control antibody at a dose of 100 μg / mL.

[0182] Figure 23 : Antitumor effect of mAb104 in NCI-N87 xenografts. Mice (n = 5) were treated with 1 mg mAb104, trastuzumab, pertuzumab, isotype control or left untreated. Tumor volume at the start of treatment was 100-120 mm 3 . Data shown in the growth curve represent mean tumor volume ± S.E. * p ≤ 0.01, control vs. mAb104.

[0183] Figure 24 : Antitumor effect of mAb104 in NCI-N87 xenografts. Mice (n = 5) were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, isotype control or left untreated. The tumor volume at the start of treatment was 100 - 120 mm 3 . Growth curves (A) and survival curves (B) are shown. Data shown in the growth curves represent mean tumor volume ± S.E. The endpoint for survival analysis was a tumor volume > 1000 mm 3 or moribund state. * p < 0.001, control vs mAb104; ** p < 0.0001 control vs treatment groups.

[0184] Figure 25 : Antitumor effect of mAb104 in NCI-N87 xenografts. Mice (n = 5) were treated with 0.1 mg mAb104, trastuzumab, pertuzumab, isotype control or left untreated. The tumor volume at the start of treatment was 100 - 120 mm 3 . Growth curves (A) and survival curves (B) are shown. Data shown in the growth curves represent mean tumor volume ± S.E. The endpoint for survival analysis was a tumor volume > 1000 mm 3 or moribund state. * p ≤ 0.001, control vs mAb104; ** p < 0.0002 control vs treatment groups.

[0185] Figure 26 : Antitumor effect of mAb104 in OE-19 xenografts. Mice (n = 5) were treated with 1 mg mAb104, trastuzumab, pertuzumab, isotype control or left untreated. The tumor volume at the start of treatment was 100 - 120 mm 3 . Data shown in the growth curves represent mean tumor volume ± S.E. * p ≤ 0.0001, control vs mAb104.

[0186] Figure 27 : Antitumor effect of mAb104 in OE-19 xenografts. Mice (n = 5) were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, isotype control. The tumor volume at the start of treatment was 100 - 120 mm 3 . Growth curves (A) and survival curves (B) are shown. Data shown in the growth curves represent mean tumor volume ± S.E. The endpoint for survival analysis was a tumor volume > 1000 mm 3 or moribund state. * p < 0.001, control vs trastuzumab.** p < 0.006, control vs. treatment group.

[0187] Figure 28 : Antitumor effect of the combination of mAb104 and trastuzumab in NCI-N87 xenografts. Mice (n = 5) were treated with a total dose of 0.5 mg of mAb104 + trastuzumab, trastuzumab + pertuzumab, or isotype control. Tumor volume at the start of treatment was 100 - 120 mm 3 The data shown in the growth curves represent the mean tumor volume ± S.E. * p < 0.0001, control vs. mAb104; ** p < 0.001, trastuzumab vs. trastuzumab / mAb104.

[0188] Figure 29 : Antitumor effect of the combination of mAb104 and trastuzumab in OE-19 xenografts. Mice (n = 5) were treated with a total dose of 0.5 mg of mAb104 + trastuzumab, trastuzumab + pertuzumab, or isotype control. Tumor volume at the start of treatment was 100 - 120 mm 3 Growth curves (A) and survival curves (B) are shown. The data shown in the growth curves represent the mean tumor volume ± S.E. The endpoint of the survival analysis was a tumor volume > 1000 mm 3 or moribund state. * p < 0.0001, control vs. trastuzumab / mAb104; ** p < 0.0001, control vs. trastuzumab / pertuzumab; *** p < 0.0001, trastuzumab vs. trastuzumab / mAb104; ± p < 0.0005, control vs. treatment group.

[0189] Figure 30-1 and 30-2 : Effects of anti-HER2 monotherapy (0.5 mg dose) (A - C) or in combination with trastuzumab (D - F) on (A and D) Ki67 proliferation, (B and E) downstream signal transduction by phosphorylated-Akt staining, and (C and F) the vasculature by podoplanin staining in NCI-N87 xenograft tumors were evaluated by immunohistochemistry.

[0190] Figure 31-1 and 31-2: Evaluation of the effect of OE-19 xenograft tumor anti-HER2 monotherapy (0.5 mg dose) (A to C) or in combination with trastuzumab (D to F) on (A and D) proliferation by Ki67; (B and E) downstream signal transduction by phosphorylated-Akt staining and (C and F) effect on vasculature by podoplanin staining.

[0191] Figure 32 : For determination of 89 The binding assay of the immunoreactive fraction of the 89 Zr-labelled anti-HER antibody. A) shows a conventional plot of specific binding to total applied radioactivity against increasing cell concentration. B) and C) are double-reciprocal plots of the same data as in A), allowing determination of the immunoreactive fraction for conditions representative of infinite antigen excess.

[0192] Figure 33 : Scatchard plot of the binding in A) 89 Zr-labelled mAb104 and B) 89 Zr-labelled Herceptin / Trastuzumab binding to NCI-N87 gastric carcinoma cells. The abscissa represents the concentration of specifically bound antibody, and the ordinate is the ratio of the concentration of specifically bound antibody to the concentration of free reactive antibody. The binding capacity per cell is determined by the intercept value at the abscissa, and the association constant is determined by the slope of the line.

[0193] Figure 34 . A) Biodistribution of Zr-labelled mAb104 in mice bearing NCI-N87 gastric carcinoma xenografts overexpressing HER2. B) Biodistribution of Zr-labelled mAb104 and isotype control in blood and tumors of mice bearing NCI-N87 xenografts. High specific tumor uptake was demonstrated with mAb104. (Data mean ± SEM, n = 5).

[0194] Sequence Listing Key

[0195] SEQ ID NO:1: HER2 / ErbB2 epitope sequence

[0196] SEQ ID NO:2: VH of mAb104

[0197] SEQ ID NO:3: VL of mAb104

[0198] SEQ ID NO:4: VH of mAb106

[0199] SEQ ID NO:5: VL of mAb106

[0200] SEQ ID NO:6: Consensus sequence of VH CDR1

[0201] SEQ ID NO:7: Consensus sequence of VH CDR2

[0202] SEQ ID NO:8: Consensus sequence of VH CDR3

[0203] SEQ ID NO:9: Consensus sequence of VL CDR1

[0204] SEQ ID NO:10: Consensus sequence of VL CDR2

[0205] SEQ ID NO:11: Consensus sequence of VL CDR3

[0206] SEQ ID NO:12: Consensus sequence of VH

[0207] SEQ ID NO:13: Consensus sequence of VL

[0208] SEQ ID NO:14: VH CDR1 of mAb104 SEQ ID NO:15: VH CDR1 of mAb106 SEQ ID NO:16: VH CDR2 of mAb104 SEQ ID NO:17: VH CDR2 of mAb106 SEQ ID NO:18: VH CDR3 of mAb104 SEQ IDNO:19: VH CDR3 of mAb106 SEQ ID NO:20: VL CDR1 of mAb104 SEQ ID NO:21: VLCDR1 of mAb106 SEQ ID NO:22: VL CDR2 of mAb104 SEQ ID NO:23: VL CDR2 of mAb106 SEQ ID NO:24: VL CDR3 of mAb104 SEQ ID NO:25: VL CDR3 of mAb106 SEQ ID NO:26: Cyclic peptide sequence for immunization SEQ ID NO:27: Sequence of HER2 / ErbB2

[0209] SEQ ID NO:28: Light chain primer sequence

[0210] SEQ ID NO:29: Light chain primer sequence

[0211] SEQ ID NO:30: Light chain primer sequence

[0212] SEQ ID NO:31: Light chain primer sequence

[0213] SEQ ID NO:32: Light chain primer sequence

[0214] SEQ ID NO:33: Light chain primer sequence

[0215] SEQ ID NO:34: Light chain primer sequence

[0216] SEQ ID NO:35: Light chain primer sequence

[0217] SEQ ID NO:36: Light chain primer sequence

[0218] SEQ ID NO:37: Light chain primer sequence

[0219] SEQ ID NO:38 Light chain primer sequence

[0220] SEQ ID NO:39: Light chain primer sequence

[0221] SEQ ID NO:40: Light chain primer sequence

[0222] SEQ ID NO:41: Heavy chain primer sequence

[0223] SEQ ID NO:42: Heavy chain primer sequence

[0224] SEQ ID NO:43: Heavy chain primer sequence

[0225] SEQ ID NO:44: Heavy chain primer sequence

[0226] SEQ ID NO:45: Heavy chain primer sequence

[0227] SEQ ID NO:46: Heavy chain primer sequence

[0228] SEQ ID NO:47: Heavy chain primer sequence

[0229] SEQ ID NO:48: Heavy chain primer sequence

[0230] SEQ ID NO:49: Heavy chain primer sequence

[0231] SEQ ID NO:50: Heavy chain primer sequence

[0232] SEQ ID NO:51: Heavy chain primer sequence

[0233] SEQ ID NO:52: Heavy chain primer sequence

[0234] SEQ ID NO:53: Heavy chain primer sequence

[0235] SEQ ID NO:54: Light chain primer sequence

[0236] SEQ ID NO:55: Light chain primer sequence Detailed implementation manners

[0237] General Terms

[0238] Throughout the specification, unless otherwise specifically stated or the context otherwise requires, reference to a single step, composition of matter, group of steps, or group of compositions of matter shall be considered to include one and more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter.

[0239] Those skilled in the art will understand that the present invention is susceptible to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions, and compounds mentioned or pointed out individually or jointly in this specification, and any and all combinations of any two or more of said steps or features.

[0240] The present invention is not limited to the scope of the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

[0241] Unless otherwise specifically stated, necessary changes should be made thereto and then applied to any other instances of the present invention.

[0242] Unless otherwise clearly defined, all technical and scientific terms used herein shall be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0243] Unless otherwise indicated, the recombinant proteins, recombinant DNA techniques, molecular biology, microbiology, cell culture, and immunology techniques used in this disclosure are standard methods well known to those skilled in the art. These techniques are described and explained in the following source documents, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al., (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al., (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0244] The descriptions and definitions of variable regions and their parts, immunoglobulins, antibodies and their fragments in this text can be further clarified by the discussions in Kabat, 1987 and / or 1991, Bork et al., 1994 and / or Chothia and Lesk, 1987 and / or 1989 or Al-Lazikani et al., 1997 or the IMGT numbering of Lefranc M.-P., (1997) Immunology 5Today 18, 509.

[0245] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean including the stated element, integer or step, or group of elements, integers or steps, but not excluding any other element, integer or step, or group of elements, integers or steps.

[0246] As used herein, the term "derived from" shall be used to indicate that the specified integer can be obtained from a particular source, although not necessarily directly from that source.

[0247] In the context of a peptide sequence, the terms "consisting of" or "consisting essentially of" refer to a peptide sequence having a determined number of residues that is not covalently linked to a larger product.

[0248] Unless otherwise specifically stated, any example herein should be understood to apply mutatis mutandis to any other example.

[0249] Selected Definitions

[0250] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. The terms "a (or an)" and the terms "one or more" and "at least one" are used interchangeably herein.

[0251] In addition, "and / or" as used herein should be regarded as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" used in a phrase such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0252] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in connection with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Generally, the term "about" is used herein to change a numerical value above and below the value by up to 10% (%).

[0253] It is to be understood that the HER2 binding proteins and antibodies, nucleic acids, cells, and vectors of the present invention are in isolated form. "Isolated" means a polypeptide, antibody, polynucleotide, vector, or cell that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, or cells include those that have been purified to the extent that they are no longer in their naturally occurring form. In some aspects, the isolated antibodies, polynucleotides, vectors, or cells are substantially pure. In some aspects, the isolated antibodies, polynucleotides, vectors, or cells are "recombinant".

[0254] The term "HER2" as used herein is to be understood to mean Figure 1 the human HER2 receptor as shown in, particularly, domain II of the HER2 receptor represented by amino acid residues 190 - 269 of the wild-type HER2 sequence (Coussens L et al., (1985) Science 230(4730):1132 - 9). The term HER2 may be used interchangeably with ErbB2.

[0255] The term "abnormal expression" or "abnormally expressed" is intended to include a state in which there is an abnormal (usually increased) amount / level of a protein, regardless of the direct cause of the abnormal amount or level. Abnormal expression includes and contemplates any situation or alteration in which the protein expression or post-translational modification mechanism in a cell is affected or otherwise disrupted due to enhanced expression or an increased level or amount of the protein, including situations in which an altered protein, such as a mutant protein or a variant caused by a sequence change, deletion, insertion, or altered folding, is expressed. In the present invention, abnormal expression is associated with HER2 expression observed in neoplastic, hyperproliferative, or abnormal cells rather than wild-type or normal cells.

[0256] As used herein, the term "affinity" refers to the strength of binding of a single molecule to its ligand and is typically expressed as the equilibrium dissociation constant (KD) of the reversible binding of two reagents. It is determined by the Koff / Kon ratio between the HER2-binding protein and HER2. KD is inversely related to the affinity. The KD value is related to the concentration of the HER2-binding protein, and thus the lower the KD value (the lower the concentration), the higher the affinity of the binding protein. The affinity of the HER2-binding protein of the present invention for HER2 can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or higher.

[0257] As used herein, the term "binding" with respect to the interaction of a HER2-binding protein with a target means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the target. For example, the HER2-binding protein recognizes and binds to a specific protein structure, rather than proteins in general.

[0258] The term "binding protein" as used herein is intended to describe a member of a pair of molecules that have binding specificity for each other. The members of a specific binding pair can be of natural origin or produced wholly or in part synthetically. One member of the pair of molecules has a region or cavity on its surface that specifically binds to the specific spatial and polar organization of the other member of the pair of molecules and is thus complementary to it. Thus, the members of the pair have the property of specifically binding to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application relates to antigen-antibody type reactions.

[0259] The term "antibody" describes immunoglobulins that are produced naturally or in part or in whole synthetically. The term also includes any polypeptide or protein having a binding domain homologous to an antibody binding domain. The term also includes CDR-grafted antibodies. An "antibody" is any immunoglobulin that binds to a specific epitope, including antibodies and their fragments. The term includes polyclonal, monoclonal, and chimeric antibodies, the last-mentioned antibodies being described in further detail in U.S. Patents Nos. 4,816,397 and 4,816,567. The term "antibody" includes wild-type immunoglobulin (Ig) molecules, which typically comprise four full-length polypeptide chains, two heavy (H) chains and two light (L) chains, or their equivalent Ig homologs (e.g., camelid nanobodies, which comprise only heavy chains); full-length functional mutants, variants, or derivatives thereof that retain the essential epitope-binding characteristics of the Ig molecule, and includes bispecific, bivalent, multispecific, and dual variable domain antibodies; the immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Since antibodies can be modified in a variety of ways, the term "antibody" should be construed to cover any specific binding member or substance having a binding domain of the desired specificity. Thus, the term includes antibody fragments, derivatives, functional equivalents, and homologs of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Thus includes chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and in U.S. Patents Nos. 4,816,397 and 4,816,567. The meaning of the term "antibody" also includes any "antibody fragment".

[0260] "Antibody fragment" refers to a molecule comprising at least one non-full-length polypeptide chain, including (i) Fab fragment, which is a monovalent fragment consisting of variable light chain (VL), variable heavy chain (VH), constant light chain (CL) and constant heavy chain 1 (CH1) domains; (ii) F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) the heavy chain portion of the Fab(Fd) fragment, which consists of VH and CH1 domains; (iv) variable fragment (Fv) fragment, which consists of VL and VH domains of a single arm of an antibody, (v) domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E.S. et al., Nature 341, 544-546 (1989)); (vi) camelid antibody; (vii) isolated complementarity determining region (CDR); (viii) single-chain Fv fragment, in which the VH domain and the VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); (ix) diabody, which is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to pair between the two domains on the same chain, thus forcing the domains to pair with complementary domains on the other chain and generating two antigen-binding sites (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) linear antibody, which comprises a pair of tandem Fv fragments (VH-CH1-VH-CH1), and the tandem Fv fragments together with a complementary light chain polypeptide form a pair of antigen-binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson, J Immunol Methods 242:193-204 9 (2000)); and (xii) other non-full-length portions of the heavy and / or light chains, or mutants, variants or derivatives thereof, alone or in any combination.

[0261] As used herein, the term "antigen-binding fragment" includes Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), VL and VH domain fragments, domain antibodies, trispecific (Fab3), bispecific (Fab2), diabodies ((VL-VH)2 or (VH-VL)2), triabodies (trivalent), tetra-bodies (tetravalent), minibodies ((scFv-CH3)2), bispecific single-chain Fv (Bis-scFv), IgGδCH2, scFv-Fc, and (scFv)2-Fc. A "Fab fragment" consists of the monovalent antigen-binding fragment of an antibody molecule and can be generated by digesting the whole antibody molecule with papain to produce a fragment consisting of the intact light chain and a portion of the heavy chain. The "Fab' fragment" of an antibody molecule can be obtained by treating the intact antibody molecule with pepsin and then reducing it to produce a molecule consisting of the intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained for each antibody molecule treated in this way. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments joined together by two disulfide bonds and is obtained by treating the intact antibody with pepsin without subsequent reduction. An "Fv fragment" is a genetically engineered fragment containing the variable regions of the light and heavy chains expressed as two chains. A "single-chain antibody" (SCA) is a genetically engineered single-chain molecule containing the variable regions of the light and heavy chains joined by a suitable flexible polypeptide linker.

[0262] As used herein, "antibody variable region" refers to the portions of the light and heavy chains of an antibody molecule that include the amino acid sequences of the complementarity-determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the framework regions (FRs). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. According to the methods used in the present invention, the amino acid positions assigned to the CDRs and FRs may be according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or Chotia and Lesk (1987 J. Mol. Biol. 196:901-917). The amino acid numbering of an antibody or antigen-binding fragment is also according to Kabat numbering.

[0263] As used herein, the term "constant region" (CR) refers to the portion of an antibody molecule that confers effector function. The constant regions of the subject humanized antibodies are derived from human immunoglobulins. The heavy chain constant region can be selected from any one of the following five isotypes: α, δ, ε, γ, or μ. In addition, the heavy chains of the various subclasses (e.g., the IgG subclasses of the heavy chain) are responsible for different effector functions, and thus, by selecting the desired heavy chain constant region, antibodies with the desired effector function can be generated. Exemplary heavy chain constant regions are γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4). The light chain constant region can be of the κ or λ type, preferably the κ type.

[0264] The "framework region" (hereinafter referred to as FR) is those variable domain residues other than the CDR residues. Each variable domain of a naturally occurring antibody typically has four FRs that are identified as FR1, FR2, FR3, and FR4.

[0265] As used herein, the term "complementary determining region" (syn CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable domain whose presence is essential for antigen binding. Each variable domain typically has three CDR regions, designated CDR1, CDR2, and CDR3. Each complementary determining region may include amino acid residues from the CDR regions as defined by Kabat (i.e., approximately residues 24-34 or 24-39 (L1), 50-56 or 55-61 (L2), and 89-97 or 93-102 (L3) in the light chain variable domain and 31-35 or 26-35 (H1), 50-65 or 50-66 (H2), and 95-102 or 97-108 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and / or those residues from the "hypervariable loops", i.e., approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol Biol. 196:901-917). In some cases, the complementary determining region may include amino acids from the CDR regions as defined by Kabat and the hypervariable loops. One of skill in the art will appreciate some variation in the positioning of the FRs, e.g., as a result of mutations (e.g., deletions and / or insertions), e.g., up to 5 residue changes, or 4 residue changes, or 2 residue changes, or 1 residue change (e.g., as in the exemplary antibodies herein).

[0266] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced from any animal, e.g., mouse, rat, rabbit, pig, etc., or can be produced synthetically and are partially or fully human sequences.

[0267] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species (e.g., murine) or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species (e.g., primate) or belonging to a particular antibody class or subclass, as well as to the corresponding sequences in fragments of these antibodies, so long as they exhibit the desired biological activity.

[0268] The term "humanized antibody" should be understood to refer to a chimeric molecule, typically prepared using recombinant techniques, having an epitope-binding site derived from an immunoglobulin of a non-human species and the remaining immunoglobulin structure of a molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site preferably comprises complementarity-determining regions (CDRs) from a non-human antibody, which are grafted into appropriate framework regions in the variable domains of a human antibody and the remaining regions from a human antibody.

[0269] As used herein, the term "human antibody" in relation to antibody molecules and binding proteins refers to an antibody having variable (e.g., VH, VL, CDR, and FR regions) and constant antibody regions, which are derived from or correspond to sequences found in humans (e.g., human germline or somatic).

[0270] As used herein, the term "specifically binds" means that a binding protein or antibody reacts or binds to a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it reacts or binds to other cells or substances. It will also be understood from reading this definition that, for example, an antibody that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specifically binds" does not necessarily require exclusive or undetectable binding to another molecule, which is included in the term "selectively binds". Usually, but not necessarily, binding refers to specifically binding.

[0271] As used herein, the term "proliferative cell disorder" and its grammatical variations, when used in reference to cells, tissues, or organs, refer to any undesirable, excessive, or abnormal cell, tissue, or organ growth, proliferation, differentiation, or survival. Undesirable cell proliferation disorders include diseases and physiological disorders, including benign proliferative disorders characterized by an undesirable, excessive, or abnormal number of cells, cell growth, cell proliferation, cell survival, or differentiation in a subject. Specific examples of such disorders include metastatic and non-metastatic tumor formation, tumors, and cancers (malignant tumors).

[0272] The term "identity" and its grammatical variants refer to two or more reference entities being the same. Thus, when two antibody sequences are identical, they have the same amino acid sequence at least within a reference region or portion. When two nucleic acid sequences are identical, they have the same polynucleotide sequence at least within a reference region or portion. Identity can be over a defined region (region or domain) of the sequence. The % identity of polynucleotides is determined by GAP (Needleman and Wunsch, J. Mol Biol. 48:444 - 453, 1970) analysis (GCG program), where the gap creation penalty = 5 and the gap extension penalty = 0.3. Unless otherwise stated, the query sequence length is at least 45 nucleotides, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence length is at least 100 nucleotides, and the GAP analysis aligns the two sequences over a region of at least 100 nucleotides. Most preferably, the two sequences are aligned over their entire length.

[0273] The term "isolated", as applied to DNA, RNA, or protein, refers to a polynucleotide / polypeptide that is at least partially separated from the polynucleotide / polypeptide sequences with which it is associated or linked in its natural state. Preferably, the isolated polynucleotide / polypeptide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free of other components with which it is naturally associated.

[0274] The term "nucleic acid" as used herein may be used interchangeably with the term "polynucleotide".

[0275] The term "pharmaceutical composition" as used herein refers to any composition containing at least one therapeutic or bioactive agent and adapted for administration to a patient. Any of these formulations can be prepared by methods well known and accepted in the art. See, for example, Gennaro, A.R., ed., Remington: The Science and Practice of Pharmacy, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

[0276] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, and / or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0277] "Subject" refers to any subject in need of diagnosis, prognosis or treatment, particularly mammalian subjects. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, bears, chickens, amphibians, reptiles, etc., and may be used interchangeably with the term "patient" where appropriate. Preferably, the subject is a primate. In particular, the subject is a human.

[0278] As used herein, reference to "similar" binding levels is to be understood to mean that an antibody binds an antigen at a level within about 30% or 25% or 20% of the level at which it binds another antigen. The term may also refer to an antibody binding an antigen at a level within about 30% or 25% or 20% of the level at which another antibody binds the same antigen.

[0279] As used herein, reference to "substantially the same level" of binding is to be understood to mean that an antibody binds an antigen at a level within about 15% or 10% or 5% of the level at which it binds another antigen. The term may also refer to an antibody binding an antigen at a level within about 5% or 4% or 3% of the level at which another antibody binds the same antigen.

[0280] The term "competitive inhibition" is to be understood to mean that the protein of the invention reduces or prevents the binding of the resulting antibody (e.g., mAb104) to domain II of human HER2 or a fragment thereof. It will be apparent from the foregoing that the protein need not completely inhibit antibody binding, only reduce binding by a statistically significant amount, e.g., at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, an antibody is exposed to HER2 or a fragment thereof in the presence or absence of the protein. If the antibody binding in the presence of the protein is less than the antibody binding in the absence of the protein, the protein is considered to competitively inhibit antibody binding. In one example, the protein and the antibody are substantially simultaneously exposed to HER2. Other methods for determining competitive inhibition of binding will be apparent to those skilled in the art and / or are described herein. In one example, the antigen-binding domain of the protein competitively inhibits antibody binding.

[0281] In the context of two epitopes, "overlapping" means that the two epitopes share a sufficient number of amino acid residues to allow an antibody that binds one epitope to competitively inhibit the binding of an antibody that binds the other epitope. For example, the epitopes share at least one or two or three or four or five or six or seven or eight or nine or ten amino acids.

[0282] As used herein, the term "substantially non-binding" is understood to mean that a protein (such as an antibody) binds to a candidate antigen at a level that is 10% or 8% or 6% or 5% above background. The background can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (such as an isotype control antibody) and / or the level of binding detected in the presence of a negative control antigen. Binding levels are detected using a biosensor assay (such as Biacore), in which the protein is immobilized and contacted with the antigen.

[0283] The term "therapeutically effective amount" is understood to mean an amount of an antibody or antigen-binding fragment sufficient to reduce or inhibit one or more symptoms of a cell proliferative disorder to a level below that observed and accepted as a clinical feature of the disorder. One of ordinary skill in the art will appreciate that such an amount will vary depending on the antibody, fragment, and / or the type or severity or level of the particular subject and / or disease being treated. Accordingly, the term should not be construed as limiting the invention to a specific amount.

