Anti-HER2 binding molecules

Abstract

The present disclosure is directed to binding proteins to the extracellular domain (ECD) of HER2 / ErbB2. More specifically, the binding proteins bind to conformational epitopes that are exposed in cells in response to HER2 amplification or activation.

Description

[Technical Field]

[0001] All documents cited or referenced herein, and all documents cited or referenced within the documents cited herein, are hereby incorporated by reference in their entirety, along with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or within any documents incorporated by reference herein.

[0002] The entire contents of the electronic sequence listing submission are incorporated by reference in their entirety for all purposes.

[0003] This application claims priority to Australian Provisional Patent Application No. AU2019900973, filed 22 March 2019, the entire contents of which are incorporated herein by reference.

[0004] The present disclosure is directed to binding proteins to the extracellular domain (ECD) of HER2 / ErbB2. More specifically, the binding proteins bind to conformational epitopes that are exposed in cells in response to HER2 amplification or activation. [Background technology]

[0005] The ErbB family of receptors includes four homologous proteins present on the cell surface: epidermal growth factor receptor (EGFR; also known as ErbB1; HER1); ERBB2 (HER2; also known as Neu); ERBB3 (or HER3); and ERBB4 (or HER4).

[0006] HER2 overexpression and amplification have been confirmed in many types of cancer, 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 carcinoma. HER2 overexpression and amplification have been shown to be associated with poor prognosis in breast cancer and gastric / gastroesophageal junction (GEJ) cancer (Nagaraja V. et al., (2016), Eur. J. Surg. Oncol. 42(1):140-8); however, the impact on other tumor types has not been clearly defined.

[0007] The HER2 antibody market exceeded $10 billion in 2017, and future sales of HER2 antibodies are expected to increase as new clinical indications and markets emerge. Response to HER2 targeting in patients with HER2 overexpression / amplification is influenced by several clinicopathological features, including tumor histology, degree of anaplasia, stage of disease, and the presence of underlying risk factors. HER2 amplification or overexpression also serves as a predictive biomarker for anti-HER2 therapy in various tumor types, including breast, gastric, and gynecological 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 The extracellular domain of HER2 cannot bind known natural ligands (Klapper LN. et al., (1999), PNAS, 96(9):4995-5000). Unlike other members of the ErbB family, HER2 adopts a conformation that favors the oligomerization and activation of HER2 kinase in the absence of ligand (Garrett TP. et al., (2002), Cell, 110(6):763-73), unlike other members of the ErbB family. Open HER2 (Cho H.S. et al., (2003), Nature, 421(6924):756-60) makes the dimerization arms permanently available for homodimeric or heterodimeric interactions between monomeric family members, as well as for conformational changes and oligomerization of existing inactive dimers (Maruyama I.N. et al., (2014), Cells, 3(2):304-30), leading to autophosphorylation of the intracellular kinase domain and signal transduction. Overexpression of HER2 increases the affinity of EGF and neuregulin to the receptor and decreases the rate of ligand dissociation from the active dimer. Overexpression of HER2 has also been shown to affect the recycling and degradation rates of HER2-containing heterodimers: EGFR-HER2 heterodimers undergo endocytic recycling rather than degradation, resulting in prolonged EGFR signaling (Huang G. et al., (1999), J. Cell Biochem. 74(1):23-30). Of the heterodimers formed upon ligand activation, the HER2-HER3 heterodimer appears to be the most potent signaling complex in HER2-amplified tumors (Tzahar E. et al., (1996), Mol. Cell Biol. 16(10):5276-87). Signaling 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 shown that HER2 overexpression leads to malignant transformation, the development of anti-apoptotic properties, increased invasiveness, and drug resistance. HER2 hetero-oligomerization-mediated receptor activation may be an important mechanism promoting the cell proliferation observed in HER2-overexpressing cells (Wolf-Yadlin A. et al., (2006), Mol. Syst. Biol. 2(1):54).

[0009] Anti-HER2 directed therapy Many different approaches to inhibiting HER2 have been attempted, and many of these have entered clinical practice.

[0010] (i) Monoclonal antibodies against domain IV of HER2: Early approaches to HER2 inhibition used monoclonal antibodies against the extracellular domain, a prominent example of which 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 BK. et al., (2015), Mol. Oncol. 9 (2): 398-408; Mahdavi M. et al., (2015), Monoclonal Antib Immunodiagn Immunother, 34 (3): 213-21; Ceran C. et al., (2012), Cancer Cell, 6 (2): 117-27.