[0284] As used herein, the terms "treat", "treating", "treatment" and their grammatical variations mean subjecting an individual patient to a protocol, course, process or therapy in which a physiological response or outcome is desired in the patient. Since each patient being treated may not respond to a particular treatment protocol, course, process or treatment, treatment need not achieve the desired physiological response or outcome in every patient or patient population. Accordingly, a given patient or patient population may not respond or may respond inadequately to treatment.

[0285] The terms "tumor" or "cancer" are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival is greater than that of normal counterpart cells (such as a cell proliferative or differentiative disorder). Typically, the growth is uncontrolled.

[0286] The terms "104 antibody" or "mAb104" and any variants not specifically listed may be used interchangeably herein and, as used throughout this application and the claims, refer to a protein material comprising a single or multiple proteins and extending to those proteins having the amino acid sequence data described herein and the activity profile set forth herein and in the claims. Accordingly, proteins showing substantially equivalent or altered activity are also contemplated. These modifications can be intentional, such as those obtained by site-directed mutagenesis, or can be accidental, such as those obtained by mutation in a host that is a producer of the complex or its designated subunit. In addition, the terms "104 antibody" or "mAb104" are intended to include within their scope the proteins specifically listed herein as well as all substantially homologous analogs and allelic variants. Detailed Description

[0288] The present invention is not limited to the scope of the specific embodiments described herein, which are for illustrative purposes only.

[0289] Antibody Production

[0290] The general method for preparing monoclonal antibodies by hybridoma is well known. Immortalized antibody-producing cell lines can also be generated by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890. A panel of monoclonal antibodies produced against HER2 can be screened for various properties; i.e., isotype, epitope, affinity, etc., as described herein. Of particular interest are monoclonal antibodies that bind to domain II of aberrantly expressed HER2. Such monoclonal antibodies can be readily identified in assays of specific binding member activity. High affinity antibodies are also useful when immunoaffinity purification of natural or recombinant specific binding members is possible. Monoclonal antibodies useful for practicing the present invention can be generated by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma secreting antibody molecules having the appropriate antigen specificity. The culture is maintained under conditions and for a time sufficient for the hybridoma to secrete the antibody molecules into the medium. The medium containing the antibody is then collected. The antibody molecules can then be further isolated by well-known techniques.

[0291] The antibodies of the present invention can also be generated by immunizing an animal with a purified antigen corresponding to a cyclic peptide comprising residues 277 to 312 or residues 293 to 309 of mature normal or wild-type human HER2.

[0292] The HER2-binding proteins of the present invention can also be synthesized by standard techniques such as solid-phase peptide synthesis and / or native protein ligation.

[0293] Other suitable techniques for use in antibody methods include affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein A column, or any combination of these techniques. Antibody isotypes can be determined using ELISA assays, for example, human Ig can be identified using mouse Ig-absorbed anti-human Ig.

[0294] Recombinant Antibody Production

[0295] The antibodies and antigen-binding fragments of the invention can also be recombinantly produced using techniques and materials readily available to those skilled in the art.

[0296] The variable domains can be derived from any germline or rearranged human variable domain, or can be synthetic variable domains based on consensus sequences of known human variable domains. Using recombinant DNA techniques, the CDR-derived sequences of the invention can be introduced into all components of a variable domain lacking the CDR regions. For example, Marks et al. (Bio / Technology, 1992, 10:779-783) describe a method for generating all components of an antibody variable domain, where consensus primers directed against or adjacent to the 5' end of the variable domain region are used in combination with consensus primers directed against the third framework region of the human VH gene to provide all components of a VH variable domain lacking one or more CDRs. Marks et al. further describe how these all components are combined with the CDRs of a particular antibody. Using similar techniques, the CDR-derived sequences of the invention can be shuffled with all components of a VH or VL domain lacking one or more CDRs, and the shuffled complete VH or VL domain is combined with the homologous VL or VH domain to provide the antibodies of the invention. The all components can then be displayed in a suitable host system such as the phage display system of WO92 / 01047 so that suitable specific binding members can be selected. The all components can be composed of any composition upwards from 10 4 individual members, for example from 10 6 to 10 8 or 10 10 members. Stemmer (Nature, 1994, 370:389-391) also discloses similar shuffling or combination techniques, who describes techniques related to the β-lactamase gene, but observes that the method can be used to generate antibodies.

[0297] Antibody affinity can also be matured using selection and / or mutagenesis methods known in the art.

[0298] The recombinant antibodies of the invention can also be produced by phage display methods as disclosed in US 5,969,108.

[0299] The antibodies of the present invention may also include an antibody constant region or a portion thereof. For example, an antibody based on SEQ ID NO: 3 or 5 may be linked at its C-terminus to an antibody light chain constant domain comprising a human CK or Cλ chain. Similarly, an antibody based on SEQ ID NO: 2 or 4 may be linked at its C-terminus to all or a portion of an immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE, IgD, and IgM) and any isotype subclass, particularly IgG1, IgG2b, and IgG4.

[0300] For recombinant production, it is preferred to isolate the nucleic acid encoding the antibody of the present invention and insert it into a replicable vector for further cloning (DNA amplification) or for expression. The DNA encoding the antibody is readily isolated or synthesized using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to the DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding the antibody of the present invention or a fragment thereof (e.g., derived from the information provided herein), enhancer elements, a promoter, and a transcription termination sequence.

[0301] (i) Signal sequence component. The antibodies of the present invention can be produced not only directly by recombination but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence preferably selected is one that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leader sequences. For yeast secretion, the native signal sequence can be replaced by, for example, the yeast invertase leader sequence, the α-factor leader sequence, or the acid phosphatase leader sequence, the Candida albicans glucoamylase leader sequence, or the signal described in WO 90 / 13646. In mammalian cell expression, mammalian signal sequences as well as viral secretion leader sequences, such as the herpes simplex gD signal, are available. The DNA of such a precursor region is ligated in-frame with the DNA encoding the antibody.

[0302] (ii) Promoter component. Expression and cloning vectors generally contain a promoter that is recognized by the host organism and operably linked to the antibody nucleic acid. Promoters suitable for prokaryotic hosts include the phoA promoter, the β-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters for bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody.

[0303] Known promoters are used in eukaryotes. In fact, all eukaryotic genes have an AT-rich region located approximately 25 - 30 bases upstream of the transcription start site. Another sequence found 70 - 80 bases upstream of the transcription start point in many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal for adding a poly-A tail to the 3' end of the coding sequence. All these sequences are appropriately inserted into eukaryotic expression vectors. Examples of suitable promoter sequences for yeast hosts include the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters are inducible promoters that have the additional advantage of transcription being controlled by growth conditions, which are the promoter regions of alcohol dehydrogenase 2, iso-cytochrome C, acid phosphatase, degradative enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used in conjunction with yeast promoters.

[0304] Antibody transcription from a vector in mammalian host cells is controlled by a promoter obtained, for example, from a viral genome, such as polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), CMV, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, most preferably simian virus 40 (SV40), from a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, from a heat shock promoter, provided that these promoters are compatible with the host cell system.

[0305] (iii) Enhancer element components. Transcription of DNA encoding the antibodies of the present invention in higher eukaryotes is generally increased by inserting enhancer sequences into the vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are now known. However, enhancers from eukaryotic cell viruses are commonly used. Examples include the SV40 enhancer (bp 100-270) located on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located on the late side of the replication origin, and the adenovirus enhancer. See also Yaniv (1982) Nature 297:17-18 regarding enhancer elements for activating eukaryotic promoters. The enhancer can be spliced into the vector at the 5' or 3' position of the antibody-encoding sequence, but is preferably located at the 5' site of the promoter.

[0306] (iv) Transcription termination components. Expression vectors for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences required for termination of transcription and stabilization of the mRNA. These sequences are typically obtained from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed into polyadenylated segments in the untranslated portion of the mRNA encoding the antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0307] (v) Selection and transformation of host cells. Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotes, yeasts, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as the Enterobacteriaceae, such as the genus Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, and the genus Bacillus, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Pseudomonas aeruginosa, and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative and not restrictive.

[0308] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used in lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces marxianus, and Kluyveromyces marxianus; Yarrowia (EP 402,226); Pichia (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces hosts such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Torulopsis, and Aspergillus hosts, such as Aspergillus nidulans and Aspergillus niger.

[0309] Suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants, as well as the corresponding permissive insect host cells, have been identified from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of virus strains for transfection are publicly available, such as the L-I variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses can be used as the viruses of the present invention, particularly for transfecting Spodoptera frugiperda cells.

[0310] Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney cell lines (293 or subclones of 293 cells for growth in suspension culture, Graham et al. (1977) Gen Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO, Urlaub et al. (1980) Proc. Natl. Acad. Sci USA 77:4216); mouse Sertoli cells (TM4, Mather (1980) Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al. (1982) Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and PER.C6 TM (Crucell NV).

[0311] Functionally Equivalent Antibodies

[0312] The invention also contemplates antibodies or antigen-binding fragments thereof that have one or more amino acid additions, deletions or substitutions in the heavy and light chain variable region sequences of the antibodies of the invention, but still retain the function of the antibodies of the invention. These modifications can be intentional, for example by site-directed mutagenesis, or can be accidental, such as those obtained by mutations in the host expressing the antibody.

[0313] Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, the polynucleotides of the invention can be mutagenized in vitro. Such in vitro mutagenesis techniques include subcloning the polynucleotide into a suitable vector, transforming the vector into a "mutant" strain such as Escherichia coli XL-1 Blue (Stratagene) and propagating the transformed bacteria for an appropriate number of generations. The products derived from the mutant / altered DNA can be readily screened using the techniques described herein to determine whether they have receptor binding and / or inhibitory activity.

[0314] When designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the feature to be modified. The mutation site(s) can be modified individually or in tandem, e.g., by (1) first making conservative amino acid substitutions and then more radical substitutions based on the results obtained, (2) deleting the target residue(s) or (3) inserting additional residue(s) adjacent to the site(s) of interest.

[0315] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably from about 1 to 10 residues, and typically about 1 to 5 contiguous residues.

[0316] Substitution mutants have at least one amino acid residue removed and a different residue inserted in its place in an antibody and / or immunoglobulin chain molecule (including in the variable region). Sites of greatest interest for substitution mutagenesis include those identified as important for antigen binding. These sites, particularly those belonging to the sequences of at least three other identical conserved sites of a human antibody and / or immunoglobulin chain, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in the table under the heading "Exemplary Substitutions".

[0317] The invention also includes conservative amino acid substitutions. These refer to the amino acid substitutions listed in the table below.

[0318] Exemplary Substitutions

[0319]

[0320]

[0321] Amino acids herein are preferably in the "L" isomeric form. However, residues of the D isomeric form can replace any L - amino acid residue provided that the polypeptide retains the desired functional properties for immunoglobulin binding. Modifications also include structural and functional analogs, e.g., peptidomimetics having synthetic or non - natural amino acids or amino acid analogs and derivative forms.

[0322] Chimeric Antibodies

[0323] Chimeric antibodies are prepared by recombinant methods by combining the variable light and heavy chain regions (VL and VH) obtained from antibody-producing cells of one species with the constant light and heavy chain regions from another species. Typically, chimeric antibodies utilize rodent or rabbit variable regions and human constant regions to produce antibodies that are predominantly of human construction. For example, a chimeric antibody includes the variable region of a murine antibody according to any of the embodiments herein fused to a human constant region. The production of such chimeric antibodies is known in the art and can be achieved by standard means (such as described, for example, in Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567 and 4,816,397). It is also contemplated that the human constant region of the chimeric antibodies of the invention can be selected from the IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgGl0, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19 constant regions.

[0324] Humanized and Human Antibodies

[0325] The antibodies of the present invention can be humanized antibodies or human antibodies. A humanized form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, immunoglobulin chain, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of an antibody) that contains minimal sequences derived from a non-human immunoglobulin. A humanized antibody includes a human immunoglobulin (recipient antibody) in which the residues of the complementarity determining regions (CDRs) of the recipient are replaced with the residues of the CDRs of a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, the Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human residues. A humanized antibody can also include residues that are neither present in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will include substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the FR regions are the FR regions of a human immunoglobulin consensus sequence. A humanized antibody optimally will also include at least a portion of the immunoglobulin constant region (Fc), usually a portion of the constant region (Fc) of a human immunoglobulin (Jones et al., (1986) Nature, 321:522-525; Riechmann et al. (1988), Nature 332:323-329; and Presta (1992) Curr Op Struct Biol, 2:593-59).

[0326] Methods for humanizing non-human antibodies are known in the art. Typically, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are usually taken from the "import" variable domain. Humanization generally can be performed according to the methods of Jones et al., supra; Riechmann et al., supra; Verhoeyen et al. (1988) Science, 239:1534-1536), by substituting the rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially no intact human variable domain has been replaced with the corresponding sequences from a non-human species. In effect, a humanized antibody is generally a human antibody in which some CDR residues and possibly some FR residues are replaced with residues from analogous sites in a rodent antibody.

[0327] A variety of techniques known in the art can also be used to generate human antibodies, including phage display libraries (Hoogenboom and Winter (1991) J Mol Biol, 227:381; Marks et al. (1991), J Mol Biol, 222:581). The techniques of Cole et al. and Boerner et al. are also applicable to the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al. (1991) J Immunol, 147:86-95). Similarly, human antibodies can be prepared by introducing the human immunoglobulin locus into transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production is observed, which is very similar in all respects to that seen in humans, including gene rearrangement, assembly, and all components of the antibody. This method is described, for example, in U.S. Patents Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.

[0328] Fully human antibodies that recognize a selected epitope can also be generated using a technique called "directed selection." In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to direct the selection of a fully human antibody that recognizes the same epitope (Jespers et al., Bio / technology 12:899-903 (1988)).

[0329] Antibody affinity can also be matured using selection and / or mutagenesis methods known in the art. The affinity of the preferred affinity-matured antibody is 5-fold, more preferably 10-fold, and even more preferably 20 or 30-fold that of the starting antibody (usually murine, humanized, or human) from which the mature antibody was prepared.

[0330] Humanized-like and Primateized Proteins

[0331] The HER2-binding protein of the present invention can be a humanized-like protein. The term "humanized-like protein" refers to a protein prepared by the method described in WO2007 / 019620. The humanized-like HER2-binding protein includes the variable region of an antibody, wherein the variable region includes the FR from the variable region of an antibody of a New World primate and the CDR from the variable region of an antibody of a non-New World primate. For example, the humanized-like HER2-binding protein includes the variable region of an antibody, wherein the variable region includes the FWR from the variable region of an antibody of a New World primate and the CDR from a murine antibody, such as as described herein. In one example, the humanized-like HER2-binding protein is a HER2-binding antibody, wherein one or both variable regions are humanized-like.

[0332] The HER2-binding protein of the present invention can be a primatized protein. "Primatized protein" includes the variable region from an antibody produced after immunizing a non-human primate (e.g., cynomolgus monkey). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to produce a primatized antibody. Exemplary methods for producing primatized antibodies are described in US 6113898.

[0333] Deimmunized Antibodies and Proteins

[0334] The present invention also relates to deimmunized antibodies or HER2-binding proteins. A deimmunized antibody has one or more epitopes, e.g., B-cell epitopes or T-cell epitopes that are removed (i.e., mutated), thereby reducing the likelihood of a subject generating an immune response against the antibody or protein. Methods for producing deimmunized antibodies and proteins are known in the art and are described, for example, in WO00 / 34317, WO 2004 / 108158, and WO 2004 / 064724.

[0335] Based on the description herein, the method of introducing suitable mutations and expressing and assaying the resulting protein will be apparent to those skilled in the art.

[0336] Antibody Variable Regions Containing Proteins

[0337] Single domain antibody

[0338] In some embodiments, the HER2-binding protein of the present invention is a single domain antibody (which can be used interchangeably with the term "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain that includes all or part of the variable region of an antibody heavy chain. In certain instances, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US6248516; WO 90 / 05144 and / or WO 2004 / 058820).

[0339] Diabody, triabody, tetrabody

[0340] Exemplary HER2-binding proteins that include antibody-antigen binding domains are diabodies, triabodies, tetra-bodies, and higher-order protein complexes such as those described in WO 98 / 044001 and WO 94 / 007921.

[0341] For example, a diabody is a protein that includes two associated polypeptide chains, each polypeptide chain including the structure VL-X-VH or VH-X-VL, where VL is the variable region of an antibody light chain, VH is the variable region of an antibody heavy chain, X is a linker that includes residues insufficient to permit association of VH and VL in a single polypeptide chain or is absent, and where the VH of one polypeptide chain binds to the VL of the other polypeptide chain to form an antigen-binding site, i.e., an Fv molecule capable of specifically binding one or more antigens. The VL and VH can be the same in each polypeptide chain, or the VL and VH can be different in each polypeptide chain so as to form a bispecific dimer antibody (i.e., including two Fvs with different specificities).

[0342] Single-chain Fv (scFv) fragments

[0343] Those skilled in the art know that an scFv includes the VH and VL regions in a single polypeptide chain. The polypeptide chain also includes a polypeptide linker between VH and VL, which enables the scFv to form the structure required for antigen binding (i.e., to enable the VH and VL of the single polypeptide chain to associate with each other to form an Fv). For example, the linker includes more than 12 amino acid residues, where (Gly 4 Ser) 3 is one of the more favorable linkers for an scFv.

[0344] The invention also contemplates disulfide-stabilized Fv (or diFv or dsFv), where a single cysteine residue is introduced into the FR of VH and the FR of VL, and the cysteine residues are linked by a disulfide bond to produce a stabilized Fv (see, for example, Brinkmann et al., (1993) Proc Natl Acad Sci USA 90:547-551).

[0345] Alternatively or additionally, the invention provides dimeric scFv, i.e., a protein that includes two scFv molecules linked by non-covalent or covalent linkages, such as by a leucine zipper domain (e.g., derived from Fos or Jun) (see, for example, Kruif and Logtenberg, 1996). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to permit the two scFvs to form and bind to an antigen, e.g., as described in US20060263367.

[0346] For a review of scFv, see Ahmad ZA et al., (2012) Clinical and Developmental Immunology doi:10.1155 / 2012 / 980250.

[0347] Minibody

[0348] Those skilled in the art will recognize that minibodies include the VH and VL domains of an antibody fused to the (CH2 and / or (CH3 domains of an antibody. Optionally, the minibody includes a hinge region between VH and VL, and sometimes this conformation is referred to as a flexible minibody. Minibodies do not include CH1 or CL. In one example, the VH and VL domains are fused to the hinge region and CH3 domain of an antibody. At least one variable region of the minibody binds HER2 in the manner of the present invention. Exemplary minibodies and methods for their preparation are described, for example, in WO 94 / 09817.

[0349] Other protein-containing antibody variable regions

[0350] The present invention also contemplates other variable regions containing HER2-binding proteins, such as:

[0351] (i) "Key and hole" bispecific proteins, as described in US 5,731,168;

[0352] (ii) Heteroconjugate proteins, such as those described in US 4,676,980;

[0353] (iii) Heteroconjugate proteins produced using chemical crosslinkers, such as those described in US 4,676,980;

[0354] (iv) Fab'-SH fragments, such as those described in Shalaby (1992) j Exp Med 1; 175(1):217-25;

[0355] (v) Single-chain Fab; or

[0356] (vi) Fab3 (for example, as described in EP 19930302894).

[0357] Non-Antibody-Based Antigen-Binding Domains Containing Proteins

[0358] Immunoglobulins and immunoglobulin fragments

[0359] Examples of compounds of the present invention are proteins comprising variable regions of immunoglobulins, such as T cell receptors or heavy chain immunoglobulins (e.g., IgNA, camel antibodies).

[0360] The term "immunoglobulin" is to be understood as including antigen-binding proteins that include immunoglobulin domains. Exemplary immunoglobulins are antibodies. Additional proteins included by the term "immunoglobulin" include domain antibodies, camelid antibodies, and antibodies from cartilaginous fish (i.e., immunoglobulin new antigen receptor (IgNAR)). Generally, camel antibodies and IgNAR include VH, yet lack VL and are commonly referred to as heavy-chain immunoglobulins. Other "immunoglobulins" include T-cell receptors.

[0361] Heavy-chain immunoglobulin

[0362] Heavy-chain immunoglobulins differ structurally from many other forms of immunoglobulins (e.g., antibodies) in that they include heavy chains but no light chains. Thus, these immunoglobulins are also referred to as "heavy-chain only antibodies". Heavy-chain immunoglobulins are found, for example, in camelids and cartilaginous fish (also known as IgNAR).

[0363] The variable regions present in naturally occurring heavy-chain immunoglobulins are commonly referred to as "VHH domains" in camel Ig and V-NAR in IgNAR in order to distinguish them from the heavy-chain variable regions (referred to as "VH domains") present in conventional 4-chain antibodies and the light-chain variable regions (referred to as "VL domains") present in conventional 4-chain antibodies.

[0364] Heavy-chain immunoglobulins do not require light chains to bind the relevant antigen with high affinity and high specificity. This means that single-domain binding fragments can be derived from heavy-chain immunoglobulins, which are easy to express and are generally stable and soluble. General descriptions of heavy-chain immunoglobulins from camelids, their variable regions, and methods for their production and / or isolation and / or use are found, inter alia, in the following references WO 94 / 04678, WO 97 / 49805, and WO 97 / 49805.

[0365] General descriptions of heavy-chain immunoglobulins from cartilaginous fish, their variable regions, and methods for their production and / or isolation and / or use are found, inter alia, in WO2005 / 118629.

[0366] V-like protein

[0367] An example of the HER2-binding protein of the present invention is a T cell receptor. The T cell receptor has two V domains that associate to form a structure analogous to an antibody Fv module. Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991 describes how the two V domains of a T cell receptor (designated alpha and beta) can be fused and expressed as a single-chain polypeptide, and how surface residues can be altered to reduce the hydrophobicity directly analogous to an antibody scFv. Other publications describing the production of single-chain T cell receptors or multimeric T cell receptors comprising two V-alpha and V-beta domains include WO 1999 / 045110 or WO 2011 / 107595.

[0368] Other non-antibody proteins comprising antigen-binding domains include proteins having V-like domains, which are generally monomeric. Examples of proteins comprising such V-like domains include CTLA-4, CD28, and ICOS. Further disclosures of proteins comprising such V-like domains are included in WO 1999 / 045110.

[0369] Adnectins

[0370] In one instance, the HER2-binding protein of the present invention is an adnectin.

[0371] Adnectins are based on the tenth type III fibronectin (10Fn3) domain of human fibronectin, wherein the loop regions are altered to confer antigen binding. For example, three loops at one end of the beta-sandwich of the 10Fn3 domain can be engineered to enable an adnectin to specifically recognize an antigen. For more details, see US20080139791 or WO2005 / 056764.

[0372] Anticalins

[0373] In another instance, the HER2-binding protein of the present invention is an anticalin. Anticalins are derived from lipocalins, which are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have a rigid beta-sheet secondary structure with multiple loops at the open end of a conical structure, which can be engineered to bind an antigen. Such engineered lipocalins are called anticalins. For further description of anticalins, see US 7250297B1 or US 20070224633.

[0374] Affibody

[0375] In another embodiment, the HER2-binding protein of the present invention is an affibody. An affibody is a scaffold derived from the Z domain (antigen-binding domain) of protein A from Staphylococcus aureus, which can be engineered to bind an antigen. The Z domain consists of a three-helix bundle of approximately 58 amino acids. A library is generated by randomization of surface residues. For more details, see EP1641818.

[0376] Avimers

[0377] In another embodiment, the HER2-binding protein of the present invention is an Avimer. An Avimer is a multi-domain protein derived from the A-domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide-bonded structure. Diversity is generated by shuffling the natural variation exhibited by the A-domain family. For more details, see WO 2002088171.

[0378] Designed ankyrin repeat proteins (DARPins)

[0379] In another example, the HER2-binding protein of the present invention is a designed ankyrin repeat protein (DARPin). DARPins are derived from Ankyrin, which is a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. They can be engineered to bind different target antigens by randomizing the residues in the first α-helix and β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For more details, see US20040132028.

[0380] Other Non-Antibody Polypeptides

[0381] Other non-antibody proteins comprising a binding domain include those based on human γ-crystallin and human ubiquitin (affilins), the kunitz-type domain of human protease inhibitors, the PDZ domain of the Ras-binding protein AF-6, scorpion toxins (charybdotoxin), and C-type lectin domains (tetranectins).

[0382] Constant Region

[0383] The present invention encompasses HER2-binding proteins comprising variable and constant regions or domains thereof (such as Fc, CH2, and / or CH3 domains). Those skilled in the art, given the disclosure herein and the references discussed herein, will appreciate the meaning of the terms constant region and constant domain.

[0384] The constant region sequences useful for generating the HER2-binding proteins of the present invention can be obtained from many different sources. In some instances, the constant region of the HER2-binding protein or portions thereof are derived from human antibodies. Additionally, the constant domain or portions thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one instance, the human isotype IgG1 is used.

[0385] The various constant region gene sequences can be obtained in the form of publicly available deposits or their sequences can be obtained from publicly available databases. Constant regions can be selected that have specific effector functions (or lack specific effector functions) or have specific modifications to reduce immunogenicity.

[0386] In one instance, the proteins of the present invention have or exhibit effector functions that promote or effect at least partial depletion, substantial depletion, or elimination of cells expressing HER2. Such effector functions can enhance the binding affinity for Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0387] In one instance, the HER2-binding protein is capable of inducing enhanced levels of effector function.

[0388] In one instance, the level of effector function induced by the constant region is enhanced relative to the wild-type Fc region of an IgG1 antibody or the wild-type Fc region of an IgG3 antibody.

[0389] In another instance, the constant region is modified to increase the level of effector function it is capable of inducing as compared to an unmodified constant region. Such modifications can be at the amino acid level and / or secondary structure level and / or tertiary structure level and / or in the glycosylation of the Fc region.

[0390] Those skilled in the art will understand that greater effector function can manifest in any of a variety of ways, such as a higher level of effect, a more persistent effect, or a faster rate of effect. Exemplary constant region modifications include amino acid substitutions such as S239D / I332E, according to the EU index numbering of Kabat, or S239D / A330L / I332E, according to the EU index numbering of Kabat.

[0391] Other amino acid substitutions that increase the ability of the Fc region to induce effector function are known in the art and / or are described, for example, in US 6737056 or US 7317091.

[0392] In one example, the glycosylation of the constant region is altered to increase its ability to induce enhanced effector functions. In some examples, the Fc region according to the present invention comprises a carbohydrate structure lacking (directly or indirectly) fucose linked to the Fc region, i.e., the Fc region is "afucosylated". These variants may have an improved ability to induce ADCC. Methods for generating afucosylated antibodies include expressing a HER2-binding protein in a cell line that cannot express α-1,6-fucosyltransferase (FUT8) (e.g., as described in Yumane-Ohnuki et al., 2004). Other methods include using cell lines that inherently produce antibodies capable of inducing enhanced effector functions (e.g., duck embryo-derived stem cells used for the production of viral vaccines, WO2008 / 129058; avian recombinant protein production in cells, WO 2008 / 142124).

[0393] The HER2-binding protein may also comprise an Fc region capable of inducing enhanced levels of CDC. For example, a hybrid of IgG1 and IgG3 produces antibodies with enhanced CDC activity (Natsume et al., 2008).

[0394] Methods for determining the ability of an antibody or its antigen-binding fragment to induce effector functions are known in the art and / or described herein.

[0395] In another example, the protein comprises one or more amino acid substitutions that increase the half-life of the HER2-binding protein. For example, the HER2-binding protein comprises a constant region that comprises one or more amino acid substitutions that increase the affinity of the constant region for the neonatal Fc region (FcRn). For example, the constant region has an increased affinity for FcRn at a lower pH (e.g., about pH 6.0) to promote Fc / FcRn binding in the endosome. In one example, the constant region has an increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which promotes the re-release of Fc into the blood after cell recycling. These amino acid substitutions can be used to extend the half-life of the protein by reducing clearance from the blood.

[0396] According to the EU numbering system, exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US 7083784.

[0397] The HER2-binding protein of the present invention may comprise an IgG4 constant region or a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" should be understood to refer to an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange or to form half antibodies. "Fab arm exchange" refers to a type of protein modification of human IgG4, in which the IgG4 heavy chain and the attached light chain (half molecule) exchange with another IgG4 molecule to form a heavy-light chain pair. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. "Half antibodies" are formed when IgG4 antibodies dissociate to form two molecules each containing a single heavy chain and a single light chain.

[0398] In one example, according to the Kabat system, the stabilized IgG4 constant region comprises a proline at position 241 in the hinge region. This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, this residue is typically serine. After substitution of serine with proline, the IgG4 hinge region comprises the sequence CPPC. In this regard, those skilled in the art will recognize that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, conferring mobility on the two Fab arms of the antibody. The hinge region comprises cysteine residues that participate in inter-heavy chain disulfide bonds. The hinge region is generally defined as the stretch from Glu226 to Pro243 of human IgGl1 according to the Kabat numbering system. By placing the first and last cysteine residues that form the inter-heavy chain disulfide bond (S-S) in the same position, the hinge regions of other IgG isotypes can be aligned with the IgGl1 sequence (see, for example, WO 2010 / 080538).

[0399] Modified Proteins

[0400] The present invention provides HER2-binding proteins having at least 80% identity to the sequences of the present invention and having the same functional properties as described or claimed herein.

[0401] In one example, the HER2-binding protein of the present invention comprises a sequence having at least 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% identity to the VL sequence disclosed herein, such as SEQ ID NO: 3.

[0402] In another example, the HER2-binding protein of the present invention comprises a sequence having at least 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% identity to the VH of the present invention herein, such as SEQ ID NO: 2.

[0403] The present invention also provides a nucleic acid encoding the aforementioned protein or a nucleic acid that hybridizes thereto under moderately to highly stringent conditions.

[0404] The present invention also includes a nucleic acid encoding a protein comprising the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, which sequences are different from the exemplary sequences herein due to the degeneracy of the genetic code.

[0405] The present invention also includes a nucleic acid encoding a protein comprising the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, which sequences are different from the exemplary sequences herein due to the degeneracy of the genetic code.

[0406] The % identity of a nucleic acid or polypeptide is determined by GAP (Needleman and Wunsch 1970) analysis (GCG program), with a gap creation penalty = 5 and a gap extension penalty = 0.3. The query sequence is preferably at least 50 residues in length, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues in length, and the GAP analysis aligns the two sequences over a region of at least 100 residues. In one instance, the two sequences are aligned over their entire length.

[0407] Modified glycosylation

[0408] The glycosylation pattern of an antibody can be altered from the original glycosylation pattern of a reference antibody. Alteration refers to deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites not present in the antibody, and / or adding one or more carbohydrate moieties to the original glycosylation pattern of the reference antibody. Glycosylation of an antibody is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Alteration to add a glycosylation site to an antibody is conveniently achieved by altering the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alteration can also be made by adding or substituting one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

[0409] Modified glycoforms of the antibodies of the present invention can be used for a variety of purposes, including but not limited to enhancing or reducing effector function and / or modifying the half-life of the antibody (see, for example, WO / 2007 / 010401). Such alterations can result in a decrease or increase in C1q and CDC binding or FcγR and ADCC binding. For example, substitutions can be made in one or more amino acid residues of the heavy chain constant region, resulting in an alteration of effector function while maintaining the ability to bind antigen compared to the modified antibody, see US5,624,821 and US 5,648,260. Engineered glycoforms can be generated by any method known to those skilled in the art, such as by using engineered or variant expression strains, by co-expression with one or more enzymes, such as β(l,4)-N-acetylglucosaminyltransferase III (GnTIl 1), by expressing the antibody or fragment thereof in various organisms or cell lines from various organisms, or by modifying the carbohydrate after expression of the antibody or fragment. Methods for generating engineered glycoforms are known in the art and include, but are not limited to, Umana et al., 1999, Nat. Biotechnol 17:176-180; Davies et al., 2007 Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Patent No. 6,602,684; U.S. Patent No. 10 / 277,370; U.S. Patent No. 10 / 113,929; PCT WO 00 / 61739A1; PCT WO 01 / 292246A1; PCT WO 02 / 311140Al; PCT WO 02 / 30954A1; Technology (Biowa, Inc. Princeton, N.J.); GlycoMAb TM Glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, for example, WO 00061739; EA01229125; US20030115614; Cong et al. (2004) JMB, 336:1239-49.

[0410] Effector function

[0411] The antibodies of the invention may need to be modified with respect to effector function, for example, in order to enhance the antigen - dependent cell - mediated cytotoxicity (ADCC) and / or complement - dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residues can be introduced in the Fc region, thereby allowing the formation of inter - chain disulfide bonds in this region. The resulting homodimeric antibodies may have improved internalization ability and / or increased complement - mediated cell killing and antibody - dependent cell cytotoxicity (ADCC). See Caron et al., J. J. ExpMed. 176:1191 - 1195 (1992) and Shopes, B. J. Immunol. 148:2918 - 2922 (1992). Homodimeric antibodies with enhanced anti - tumor activity can also be prepared using the heterobifunctional cross - linkers described by Wolff et al., Research 53:2560 - 2565 (1993). Alternatively, antibodies with a dual Fc region can be engineered, thereby enhancing complement lysis and ADCC capabilities. See Stevenson et al., Anti - Cancer Drug Design 3:219 - 230 (1989).

[0412] Half - life

[0413] To increase the serum half - life of an antibody, a salvage receptor - binding epitope can be incorporated into the antibody (especially antibody fragments), for example, as described in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor - binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in - vivo serum half - life of the IgG molecule. Alternatively, the antibody half - life can be increased by polyethylene glycolylation.

[0414] Determining the Activity of the Binding Proteins of the Invention

[0415] Binding assay

[0416] One form of such an assay is an antigen - binding assay, for example, as described in Scopes (1994) Protein Purification: principles and practice Springer - Verlag. This method generally involves labeling the HER2 - binding protein and contacting it with a fixed antigen or a fragment thereof, for example, as Figure 1A protein comprising residues 293 to 309 of mature normal or wild-type human HER2 as shown. After washing to remove non-specifically bound proteins, the amount of the label is detected, and as a result, the amount of the bound protein is detected. Of course, the HER2-binding protein can be immobilized and the antigen labeled. A panning assay can also be used. The examples herein describe a binding assay based on flow cytometry.

[0417] HER2-binding proteins that competitively inhibit the binding of the HER2 antibodies of the present invention to the epitope can be screened and identified using conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0418] Competitive binding assay

[0419] Assays for determining HER2-binding proteins that competitively inhibit the binding of the antibodies of the present invention (e.g., mAb104) will be apparent to those skilled in the art. For example, the antibodies of the present invention are conjugated to a detectable label, such as a fluorescent label or a radioactive label. Then the labeled antibody and the tested HER2-binding protein are mixed and contacted with HER2 or a peptide comprising its epitope (e.g., residues 293 to 309 corresponding to domain II of human HER2). Then the level of the labeled antibody is determined and compared to the level determined when the labeled antibody is contacted with HER2 or a peptide comprising its epitope in the absence of the HER2-binding protein. If the level of the labeled antibody is reduced in the presence of the tested HER2-binding protein compared to the absence of the HER2-binding protein, the HER2-binding protein competitively inhibits the binding of the antibody.

[0420] Optionally, the tested HER2-binding protein is conjugated with a label different from the antibody. This allows detection of the binding level of the tested HER2-binding protein to the protein or epitope.

[0421] In another example, the tested HER2-binding protein is allowed to bind to HER2 or a peptide comprising its epitope before contacting HER2 or a peptide comprising its epitope with the antibodies described herein. A decrease in the amount of bound antibody in the presence of the HER2-binding protein compared to the absence of the HER2-binding protein indicates that the HER2-binding protein competitively inhibits the binding of the antibody to HER2. An alternative assay can also be performed using a labeled HER2-binding protein, and the antibody is first allowed to bind to HER2 or a peptide comprising its epitope. In this case, a decrease in the amount of labeled HER2-binding protein bound to HER2 or a peptide comprising its epitope in the presence of the antibody compared to the absence of the antibody indicates that the HER2-binding protein competitively inhibits the binding of the antibody to HER2.

[0422] Affinity assay

[0423] Optionally, the dissociation constant (Kd), association constant (Ka), or binding constant (KD, i.e., Ka / Kd) of the HER2-binding protein to HER2 or its epitope-containing peptide is determined. In one embodiment, these constants of the HER2-binding protein are measured by a radiolabeled or fluorescent-labeled HER2-binding assay. The assay equilibrates the HER2-binding protein with a minimal concentration of labeled HER2 in the presence of a titration series of unlabeled HER2. After washing away the unbound HER2, the amount of label is determined. According to another embodiment, the constants are measured by using surface plasmon resonance assays, e.g., using a BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized HER2 or a region thereof.

[0424] Protein Detection Assays

[0425] One example of the invention detects the presence of HER2 or cells expressing HER2 (e.g., breast cancer cells). Any of a variety of techniques known to those of skill in the art are used to determine the amount, level, or presence of the protein or cell, e.g., techniques selected from the group consisting of flow cytometry, immunohistochemistry, immunofluorescence, immunoblotting, Western blot, dot blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g., LC MS / MS), biosensor technology, evanescent fiber technology, or protein chip technology.

[0426] In one example, the assay for determining the amount or level of the protein is a semi-quantitative assay. In another example, the assay for determining the amount or level of the protein is a quantitative assay.

[0427] For example, the protein is detected by an immunoassay, e.g., using an assay selected from the group consisting of immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), Western blotting, radioimmunoassay (RIA), biosensor assay, protein chip assay, and immunostaining assay (e.g., immunofluorescence).

[0428] Standard solid-phase ELISA or FLISA formats are particularly useful in assaying protein concentrations from various samples.

[0429] In one form, an ELISA or FLISA comprises immobilizing a HER2-binding protein of the invention or a protein that binds to different epitopes of HER2 on a solid matrix such as a membrane, polystyrene or polycarbonate micropore, polystyrene or polycarbonate impregnated sheet, or glass support. The sample is then brought into physical association with the immobilized protein and HER2 is bound or "captured". The bound HER2 is then detected using a second labeled compound that binds to a different epitope of HER2. Alternatively, a third labeled antibody that binds to a second (detection) antibody can be used. It will be apparent to those skilled in the art that the assay formats described herein are suitable for high-throughput formats such as automation of screening processes or microarray formats. In addition, variations of the above assays will be apparent to those skilled in the art, such as competitive ELISA.

[0430] In another example, a polypeptide is detected intracellularly or on cells using methods known in the art such as immunohistochemistry or immunofluorescence. Methods using immunofluorescence are exemplary because they are quantitative or at least semi-quantitative. Methods for quantifying the degree of fluorescence of stained cells are known in the art and are described, for example, in Cuello, 1984.

[0431] Biosensor devices typically employ an electrode surface in combination with a current or impedance measuring element that is incorporated into a device that binds to an assay matrix (such as described in US 5567301). A HER2-binding protein of the invention is incorporated onto the surface of the biosensor device and a biological sample is brought into contact with the device. A change in the current or impedance detected by the biosensor device indicates the binding of the protein to the HER2-binding protein. Some forms of biosensors known in the art also rely on surface plasmon resonance (SPR) to detect protein interactions, whereby a change in the reflected surface plasmon resonance surface indicates the binding of a protein to a ligand or antibody (US 5485277 and US 5492840).

[0432] Because such systems are readily adaptable to the micron or nanoscale, biosensors are particularly useful for high-throughput analysis. In addition, such systems are conveniently adapted to incorporate several detection reagents, allowing multiplexing of diagnostic reagents in a single biosensor unit. This allows the simultaneous detection of several proteins or peptides in a small volume of body fluid.

[0433] Binding of a protein to HER2 can also be detected by flow cytometry in the examples herein.

[0434] Epitopes Bound by the Binding Proteins of the Invention

[0435] The present inventors have generated binding molecules that are specific for conformationally exposed epitopes in domain II of human HER2 that are present in neoplastic, hyperproliferative, or abnormal cells but not in wild-type or normal cells. The conformationally exposed epitope is located in the distal portion of domain II, flanked by disulfide bonds, allowing for flexible variation in this region and the exposure of the epitope has been shown to bind to mAb104. In particular, the epitope appears to be exposed in cells responsive to HER2 amplification or activation. What is particularly surprising about the antibodies of the present invention is that they do not block the binding of pertuzumab or trastuzumab to the extracellular domain of HER2, indicating that this epitope region of domain II allows antibody binding without blocking the binding of these antibodies when conformationally exposed, potentially allowing for dual therapeutic approaches.

[0436] The crystal structure of pertuzumab in complex with HER2 has been determined (see Franklin MC et al., (2004) Cancer Cell Vol. 5: 317 - 328). Pertuzumab binds to HER2 near the center of domain II, sterically blocking the binding pocket necessary for receptor dimerization and signal transduction. It is understood that the CDR H3 of pertuzumab makes hydrophobic and hydrogen bond contacts with residues Lys311 and His296 of HER2. His296 is completely buried in the pertuzumab binding. Since pertuzumab specifically inhibits HER2 heterodimerization by blocking the pocket that accepts the heterodimeric receptor ligand on HER2, without wishing to be bound by theory, the present inventors hypothesized that the binding molecules of the present invention (such as mAb104) might bind spatially to different faces of the epitope loop in HER2. The inventors hypothesized that the epitope might be revealed in a subset of receptors on the cancer cell surface when the receptor undergoes a conformational change due to redox disulfide bond switching or abnormal expression in cancer conditions of HER2 overexpression or hypoxia, or when HER2 binds to a dimerization ligand and undergoes a conformational change to reveal the loop that is bound by the current binding molecule, making it more accessible. Additionally, it is known that within the epitope region, the antibody does not bind to every amino acid in this region, and due to the conformational nature of the mAb104 epitope, this can explain why closely opposed epitopes do not affect the binding of the two antibodies.

[0437] In one example, the HER2 binding protein inhibits HER2 heterodimerization.

[0438] Antibody Conjugates

[0439] The present invention also provides a HER2-binding protein as described herein, conjugated to a moiety. The moiety can include, but is not limited to, a detectable or functional label. In some embodiments, the moiety is selected from the group consisting of radioisotopes, detectable labels, therapeutic compounds, colloids, toxins, nucleic acids, peptides, proteins, compounds that increase the half-life of the HER2-binding protein in a subject, and mixtures thereof. As will be understood by those skilled in the art, the moiety can be classified as one or more of the above-listed categories. For example, the moiety can be classified as a therapeutic compound and a toxin.

[0440] In some embodiments, the moiety is a radioisotope. Suitable radioisotopes include the isotopes 3 H, 14 C, 32 P, 33 P, 35 S, 36 Cl, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Ga, 68 Ga, 89 Zr, 90 Y, 121 I, 124 I, 125 I, 131 I, 111 In, 177 Lu, 211 At, 198 Au, 67 Cu, 223 Ra, 225 Ac, 213 Bi, 99 Tc and 186 Re, which can be linked to the antibodies of the present invention using conventional chemical methods known in the art of antibody imaging.

[0441] In some embodiments, the moiety is a detectable label. Suitable detectable labels include, but are not limited to, radiolabels, such as the isotopes 3 H, 14 C, 32 P, 33 P, 35 S, 36 Cl, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Ga, 68 Ga,89 Zr, 90 Y, 121 I, 124 I, 125 I, 131 I, 111 In, 177 Lu, 211 At, 198 Au, 67 Cu, 223 Ra, 225 Ac, 213 Bi, 99 Tc and 186 Re, which can be linked to the antibodies of the present invention using conventional chemical methods known in the field of antibody imaging. Labels also include fluorescent labels (e.g., fluorescein, rhodamine, Texas red, phycoerythrin) and labels conventionally used in the field for MRI-CT imaging. They also include enzyme labels such as horseradish peroxidase, β-glucuronidase, β-galactosidase, urease, catalase, alkaline phosphatase, and chloramphenicol transferase. Labels also include peptide tags such as T7-, his-, myc-, HA-, and FLAG-tags. Labels also include chemical moieties such as biotin, which can be detected by binding to a specific homologous detectable moiety such as labeled avidin. Labels also include electron-dense reagents; energy transfer molecules; paramagnetic labels, chemiluminescence (imidazole, luciferase); and bioluminescent agents.

[0442] In some embodiments, the moiety is a nucleic acid. Suitable nucleic acids include double-stranded DNA, single-stranded DNA, siRNA, DNAzyme, or ribozyme.

[0443] In some embodiments, the moiety is a therapeutic compound. Suitable therapeutic compounds include compounds capable of altering a biological response (e.g., but not limited to inhibiting or preventing the expression activity of a cell, causing the destruction of a cell, or otherwise affecting the function of a cell). Such therapeutic compounds include, for example but not limited to, chemical ablatives, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents and / or drugs, and include but are not limited to the following: 4-deacetylvinblastine-3-carbohydrate; 5-fluoro-2'-deoxyuridine; 5-fluorouracil; 5-fluorouracil decarb; 6-mercaptopurine; 6-thioguanine; abrin; abrin A chain; actinomycin D; 1-dehydrotestosterone; doxorubicin; ricin; alkylating agents; alkylphosphocholines; aminopterin; angiogenin; angiostatin; anthracyclines; anthranilamycin; antiangiogenic agents; antifolates; antimetabolites; antimitotics; antibiotics; cytarabine; asparaginase auristatin derivatives (see, e.g., but not limited to U.S. Patent Publications 2008 / 0300192, 2009 / 0018086, 2009 / 0018086, and 2009 / 0111756, each of which is incorporated herein by reference in its entirety); auristatin E (see, e.g., but not limited to U.S. Patent No. 5,635,483, which is incorporated herein by reference in its entirety); auristatin E valeryl benzyl hydrazone; auristatin F phenylenediamine; auristatin; auromycins; dichlorophenol mustard; bismuth; bleomycin; busulfan; calicheamicin; carboplatin; carminomycin; carmustine; cc-1065 compounds (see, e.g., but not limited to U.S. Patent Nos. 5,475,092, 5,585,499, 5,846,545, 6,534,660, 6,586,618, 6,756,397, 7,049,316, 7,329,760, 7,388,026, 7,655,660, and 7,655,661, U.S. Patent Publications 2007 / 0135346, 2008 / 0260685, 2009 / 0281158, and 2009 / 0318668, and PCT Publication No. WO 2009 / 017394, each of which is incorporated herein by reference in its entirety); chlorambucil; cis-dichlorodiamine platinum (cisplatin); cladribine; colchicine; carbetocin; crotoxin; curicin; cyclophosphamide; cytarabine; cytochalasin B; cytarabine; cytotoxins; dacarbazine; dactinomycin; daunorubicin; dianthin; dibromomannitol; dihydroxy anthracin dione; diphtheria toxin; dolastatin-10; docetaxel; doxorubicin; doxorubicin hydrazide;Duocarmycins (see, e.g., but not limited to, U.S. Pat. No. 7,214,685, which is incorporated herein by reference in its entirety); emetine; endostatin; enediyenes; enonomycin; epirubicin; esperamicin compounds (see, e.g., but not limited to, U.S. Pat. No. 4,675,187, which is incorporated herein by reference in its entirety); ethidium bromide; etoposide; fludarabine gelonin; gefitinib, gemcitabine; glucocorticoids; gramicidin D; granulocyte colony stimulating factor; granulocyte macrophage colony stimulating factor; idarubicin; intercalating agents; interleukin-1; interleukin-2; interleukin-6; lidocaine; lomustine; lymphokines; maytans inols) (see, e.g., but not limited to, U.S. Patents 4,137,230; 4,151,042; 4,162,940; 4,190,580; 4,225,494; 4,228,239; 4,248,870; 4,256,746; 4,260,608; 4,263,294; 4,264,596; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,320,200; 4,322,348; 4,331,598; 4,360,462; 4,361,6 50, 4,362,663, 4,364,866, 4,371,533, 4,424,219, 4,450,234, 5,141,736 and 5,217,713, each of which is incorporated herein by reference in its entirety); nitrogen mustard; melphalan (and other related nitrogen mustards); methotrexate; minor groove binders; mithramycin; mitogellin; mitomycin C; mitomycin; mitoxantrone; MMAF-dimethylaminoethylamine; MMAF-Nt-butyl; MMAF-tetraethylene glycol; modeccin A chain; momordica charantia inhibitor; monomethyl auristatin E (MMAE) (see, for example, but not limited to, U.S. Pat. Nos. 6,884,8 69, 7,098,308, 7,256,257 and 7,423,116, and U.S. Patent Publication Nos. 2003 / 0083263, 2004 / 0157782, 2005 / 0009751, 2005 / 0113308 and 2006 / 0229253, each of which is incorporated herein by reference in its entirety); monomethyl auristatin F (MMAF) (see, for example, but not limited to, U.S. Patent No. 7,498,298, and U.S. Patent Publication Nos. 2008 / 0226657, 2008 / 0248051, 2008 / 0248053 and 2009 / 0047296, each of which is incorporated herein by reference in its entirety); morpholinodoxorubicin;N2'-deacetyl-N2'-(c-mercapto-1-oxopropyl)-maytansine (DM1) (see, e.g., but not limited to, U.S. Patent No. 5,208,020, which is incorporated herein by reference in its entirety); N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) (see, e.g., but not limited to, U.S. Patent No. 7,276,497, which is incorporated herein by reference in its entirety); neocarzinostatin; nerve growth factor (and other growth factors); onapristone; paclitaxel; PE40; phenomycin; pokeweed antiviral protein (PAPI, PAPII, and PAP-S); platelet-derived growth factor; plicamycin; prednisone; procaine; procarbazine; propranolol; Pseudomonas exotoxin A; puromycin; pyrrolobenzodiazepine, radioisotopes (e.g., such as but not limited to At; 211 , Bi 212 , Bi 213 , Cf 252 , I 125 , I 131 , In 111 , Ir 192 , Lu 177 , P 32 , Re 186 , Re 188 , Sm 153 , Y 90 and W 188 ); restrictocin; restrictin; ricin A; ricin; saponin inhibitor; saponin; streptozocin; suramin; tamoxifen; taxanes; taxoids; tacrolimus; teniposide; tetracaine; thiotepa chlorambucil; thiotepa; thrombogenic agents; tissue plasminogen activator; topoisomerase I inhibitors; topoisomerase II inhibitors; toxotere; tricothecenes; tumor necrosis factor; vinblastine; vinca alkaloids; Vinca; vincristine; vindesine; vinorelbine; yttrium; alpha-interferon; alpha-sarcina; and beta-interferon, as well as their analogs, homologs, fragments, variants, and derivatives (see also Garnett (2001) Advanced drug Delivery Reviews 53:171-216, which is incorporated herein by reference in its entirety).

[0444] In a preferred embodiment, the therapeutic compound is selected from the group consisting of auristatin or its derivatives, maytansine or its derivatives (also known as maytansinol), or pyrrolobenzodiazepine or its derivatives. In one example, the therapeutic agent is N2′-deacetyl-N2′-(c-mercapto-1-oxopropyl)-maytansine (DM1). In another example, the therapeutic agent is monomethyl auristatin E (MMAE). In another example, the therapeutic agent is pyrrolobenzodiazepine.

[0445] The present invention also contemplates immunotoxin conjugates such as those described in WO 93 / 21232.

[0446] Suitable colloids include colloidal gold and gold nanoparticles. The HER2-binding protein can be conjugated to the colloid by techniques known to those skilled in the art (see Jazayeri et al. (2016) Sensing and Bio-Sensing Research, 9:17-22).

[0447] In some embodiments, the moiety is a toxin. Suitable toxins include, but are not limited to, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or enzymatically active fragments of such toxins. The enzymatically active toxins and their fragments used are diphtheria A chain, the non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, caryophyllin protein, pokeweed antiviral proteins (PAPI, PAPII, and PAP-S), momordica inhibitor, curcin, croton toxin, saponaria inhibitor, cladostephus protein, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0448] In some embodiments, the moiety is a compound that increases the half-life of the HER2-binding protein in a subject. Suitable compounds that increase the half-life of the HER2-binding protein in a subject include PEG, recombinant PEG mimetics (including flexible polypeptides such as XTEN, elastin-like polypeptides, gelatin-like polypeptides, and (Pro-Ala-Ser) n )), carbohydrates (such as dextran, hydroxyethyl starch, polysialic acid, and hyaluronic acid), and peptides / polypeptides (such as albumin and the Fc protein of IgG).

[0449] In addition, the HER2-binding proteins of the present invention, particularly antibodies and fragments thereof, can be conjugated to a secondary antibody to form an antibody heteroconjugate (see, e.g., but not limited to, U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety), can be administered alone or in combination with another agent (e.g., but not limited to the agents described above) (with or without an agent linked or conjugated thereto), and / or can be conjugated to an anticancer prodrug-activating enzyme capable of converting a prodrug into its active form.

[0450] As will be understood by those skilled in the art, the foregoing moieties and other suitable moieties can be conjugated or linked to the HER2-binding proteins of the present invention, particularly antibodies and fragments thereof, in any suitable manner to produce antibody conjugates. For example, but not limited to, in various embodiments of the present invention, the HER2-binding protein and the moiety can be covalently linked and / or can be conjugated using linkers, spacers, and / or extension compounds, which are cleavable, non-cleavable, and result in the internalization of the reagent by target cells in various embodiments of the present invention.

[0451] For example, such linkers, spacers, and / or extension compounds include, but are not limited to, the following: aminobenzoic acid spacers (see, e.g., but not limited to, U.S. Patent Nos. 7,091,186 and 7,553,816, each of which is incorporated herein by reference in its entirety); maleimidocaproyl; p-aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers (see, e.g., but not limited to, U.S. Patent No. 6,214,345, which is incorporated herein by reference in its entirety); maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valine-citrulline; 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) (see, e.g., but not limited to, Yoshitake et al., (1979) Eur. J. Biochem., 101, 395-399, which is incorporated herein by reference in its entirety); 4-(2-pyridyldithio)butyric acid N-succinimidyl ester (SPDB) (see, e.g., but not limited to, U.S. Patent No. 4,563,304, which is incorporated herein by reference in its entirety); 4-(2-pyridylthio)valeric acid N-succinimidyl ester (SPP); valine-citrulline; and other linkers, spacers, and / or extension compounds (see, e.g., but not limited to, U.S. Patent Nos. 7,090,843, 7,223,837, and 7,659,241, as well as U.S. Patent Publications 2004 / 0018194, 2004 / 0121940, 2006 / 0116422, 2007 / 0258987, 2008 / 0213289, 2008 / 0241128, 2008 / 0311136, 2008 / 0317747, and 2009 / 0010945, each of which is incorporated herein by reference in its entirety).

[0452] Generally, techniques for conjugating the above-described moieties and other moieties to the HER2-binding proteins of the present invention, particularly antibodies and fragments thereof, are known in the art. In various embodiments of the present invention, the HER2-binding protein and the moiety can be covalently conjugated and / or attached via lysine or cysteine residues present in the HER2-binding protein. In one embodiment, the MMAE moiety is attached by conjugation to a cysteine residue. In one embodiment, the DM1 moiety is attached by conjugation to a lysine residue. In one embodiment, the PBD (pyrrolobenzodiazepine) moiety is attached by conjugation to a cysteine residue. Suitable conjugation chemistries are reviewed in Jain et al. (2015) Pharmaceutical Research, 32:3526.See also, for example but not limited to, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds), pp. 303-16 (Academic Press 1985) and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” Immunol. Rev., 62:119-58 (1982), Parslow et al. (2016) Biomedicines, 4, 14, each of which is incorporated herein by reference in its entirety.

[0453] In embodiments where the linked moiety is a peptide or polypeptide, the conjugate can be a fusion protein in which the HER2 binding protein and the peptide or polypeptide form a single continuous polypeptide chain. These fusion proteins can be produced using techniques known in the art, including recombinant or synthetic techniques.

[0454] In addition, antibodies (including fragments thereof) and regulatory specific binding members, and drugs that modulate the production or activity of antibodies and / or their subunits can have certain diagnostic applications and can be used, for example, to detect and / or measure conditions such as cancer, pre-cancerous lesions, conditions associated with or caused by hyperproliferative cell growth, etc. For example, specific binding members, antibodies or their subunits can be used to generate polyclonal and monoclonal antibodies against themselves in a variety of cell culture media using known techniques such as hybridoma technology, for example, by fusing murine spleen lymphocytes and myeloma cells. Similarly, small molecules that mimic or antagonize the activity of the specific binding members of the present invention can be discovered or synthesized and can be used in diagnostic and / or therapeutic regimens.

[0455] Radioactively labeled specific binding members, particularly antibodies and their fragments, can be used in in vitro diagnostic techniques and in vivo radioimaging techniques as well as radioimmunotherapy. In the case of in vivo imaging, the specific binding members of the present invention can be conjugated to imaging agents rather than radioisotopes, including but not limited to magnetic resonance image enhancing agents, where, for example, antibody molecules are loaded with a large number of paramagnetic ions through chelating groups. Examples of chelating groups include EDTA, porphyrins, polyamine crown ethers, and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanum, holmium, and terbium. In another aspect of the present invention, radioactively labeled specific binding members, particularly antibodies and their fragments, particularly radioimmunoconjugates can be used in radioimmunotherapy, particularly as radioactively labeled antibodies for cancer treatment. In a further aspect, radioactively labeled specific binding members, particularly antibodies and their fragments can be used in radioimmunoguided surgery techniques, where they can identify and indicate the presence and / or location of cancer cells, pre-cancerous cells, tumor cells, and hyperproliferative cells before, during, or after surgical removal of these cells.

[0456] Competitive Inhibition

[0457] Antibodies that competitively inhibit the binding of the HER2 antibodies of the present invention to the epitope can be screened and identified using conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0458] Compositions of the Invention

[0459] HER2-binding proteins according to the invention, including their conjugates, will generally be administered in the form of a pharmaceutical composition, which may include at least one component other than the HER2-binding protein, HER2 antibody or antigen-binding fragment thereof. Thus, the pharmaceutical compositions according to the invention, as well as the uses according to the invention, may, in addition to the active ingredient, also include pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art. These substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, such as intravenously.

[0460] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may include solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions generally include liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oils. It may include physiological saline solutions, dextrose or other sugar solutions or diols such as ethylene glycol, propylene glycol or polyethylene glycol.

[0461] For intravenous, injection or injection at the site of pain, the active ingredient will be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the relevant art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. As needed, preservatives, stabilizers, buffers, antioxidants and / or other additives may be included.

[0462] In some embodiments, liposomes and / or nanoparticles may also be used with the active ingredient. The formation and use of liposomes are generally known to those skilled in the art. Liposomes can be formed from phospholipids dispersed in an aqueous medium and spontaneously forming multilamellar concentric bilayer vesicles (also known as multilamellar vesicles (MLV)). MLV generally may have a diameter of 25 nm to 4 μm. Sonication of MLV results in the formation of small unilamellar vesicles (SUV) with a diameter in the range of 200 to 500 angstroms, containing an aqueous solution in the core. When dispersed in water, phospholipids can form various structures different from liposomes, depending on the molar ratio of lipid to water. At low ratios, liposomes are the preferred structure. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes may exhibit low permeability to ions and polar substances, but undergo a phase transition at high temperatures, which significantly alters their permeability. The phase transition involves a change from a closely packed ordered structure called the gel state to a loosely packed disordered structure called the fluid state.

[0463] The HER2-binding protein or a composition comprising the same may be administered alone or in combination with other therapies, therapeutic agents or medicaments, either simultaneously or sequentially, depending on the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the HER2-binding protein described herein and other reagents or therapeutic agents (such as anti-cancer agents or therapeutic agents, hormones, other anti-HER2 agents or antibodies, or anti-EGFR agents or antibodies). More generally, these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational regulators, cell growth or division inhibitors (such as anti-mitotic agents) or signal transduction inhibitors. Other treatments or therapies may include administration of a suitable dose of a pain relief drug, such as a non-steroidal anti-inflammatory drug (such as aspirin, paracetamol, ibuprofen or ketoprofen) or an opioid preparation, such as morphine or an anti-emetic. The composition may be administered in combination (either sequentially (i.e., before or after) or simultaneously) with tyrosine kinase inhibitors (including but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosourea, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytarabine, cyclophosphamide, etoposide, carmustine, lomustine and / or other chemotherapeutic agents. Thus, these agents may be anti-HER2 specific agents or tyrosine kinase inhibitors, such as lapatinib, afatinib, AG1478, ZD1839, STI571, OSI-774 or SU-6668, or may be more common anti-cancer and anti-tumor agents, such as doxorubicin, cisplatin, temozolomide, nitrosourea, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytarabine, cyclophosphamide, etoposide, carmustine or lomustine. In addition, the composition may be used in combination with a hormone (such as dexamethasone), an immunomodulator (such as interleukin), a tumor necrosis factor (TNF) or other cytokines or angiogenesis inhibitors that stimulate an immune response and reduce or eliminate cancer cells or tumors.

[0464] In some instances, the HER2-binding protein or a composition comprising the same is combined with a chemotherapeutic agent, a radioimmunotherapeutic agent or an immunotherapeutic agent. In one instance, the immunotherapeutic agent is a checkpoint inhibitor. In another instance, the checkpoint inhibitor is selected from ipilimumab (CTLA4), nivolumab (PD-1), pembrolizumab (PD-1), atezolizumab (PD-L1), avelumab (PD-L1), durvalumab (PD-L1) and cemiplimab (PD-1).

[0465] In some instances, the HER2-binding protein or a composition comprising the same is administered together with an immunosuppressive drug.

[0466] In some instances, the HER2-binding protein or a composition comprising the same is administered together with an immunomodulator. Examples of suitable immunomodulators include interleukins (such as IL-2, IL-7, IL-12), cytokines (such as interferons, G-CSF), chemokines (such as CCL3, CCL26 and CXCL7), and immunomodulatory imide drugs (such as thalidomide).

[0467] The HER2-binding protein of the present invention can be administered to a patient in need of treatment via any suitable route, typically by injection into the bloodstream or CSF, or directly into the tumor site. The exact dose will depend on many factors, including whether the antibody is for diagnostic or therapeutic use, the size and location of the tumor, the exact nature of the HER2-binding protein (whether it is a whole antibody, fragment, diabody, etc.), and the nature of the detectable or functional label attached to the antibody. When a radionuclide is used for treatment, a suitable minimum single dose is about 45 mCi / m 2 , and the maximum is about 250 mCi / m 2 . The preferred dose range is 15 - 40 mCi, and the further preferred dose range is 20 to 30 mCi or 10 to 30 mCi. Such treatment may require bone marrow or stem cell replacement. The typical antibody dose for tumor imaging or tumor treatment is 0.5 to 40 mg, preferably 1 to 4 mg F(ab') 2 form of the antibody. The naked antibody is preferably administered at a dose of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is the dose for a single treatment of an adult patient, which can be adjusted proportionally for children and infants, and also for other antibody forms proportional to the molecular weight. The treatment can be repeated at intervals of daily, twice a week, weekly or monthly, according to the judgment of the physician.

[0468] Examples of suitable angiogenesis inhibitors (anti-angiogenic agents) include, but are not limited to, urokinase inhibitors, matrix metalloproteinase inhibitors (such as marimastat, neovastat, BAY 129566, AG3340, BMS275291, and similar agents), inhibitors of endothelial cell migration and proliferation (such as TNP470, squalamine, 2-methoxyestradiol, combretastatin, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ), and similar agents), antagonists of angiogenic growth factors (such as ZD6474, SU6668, antibodies against angiogenic agents and / or their receptors (such as VEGF, bFGF, and angiopoietin 1), thalidomide, thalidomide analogs (such as CC5013), Sugen 5416, SU5402, anti-angiogenic ribozymes (such as angiozyme), interferon α (such as interferon α2a), suramin, and similar drugs), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (such as SU011248), endothelial-specific integrin / survival signal transduction inhibitors (such as vitaxin and similar drugs), copper antagonists / chelators (such as tetrathiomolybdate, captopril, and similar drugs), carboxyamidotriazole (CAI), ABT627, CM101, interleukin 12 (IL12), IM862, PNU145156E, and nucleotide molecules that inhibit angiogenesis (such as antisense-VEGF-cDNA, cDNA encoding angiostatin, cDNA encoding p53, and cDNA encoding a defective VEGF receptor 2) and similar agents. Other examples of inhibitors of angiogenesis, neovascularization, and / or other vasculogenesis are anti-angiogenic heparin derivatives and related molecules (such as heparinase III), temozolomide, NK4, macrophage migration inhibitory factor (MIF), cyclooxygenase 2 inhibitors, hypoxia-inducible factor 1 inhibitors, anti-angiogenic soy isoflavones, oltipraz, fumagillin and its analogs, somatostatin analogs, pentosan polysulfate, tecarfarin sodium, dalteparin, tumstatin, thrombospondin, NM3, carbetocin, vasohibin, avastatin, antibodies against other related targets (such as anti-α-v / β-3 integrin and anti-kininostatin mAb), and similar agents.

[0469] Measuring Cell Viability and Proliferation

[0470] Cytotoxicity and viability (apoptosis, lysis, growth proliferation, etc.) can be measured in a variety of ways based on calorimetric, luminescent, radiometric or fluorescent assays known in the art and as exemplified herein. Colorimetric techniques for determining cell viability include, for example, trypan blue exclusion. Briefly, cells are stained with trypan blue and counted using a hemocytometer. Live cells exclude the dye, while dead and dying cells take up the blue dye and are easily distinguishable under an optical microscope. Neutral red is adsorbed by live cells and concentrated in cell lysosomes; the number of live cells can be determined by quantifying the number of neutral red-stained cells using an optical microscope.

[0471] Fluorescent techniques for determining cell viability include, for example, propidium iodide, a fluorescent DNA intercalator. Propidium iodide is excluded from live cells but stains the nuclei of dead cells. Flow cytometry of propidium iodide-labeled cells can then be used to quantify live and dead cells. Release of lactate dehydrogenase (LDH) indicates structural damage and death of cells and can be measured by spectrophotometric enzyme assay. Incorporation of bromodeoxyuridine (BrdU) into newly synthesized DNA is detected with an antibody labeled with a fluorescent dye. The fluorescent dye Hoechst 33258 labels DNA and can be used to quantify cell proliferation (e.g., flow cytometry). Quantitative incorporation of the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA-SE) provides cell division analysis (e.g., flow cytometry). This technique can be used in vitro or in vivo. 7-Aminoactinomycin D (7-AAD) is a fluorescent intercalator that undergoes a spectral shift upon binding to DNA and can provide cell division analysis (e.g., flow cytometry).

[0472] Radiometric measurement techniques for determining cell proliferation include, for example, 3 [3H]-thymidine, which is incorporated into newly synthesized DNA of live cells and is often used to assay cell proliferation. Release of chromium ([51Cr) from dead cells can be quantified by scintillation counting to quantify cell viability. 51 Cr) to quantify cell viability.

[0473] Luminescent techniques for determining cell viability include, for example, the CellTiter-Glo Luminescent Cell Viability Assay (Promega Madison Wis.). This technique quantifies the amount of ATP present to determine the number of live cells.

[0474] Commercially available kits for determining cell viability and cell proliferation include, for example, Cell Proliferation Biotrak ELISA (Amersham Biosciences Piscataway, N.J.); Guava ViaCount Assay, which provides rapid cell counting and viability determination based on differential uptake of fluorescent reagents (Guava Technologies, Hayward, Calif.); CyQUANT® Cell Proliferation Assay Kit (Molecular Probes, Inc., Eugene, Oreg.); and CytoLux Assay Kit (PerkinElmer Life Sciences Inc., Boston, Mass.). The DELFIA Assay Kit (PerkinElmer Life Sciences Inc., Boston, Mass.) can be used to determine cell proliferation and viability using time-resolved fluorometry. Quantitative cell proliferation assays are fluorescence-based assays that measure the fluorescence of DNA-dye complexes from lysed cells (Stratagene, La Jolla, Calif.). The CellTiter-Glo Cell Viability Assay is a luminescence assay for measuring cell viability (Promega, Madison Wis.).

[0475] Applications

[0476] (i) Diagnostic and therapeutic uses

[0477] The unique specificity of the HER2-binding proteins of the present invention, particularly antibodies or fragments thereof, whereby the binding proteins recognize HER2 / ErbB2 epitopes that are found in neoplastic, hyperproliferative or abnormal cells and are not detectable in normal or wild-type cells, and wherein the proteins bind to amplified EGFR and not to wild-type HER2, provides diagnostic and therapeutic uses for the identification, characterization, targeting and treatment, reduction or elimination of a variety of neoplastic cell types and tumor types (e.g., head and neck, breast, lung, bladder or prostate tumors and gliomas), without the problems associated with normal tissue uptake that may occur with previously known HER2 antibodies.

[0478] Thus, using the binding proteins of the present invention, particularly antibodies or fragments thereof, cells that overexpress HER2 (e.g., by amplification) can be identified, isolated, characterized, targeted and treated or eliminated.

[0479] Thus, the HER2-binding proteins (e.g., antibodies) of the present invention can specifically classify the nature of HER2 tumors or tumorigenic cells by staining or otherwise identifying those tumors or cells in which HER2 overexpression is present. In addition, the antibodies of the present invention, as exemplified by mAb104, demonstrate significant in vivo anti-tumor activity against tumors containing amplified HER2 and HER2-positive xenografts.

[0480] As described above, the inventors have found that when expressed in normal cells, the HER2-binding proteins of the present invention recognize the tumor-associated form of HER2, but not the normal wild-type receptor. It is believed that antibody recognition depends on a conformation responsive to HER2 amplification or activation, which exposes an epitope for binding.

[0481] It is shown that mAb104 inhibits the growth of overexpressed (e.g., amplified) HER2 xenografts of human tumors and induces necrosis within such tumors.

[0482] (ii) Therapeutic HER2-binding proteins and their uses

[0483] In vivo characteristics, particularly with respect to tumors: The blood ratio and clearance rate of the HER2-binding proteins of the present invention will be at least comparable to mAb104. After administration to a human or animal subject, such specific binding members will exhibit a peak tumor-to-blood ratio > 1:1. Preferably, at such a ratio, the specific binding member will also have a tumor-to-organ ratio greater than 1:1, preferably greater than 2:1, more preferably greater than 5:1. Preferably, at such a ratio, the binding protein will also have an organ-to-blood ratio < 1:1 in organs remote from the tumor site. These ratios exclude the organs in which the administered binding protein is catabolized and secreted. Thus, in the case of scFv and Fab, the binding member will be secreted by the kidneys. In the case of whole IgG, clearance will be at least in part via the liver. The peak localization ratio of the intact antibody typically reaches between 10 and 200 h after administration of the HER2-binding protein. More specifically, the ratio can be measured in tumor xenografts of approximately 0.2 to 1.0 g formed subcutaneously on one side of athymic nude mice.

[0484] The HER2-binding proteins (e.g., antibodies) of the present invention can be labeled with detectable or functional labels or moieties. As will be understood by those skilled in the art, the labels can be defined under more than one category. Detectable labels include, but are not limited to, radioactive labels such as isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co,59 Fe, 90 Y, 121 1, 124 1, 125 1, 131 I, πι In, 211 At, 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc and 186 Re, which can be linked to the antibodies of the present invention using conventional chemical methods known in the field of antibody imaging. Labels also include fluorescent labels and labels conventionally used in MRI-CT imaging in the art. They also include enzyme labels such as horseradish peroxidase. The label also includes chemical moieties such as biotin, which can be detected by binding to a specific homologous detectable moiety such as labeled avidin.

[0485] Functional labels include substances designed to target tumor sites to cause tumor tissue destruction. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the tumor site.

[0486] In addition, antibodies, including polyclonal and monoclonal antibodies, and drugs that regulate the production or activity of binding proteins, antibodies, and / or their subunits can have certain diagnostic applications and can be used, for example, to detect and / or measure conditions such as cancer, pre-cancerous lesions, conditions associated with or caused by hyperproliferative cell growth, etc. For example, HER2 binding proteins, antibodies, or their subunits can be used to generate polyclonal and monoclonal antibodies against themselves in a variety of cell culture media by known techniques such as hybridoma technology using, for example, fused mouse spleen lymphocytes and myeloma cells. Similarly, small molecules that mimic or antagonize the activity of the HER2 binding protein of the present invention can be discovered or synthesized and can be used in diagnostic and / or therapeutic regimens.

[0487] Radioactively labeled HER2-binding proteins, especially antibodies and their fragments, can be used in in vitro diagnostic techniques and in vivo radioimaging techniques as well as radioimmunotherapy. In the case of in vivo imaging, the HER2-binding proteins of the present invention can be conjugated to imaging agents rather than radioactive isotopes, including but not limited to magnetic resonance image enhancers, where for example the antibody molecule is loaded with a large number of paramagnetic ions through a chelating group. Examples of chelating groups include EDTA, porphyrin, polyamine crown ether and polyoxime. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanum, holmium and terbium. In another example of the present invention, radioactively labeled HER2-binding proteins, especially antibodies and their fragments, especially radioimmunoconjugates can be used for radioimmunotherapy, especially as radioactively labeled antibodies for cancer treatment. In a further example, radioactively labeled HER2-binding proteins, especially antibodies and their fragments can be used in radioimmunoguided surgical techniques, where they can identify and indicate the presence and / or location of cancer cells, pre-cancerous cells, tumor cells and hyperproliferative cells before, during or after surgical removal of these cells.

[0488] The immunoconjugates or antibody fusion proteins of the present invention, wherein the HER2-binding proteins of the present invention, especially antibodies and their fragments are conjugated or linked to other molecules or reagents also include but are not limited to binding proteins conjugated to chemical ablating agents, toxins, immunomodulators, cytokines, cytotoxic drugs, chemotherapeutic agents or drugs.

[0489] The efficacy of radioimmunotherapy (RAFT) has been demonstrated using various antibody immunoconjugates. It has been evaluated in colorectal cancer 131 I-labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 (Behr TM et al. (2002) Cancer 94 (Suppl 4): 1373-81) and has been evaluated in medullary thyroid cancer with 90 the same antibody labeled with

[0490] Radioimmunoguided surgery (RIGS) has demonstrated efficacy and usefulness, including the use of anti-CEA antibodies and antibodies against tumor-associated antigens (Kim JC et al. (2002) Int J Cancer 97(4):542-7; Schneebaum S et al. (2001) World J Surg 25(12):1495-8; Avital S et al. (2000) Cancer 89(8):l092-8; McLosh DG et al. (1997) Cancer Biothcr Radiopharai 12(4):2S7-94).

[0491] The HER2-binding proteins (e.g., antibodies) of the present invention can be administered to a patient in need of treatment via any suitable route, typically by injection into the bloodstream or CSF, or directly into the tumor site. The exact dose will depend on many factors, including whether the antibody is for diagnostic or therapeutic use, the size and location of the tumor, the exact nature of the antibody (whether whole antibody, fragment, diabody, etc.), and the nature of the detectable or functional label attached to the antibody. When a radionuclide is used for treatment, a suitable maximum single dose is about 45 mCi / m 2 , up to about 250 mCi / m 2 . Preferred dose ranges are 15 - 40 mCi, and further preferred dose ranges are 20 to 30 mCi or 10 to 30 mCi. Such treatment may require bone marrow or stem cell replacement. Typical antibody doses for tumor imaging or tumor treatment are 0.5 to 40 mg, preferably 1 to 4 mg F(ab') 2 form of the antibody. Naked antibodies are preferably administered at a dose of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is the dose for a single treatment of an adult patient, which can be adjusted proportionally for children and infants, and also for other antibody forms proportional to the molecular weight. Treatment can be repeated at intervals of daily, twice weekly, weekly, or monthly, at the discretion of the physician.

[0492] These formulations may include a second binding protein, such as the EGFR-binding protein or HER2-binding protein of the present invention. In a particularly preferred form, the second binding protein is trastuzumab.

[0493] (iii) Anti-cancer treatment

[0494] The HER2-binding proteins (e.g., antibodies) of the present invention can be used in a variety of applications, including research, diagnostic, and therapeutic applications. In one example, the present invention provides a method of treating or preventing a disorder in a subject. As used herein, a "disorder" is a disruption or interference with normal function.

[0495] (iv) Diagnostic assays

[0496] The present invention also relates to a variety of in vitro or in vivo diagnostic applications, including methods for detecting the presence of aberrantly expressed HER2 by reference to the ability of the HER2-binding proteins (such as antibodies) of the present invention to recognize aberrantly expressed HER2. Diagnostic applications of the antibodies of the present invention include in vitro and in vivo applications well-known and standard to those skilled in the art and based on this specification. Diagnostic assays and kits for in vitro assessment and evaluation of HER2 status, particularly with regard to aberrant HER2 expression, can be used to diagnose, evaluate, and monitor patient samples, including those known or suspected of having cancer, pre-cancerous conditions, conditions associated with hyperproliferative cell growth, or from tumor samples. Assessment and evaluation of HER2 status can also be used to determine a patient's suitability for a drug clinical trial or for the administration of a particular chemotherapeutic agent or specific binding member (particularly an antibody) of the present invention (including combinations thereof) in combination with different agents or antibodies. This type of diagnostic monitoring and evaluation has been practiced in the art using antibodies against the HER2 protein in breast cancer (Hercep Test, Dako Corporation), where the assay is also used to evaluate a patient's antibody therapy with Herceptin. In vivo applications include tumor imaging or assessment of an individual's cancer status, including radioimaging.

[0497] The presence of HER2 in cells can be determined in in vitro or in vivo immunological methods known to those skilled in the art. For example, the HER2 receptor forms a complex with one or more antibodies, and one member of the complex is labeled with a detectable marker. The most commonly used labels for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, etc. Many fluorescent materials are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramine, Texas red, AMCA blue, and fluorescein yellow. Anti-HER2 antibodies can also be labeled with radioactive elements or enzymes. Radioactive labels can be detected by any currently available counting procedure. Preferred isotopes can be selected from 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 1, 125 1, 131 I 5 111 In, 211 At, 198 Au, 67 Cu,225 Ac, 213 Bi, 99 Tc, and 186 Re. Enzyme labels are also useful and can be detected by any of the currently used colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particles by reaction with a bridging molecule such as carbodiimide, diisocyanate, glutaraldehyde and the like. Many enzymes that can be used in these methods are known and available. Preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090, 3,850,752 and 4,016,043 are mentioned by way of example of alternative labeling materials and methods disclosed therein.

[0498] Kits

[0499] The invention also contemplates the use of a therapeutic or diagnostic kit comprising the HER2-binding protein of the invention for such therapeutic methods. Such kits generally contain a pharmaceutically acceptable formulation of at least one HER2-binding protein (such as an antibody or fragment) of the invention in a suitable container device. The kit can be used to detect the presence of the HER2 receptor in a biological sample. The antibody compositions of the invention can be provided in liquid or lyophilized form, alone or in combination with other antibodies specific for other epitopes. The antibody, labeled or unlabeled, can be included in the kit together with auxiliary components such as buffers, such as Tris, phosphate and carbonate, stabilizers, excipients, antimicrobial agents and / or inert proteins, such as bovine serum albumin. For example, the antibody can be provided as a lyophilized mixture with the auxiliary components, or the auxiliary components can be provided separately for the user to combine. Based on the amount of active antibody, these auxiliary substances are generally present in an amount less than about 5 wt%, and based on the antibody concentration, generally in an amount of at least about 0.001 wt% in total. When a secondary antibody capable of binding to the antibody is used, such an antibody can be provided in the kit, for example, in a separate vial or container. The secondary antibody, if present, is generally labeled and can be formulated in a manner similar to the antibody preparations herein.

[0500] Commercial test kits suitable for use by medical professionals can be prepared to determine the presence or absence of abnormal expression of HER2, including but not limited to amplified HER2, in suspected target cells. Depending on the test techniques discussed above, one class of such kits will contain at least a labeled HER2 or its binding partner, such as an antibody specific therefor, and of course depending on the guidance of the selected method, such as "competitive", "sandwich", "DASP", etc. The kit can also contain peripheral reagents such as buffers, stabilizers, etc.

[0501] Thus, test kits can be prepared for demonstrating the presence of cells or cells capable of aberrantly expressing HER2, and include:

[0502] (a) A predetermined amount of at least one labeled immunochemically reactive component obtained by directly or indirectly linking a HER2-binding protein or its specific binding partner as described herein to a detectable label;

[0503] (b) Other reagents; and

[0504] (c) Instructions for using the kit.

[0505] More specifically, the diagnostic test kit can include:

[0506] (a) A known amount of the HER2-binding protein (or binding partner) as described above, which is typically bound to a solid phase to form an immunosorbent, or alternatively, bound to a suitable tag, or a plurality of such end products, etc. (or its binding partner), one of each;

[0507] (b) Other reagents, if necessary; and

[0508] (c) Instructions for using the test kit.

[0509] In another example, test kits can be prepared and used for the above purposes, which operate according to a predetermined protocol (e.g., "competitive", "sandwich", "double antibody", etc.) and include:

[0510] (a) A labeled component obtained by conjugating a HER2-binding protein with a detectable label;

[0511] (b) One or more additional immunochemical reagents, wherein at least one reagent is a ligand or an immobilized ligand, and the ligand is selected from the group consisting of:

[0512] (i) A ligand capable of binding to the labeled component (a);

[0513] (ii) A ligand capable of binding to the binding partner of the labeled component (a);

[0514] (iii) A ligand capable of binding to at least one component to be assayed; and

[0515] (iv) A ligand capable of binding to at least one binding partner of at least one component to be assayed; and

[0516] (c) Instructions for implementing a protocol for detecting and / or assaying an immunochemical reaction between HER2, the HER2-binding protein, and its specific binding partner.

[0517] Based on the above, an assay system can be prepared for screening potential drugs that effectively modulate HER2 activity, HER2 aberrant expression, and / or the activity or binding of HER2-binding proteins. A receptor or binding protein can be introduced into the test system, or a prospective drug can be introduced into the resulting cell culture, and then the culture is examined to observe any changes in the S-phase activity of the cells, either due to the addition of the prospective drug alone or due to the action of a known reagent in an added amount.

[0518] This application also relates to the following embodiments:

[0519] Embodiment 1. A HER2 / ErbB2-binding protein comprising an antigen-binding domain, wherein the antigen-binding domain specifically binds to an epitope within Domain II of HER2, comprising residues 293 to 309 of the mature normal or wild-type human HER2 sequence according to Figure 1 (SEQ ID NO: 27), and the epitope is exposed in response to HER2 amplification or activation, and wherein the epitope is expressed in neoplastic, hyperproliferative, or abnormal cells but not in normal or wild-type cells.

[0520] Embodiment 2. The HER2-binding protein according to Embodiment 1, wherein the epitope comprises the amino acid sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1).

[0521] Embodiment 3. The HER2-binding protein according to Embodiment 1 or 2, wherein the HER2-binding protein is an antibody or an antigen-binding fragment thereof, comprising:

[0522] (i) a heavy-chain variable region sequence (VH) having at least 55% identity with the heavy-chain variable region sequence of mAb104 (SEQ ID NO: 2); and / or

[0523] (ii) a light-chain variable region sequence (VL) having at least 50% identity with the light-chain variable region sequence of mAb104 (SEQ ID NO: 3).

[0524] Embodiment 4. The HER2-binding protein according to any one of the foregoing embodiments, wherein the binding protein comprises:

[0525] (i) a VH CDR1 having the following sequence:

[0526] GYX 7 FTX 8 YX 9 MX 10 (SEQ ID NO: 6)

[0527] wherein X7 is S or T; X 8 is G or D; X 9 is F or G; X 10 is H or N;

[0528] (ii) has a VH CDR2 with the following sequence:

[0529] X 19 INX 20 YX 21 GX 22 X 23 X 24 YX 25 X 26 X 27 FKX 28 (SEQ ID NO: 7)

[0530] where X 19 is R or W; X 20 is P or T; X 21 is N or T; X 22 is D or K; X 23 is I or P; X 24 is R or T; X 25 is N or D; X 26 is Q or D; X 27 is N or D; and X 28 is D or G;

[0531] (iii) has a VH CDR3 with the following sequence:

[0532] X 50 X 51 X 52 X 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 X 61 FX 62 Y(SEQ ID NO: 8)

[0533] where X 50 is absent or is R; X 51 is absent or is F; X 52 is absent or is L; X 53 is absent or is N; X 54 is absent or is T; X 55 is absent or is V; X 56 is absent or is A; X 57 is absent or is G; X 58Absent or R; X 59 Absent or S; X 60 Is L or V; X 61 Is N or Y; and X 62 Is A or D;

[0534] And / or

[0535] (iv) Having the following sequence for VL CDR1:

[0536] X 14 X 15 SX 16 SX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 (SEQ ID NO: 9)

[0537] Where X 14 Is K or S; X 15 Is S or V; X 16 Is Q or S; X 17 Is L or absent; X 18 Is L or absent; X 19 Is D or absent; X 20 Is S or absent; X 21 Is D or absent; X 22 Is G or absent; X 23 Is K or V; X 24 Is T or G; X 25 Is F or S; X 26 Is L or M; and X 27 Is N or Y;

[0538] (v) Having the following sequence for VL CDR2:

[0539] LX 35 SX 36 LX 37 S(SEQ ID NO: 10)

[0540] X 35 Is D or E; X 36 Is K or T; X 37 Is S or A; and

[0541] (vi) Having the following sequence for VL CDR3:

[0542] X49 QX 50 X 51 X 52 X 53 PX 54 T (SEQ ID NO: 11)

[0543] wherein X 49 is W or Q; X 50 is G or W; X 51 is T or S; X 52 is H or S; X 53 is F or N; and X 54 is W or P.

[0544] Embodiment 5. The HER2 binding protein according to any one of the foregoing embodiments, wherein the binding protein comprises the heavy chain variable region sequence (VH) shown below:

[0545] X 1 X 2 QLX 3 QSGPELX 4 KPGX 5 X 6 VKISCKAS GYX 7 FTX 8 YX 9 MX 10 WVX 11 QX 12 X 13

[0546] X 14 X 15 X 16 LX 17 WX 18 G X 19 INX 20 YX 21 GX 22 X 23 X 24 YX 25 X 26 X 27 FKX 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 SX 37 STAYX 38 X 39 X 40 X 41 X 42 LX 43 X 44 EDX 45 AX 46 X 47 X 48 CAX 49 X 50 X 51

[0547] X 52 X 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 X 61 FX 62 Y WGQGTX 63 X64 TVSX 65 (SEQ ID NO: 12)

[0548] Wherein

[0549] X 1 is E or Q; X 2 is V or I; X 3 is Q or V; X 4 is V or K; X 5 is A or E; X 6 is S or T; X 7 is S or T; X 8 is G or D; X 9 is F or G; X 10 is H or N; X 11 is R or K; X 12 is S or A; X 13 is H or P; X 14 is V or G; X 15 is R or K; X 16 is S or G; X 17 is E or K; X 18 is I or M; X 19 is R or W; X 20 is P or T; X 21 is N or T; X 22 is D or K; X 23 is I or P; X 24 is R or T; X 25 is N or D; X 26 is Q or D; X 27 is N or D; X 28 is D or G; X 29 is K or R; X 30 is A or F; X 31 is S or A; X 32 is L or F; X 33 is T or S; X 34 is V or L; X 35 is D or E; X 36 is K or T; X 37 is S or A; X 38 is M or L; X 39 is E or Q; X 40 is L or I; X 41 is H or N; X 42 is R or N; X 43 is T or K; X 44 is S or N; X 45 is S or M; X 46 is V or T; X 47 is F or Y; X 48is Y or F; X 49 is S or R; X 50 does not exist or is R; X 51 does not exist or is F; X 52 does not exist or is L; X 53 does not exist or is N; X 54 does not exist or is T; X 55 does not exist or is V; X 56 does not exist or is A; X 57 does not exist or is G; X 58 does not exist or is R; X 59 does not exist or is S; X 60 is L or V; X 61 is N or Y; X 62 is A or D; X 63 is P or T; X 64 is V or L; and X 65 is A or S;

[0550] and / or

[0551] The light chain variable region sequence (VL) is as follows:

[0552] X 1 IVX 2 TQSPX 3 X 4 X 5 SVX 6 X 7 GX 8 X 9 X 10 X 11 X 12 X 13 C X 14 X 15 SX 16 SX 17 X 18 X 19 X 20 X 21

[0553] X 22 X 23 X 24 X 25 X 26 X 27 WX 28 X 29 QX 30 PX 31 X 32 SPKX 33 X 34 IY LX 35 SX 36 LX 37 S GVPX 38 RFX 39 GSGSGTX 4 0 X 41 X 42 LX 43 ISX 44 X 45 EAEDX 46 X 47 X 48 YY CX 49 QX 50 X 51 X 52 X 53 PX 54 T FGX 55 GTKLEX 56 KR (SEQ ID NO: 13)

[0554] wherein

[0555] X 1 is D or Q; X 2 is I or L; X 3 is L or A; X 4 is T or L; X 5 is L or M; X 6 is T or S; X 7 is F or P; X 8 is Q or E; X 9 is P or K; X 10 is A or V; X 11 is S or T; X 12 is I or M; X 13 is S or T; X 14 is K or S; X 15 is S or V; X 16 is Q or S; X 17 is L or absent; X 18 is L or absent; X 19 is D or absent; X 20 is S or absent; X 21 is D or absent; X 22 is G or absent; X 23 is K or V; X 24 is T or G; X 25 is F or S; X 26 is L or M; X 27 is N or Y; X 28 is L or Y; X 29 is L or Q; X 30 is R or K; X 31 is G or R; X 32 is Q or S; X 33 is R or P; X 34 is L or W; X 35 is V or T; X 36 is K or N; X 37 is D or A; X 38 is D or P; X 39是 T or S; X 40 is D or S; X 41 is F or Y; X 42 is T or S; X 43 is K or T; X 44 is R or S; X 45 is V or M; X 46is L or A; X 47 is G or A; X 48 is V or T; X 49 is W or Q; X 50 is G or W; X 51 is T or S; X 52 is H or S; X 53 is F or N; X 54 is W or P; X 55 is G or A; and X 56 is I or L.

[0556] Embodiment 6. The HER2-binding protein according to any one of the foregoing embodiments, wherein the VH comprises:

[0557] (i) a CDR1 sequence selected from GYSFTGYFMH (SEQ ID NO: 14) or GYTFTDYGMN (SEQ ID NO: 15);

[0558] (ii) a CDR2 sequence selected from RINPYNGDIRYNQNFKD (SEQ ID NO: 16) or WINTYTGKPTYDDDFKG (SEQ ID NO: 17);

[0559] (iii) a CDR3 sequence selected from LNFAY (SEQ ID NO: 18) or RFLNTVAGRSVYFDY (SEQ ID NO: 19); and

[0560] wherein the VL comprises:

[0561] (iv) a CDR1 sequence selected from KSSQSLLDSDGKTFLN (SEQ ID NO: 20) or SVSSSVGSMY (SEQ ID NO: 21);

[0562] (v) a CDR2 sequence selected from LVSKLDS (SEQ ID NO: 22) or LTSNLAS (SEQ ID NO: 23); and

[0563] (vi) a CDR3 sequence selected from WQGTHFPWT (SEQ ID NO: 24) or QQWSSNPPT (SEQ ID NO: 25).

[0564] Embodiment 7. The HER2-binding protein according to any one of the foregoing embodiments, wherein the HER2-binding protein comprises:

[0565] a heavy chain variable region sequence (VH) having CDR1, CDR2, and CDR3 sequences that respectively comprise or consist of the following:

[0566] (i) SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18; or

[0567] (ii) SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19;

[0568] and / or

[0569] a light chain variable region sequence (VL) having CDR1, CDR2, and CDR3 sequences that respectively comprise or consist of the following:

[0570] (i) SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24; or

[0571] (ii) SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25.

[0572] Embodiment 8. The HER2-binding protein according to any one of the preceding embodiments, wherein the HER2-binding protein comprises CDRs having sequences comprising SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24.

[0573] Embodiment 9. The HER2-binding protein according to any one of the preceding embodiments, wherein the HER2-binding protein comprises CDRs having sequences comprising SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, and SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25.

[0574] Embodiment 10. The HER2-binding protein according to any one of the preceding claims, wherein the HER2-binding protein comprises a VH and / or a VL, the VH comprises a sequence having at least 55% identity to the sequence shown in SEQ ID NO: 2, the VL comprises a sequence having at least 50% identity to the sequence shown in SEQ ID NO: 3, or a humanized, chimeric, or deimmunized form thereof.

[0575] Embodiment 11. A HER2-binding protein according to any one of the foregoing requirements, wherein the HER2-binding protein comprises a VH and / or a VL, the VH comprises a sequence having at least 55% identity to the sequence shown in SEQ ID NO: 4, the VL comprises a sequence having at least 50% identity to the sequence shown in SEQ ID NO: 5, or a humanized, chimeric or deimmunized form thereof.

[0576] Embodiment 12. The HER2-binding protein according to any one of the foregoing embodiments, wherein the HER2-binding protein comprises:

[0577] (i) a VH shown in SEQ ID NO: 2 and a VL shown in SEQ ID NO: 3; or

[0578] (ii) a VH shown in SEQ ID NO: 4 and a VL shown in SEQ ID NO: 5.

[0579] Embodiment 13. The HER2-binding protein according to any one of Embodiments 3 to 12, wherein the antigen-binding fragment is:

[0580] (i) a single-chain Fv fragment (scFv);

[0581] (ii) a dimeric scFv (di-scFv);

[0582] (iii) at least one of (i) and / or (ii) linked to a heavy-chain constant region or Fc or heavy-chain constant domain (CH)2 and / or CH3; or

[0583] (iv) at least one of (i) and / or (ii) linked to a protein that enhances the antibody half-life.

[0584] Embodiment 14. The HER2-binding protein according to any one of the foregoing embodiments, wherein the antigen-binding fragment is:

[0585] (i) a diabody;

[0586] (ii) a triabody;

[0587] (iii) a tetrabody;

[0588] (iv) a Fab;

[0589] (v) an F(ab′)2;

[0590] (vi) an Fv; or

[0591] (vii) at least one of (i) to (vi) linked to a heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3; or

[0592] (viii) at least one of (i) to (vi) linked to a protein that enhances the antibody half-life.

[0593] Embodiment 15. The HER2-binding protein according to any one of the foregoing embodiments, which is conjugated to a detectable or functional moiety.

[0594] Embodiment 16. The HER2-binding protein according to any one of Embodiments 1 to 14, which is conjugated to a drug.

[0595] Embodiment 17. A combination composition comprising (i) a HER2-binding protein according to any one of Embodiments 1 to 14 and (ii) an anti-HER2 antibody, a chemotherapeutic agent, a radioimmunotherapeutic agent, or an immunotherapeutic agent, or a combination thereof.

[0596] Embodiment 18. A composition comprising a HER2-binding protein according to any one of Embodiments 1-16 and a suitable carrier.

[0597] Embodiment 19. A method of treating cancer expressing HER2 in a subject, comprising administering to a subject in need thereof a HER2-binding protein according to any one of Embodiments 1 to 16, the combination according to Embodiment 17, or the composition according to Embodiment 18.

[0598] Embodiment 20. A method for detecting HER2 in a biological sample, the method comprising contacting the sample with a HER2-binding protein or antibody according to any one of Embodiments 1 to 16 and detecting the complex, wherein detecting the complex indicates HER2 expression in the sample.

[0599] Those skilled in the art will understand that many variations and / or modifications can be made to the present invention as shown in the specific embodiments without departing from the scope of the present invention as broadly described. Accordingly, this embodiment is considered illustrative in all respects and not restrictive.

[0600] The present invention is further described in the following non-limiting examples.

[0601] Examples

[0602] Those skilled in the art should understand that various changes and / or modifications can be made to the above embodiments without departing from the broad scope of the present disclosure. Accordingly, this embodiment is considered illustrative in all respects and not restrictive.

[0603] Materials and Methods

[0604] Cell Lines and Culture Conditions

[0605] The parental lines were obtained from the American Type Culture Collection (ATCC, USA), Asterand Bioscience (USA), Ludwig Institute for Cancer Research or the Australian Cell Bank (Australia). The cells were incubated in the medium recommended by the supplier in a 37 °C incubator with 5% CO 2 cultivation. All media were supplemented with 10% fetal calf serum (FCS) (CSL, Melbourne, Victoria, Australia), 2 mM glutamine (Sigma Chemicals Co, St Louis, MO, USA) and 2 mM penicillin / streptomycin (Life Technologies, Grand Island, NY, USA). The cells were passaged and their medium was changed when they reached 80% confluence. The cells were used for experiments in the exponential growth phase. To passage adherent cells, the medium was removed and an appropriate volume (based on the growth surface area) of PBS solution containing 2 mM EDTA and trypsin (Life technologies TM , Australia) was added. The cell lines are described in Table 1.

[0606] Table 1 Cell Lines

[0607]

[0608] Antibodies and Antigens

[0609] The primary antibodies were purchased from the commercial sources listed in Table 2 below or purified from hybridoma supernatants using protein-G affinity chromatography.

[0610] Table 2 Antibodies Used in This Application

[0611]

[0612] Antigens

[0613] Linear and cyclized peptide immunogens and unrelated control peptides were chemically synthesized by Mimitopes Pty Ltd (Clayton, Australia) and conjugated to keyhole limpet hemocyanin (KLH). The linear peptide sequence is H-CPLHNQEVTAEDGTQR-NH2, and the cyclic peptide sequence is H-GCPLHNQEVTAEDGTQRC-NH 2 , where H- at the N-terminus refers to the free amine. A control unrelated peptide H-LEEKKGNYVVTDHC-NH 2 conjugated to KLH was also prepared.

[0614] Based on a previously published method, the HER2 extracellular domain (HER2-ECD) was generated in the laboratory of academic collaborator Professor A.W. Burgess (The Walter and Eliza Hall Institute of Medical Research) (Xu Y, Soo P, Walker F, Zhang HH, Redpath N, Tan CW et al., LRIG1 extracellular domain: Structure and function analysis. Journal of molecular biology. 2015;427(10):1934-48.). Briefly, synthetic DNA corresponding to human HER2-ECD was cloned into an expression vector, expressed in Hi5 insect cells, and purified by anti-FLAG M2 beads (Sigma-Aldrich). The protein was further purified by gel filtration in 20 mM Tris–HCl (pH 8.5) and 100 mM NaCl.

[0615] Cell Biology Reagents and Sources

[0616] Details of the reagents are provided in the table below.

[0617] Table 3 Reagents used

[0618]

[0619] Media and Solutions

[0620] Agarose (1-1.5%): Dissolve agarose in 1×TAE to a final concentration of 1-1.5% w / v.

[0621] Agarose buffer 1x: 10 mM BisTris HCl pH 6.5; 0.2 mM EDTA; 100 mM NaCl.

[0622] Blocking buffer: 5% w / v non-fat milk powder (Fonterra, Mount Waverly, Australia), 0.1% v / v Tween 20 (ICN Biomedicals) in Tris-buffered saline (TBS; 20 mM Tris-HCl, 150 mM NaCl).

[0623] Diethanolamine-HCl buffer: Diethanolamine (10% or 0.1 M), MgCl2.6H2O (1 mM), NaN3

[0624] DMEM-10: Dulbeccos modified eagles medium supplemented with 10% FCS, 2 mM L-alanyl-L-glutamine GlutaMAX TM ), 100 U / ml penicillin and 100 mg / ml streptomycin.

[0625] EDTA-PBS: PBS solution with 2 mM EDTA, without Ca 2+ or Mg 2+ .

[0626] EDTA-PBS-3% FCS: EDTA-PBS supplemented with 3% FCS.

[0627] MATRIGEL TM Matrix:

[0628] Composition MATRIGEL TM Matrix (product information)

[0629]

[0630] RIPA buffer: 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 10 mM NaF and protease inhibitor (pH 7.5).

[0631] RPMI-10 medium: RPMI-1640 containing 10% fetal bovine serum (FCS), 2 mM L-alanyl-L-glutamine (GlutaMAX TM ), 100 U / ml penicillin and 100 mg / ml streptomycin.

[0632] Running buffer (Western blot): Dilute SDS running buffer (Invitrogen) in milliQ H2O to 1× running buffer.

[0633] TBS (10×): 24.2 g Base; 80 g sodium chloride; 970 ml H2O, adjust pH to 7.5 with HCl, total volume 1000 ml.

[0634] TBS-T: Supplemented with 0.05% 20 of 1× TBS.

[0635] Cell Biology Instruments

[0636] The following table provides detailed information on cell biology instruments.

[0637] Table 4 Instrument Details

[0638]

[0639] Immunization of Mice and Monoclonal Antibody Production

[0640] Female BALB / c mice were immunized with 30 μg of a peptide containing the conformationally exposed region of the HER2 extracellular domain, as determined by structural modeling, the sequence H-GCPLHNQEVTAEDGTQRC-NH 2 (SEQ ID NO: 26) synthesized as a cyclic peptide and conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. Injections were made intraperitoneally at four-week intervals. Antigen was prepared in phosphate-buffered saline (PBS, pH 7.2) and then mixed with Freund's complete adjuvant (Sigma, St. Louis, MO) (Flies DB, Chen L. A simple and rapid vortex method for preparing antigen / adjuvant emulsions for immunization. Journal of immunological methods. 2003;276(1):239 - 42) for the first injection and Freund's incomplete adjuvant for the second injection. Then two separate booster injections of the peptide immunogen were given. Three days after the last immunization, the mice were sacrificed, and splenocytes from the hyperimmunized mice were harvested and fused with the mouse myeloma cell line SP2 / 0 at a ratio of 1:50 to generate hybridomas (Yokoyama WM, Christensen M, Santos GD, Miller D, Ho J, Wu T et al., Production of monoclonal antibodies. Current protocols in immunology. 2006:2.5.1 - 2.5.29). The fused cells were grown in complete RPMI medium supplemented with 10% FCS and additives. The supernatants of the growing cells were screened using indirect ELISA.

[0641] Enzyme-Linked Immunosorbent Assay (ELISA)

[0642] A polystyrene 96-well plate was coated overnight at 40 °C with 3 μg / mL HER2-ECD peptide-KLH antigen as linear or cyclic peptide, negative control-KLH conjugated peptide, or recombinant HER2 ECD in PBS. The plate was blocked for 1 h at room temperature (RT) with PBS containing 3% FCS. The plate was incubated for 1 h with serial dilutions of hybridoma supernatant starting at a 1:50 dilution and appropriate controls. After washing three times, the plate was incubated with anti-mouse IgG HRP conjugate (1:2000 dilution) for 1 h at RT. After washing three more times, phosphatase activity was measured using pNPP substrate and absorbance (OD) was read at 405 nm using a Versamax microplate reader (Molecular Devices) with Softmax Pro 4.8 software.

[0643] After identifying and purifying antibodies from positive hybridomas from 4 candidate clones (referred to herein as mAb104, mAb105, mAb106, or mAb107), they were evaluated by ELISA. A polystyrene 96-well plate was coated for 1 h at RT with 3% FCS in PBS. The final peptide concentration of 1 mg / mL was achieved by diluting the peptide in 0.1% acetic acid. This peptide solution was further diluted to 30 μg / ml in 3% FCS-PBS. The plate was incubated for 1 h at RT in dilution buffer (3% FCS-PBS) with linear or cyclic peptide immunogens conjugated to KLH, HER2-ECD, or negative control peptide-KLH. After washing the plate three times with 0.05% Tween 20-PBS, the wells were incubated for 1 h with 10 μg / mL mAb104, mAb105, mAb106, or mAb107. After washing, the plate was incubated with anti-mouse Ig-alkaline phosphatase (Sigma A-3688) (1:3000 dilution) for 1 h at RT. After washing three more times, phosphatase activity was measured using pNPP substrate and absorbance of optical density was read at 405 nm.

[0644] A polystyrene 96-well plate was coated overnight at 40 °C with a PBS solution of 50 μl / well of 3 μg / mL recombinant ErbB2 ECD, ErbB3 ECD, ErbB4 ECD, or EGFR501. The plate was blocked for 1 h at room temperature (RT) with PBS containing 3% FCS. The plate was incubated for 1 h at RT with serial dilutions of 10 μg / mL purified antibody and appropriate controls. After washing three times, the plate was incubated with anti-mouse IgG AP conjugate (1:2000 dilution) for 1 h at RT. After washing three more times, phosphatase activity was measured using pNPP substrate and absorbance (OD) was read at 405 nm using a SPECTROstar microplate reader (BMG LABTECH, Victoria, Australia).

[0645] FACS Analysis

[0646] Cells (1×10 4 ) were seeded in 96-well plates and incubated with 10 μg / ml anti-HER2 antibody or IgG1 isotype control antibody at 4 °C for 1 h. Humanized antibodies were detected for trastuzumab and pertuzumab using Alexa-488-conjugated anti-human IgG antibody. Bound mAb104, mAb105, mAb106, or mAb107 or murine isotype control LMH-3 were detected using Alexa-488-conjugated anti-mouse IgG antibody and fluorescence was read on a Becton Dickinson FACScan (version CellQuestPro 4.0.2). Negative controls included secondary antibody alone and cell background fluorescence alone.

[0647] Biosensor Analysis

[0648] Surface plasmon resonance (SPR) kinetic analysis was performed using a carboxymethyl dextran-coated sensor chip (CM5-S, GE LifeSciences) in a BIAcore TM T200 system. The test channel was derivatized to 200 response units (RU) of HER2-ECD using standard amine coupling chemistry (0.05 M NHS / 0.2 M EDC). The blank control channel for correction of refractive index effects was derivatized with ethanolamine.

[0649] Samples of mAb104, mAb106, pertuzumab, or trastuzumab anti-HER2 antibodies were diluted in PBS / 0.005% Tween 20 buffer to concentrations from 320 μg / mL to 0 μg / mL, two-fold dilutions (2133 to 0 nM). Samples were injected at 45 μL / min for 200 s (30 μL, 30 μL, 10 μL / min) with PBS buffer containing 0.005% Tween-20 over immobilized HER2-ECD, using PBS / 0.005% Tween 20 as the running buffer. After the injection phase, dissociation was monitored by flowing the running buffer over the chip surface for 600 s. Bound antibody was eluted and the chip surface was regenerated between samples by injecting 30 μl of 50 mM NaOH at 30 μL / min for 30 s.

[0650] Western Blot Analysis

[0651] The reactivity of the anti-HER2 monoclonal antibody against native HER2 was determined using Western blot. Trypsin-digested cells were lysed with RIPA buffer [50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 200 mM Na 3 VO 4 , 0.5% deoxycholate, 0.05% SDS, 10 mM NaF and protease inhibitor cocktail set I (Calbiochem, CA, USA)] for 20 min and centrifuged at 17,000 rpm for 15 min. 10 μg of cell lysate protein was run on a 4-12% gradient Nu-PAGE gel and electrotransferred to a nitrocellulose membrane using a 2 gel transfer apparatus (ThermoFisher). The presence of EGFR and HER2 was evaluated by probing the blot with the respective antibodies using commercially available antibodies and HER2 conjugated with mAb104. The blot was visualized on a Storm 804 Phosphoimager (Amersham Bioscience) and analyzed using ImageQuant TL image analysis software (version 2005).

[0652] Immunohistochemistry

[0653] To confirm the tumor selectivity of mAb104, an immunohistochemistry method was developed and used to screen the reactivity of mAb104 against a series of normal and tumor tissue types. Variations in antigen retrieval, primary antibody concentration, and incubation time were evaluated prior to the optimized conditions described above. Only the final protocol is briefly discussed below. The slides were placed in an oven at 60 °C for 30 min and transferred to a xylene bath, and the bath was changed after 10 min. The slides were then rehydrated in two changes of 100% ethanol for 10 min each, followed by rehydration in a 70% ethanol bath for 10 min. The slides were rinsed three times in double-distilled (dd)H 2 O, with each wash lasting approximately 2 min. The slides were then quenched in 3% H 2 O 2 for 20 min. Antigen retrieval was achieved by treating the slides in a 10% (v / v) EDTA buffer bath at 100 °C for 30 min. After cooling and washing with phosphate-buffered saline (PBS), the slides were blocked with a protein blocker (SuperBlock TM T20, ) Incubate for 60 min pre - incubation. Then incubate the slides with the primary antibody mAb104 (2.5 μg / mL) for 60 min at room temperature. After staining with the primary antibody, detect the bound antibody with a secondary anti - mouse antibody conjugated with streptavidin - horseradish peroxidase (HRP) (Dakocytomation, Carpinteria, CA, USA). Detect the bound antibody with 3,3′ - diaminobenzidine (DAB) substrate and counterstain with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrate and mount in ethanol and xylene.

[0654] Using this method, the expression of these proteins was examined in 11 normal human tissues and 10 common tumor types (intraductal breast carcinoma, mesothelioma, colorectal and gastric adenocarcinoma, renal cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, prostatic adenocarcinoma, and glioblastoma multiforme) from 9 - 27 different human donors using tissue microarrays (TMAs). Tumor and normal tissues were not from the same patient (i.e., not matched). Human tissues were obtained from the Department of Anatomical Pathology, Austin Health (Melbourne, Australia). This study was approved by the Austin Health Human Research and Ethics Committee.

[0655] Ki - 67: The expression of the human protein Ki - 67 protein is strictly related to cell proliferation and is present in all active stages of the cell cycle (Gerdes J, editor Ki - 67 and other proliferation markers useful in immunohistological diagnostic and prognostic evaluations in human malignancies, Seminars in Cancer Biology (1990)). Recover the antigen for 20 min in 10% (v / v) citrate buffer (pH 6.0) in water at 100 °C. After cooling, block non - specific binding sites with a protein blocker (SuperBlock TM T20, ) for 20 min. Incubate with a rabbit anti - human Ki - 67 primary antibody (RM - 9106 - S1, )Dilute it 1:100 in blocking buffer and incubate at room temperature for 2 h. After washing away the excess antibody, detect the bound antibody using the appropriate type of secondary antibody (Dakocytomation, Carpinteria, CA, USA) for 30 min at room temperature. Detect the bound antibody using 3,3'-diaminobenzidine (DAB) substrate. Then the slides are counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated in ethanol and xylene, and mounted.

[0656] Apoptosis : Use the terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay to detect apoptotic cells with an in situ cell death detection kit, fluorescein (11684795910 Roche, USA). Deparaffinize the slides made from paraffin-embedded tissues and rehydrate them as described above, and rinse three times in ddH 2 O for about 2 min each wash. Incubate the tissue sections in the proteinase K working solution at room temperature for 20 min, then rinse twice in PBS washes. Prepare positive and negative controls according to the product specifications. Add 100 μl of the TUNEL reaction mixture or 100 μl of the control labeling solution for the negative control to each slide and incubate in a humidified chamber at 37 °C for 60 min. After the incubation period, wash the slides three times with PBS. After applying to avoid evaporation loss, add 50 μl of Covertor-POD to the slides with coverslips and incubate in a humidified chamber at 37 °C for 30 min. After washing three times with PBS, add 50 - 100 μl of DAB substrate to the slides and incubate at room temperature for 10 min. Wash the slides with PBS and analyze under an optical microscope.

[0657] Podocalyxin: Deparaffinize and rehydrate the slides made from paraffin-embedded tissues as before. Quench the slides in 3% H 2 O 2 for 20 min at room temperature, then retrieve the antigen by incubating the slides in 10% (v / v) citrate buffer (pH 6.0) in a 100 °C water bath for 20 min. Add 15 μg / mL goat anti-mouse podocalyxin primary antibody (Catalog No. AF1556, R&D ) to each slide and incubate at room temperature for 2 h. Then wash the sections and detect the bound antibody using anti-goat HRP, counterstain with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrate in ethanol and xylene, and mount.

[0658] p-Akt: The slides generated from paraffin-embedded tissues were dewaxed by heating to 60 °C and rehydrated in xylene and graded alcohols. After washing the slides three times in ddH 2 O, antigen retrieval was performed by incubating the slides in 0.01 M citrate buffer (pH 6.0) in a 95% water bath for 20 min. Once the slides were cooled, they were washed in PBS and 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, Tween 20 (0.1%; TBS-T). Endogenous peroxidase activity was quenched by incubating in TBS-T containing 3% hydrogen peroxide for 15 min at room temperature. The sections were then incubated overnight at 4 °C with a primary antibody (rabbit polyclonal phospho-Akt (Ser473; Cell Signaling Technology, Beverly, MA, catalog number 9277, IHC specific) diluted 1:100 in TBS-T. After washing the slides three times in TBS-T, with each wash lasting approximately two min, the slides were incubated for 1 h in a rabbit biotinylated secondary antibody at a dilution of 1:200. Bound antibodies were detected with DAB substrate and counterstained in hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated in ethanol and xylene, and mounted.

[0659] Cell Proliferation Assays

[0660] Cells (1 × 10 4 ) in serum-free medium were seeded in 96-well microtiter plates and allowed to adhere overnight. The next day, serial dilutions of the antibody with appropriate controls were added and measured at time 0 (T = 0). The remaining cell plates were incubated for 3 to 5 days. Cell viability was assessed using the MTS colorimetric viability assay with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) as the substrate (Promega, Australia). Absorbance was measured at 490 nm using a VersaMax microplate reader (Molecular Devices, USA) and SoftMax Pro 5.4.1 software (Molecular Devices, USA). Absorbance at 630 nm was also measured as background and subtracted from the 490 nm readings. The experiments were performed in triplicate and repeated for 2 to 3 independent runs. All data were normalized to the signal at the time of compound addition (T = 0). Dose-response curves were analyzed using GraphPad prism 4.03 (Graphpad Software Inc, La Jolla, CA, USA).

[0661] Downstream Signaling

[0662] Seed cells (1×10 6 ) in duplicate into 6-well plates and allow them to establish overnight. Discard the medium in each well and replace it with serum-free medium containing the required antibody at a total concentration of 10 mg / mL. At the designated time point (24 h), treat half of the wells with 100 ng EGF for 10 min at room temperature. Terminate the reaction by washing with ice-cold PBS and lyse for 30 min with RIPA buffer [50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 200 mM Na 3 VO 4 , 0.5% deoxycholate, 0.05% SDS, 10 mM NaF and protease inhibitor cocktail set 1m, CA, USA]. Then centrifuge at 17,000 rpm for 15 min. Determine the total protein concentration using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hemel Hempstead, UK). Assess MAPK activation by Western blotting using commercial antibodies against HER2 (#4290), pHER2 (#2243), HER3 (#12708), pHER3 (#4791), EGFR (#4267), pEGFR (#3777), AKT (#4691), pAKT (#4060), ERK (#4695) and pERK (#4370) purchased from Cell Signaling Technology. The anti-GAPDH (AbC-1001) antibody was purchased from AbClon. Bands were visualized using AbSignal (AbClon, AbC-3001).

[0663] Detection of Cell Death by Enzyme-Linked Immunosorbent Assay (ELISA)

[0664] Cell death and apoptosis were evaluated using ELISA analysis (Cell Death Detection ELISA Plus kit; Roche Molecular Biochemicals) (Holdenrieder S, Stieber P, Bodenmüller H, Fertig G, Fürst H, Schmeller N et al., Nucleosomes in serum as a marker for cell death. Clinical Chemistry and Laboratory Medicine. 2001;39(7):596 - 605) according to the manufacturer's instructions. Briefly, cells were cultured in 96 - well plates and allowed to establish overnight. Cells were treated with trastuzumab, pertuzumab, and mAb104 as single therapies and in combination in serum - free (1%) growth medium for 24 h. The plates were centrifuged at 200×g for 10 min at 4°C. The supernatant was carefully removed, 200 μl of the manufacturer's lysis buffer was added, and the mixture was incubated at room temperature for 30 min. After incubation, the plates were centrifuged, and 20 μl of the supernatant and cell lysate solution were placed in triplicate into streptavidin - coated microtiter plates. Another 80 μL of the immunoreagent containing anti - histone - biotin and anti - DNA - POD mixture was added to the supernatant. The plates were incubated in an orbital shaker at room temperature for 2 h. Using ABTS (2,2'-azino - bis - 3 - ethyl - benzothiazoline - 6 - sulfonic acid) as the substrate, the degree of apoptosis was photometrically quantified using Softmax Pro 4.8 software on a Versamax microplate reader (Molecular Devices) at a wavelength of 405 nm and a reference wavelength of 490 nm.

[0665] Apoptosis Analysis

[0666] Cell viability was determined using propidium iodide (PI) uptake and annexin V binding. Briefly, cells (5×10 4 ) were cultured in 24 - well plates and allowed to establish overnight. Cells were treated with the antibody as single therapy and in combination with appropriate controls or in medium for 24 h. The cells were transferred to 96 - well plates, washed 3 times with cold PBS, and then resuspended in binding buffer containing 2.5 μl of FITC - annexin V and 2.5 μl of PI in the dark with gentle agitation at room temperature for 15 min. After incubation, an additional 150 μl of binding buffer was added, and then flow cytometry analysis was performed.

[0667] Migration (Wound Healing) Assay

[0668] To evaluate the effect of mAb014 on cell migration, OE - 19 cells (1×105 ) Seed the cells in 6-well plates and allow them to grow to 80% confluence. Create three parallel scratches in each well using a 100 μl pipette tip. Treat the cells with the desired antibody at 100 μg / mL or an isotype control. Take phase-contrast microscopy images immediately after treatment (designated as T0) and at 72 h.

[0669] Effect of mAb104 on ErbB Receptor Dimerization

[0670] Seed the cells in serum-free medium in 12-well plates and allow them to adhere overnight. Treat the cells in duplicate with the relevant antibody or control at 10 μg / mL for 1 h. At the designated time points, treat half of the wells with 100 ng EGF at room temperature for 10 min. Terminate the reaction by washing with ice-cold PBS, and incubate the cells with BS3 (bis(sulfosuccinimidyl)substrate ((BS3), Pierce, Rockford, IL, USA)) at room temperature for 20 min with gentle shaking according to the manufacturer's instructions (Staros JV. N-hydroxysulfosuccinimide active esters: bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry. 1982;21(17):3950 - 5). After quenching the cross-linking reaction mixture with a buffer containing 10 mM Tris-HCl, wash the cells twice with cold PBS and lyse them with RIPA buffer [50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 200 mM Na 3 VO 4 , 0.5% deoxycholate, 0.05% SDS, 10 mM NaF, and protease inhibitor mixture set 1M, CA, USA] for 30 min. Immunoprecipitate the cell lysates with the relevant antibody and perform immunoblotting for EGFR and HER2.

[0671] DNA Gel Electrophoresis

[0672] Perform DNA gel electrophoresis on a gel containing 1% (w / v) DNA-grade agarose (Bioline), where the agarose is in 1× Tris-acetate EDTA (TAE) buffer (Invitrogen) Preparation of Safe DNA Gel Stain (Invitrogen). Before loading onto the agarose gel, all DNA samples were diluted with 10× Orange G (Sigma) gel loading buffer stock solution. Electrophoresis was performed at 70 - 150 V, and 1 kb Plus DNA ladder (Invitrogen) was used as a reference for size estimation. The DNA bands were visualized and photographed on a transilluminator (Bio-Rad) under UV light.

[0673] Hybridoma cDNA Synthesis

[0674] The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, California, USA) was used to reverse transcribe (RT) 10 μL (~1 - 5 μg) of RNA into cDNA using a reaction volume of 20 μL. According to the standard method, cDNA synthesis was carried out in 200 μL thin-walled polypropylene PCR tubes (Eppendorf, USA) using a TM (BIO-RAD, USA) or (Eppendorf, USA) thermal cycler under the following reaction and cycling conditions.

[0675] cDNA Synthesis Conditions:

[0676] 25 °C for 10 min

[0677] 37 °C for 120 min

[0678] 85 °C for 5 min

[0679] Hold at 4 °C

[0680] Murine Light Chain Variable Region Primer:

[0681] (i) ATG AAG TTG CCT GTT AGG CTG TTG GTG CTG (SEQ ID NO: 28)

[0682] (ii) ATG GAG WCA GAC ACA CTC CTG YTA TGG GT (SEQ ID NO: 29)

[0683] (iii) ATG AGT GTG CTC ACT CAG GTC CTG GSG TTG (SEQ ID NO: 30)

[0684] (iv) ATG AGG RCC CCT GCT CAG WTT YTT GGM WTC TTG (SEQ ID NO: 31) (v) ATGGAT TTW CAG GTG CAG ATT WTC AGC TTC (SEQ ID NO: 32) (vi) ATG AGG TKC YYT GYT SAGYTY CTG RGG (SEQ ID NO: 33)

[0685] (vii) ATG GGC WTC AAG ATG GAG TCA CAK WYY CWG G (SEQ ID NO: 34) (viii) ATGTGG GGA YCT KTT TYC MMT TTT TCA ATT G (SEQ ID NO: 35) (ix) ATG GTR TCC WCA SCTCAG TTC CTT G (SEQ ID NO: 36)

[0686] (x) ATG TAT ATA TGT TTG TTG TCT ATT TCT (SEQ ID NO: 37)

[0687] (xi) ATG GAA GCC CCA GCT CAG CTT CTC TTC C (SEQ ID NO: 38)

[0688] (xii) ATG AAG TTT CCT TCT CAA CTT CTG CTC (SEQ ID NO: 39)

[0689] Murine Light Chain Variable Region Reverse Primer Sequence:

[0690] MKC: TGG ATG GTG GGA AGA TG (SEQ ID NO: 40)

[0691] Murine Heavy Chain Variable Region Primer:

[0692] (i) ATG AAA TGC AGC TGG GTC ATS TTC TTC (SEQ ID NO: 41)

[0693] (ii) ATG GGA TGG AGC TRA TCA TSY TCT T (SEQ ID NO: 42)

[0694] (iii) ATG AAG WTG TGG TTA AAC TGG GTT TTT (SEQ ID NO: 43)

[0695] (iv) ATG RAC TTT GWY TCA GCT TGR TTT (SEQ ID NO: 44)

[0696] (v) ATG GAC TCC AGG CTC AAM AGT TTT CCT T (SEQ ID NO: 45)

[0697] (vi) ATG GCT GTC YTR GSG CTR CTC TTC TGC (SEQ ID NO: 46)

[0698] (vii) ATG GRA TGG AGC KGG RTC TTT MTC TT (SEQ ID NO: 47)

[0699] (viii) ATG AGA GTG CTG ATT CTT TTG TG (SEQ ID NO: 48)

[0700] (ix) ATG GMT TGG GTG TGG AMC TTG CTA TTC CTG (SEQ ID NO: 49) (x) ATG GGCAGA CTT ACA TTC TCA TTC CTG (SEQ ID NO: 50)

[0701] (xi) ATG GAT TTT GGG CTG ATT TTT TTT ATT G (SEQ ID NO: 51)

[0702] (xii) ATG ATG GTG TTA AGT CTT CTG TAC CTG (SEQ ID NO: 52)

[0703] Murine Heavy Chain Variable Region Reverse Primer Sequence:

[0704] MHC: CCAGTGGATAGACAGATG (SEQ ID NO: 53)

[0705] Degenerate Forward and Reverse Primers for Murine Light Chain Variable Region

[0706] κF: GCC GAA TTC GAY ATT GTG MTS ACM CAR WCT MCA (SEQ ID NO: 54) κR: CCGGTC GAC GGA TAC AGT TGG TGC AGC ATC (SEQ ID NO: 55)

[0707] Symbolic annotation:

[0708] R = A or G, Y = C or T, M = A or C, K = G or T, S = G or C, W = A or T, H = A or T or C

[0709] B = G or T or C, D = G or A or T, N = A or C or G or T, V = G or A or C

[0710] Polymerase Chain Reaction

[0711] To amplify the DNA fragment for cloning, Pfx DNA polymerase (Invitrogen) was used according to the manufacturer's instructions. Using standard methods, a T100 (BIO - RAD, USA) or TM (Eppendorf) thermal cycler was used to perform the PCR reaction mixture in a 200 μl thin - wall polypropylene PCR tube (Eppendorf) under the following reaction and cycling conditions. 94°C for 3 min

[0712] PCR Conditions:

[0713] 94°C 3 min

[0714] (94°C 1 min → x°C a 55 - 90 s b → 72°C 2 min) for 15 - 25 cycles

[0715] 72°C / 10 min

[0716] Hold at 4°C

[0717] In Vivo Studies

[0718] NOD - SCID - IL2R - / - mice (4 - 6 weeks old, Animal Research Center, Perth, Australia) were subcutaneously injected with 5×10 6 of NCI - N87 or 8×10 6 of BT - 474 cells into the lateral region in Matrigel (BD Biosciences). Mice injected with BT - 474 cells were implanted with estrogen pellets 24 h before. The tumor volume was calculated using the formula (L × W 2 ) / 2, where "W" represents the width of the tumor and "L" represents the length of the tumor. The tumors were allowed to grow to approximately 100 mm 3 in size, and then the mice were randomly divided into different treatment groups. Tumors that failed to implant were excluded from further analysis. Treatments were administered three times a week for three weeks at the indicated doses by intraperitoneal injection. The animals were observed after treatment, and when the average tumor volume > 1000 mm 3Euthanize animals at that time, or show long-term stress symptoms. Excise the tumors after death and process them into formalin-fixed, paraffin-embedded specimen sections, collect for reverse phase protein array (RPPA), and store the excess tissue at -80 °C. At the end of the treatment, calculate the percentage of tumor growth inhibition (%TGI) as follows: %TGI = [1 - {T / T 0 / C / C 0} / 1 - {C 0 / C}] × 100 where T = the average tumor volume of the treatment at the end point, T 0 = the average tumor volume of the treatment at time 0, C = the average tumor volume of the control at the end point, C 0 = the average tumor volume of the vehicle control at time 0.

[0719] All animal study protocols were approved by the Austin Health Animal Ethics Committee (Protocol #A2015 / 05297) and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th Edition, 2013).

[0720] Reverse Phase Protein Array (RPPA)

[0721] Extract proteins from HER2-overexpressing breast PDX tumors and perform RPPA as previously described (Hennessy BT, Lu Y, Gonzalez-Angulo AM, Carey MS, Myhre S, Ju Z, et al. A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers. Clinical proteomics. 2010;6(4):129). Use lysis buffer (Cat. No. #9803, Cell Signaling Technology, Beverly, MA, USA), supplemented with protease and phosphatase inhibitors (Roche Applied Science Cat. No. #05056489001, Penzberg, Germany), to lyse the tumor samples obtained at the end of the treatment by homogenization. Use Pierce TM BCA Protein Assay Kit to determine the protein concentration, normalized to 1 mg / mL, and the samples are boiled with 2-mercaptoethanol and SDS. Send the processed protein lysates to MD Anderson Cancer Center, Houston, TX, USA for RPPA analysis as follows.

[0722] The lysate was serially diluted with lysis buffer in 5-fold serial dilutions to obtain a 1:16 dilution. The lysate was arrayed on a nitrocellulose-coated slide (Grace Biolab) in an 11×11 format. The samples were probed with 297 validated primary antibodies by a tyramide-based signal amplification method and visualized by DAB colorimetric reaction. The slides were scanned, analyzed, and quantified by an Array-Pro analyzer (Meyer Instruments, INC. Houston, TX) to generate spot intensities.

[0723] Each dilution curve was fitted with a logistic model ("hypercurve fitting", developed by the Department of Bioinformatics and Computational Biology, MD Anderson Cancer Center, Houston, TX, USA). All data were normalized by median polishing with a protein loading correction factor and transformed to linear values using the median expression level of all antibody experiments. "Red" in the heatmap indicates above median and "green" indicates below median.

[0724] Statistical Analysis

[0725] Analysis was performed using version 5.04. All p-values were two-sided and values ≤ 0.05 were considered significant.

[0726] To compare means, the Student t-test or the non-parametric Mann Whitney U test was used, where only two groups were considered. To compare between three or more groups, parametric data were analyzed by ANOVA and, if p ≤ 0.05, a post hoc test using the Bonferroni method was performed to determine which groups were significantly different. The non-parametric test used for multiple groups was the Kruskal-Wallis test, and if p ≤ 0.05, a post hoc test was performed to determine which groups were significantly different.

[0727] The survival rates of the groups were also compared, and if the log-rank test of all groups was significantly different (p ≤ 0.05), a post hoc test by further log-rank tests was performed to determine which groups were significantly different.

[0728] Example 1 - In Vitro Antibody Production and Characterization

[0729] The immunogens and immunization protocols were described previously. Through a series of immunization and screening strategies involving HER2 peptides, recombinant proteins, and cell-based assays expressing HER2, the inventors finally succeeded in generating tumor-specific monoclonal antibodies against the conformationally flexible region of domain II of HER2.

[0730] The present inventors employed an immunization strategy using linear peptides conjugated to biotin, GST, MBP, and KLH carrier proteins and Baf / 03 hematopoietic cells transfected to express erbB2 (which do not express HER members on the cell surface). ErbB2 had a cysteine mutation to expose a peptide loop, but was unsuccessful in generating any clones. Immunization with cells expressing the mutant plus recombinant mutant ECD ErbB2 did not generate mAbs that bound the peptide, but bound to different positions within the ECD of ErbB2. After immunizing once with a cyclized peptide conjugated to KLH, the inventors were able to obtain monoclonal antibody clones that recognized the peptide and bound to erbB2-expressing cells. This is summarized in the table below (Table 5).

[0731] Table 5 Results of Immunization Protocols

[0732]

[0733] Using the immunization protocol, the present inventors generated hybridoma clones that produced novel monoclonal antibodies (mAbs) against a conformationally exposed region of the HER2 extracellular domain, which are believed to be useful for binding only under conditions found in tumor cells. By immunizing mice with a peptide immunogen from the HER2 extracellular domain, these monoclonal antibodies against conformational epitopes were produced: H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 1) folds into a loop via cysteine (C) residues and is conjugated to a KLH protein. This sequence is underlined in the Figure 1 provided human HER2 sequence. This sequence was derived from the ncbi database at the following link: https: / / www.ncbi.nlm.nih.gov / protein / NP_004439.2 .

[0734] This region is within domain II, but is distant from the known epitope of pertuzumab (Franklin MC et al., (2004) Insights into ErbB signaling from the structure of the ErbB2-Pertuzumab complex. Cancer cell. 5(4):317-2).

[0735] Screening of Hybridomas

[0736] The specificity of the reactivity of hybridoma culture supernatants (designated mAb104, mAb105, mAb106, and mAb107) against the extracellular domain (ECD) of HER2 and the loop (circular) and linear peptides of the antigen to which the antibody is generated was screened using ELISA-based assays. Purified antibodies were extracted from the hybridoma supernatants using protein-G affinity chromatography. The integrity of the eluted antibodies was confirmed by SDS-PAGE analysis under reducing and non-reducing conditions. The immunoglobulin isotypes of the selected antibodies were detected using a monoclonal antibody isotyping kit (Thermo Fisher Scientific, IL, USA), and all antibodies were found to be IgG1 with K-light chains. The results of the ELISA analysis of the purified mAb are as Figure 2 shown.

[0737] Monoclonal antibodies mAb104 and mAb106 showed the strongest binding activity to all peptide configurations, while mAb105 showed the lowest binding (see Figure 2 B). Clones producing antibodies with the highest affinity were selected for further development, namely mAb104 and mAb106, and their antibodies were selected for further analysis and characterization.

[0738] FACS Detection of the Binding Ability of Hybridoma Cells

[0739] The binding extent of 10 μg / ml purified antibodies (mAb104, mAb105, mAb106, and mAb107) to cellular HER2 was tested by flow cytometry on breast cancer cell lines (BT474, SK-BR-3, and MDA-MB-453) and gastric cancer cell line (NCI-N87) overexpressing HER2.

[0740] The results are summarized in Table 6 below and represent two or more experiments.

[0741] Table 6: FACS analysis of antibodies

[0742] mAb104 mAb105 mAb106 mAb107 MDA-MB-453 + - - - BT 474 ++ - + - SK-BR-3 ++ - - - NCI-N87 +++ - +++ +++

[0743] Among all the evaluated cell lines, mAb104 showed the highest binding compared to other antibodies. Among the evaluated cell lines, mAb104 showed the highest log shift in the NCI-N87 and SK-BR-3 cell lines. No mAb105 binding was observed in any of the evaluated cell lines. For all antibodies, the binding was lower than that of the commercial HER2-binding antibody. The present inventors propose that these antibodies bind to a portion of the receptor on the cell surface, and the epitope is only exposed to a portion of the HER2 receptor population.

[0744] Binding Analysis by Western Blot

[0745] The ability of these new antibodies to bind to the HER2 protein was further evaluated in human breast cancer cell lines (BT474, SK-BR-3, and MDA-MB-453) and gastric cancer cell line (NCI-N87) by Western blot analysis. Trypsinized cells were washed, lysed, and immunoblotted with the respective purified antibodies. Anti-HER2 antibody 2242 (Cell Signaling Technology, Beverly, MA) was used as a positive control.

[0746] Consistent with the previous ELISA and FACS data, mAb104 showed the strongest binding in all cell lines (mAb104 > mAb106 > mAb107 > mAb105)( Figure 3 ), and bound to all four tested cell lines.

[0747] Sequences of mAb104 and mAb106

[0748] The PCR reactions were purified using the BioLine Isolate I I PCR and Gel Extraction Kit (BIO-52059). The purified amplification products were sent to the Monash Micromon DNA Sequencing Facility for Sanger DNA sequencing using the same primers as those used for amplifying the products with the sequencing primers.

[0749] CDRs were defined according to the Chothian and Kabat numbering systems and the amino acids in the CDRs that fell into both. The heavy and light chain variable region sequences are as Figure 4 shown.

[0750] The complementary determining region sequences for each antibody are provided below:

[0751] VH Chain of mAb104

[0752] CDR1: GYSFTGYFMH (SEQ ID NO: 14)

[0753] CDR2: RINPYNGDIRYNQNFKD (SEQ ID NO: 16)

[0754] CDR3: LNFAY (SEQ ID NO: 18)

[0755] VL Chain of mAb104

[0756] CDR1: KSSQSLLDSDGKTFLN (SEQ ID NO: 20)

[0757] CDR2: LVSKLDS (SEQ ID NO: 22)

[0758] CDR3: WQGTHFPWT (SEQ ID No: 24)

[0759] VH Chain of mAb106

[0760] CDR1: GYTFTDYGMN (SEQ ID NO: 15)

[0761] CDR2: WINTYTGKPTYDDDFKG (SEQ ID NO: 17)

[0762] CDR3: RFLNTVAGRSVYFDY (SEQ ID NO: 19)

[0763] VL Chain of mAb106

[0764] CDR1: SVSSSVGSMY (SEQ ID NO: 21)

[0765] CDR2: LTSNLAS (SEQ ID NO: 23)

[0766] CDR3: QQWSSNPPT (SEQ ID NO: 25)

[0767] BIAcore Analysis

[0768] The epitope bound by mAb104 is flanked by disulfide bonds, which suggests flexibility around the epitope site and exposure of the epitope bound by mAb104 under certain conditions or environments.

[0769] The binding characteristics and apparent affinity of purified mAb104 and mAb106 for HER2 compared to trastuzumab were detected by surface plasmon resonance (BIAcore) using a BIAcore T200. The recombinant HER2 extracellular domain was immobilized on a CM5 sensor chip, and then various concentrations of mAb104, mAb106, and a commercial anti-HER2 mAb were passed over the sensor to determine the apparent binding affinity.

[0770] As Figure 5 shown in

[0771] Table 7: Binding affinity of mAb104

[0772] Antibodies <![CDATA[K D (nM)]]> <![CDATA[Chi 2 > mAb104 2.91 0.03 mAb106 3.18 0.13 Trastuzumab 0.1 0.12 Pertuzumab 1.9 14.3

[0773] In summary, the spectra of mAb104, mAb106, and mAb107 binding to tumor cells overexpressing HER2 were determined. mAb104 consistently appeared to show stronger in vitro binding than mAb106. Based on the results of these initial screening assays, mAb104 was selected for further evaluation.

[0774] Example 2 Epitope Analysis and Competition Assays

[0775] Using the method described previously, the variable domains of the mAb104 antibody that bind to the epitopes located on domain II of HER2 were computationally predicted based on the homology modeled 3D structure of the antibody Fv domain and the known X-ray structure of human HER2 (Zhang W, Zeng X, Zhang L, Peng H, Jiao Y, Zeng J, et al. Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5). Journal of Computer-Aided Molecular Design. 2013;27(6):539-50).

[0776] The HER2 binding of the predicted mAb104 was compared with the crystal structures of the known HER2 bindings of pertuzumab and trastuzumab (Hu S, Sun Y, Meng Y, Wang X, Yang W, Fu W, et al., Molecular architecture of the ErbB2 extracellular domain homodimer. Oncotarget. 2015;6(3):1695). Without wishing to be bound by theory, it is believed that the binding of mAb104 to HER2 requires conformational changes that occur upon receptor activation, as previously shown for EGFR / HER1 (Garrett TP, Burgess AW, Gan HK, Luwor RB, Cartwright G, Walker F, et al., Antibodies specifically targeting a locally misfolded region of tumour-associated EGFR. Proceedings of the National Academy of Sciences. 2009;106(13):5082-7), where the disulfide bonds in domain II of the HER2 ECD can form and break dynamically.

[0777] Epitope Recognized by mAb104 Compared with Other Known HER2-Binding Antibodies

[0778] Antibody H2-18 (Lu et al. (2016) Oncotarget 7(41)), Chinese Patent CN 104447993 recognizes an epitope within domain I of HER2 / ErbB2. H2-18 inhibits the growth of trastuzumab-resistant breast cancer cells in vivo and in vitro and induces programmed cell death in trastuzumab-sensitive and resistant breast cancer cell lines.

[0779] Antibody A21 (Hu S et al. (2015) Oncotarget 6(3):1695-1706) appears to recognize a conformational epitope that includes a large region encompassing most of the ErbB2 EC domain I. The antibody bivalency of A21 was found to be essential for its inhibitory activity against tumour cells and for ErbB2 phosphorylation and receptor downregulation.

[0780] Trastuzumab / Herceptin (4D5) binds to the juxtamembrane region in subdomain IV of ErbB2 and can interrupt the activation of ErbB2 by metalloprotease cleavage and also block ErbB2 dimerization.

[0781] Pertuzumab (2C4) disrupts the binding between ErbB2 and other ErbB receptors directly with epitopes within or near subdomain II, and thus inhibits tumor cell growth.

[0782] The epitopes recognized by pertuzumab and mAb104 share only three amino acids, namely P294, L295, and H296, although it is understood that H296 is completely masked upon pertuzumab binding (Franklin MC et al., (2004) Cancer Cell 5:317). The epitope bound by pertuzumab consists of H245, Y252, F257, D285, V286, S288, T290, P294, L295, H296, K311, K314, and P315.

[0783] In contrast, the epitope recognized by mAb104 is the sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1).

[0784] Although both antibodies recognize epitopes within domain II of HER2, the epitopes bound by pertuzumab and mAb104 are distinct.

[0785] Without wishing to be bound by theory, the inventors postulate that pertuzumab and mAb104 bind to opposite / sides of domain II of HER2 / ErbB2, thus explaining why mAb104 does not block the binding of pertuzumab, despite being closely opposed epitopes. Conformational changes in domain II, such as those occurring during activation, hypoxic conditions, and / or aberrant expression, would allow the antibody to bind to CPLHNQEVTAEDGTQRC in domain II and mAb104 to bind to a small subset of HER2 receptors that have undergone such conformational changes. However, the required conformational changes are possible if, for example, the disulfide bond (C277 / C289 (numbering excluding the leader sequence)) preceding the mAb104 epitope (C293 / C309 (numbering excluding the leader sequence)) is transiently broken or undergoes disulfide bond conversion, allowing rearrangement to expose at least a portion of domain II that exposes the mAb104 epitope. During the rearrangement, the pertuzumab-binding epitope remains mostly undisturbed. However, in computer simulations of adsorbed recombinant HER2-ECD for ELISA capture, the structural rearrangement of domain II is fixed, and the smaller steric hindrance of mAb104 binding can lead to reduced pertuzumab binding (e.g., as shown below). The possible loss of binding affinity can be balanced by the synergistic interaction between the two antibodies.

[0786] Competition Assays

[0787] To better define the epitope of mAb104, the binding of mAb104 to HER2-ECD and its ability to interfere with the binding of the domain II-binding antibody of pertuzumab and the spatially distant domain IV epitope of trastuzumab were compared using ELISA ( Figure 6 ). In these experiments, the inventors evaluated the effect of pre-incubation with mAb014 on the binding of trastuzumab and pertuzumab ( Figure 6 B and C), and also determined the effect of pre-incubation with trastuzumab and pertuzumab on the binding of mAb104.

[0788] The inventors showed that trastuzumab and mAb104 did not affect each other's binding to HER2-ECD ( Figure 6 A and B). The present inventors also demonstrated that pre-incubation with pertuzumab did not affect mAb104 binding ( Figure 6 A). However, interestingly, the binding of pertuzumab to HER2-ECD was reduced prior to incubation with mAb104 ( Figure 6 C), indicating that the binding of mAb104 to its epitope may cause some steric hindrance to pertuzumab in some cases.

[0789] The competition between mAb104 and pertuzumab and trastuzumab for endogenous HER2 was further investigated by flow cytometry in breast (BT474 and SK-BR-3; Figure 7-1 and 7-2 ) and gastric cell lines (NCI-N87 and OE19; Figure 7-3 and 7-4 ) using the above two sequential incubation methods. Experiments in these groups used pre-incubation with a high dose (100 μg / mL) of mAb104 to maximize the variation in the observed effect on the binding of trastuzumab and pertuzumab. Incubation with a much higher dose of mAb104 did not affect the binding of trastuzumab or pertuzumab to cell surface HER2. The inconsistency in the results of mAb104 competing with pertuzumab using flow cytometry and ELISA could be explained by differences between the antigen preparations in the assays, i.e., when analyzed by flow cytometry, HER2 is present in its native conformation, while in ELISA it is partially denatured, and subsequent epitope presentation and availability.

[0790] Example 3 Binding of mAb104 to Cell Surface HER2

[0791] The present inventors examined the pattern and efficiency of mAb104 binding by FACS analysis using a panel of cell lines with differential HER2 expression.

[0792] The results are summarized in Table 8 below. The results were compared with the binding of secondary antibody only.

[0793] Binding of mAb104

[0794] Herceptin Pertuzumab mAb104 BT-474 +++ +++ - SK-BR-3 +++ +++ - NCI-N87 +++ +++ ++ OE-19 +++ +++ + MDA-MB-231 ND ND - MCF-7 ND ND -

[0795] FACS analysis of the binding of mAb104 to HER2-expressing cells BT474, SK-BR-3, NCI-N87, OE-19, MDA-MB-231, and MCF7 cells, incubated separately with 10 μg / mL trastuzumab, pertuzumab, mAb104, or secondary antibody only, and the degree of binding was analyzed by FACS. Results represent two or more experiments

[0796] In HER2-overexpressing cell lines, mAb104 showed the strongest binding to the HER2 population in the gastric cell line NCI-N87, and negligible HER2 binding was observed in low HER2-expressing cell lines (MDA-MB-231 and MCF-7).

[0797] FACS of trastuzumab (Herceptin) and pertuzumab showed greater fluorescence, indicating that they bound to a greater number of HER2 receptors on all evaluated cell lines compared to mAb104 (Table 8), and no difference in the degree of binding was observed between the two humanized antibodies on different cell lines. The findings of the present inventors support the hypothesis that mAb104 binds to a subset of receptors on the cell surface and would explain the differences observed in the degree of binding between the antibodies.

[0798] Specificity of mAb104 for HER2

[0799] To confirm the specificity of mAb104 for endogenously expressed HER2 and HER3, the present inventors performed western blot analysis using different HER2-positive and -negative cancer cell line lysates prepared under reducing conditions ( Figure 8-1 ).

[0800] The antibody 2242 was used as a positive control for total HER2. The antibody is a commercially available antibody from Cell Signaling Technology that is generated against an intracellular HER2 epitope. As shown in Figure 8A, mAb104 showed strong reactivity against multiple HER2-expressing cancer cell lines and was comparable to the control antibody that provided a positive signal representing the HER2 overexpression status of multiple cell lines. The correlation of mAb104 and control 2242 HER2 binding reflects the HER2 epitope recognized by mAb104 under reducing conditions. HER3 expression levels were very low in the cancer cell lines studied, as also observed by FACS analysis and previously in other studies (Brockhoff G, Heiss P, Schlegel J, Hofstaedter F, Knuechel R. Epidermal growth factor receptor, c-erbB2 and c-erbB3 receptor interaction, and related cell cycle kinetics of SK-BR-3and BT474 breast carcinoma cells. Cytometry Part A. 2001;44(4):338-48.).

[0801] The specificity of HER2 / ErbB2 was evaluated by ELISA. As Figure 8-2 shown, mAb104 was specific for ErbB2 / HER2 and did not bind to the extracellular domain of EGFR / HER1, or the ECD of ErbB3 / HER3 or ErbB4 / HER4.

[0802] Efficacy of mAb104 in breast cancer in vitro

[0803] Example 4 Anti-proliferative effect of mAb104

[0804] The effect of mAb104 as a single therapy and in combination with trastuzumab or pertuzumab on the proliferation of HER2-overexpressing breast cancer cell lines was determined by the MTS cell proliferation assay using increasing concentrations up to a maximum concentration of 100 μg / mL in serum-deprived conditions (1% FCS) (Figure 9).

[0805] Compared with the isotype control antibody, trastuzumab significantly reduced the proliferation of BT-474 (Figure 9B) and SK-BR-3 (Figure 9A) (p = 0.0006 and p = 0.0005, respectively; two-sided), while pertuzumab monotherapy had no significant anti-proliferative activity in the cell lines evaluated (p = 0.22 and p = 0.15, respectively; two-sided, see Figures 9A and 9B). These findings are consistent with other studies (Brockhoff G, Heckel B, Schmidt-Bruecken E, Plander M, Hofstaedter F, Vollmann A et al., Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK-BR-3 breast cancer cell proliferation. Cell proliferation. 2007;40(4):488-507; Tokuda Y, Ohnishi Y, Shimamura K, Iwasawa M, Yoshimura M, Ueyama Y et al., In vitro and in vivo anti-tumour effects of a humanised monoclonal antibody against c-erbB-2 product. British journal of cancer. 1996;73(11):1362; Yamashita-Kashima Y, Iijima S, Yorozu K, Furugaki K, Kurasawa M, Ohta M et al., Pertuzumab in combination with Trastuzumab shows significantly enhanced antitumour activity in HER2-positive human gastric cancer xenograft models. Clinical Cancer Research. 2011;17(15):5060-70; Nahta R, Hung M-C, Esteva FJ. The HER-2-targeting antibodies Trastuzumab and Pertuzumab synergistically inhibit the survival of breast cancer cells. Cancer research.2004; 64(7):2343 - 6; Gong SJ, Jin CJ, Rha SY, Chung HC. Growth inhibitory effects of Trastuzumab and chemotherapeutic drugs in gastric cancer cell lines. Cancer letters. 2004; 214(2):215 - 24; Ko B - K, Lee S - Y, Lee Y - H, Hwang I - S, Persson H, Rockberg J et al., Combination of novel HER2 - targeting antibody 1E11 with Trastuzumab shows synergistic antitumour activity in HER2 - positive gastric cancer. Molecular oncology. 2015; 9(2):398 - 408; Tomioka H, Mukohara T, Kataoka Y, Ekyalongo RC, Funakoshi Y, Imai Y et al., Inhibition of the mTOR / S6K signal is necessary to enhance fluorouracil - induced apoptosis in gastric cancer cells with HER2 amplification. International journal of oncology. 2012; 41(2):551 - 8).

[0806] Compared with the isotype control antibody, mAb104 did not show any significant growth inhibition in either cell line SK - BR - 3 and BT - 474 (p = 0.33 and p = 0.2, respectively; two - tailed) (Figures 9A and 9B).

[0807] As shown in Figures 9C and 9D, the combination of Trastuzumab and Pertuzumab significantly inhibited proliferation in the cell lines evaluated (BT474 p = 0.0008 and SK - BR - 3 p = 0.0007; two - tailed); however, there was no statistically significant difference compared with Trastuzumab monotherapy (BT - 474 p = 0.59 and SK - BR - 3 p = 0.51; two - tailed).

[0808] Adding mAb104 to mAb104 or pertuzumab does not affect the anti-cell proliferation activities of trastuzumab and pertuzumab, respectively (Figure 9C-F). In the cell lines evaluated, there was no statistical difference in the anti-proliferative effects of the combination of mAb104 and trastuzumab compared to the combination of trastuzumab and pertuzumab (BT-474 p = 0.66; SK-BR-3 p = 0.47).

[0809] Therefore, unlike trastuzumab and pertuzumab, mAb104 has no detectable anti-proliferative effect on HER2-positive cell lines. The complex interplay of multiple receptor kinases and signal transduction pathways that occurs in vivo cannot always be fully replicated in vitro, and the effects of therapeutic agents that capture activated receptors that require conformational changes may be immeasurable in vitro. Antibodies targeting conformationally exposed epitopes on activated EGFR have shown significant anti-tumor activity in vivo, although they failed to show any growth inhibition or altered signal transduction in vitro [Johns TG, Perera RM, Vernes SC, Vitali AA, Cao DX, Cavenee WK et al., The efficacy of epidermal growth factor receptor–specific antibodies against glioma xenografts is influenced by receptor levels, activation status, and heterodimerization. Clinical Cancer Research. 2007;13(6):1911-25].

[0810] Example 5 Effect of mAb104 on ErbB receptors and downstream signal transduction pathways

[0811] Considering the functional differences of mAb104 observed in the proliferation assay, the present inventors attempted to detect the effects of mAb104 treatment for 24 h on the MAPK and Akt ligand-independent pathways in SK-BR-3 and BT-474 breast cancer cell lines under serum-starved conditions. To evaluate the effect of mAb104 on ligand-dependent signal transduction pathways, the cell lines were treated with anti-HER2 antibody for 24 h and then 100 ng EGF was added for 10 min.

[0812] The results of ligand-independent and ligand-dependent effects are presented in FIGS. 10 and 11, respectively. The inventors focused on EGFR-HER2 signal transduction because HER3 expression levels are very low in cancer cell lines, as demonstrated in FIG. 8, which is consistent with other studies (Brockhoff G et al., Epidermal growth factor receptor, c-erbB2 and c-erbB3 receptor interaction, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells. Cytometry Part A. 2001; 44(4): 338-48).

[0813] In this series of experiments (FIGS. 10 and 11), the inventors treated with trastuzumab and pertuzumab for 24 h and did not significantly reduce total HER2 expression in the HER2-positive breast cancer cell lines BT-474 and SK-BR-3, as previously demonstrated by others (Molina MA et al., (2001) Cancer research. 61(12): 4744-9; Lu Q et al., (2016) Oncotarget. 2016; 7(41): 67129).

[0814] The effects of anti-HER2 antibodies on the Akt and MAPK pathways were also evaluated using phospho-specific antibodies. In both breast cancer cell line cells (BT-474 and SK-BR-3), trastuzumab treatment led to a decrease in Akt phosphorylation without a change in total Akt protein levels, which represents a decrease in phosphorylation activity rather than a downregulation of Akt protein. These findings are also consistent with other studies (Lu Q et al., ibid.; Yakes FM et al., (2002) Cancer research. 62(14): 4132-41).

[0815] In BT-474 cells, trastuzumab treatment led to a decrease in MAPK activity, as shown by the decrease in phospho-MAPK (FIGS. 10C and D). In contrast, no change in MAPK activity was shown in SK-BR-3 cells (FIGS. 10A and B), which is consistent with other studies (Cuello M et al., (2001) Cancer research. 61(12): 4892-900).

[0816] As shown in FIGS. 10A and B, treatment with mAb104 as a single therapy did not result in detectable changes in the amount of total or phosphorylated proteins in the cell lines evaluated.

[0817] In BT-474 cells, the combination of trastuzumab and pertuzumab reduced the levels of phosphorylated-Akt and phosphorylated p44 / p42 MAPK to a greater extent compared to either agent alone, with no effect on total Akt or MAPK observed (FIG. 10D). In contrast, in SK-BR-3, signal transduction through the MAPK cascade was not inhibited by the combination of drugs, as shown by the unaltered levels of phosphorylated p44 / p42 MAPK, as previously described (Nahta R et al., (2004) Cancer research. 64(7):2343-6). Similar changes in the Akt and MAPK signaling cascades were observed using the combination of trastuzumab and mAb104 and the combination of trastuzumab and pertuzumab. There was no significant difference in the degree of downregulation between the two drug combinations (FIGS. 10C and D).

[0818] Under ligand-stimulated conditions (FIG. 11), trastuzumab and pertuzumab did not affect the MAPK or Akt signaling pathways in BT-474 (FIGS. 11A and B). In the SK-BR-3 cell line, pretreatment with pertuzumab resulted in a decrease in Akt phosphorylation 10 min after EGF stimulation, with no change in total Akt (FIG. 11A). These findings are consistent with other reports (Henjes F et al., (2012) Oncogenesis 1(7):e16). Trastuzumab inhibits Akt-mediated signal transduction due to the abrogation of ligand-independent signal transduction, while pertuzumab blocks ligand-induced signal transduction. Addition of EGF to mAb104-treated cells did not result in alterations in the amount of total or phosphorylated MAPK and Akt pathway proteins in the breast cancer cell lines evaluated. In both cell lines, when treated with various combinations of mAb104, there was no effect on MAPK or Akt signal transduction compared to the control antibody (FIGS. 11C and D).

[0819] Example 6 Effect of mAb104 on apoptosis in vitro

[0820] The Dead Cell Apoptosis Kit (Thermo Fisher Scientific, catalog number V13241) was used to determine the apoptosis-inducing activity of mAb104 in BT-474 and SK-BR-3 cells using flow cytometry. Early and late apoptotic cell fractions were quantified by quadrant analysis. Treatment with trastuzumab or pertuzumab did not induce any apoptosis compared to cells alone, which is consistent with previous studies (Rockhoff G et al., (2007) Cell proliferation. 40(4):488-507; Nahta R et al., (2004) Cancer Research 64(7):2343-6; Lu Q et al., ibid) (Figure 12). Treatment of SK-BR-3 cells with trastuzumab resulted in a greater number of apoptotic cells compared to BT-474 (Figure 12A-G), however this was not statistically significant (Figure 12I-O). Similarly, mAb104 did not induce any significant apoptosis in the cell lines evaluated (p = 0.494). No differences in apoptotic activity were observed between the antibodies (p = 0.726).

[0821] No increase in apoptotic activity was observed in cells exposed to treatment with a combination of trastuzumab and pertuzumab or mAb104 compared to single agent treatment. In BT-474, 89.9% of cells survived after treatment with trastuzumab alone, while 91.8% survived after treatment with trastuzumab and mAb104 (Figure 12A-G). No differences in apoptotic activity were observed between the combination of trastuzumab and pertuzumab or mAb104 (Figure 12A-F and G).

[0822] An interesting finding was that a greater number of necrotic cells were observed after treatment with mAb104 compared to cells alone and trastuzumab and pertuzumab (Figure 12H and P), however this difference was not statistically significant. In BT-474 cells, 1.8% of cells underwent necrosis, while <0.5% of cells underwent necrosis in all other groups; similarly, a higher number of SK-BR-3 cells underwent necrosis after treatment with mAb104 compared to other treatment groups (Figure 12P), however this difference was not statistically significant. Interestingly, treatment of cells with mAb104 in combination with trastuzumab did not result in an increase in the number of cells undergoing necrosis.

[0823] The complex interplay of multiple receptor kinases and signal transduction pathways occurring in vivo cannot always be fully replicated in vitro and can reflect differences in HER2 processing and function, and the influence of the tumor microenvironment on HER2 function or other factors in vivo is necessary for the functional effects of mAb104, which targets epitopes conformationally exposed on HER2. Antibodies targeting conformationally exposed epitopes on activated EGFR have shown significant antitumor activity in vivo, although they failed to show any growth inhibition or altered signal transduction in vitro (Johns TG, Perera RM, Vernes SC, Vitali AA, Cao DX, Cavenee WK, et al. The efficacy of epidermal growth factor receptor–specific antibodies against glioma xenografts is influenced by receptor levels, activation status, and heterodimerization. Clinical Cancer Research. 2007;13(6):1911-25).

[0824] Despite the lack of detectable in vitro activity of mAb104, the inventors continued to investigate its in vivo effects in a cancer xenograft model overexpressing HER2.

[0825] In vivo efficacy of mAb104

[0826] Example 7 Efficacy of mAb104 monotherapy in HER2-overexpressing / amplified ER-positive breast cancer xenografts Efficacy

[0827] The inventors evaluated the efficacy of mAb104 in mice bearing established tumor xenografts of the ER-positive, HER2-overexpressing breast cancer cell line BT-474. Once the tumor volume reached 100-120 mm 3 , a 1 mg / antibody treatment dose of mAb104, trastuzumab, pertuzumab, or control antibody was administered three times a week for three weeks.

[0828] The results are shown in Figure 13 . At the end of the treatment (day 32), all tumors in the treatment groups were significantly smaller than those in the control group (ANOVA p < 0.0006). Post hoc tests using the Bonferroni method showed that all treatment groups were significantly smaller compared to the control group (p ≤ 0.001). At day 32, the mean tumor volume was 337.2 mm3 (control group), 4.8 mm 3 (trastuzumab), 6.7 mm 3 (pertuzumab) and 48.7 mm 3 (mAb104). A sustained significant anti-tumor response was observed until the end of the study (day 39) in the trastuzumab and pertuzumab treatment groups, one week after treatment cessation. However, for mAb104, tumor growth resumed after treatment cessation. At the end of the study (day 39), no significant difference in growth inhibition was observed between the treatment groups (p = 0.14).

[0829] The inventors also evaluated the efficacy of lower doses of 0.5 mg / antibody of mAb104, trastuzumab, pertuzumab and control IgG administered three times weekly for three weeks on established BT-474 tumor xenografts (120 - 150 mm 3 ).

[0830] The results are shown in Figure 14 . Trastuzumab treatment abolished further tumor growth; pertuzumab and mAb104 reduced the tumor growth rate compared to the control group. At the end of the study (day 52), the tumors in all treatment groups were significantly smaller than those in the control group (ANOVA p < 0.038). The mean tumor volume measured was 927.5 mm 3 (control), 182.4 mm 3 (trastuzumab), 415.0 mm 3 (pertuzumab) and 469.1 mm 3 (mAb104). Post hoc tests using the Bonferroni method showed that the mice in the treatment groups had significantly smaller tumors compared to the control (trastuzumab p = 0.0035, pertuzumab p = 0.02 and mAb014 p = 0.008). At 0.5 mg / mL, mAb104 showed similar anti-tumor efficacy to pertuzumab in this model (p = 0.97, two-sided). Although treatment with trastuzumab resulted in numerically greater tumor growth inhibition, there was no significant difference between trastuzumab and pertuzumab (p = 0.22, two-sided) or mAb104 (p = 0.15, two-sided) at the end of the study (day 51).

[0831] Survival analyzed by log-rank showed that the survival rate of mice treated with anti-HER2 antibodies was significantly longer than that of the control group (p < 0.002) when the control group was excluded for ethical reasons (i.e., tumor size ≤ 1000 mm 3 ). The median survival of control group mice was 41 days, while the mice in the treatment groups did not reach the median survival at the end of the experiment (day 52).

[0832] Example 8 Efficacy of mAb104 monotherapy in HER2-overexpressing / amplified breast PDX models

[0833] The present inventors evaluated the role of mAb104 in a HER2-overexpressing / amplified patient-derived xenograft (PDX) model of breast cancer. The donor samples were untreated, and thus the tumor susceptibility to anti-HER2 therapy was assumed to be 100%. Once the tumor volumes between 100 - 120 mm measured on day 64 were determined, mice were treated with a total dose of 0.5 mg of mAb104, trastuzumab, pertuzumab, or control IgG three times a week for three weeks. The results of the tumor growth curves are shown in Figure 15A. 3 Anti-HER2 therapy had a direct effect on the growth rate of the PDX. After treatment was stopped on day 86, all anti-HER2 therapies showed equivalent anti-tumor efficacy, and the delay in tumor growth rate continued until ~ day 125, at which point the tumor growth curve began to parallel the control growth rate. At the end of the study, on day 145, the control group was culled for ethical reasons. On day 145, all treatment groups were significantly smaller than the control group (ANOVA p < 0.04) (Figure 15A). Post hoc testing using the Bonferroni method showed that mice in the treatment groups had significantly smaller tumors compared to the control (trastuzumab p = 0.02; pertuzumab = 0.02 and mAb104 p = 0.038). The mean tumor volumes were 1099.2 mm

[0834] (control), 761.2 mm 3 (mAb104), 632.8 mm 3 (trastuzumab), and 691.3 mm 3 (pertuzumab). mAb104 showed strong and equivalent anti-tumor activity to approved HER2-targeted therapies in this model, with no significant differences between anti-HER2 therapies (p = 0.547 (two-sided) trastuzumab vs. mAb104 and p = 0.754 (two-sided) pertuzumab vs. mAb104). 3 (pertuzumab). mAb104 showed strong and equivalent anti-tumor activity to approved HER2-targeted therapies in this model, with no significant differences between anti-HER2 therapies (p = 0.547 (two-sided) trastuzumab vs. mAb104 and p = 0.754 (two-sided) pertuzumab vs. mAb104).

[0835] In the survival analysis, mice in the treatment groups had significantly longer survival than the control group (p < 0.0005), and post hoc testing showed that all groups treated with anti-HER2 antibodies had significantly longer survival compared to control mice (p < 0.001). The median survival of control group mice was 145 days, while at the end of the experiment, mice in the treatment groups had not reached the median survival.

[0836] Example 9 Efficacy of mAb104 in combination with trastuzumab in ER-positive breast xenografts overexpressing / amplifying HER2 Efficacy

[0837] The combination of trastuzumab and pertuzumab has been shown in the literature to have more effective anti-tumor activity and prevent metastatic tumor spread compared to either antibody alone, independent of HER2 expression. Given the different domain II epitope binding sites of mAb104 compared to trastuzumab and pertuzumab, and the inventors' observation of the effective anti-tumor activity of mAb104 as a single therapy in vivo, the inventors proceeded to evaluate the combination of mAb104 with trastuzumab either alone or in combination with pertuzumab.

[0838] The inventors evaluated the effect of the combination of mAb104 with trastuzumab in an established BT-474 breast cancer xenograft tumor model. Each mouse received 0.25 mg trastuzumab and 0.25 mg mAb104 or pertuzumab to achieve a total dose of 0.5 mg / treatment or an equivalent control antibody, three times a week for three weeks. Once the average tumor volume was 100 - 120 mm 3 , treatment was initiated.

[0839] The results are shown in Figure 15B. Anti-tumor efficacy was evident within 10 days of the start of treatment and continued after treatment cessation. At the end of the 50th day of the study period, the control group was culled due to ethical reasons regarding tumor burden. The mean ± SD tumor volume in the trastuzumab alone group and the combination group was significantly smaller than that in the control group (ANOVA p < 0.0001). A post hoc test was then performed using the Bonferroni method. Tumors treated with the combination therapy were significantly smaller than those in the control group (p < 0.0001), measured as 88.9 mm 3 (mAb104 plus trastuzumab) and 43.6 mm 3 (trastuzumab plus pertuzumab). No complete regression of tumors was seen in any treatment group (Figure 15B). Concurrent treatment with mAb104 and trastuzumab resulted in a greater tumor reduction compared to trastuzumab alone; however, the difference in tumor size between the combination and single treatment groups was not statistically significant (by ANOVA, p = 0.09).

[0840] Survival analysis showed that mice in the two combination groups had significantly longer survival compared to the control group (p < 0.002). The median survival of the control group was 44 days, while the median survival of mice in the combination treatment groups was not reached. Log rank analysis showed no statistical difference between the two combination groups (p = 0.21, two-sided); treatment with mAb104 and trastuzumab significantly inhibited tumor growth compared to mAb104 monotherapy (p 0.04, two-sided) (Figure 15A).

[0841] Thus, this indicates that the combination of mAb104 with trastuzumab provides enhanced anti-tumor activity compared to either single therapy alone.

[0842] Example 10 Efficacy of mAb104 in combination with trastuzumab in HER2-overexpressing / amplified ER-positive breast PDX models Efficacy

[0843] The present inventors evaluated the efficacy of concurrent antibody therapy in a HER2-overexpressing / amplified breast PDX model. The donor samples were not treated with anti-HER2, so 100% tumor sensitivity to treatment was assumed. Once the tumor volume measured between 100 - 120 mm 3 was determined, mice were treated with trastuzumab at a total treatment dose of 0.5 mg three times per week, or isotype control alone or in combination with mAb104 and trastuzumab, or trastuzumab plus pertuzumab for three weeks.

[0844] At the end of treatment on day 85, a significant difference was observed between all treatment groups and the control group (p < 0.0001) (Figure 15C). Additionally, the combination group was more effective than trastuzumab alone (p = 0.001). The greater anti-tumor efficacy of the combination group persisted until the study was terminated on day 145, at which time the control group was culled due to ethical reasons related to tumor burden. On day 145, the tumors in all treatment groups remained significantly smaller than the control group (p < 0.0001). The mean tumor volume measured was 164.4 mm 3 (mAb104 plus trastuzumab) and 84.2 mm 3 (trastuzumab plus pertuzumab) (Figure 15C). The difference in tumor volume between the two combination groups did not reach statistical significance (p = 0.46, two-sided). No complete regression of tumors was seen in any treatment group.

[0845] Concurrent treatment with mAb104 and trastuzumab led to significantly greater tumor volume shrinkage compared to trastuzumab monotherapy (by ANOVA, p < 0.0001). Post hoc tests were then performed using the Bonferroni method. The tumors in the combination group were significantly smaller compared to the single agent trastuzumab (p < 0.0049).

[0846] Survival analysis showed that mice in the two combination groups had significantly longer survival compared to the control group (p < 0.0005) and mice in the single treatment groups (p = 0.0014). The median survival of the control group was 145 days and the median survival of mice in the combination treatment groups was not reached.

[0847] The results in Figures 15A and C show that after day 145, the average tumor size of trastuzumab alone was 632.8 mm 3 and that of mAb104 was 761.2 mm 3 . When mAb104 was combined with trastuzumab, the tumor size was significantly reduced to 164.4 mm 3The reduction in tumor size indicates that the combination of mAb104 and trastuzumab results in enhanced anti-tumor activity compared to single therapies with trastuzumab or mAb104 alone.

[0848] Example 11 Reverse-phase protein array (RPPA) analysis

[0849] Lysates obtained from HER2-breast PDX tumors (n = 2 / group) were collected at the completion of treatment on day 85 and analyzed by RPPA. A panel of over 300 antibodies detecting total proteins and / or their activated forms was included in this RPPA analysis. Key proteins involved in key signal transduction pathways including the phosphatidylinositol 3-kinase (PI3K) / AKT pathway, extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) pathway, Janus kinase (JAK) / signal transducer and activator of transcription (STAT) pathway, apoptosis pathway, cell cycle (including cell death and survival). The data collected were normalized for protein loading and transformed to linear values for analysis. The percent fold change was calculated as the ratio of the difference in protein expression between control groups and tumor samples treated with trastuzumab, pertuzumab or mA104 or combinations.

[0850] Although mAb104 demonstrated significant anti-tumor activity in the HER2-breast PDX model, no significant changes in protein levels were seen compared to the other antibodies evaluated (Table 9).

[0851] Table 9: Differential expression of key proteins treated with trastuzumab, pertuzumab and mAb104 as evaluated by RPPA

[0852]

[0853]

[0854]

[0855]

[0856] *ns: Not significant. This list evaluates the fold change of key pathway kinases and their downstream effectors. The fold change was calculated as the ratio of the difference in protein expression between control and treatment groups. The p-value was obtained using a t-test for the indicated comparisons.

[0857] Example 12 Immunohistochemical analysis of established tumors

[0858] Mice (n = 2) bearing established BT-474 breast tumor xenografts from each of the monotherapy and combination therapy groups were sacrificed 1 day after the last treatment, and xenograft tissue samples were obtained and prepared for IHC analysis of tumor proliferation, downstream signal transduction, and angiogenesis.

[0859] The effect of mAb104 monotherapy on tumor proliferation was detected by Ki67 staining, and the results are shown in Figure 16A. In BT-474 xenograft tumors, compared with the control group, treatment with anti-HER2 antibodies did not significantly reduce proliferation (by ANOVA, p = 0.625, and post hoc analysis demonstrated no difference between different treatment groups). The average H-scores were 102.6 (control), 83.9 (trastuzumab), 91.1 (pertuzumab), and 99.7 (mAb104).

[0860] To determine whether the anti-proliferative effect was mediated by downregulation of the Akt pathway, Akt was evaluated by phosphoprotein assay (Figure 16B). In BT-474, no significant difference in the H-score of phospho-Akt was observed between the treatment group and the control group (by ANOVA, p = 0.958, and no difference in the treatment group in post hoc analysis). The average H-scores were 129.6 (control), 124.3 (trastuzumab), 114.6 (pertuzumab), and 132.5 (mAb104).

[0861] Trastuzumab has been shown to have anti-angiogenic effects (Parakh S, (2017) Cancer treatment reviews. 59:1-21). Therefore, the inventors examined the effect of mAb104 on the microvessel density in tumor tissues by podoplanin staining (Figure 16C). Immunohistochemical staining was performed as described above. The microvessel density (%) was calculated as the ratio of the positively stained area to the total observed area in the viable region. In BT-474 xenograft tumors, although significant anti-angiogenic activity of trastuzumab was observed (p < 0.001), mAb104 and pertuzumab had no significant effect on the tumor vascular system compared with the control antibody (p = 0.987).

[0862] The combination of trastuzumab and pertuzumab significantly reduced proliferation compared with the control (p < 0.05) and compared with trastuzumab and mAb104 (p = 0.017, two-sided). The average H-scores were 129.6 (control), 7.4 (trastuzumab plus pertuzumab), and 97.6 (trastuzumab plus mAb104) (Figure 16D). These findings are similar to previous reports (Brockhoff G, (2007) Cell proliferation. 40(4):488-507).

[0863] No effects on pAkt (Figure 16E) or angiogenesis (Figure 16F) were observed in BT-474 tumor samples treated with trastuzumab plus pertuzumab or trastuzumab plus mAb104 when compared to control antibodies.

[0864] Gastric cancer

[0865] Example 13 Anti-proliferative effect of mAb104 in vitro

[0866] In vitro, trastuzumab significantly (p<0.0001) inhibited the growth of NCI-N87 and affected the proliferation of OE-19 gastric cancer cells ( Figure 17 ), which is consistent with previous reports (Gravalos C et al., (2008) Annals of oncology.19(9):1523-9). Compared to control antibodies, pertuzumab as a single therapy did not have any significant anti-proliferative activity in the cell lines evaluated (NCI-N87 p = 0.02; and OE19 p = 0.96). These findings are consistent with other studies, although incubation times and doses vary (Brockhoff G et al., (2007) Cell proliferation.40(4):488-507; Tokuda Y et al., (1996) British journal of cancer.73(11):1362; Yamashita-Kashima Y et al., (2011) Clinical Cancer Research.17(15):5060-70; Nahta R et al., (2004) Cancerresearch.64(7):2343-6; Gong SJ et al., (2004) Cancer letters.214(2):215-24; Ko B-K et al., (2015) Molecular oncology.9(2):398-408; Tomioka H et al., (2012) Internationaljournal of oncology.41(2):551-8).

[0867] Compared to the isotype control antibody, the monoclonal antibody mAb104 also did not show significant growth inhibitory effects in vitro (NCI-N87 p = 0.34; and OE19 p = 0.12) ( Figure 17)。This is consistent with other in vitro studies evaluating antibodies targeting conformationally exposed epitopes on the EGFR receptor (Johns TG et al., (2003) Proceedings of the National Academy of Sciences. 100(26):15871-6; Johns TG et al., (2007) Clinical Cancer Research. 13(6):1911-25).

[0868] The combination of trastuzumab and pertuzumab significantly inhibited proliferation in the gastric / GEJ cell lines evaluated ( Figure 18 ). However, there was no statistical difference compared to trastuzumab monotherapy. The effect of the combination of mAb104 with trastuzumab or pertuzumab on proliferation was evaluated; the addition of mAb104 did not increase the anti-proliferative effect compared to the individual antibodies alone. Significantly, the combination of mAb104 and trastuzumab was not statistically different from the combination of trastuzumab and pertuzumab (NCI-N87 p = 0.29; and OE19 p = 0.14).

[0869] Example 14 Effect of mAb104 on ErbB receptors and downstream signal transduction pathways

[0870] In the gastric cancer cell lines NCI-N87 and OE19 overexpressing HER2, treatment with anti-HER2 antibodies as monotherapy did not affect the total or phosphorylated proteins in the MAPK and AKT signaling pathways, which is consistent with previous reports (Ko B-K et al., (2015) Molecular oncology. 9(2):398-408; Tomioka H et al., (2012) International journal of oncology. 41(2):551-8)) (Figures 19A and B).

[0871] Combination treatment with trastuzumab and pertuzumab or mAb104 in the NCI-N87 gastric cancer cell line led to downregulation of phosphorylated-Akt and phosphorylated p44 / p42 MAPK, while the total protein levels remained unchanged (Figures 19C and D).

[0872] Under ligand-stimulated conditions (Figures 20A and B and 20C and D), mAb104 did not affect the MAPK or Akt signaling pathways in the cell lines evaluated. This lack of effect was also observed when mAb014 was used in combination with trastuzumab and pertuzumab (Figures 20C and D). ...

Claims

1. A HER2 / ErbB2 binding protein comprising an antigen-binding domain, wherein, the antigen-binding domain specifically binds to a cyclic peptide comprising the amino acid sequence H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 26) corresponding to an epitope within domain II of HER2, the epitope within domain II of HER2 comprising residues 293 to 309 of the mature normal or wild-type human HER2 sequence according to FIG. 1 (SEQ ID NO: 27), and the epitope being exposed in response to HER2 amplification or activation, and wherein the epitope is expressed in neoplastic, hyperproliferative or abnormal cells but not in normal or wild-type cells.

2. The HER2 binding protein according to claim 1, wherein the HER2 binding protein comprises: a heavy chain variable region sequence (VH) comprising: CDR1 consisting of the sequence shown by SEQ ID NO: 14, CDR2 consisting of the sequence shown by SEQ ID NO: 16, and CDR3 consisting of the sequence shown by SEQ ID NO: 18; and a light chain variable region sequence (VL) comprising: CDR1 consisting of the sequence shown by SEQ ID NO: 20, CDR2 consisting of the sequence shown by SEQ ID NO: 22, and CDR3 consisting of the sequence shown by SEQ ID NO:

24.

3. The HER2 binding protein according to claim 1 or 2, wherein the HER2 binding protein is an antibody or an antigen-binding fragment thereof, comprising: (i) a heavy chain variable region sequence (VH) having at least 55% identity with the heavy chain variable region sequence of mAb104 (SEQ ID NO: 2); and (ii) a light chain variable region sequence (VL) having at least 55% identity with the light chain variable region sequence of mAb104 (SEQ ID NO: 3), or a humanized form, chimeric form or deimmunized form thereof.

4. The HER2 binding protein according to any one of claims 1-3, wherein the HER2 binding protein comprises VH and VL or a humanized form, chimeric form or deimmunized form thereof, the VH comprising a sequence having at least 90% identity with the sequence shown by SEQ ID NO: 2, and the VL comprising a sequence having at least 90% identity with the sequence shown by SEQ ID NO:

3.

5. The HER2 binding protein according to claim 1, wherein the HER2 binding protein comprises: a heavy chain variable region sequence (VH) comprising: CDR1 consisting of the sequence shown by SEQ ID NO: 15, CDR2 consisting of the sequence shown by SEQ ID NO: 17, and CDR3 consisting of the sequence shown by SEQ ID NO: 19; and a light chain variable region sequence (VL) comprising: CDR1 consisting of the sequence shown by SEQ ID NO: 21, CDR2 consisting of the sequence shown by SEQ ID NO: 23, and A CDR3 consisting of the sequence shown in SEQ ID NO:

25.

6. The HER2-binding protein according to claim 1 or 5, wherein the HER2-binding protein is an antibody or an antigen-binding fragment thereof, comprising: (i) a heavy-chain variable region sequence (VH) having at least 55% identity with the heavy-chain variable region sequence of mAb106 (SEQ ID NO: 4); and (ii) a light-chain variable region sequence (VL) having at least 55% identity with the light-chain variable region sequence of mAb106 (SEQ ID NO: 5), or a humanized form, chimeric form or deimmunized form thereof as described above.

7. The HER2-binding protein according to any one of claims 1, 5 and 6, wherein the HER2-binding protein comprises VH and VL or a humanized form, chimeric form or deimmunized form thereof as described above, the VH comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 4, and the VL comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO:

5.

8. The HER2-binding protein according to claim 4, wherein the HER2-binding protein comprises the VH shown in SEQ ID NO: 2 and the VL shown in SEQ ID NO:

3.

9. The HER2-binding protein according to claim 7, wherein, the HER2-binding protein comprises the VH shown in SEQ ID NO: 4 and the VL shown in SEQ ID NO:

5.

10. The HER2-binding protein according to any one of claims 1-9, wherein, the antigen-binding fragment is: (i) a single-chain Fv fragment (scFv); (ii) a dimeric scFv (di-scFv); (iii) at least one of (i) and / or (ii) linked to a heavy-chain constant region or Fc or heavy-chain constant domain CH2 and / or CH3; or (iv) at least one of (i) and / or (ii) linked to a protein that enhances the antibody half-life.

11. The HER2-binding protein according to claims 1-9, wherein, the antigen-binding fragment is: (i) a diabody; (ii) a triabody; (iii) a tetrabody; (iv) Fab; (v) F(ab′)2; (vi) Fv; or (vii) at least one of (i) to (vi) linked to a heavy-chain constant region or Fc or heavy-chain constant domain CH2 and / or CH3; or (viii) at least one of (i) to (vi) linked to a protein that enhances the antibody half-life.

12. The HER2-binding protein according to any one of claims 1-11, which is conjugated to a detectable or functional moiety, or which is conjugated to a drug.

13. A composition comprising the HER2-binding protein according to any one of claims 1-12 and a suitable carrier.

14. Use of the HER2-binding protein according to any one of claims 1 to 12 or the composition according to claim 13 in the preparation of a medicament for treating HER2-expressing cancer.

15. Use according to claim 14, wherein the HER2-expressing cancer is selected from cholangiocarcinoma, colon cancer, lung cancer, bladder cancer, endometrial cancer, ovarian cancer, breast cancer, gastric cancer, and gastroesophageal cancer.

16. Use of the HER2-binding protein according to any one of claims 1 to 12 in the preparation of a composition or system for diagnosing or detecting breast cancer in a biological sample obtained from a subject, wherein the HER2-binding protein binds to HER2.

17. A complex comprising the HER2-binding protein according to any one of claims 1 to 12 that binds to HER2.

18. A method for producing a HER2-binding protein, which comprises immunizing a non-human animal with a cyclic peptide comprising the sequence H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 26).

19. The method according to claim 18, further comprising obtaining a hybridoma cell line from the B cells of the immunized non-human animal and purifying an antibody from the hybridoma cell line.

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