[0011] (ii) Antibodies against domain II of HER2: Pertuzumab (Adams CW. et al., (2006), 55(6):717-27), a first-in-class drug known as a HER dimerization inhibitor, binds to the extracellular dimerization domain II of HER2 (an epitope different from that of trastuzumab) and inhibits dimerization between HER receptors (Adams CW, supra).

[0012] (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 because secondary signaling may be one of the mechanisms of resistance to HER2 inhibitors (Ritter CA et al., (2007), Clin. Cancer Res. 13(16):4909-19). Two approved tyrosine kinase inhibitors exist: lapatinib, an oral small molecule reversible inhibitor that inhibits both EGFR kinase and HER2 kinase (Tevaarwerk AJ et al., (2009), Clin. Ther. 31:2332-48), and afatinib, an irreversible inhibitor of EGFR, HER2, and HER4 tyrosine kinase activity, as well as EGFR- and HER2-containing dimers (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 signaling in cells with HER2 overexpression and EGFR amplification, leading to 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).

[0013] (iv) Antibody-drug conjugates targeting HER2: High expression in HER2-positive cancer cells and low expression in normal tissues make it a target for antibody-drug conjugates (ADCs). Ado-trastuzumab emtansine (T-DM1) is the first anti-HER2 ADC approved for solid tumors. It consists of trastuzumab linked to the potent cytotoxic agent DM1, an inhibitor of microtubule dimerization. The antitumor effects of T-DM1 are 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. While T-DM1 shows maximal efficacy in tumors with high HER2 expression, it also demonstrates efficacy across a range of 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 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 composed of 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 containing the anti-HER2 antibody, HT-19, linked to an auristatin-based cytotoxic payload (AF-HPA) (Bergstrom D. et al., (2016), Can. Res. 76 (4 Supplement), P4-14-28).

[0014] (v) Monoclonal antibodies: Many new anti-HER2 monoclonal antibodies have been developed in clinical settings, including 10H8 and 8H11 (Kim AY. et al., (2013) Proceedings of the ASCO Annual Meeting), MGAH22 (margetuximab), an Fc-optimized chimeric anti-HER2 monoclonal antibody, ertumaxomab, a trifunctional antibody targeting HER2, T cell-specific CD3 antigen, and FcγI / III receptors; and CMAB302 (cipterbine), a biosimilar to trastuzumab.

[0015] (vi) Bispecific antibodies: A 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 early-stage first-in-human trials are evaluating MM-111 as monotherapy and in combination with trastuzumab and various chemotherapy regimens or lapatinib in HER2-positive solid tumors. MM-111 is also being studied in gastrointestinal malignancies. MCLA-128 is a humanized bispecific antibody with enhanced ADCC activity targeting HER2 and HER3 (Calvo E. et al., Abstract CT050 AACR, 2016). MCLA-128 blocks downstream signaling via the HER2:Her3 heterodimer, even in the presence of high heregulin concentrations. GBR 1302 is another bispecific antibody targeting CD3ε and HER2 that is being evaluated in early-phase clinical trials after demonstrating potent antitumor activity in HER2-overexpressing and non-overexpressing tumors (Moretti P. et al., (2016), the 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. Additional bispecific immunotoxins are being developed, including an anti-HER2 single-chain variable fragment (scFv) fused to diphtheria toxin-anti-EpCAM.

[0016] Despite various approaches, challenges remain. Primary resistance to single-agent trastuzumab occurs in 70% of HER2-overexpressing breast cancers (Vogel CL et al. (2002), 20(3):719-26), and the majority of patients develop resistance during treatment. Several mechanisms have been proposed (see Parakh S. et al., supra). In contrast to trastuzumab, the mechanisms of resistance to pertuzumab are poorly understood. Similar to other anti-HER2 therapies, primary and acquired resistance to T-DM1 has also developed (Tan X. et al. (2013), Can. Res. 73(8, Supplement):4629). While the mechanisms of resistance to T-DM1 appear to depend on tumor size and treatment duration, resistance has been observed even after long latency periods (Barok M. et al. (2011), Breast Cancer Res. 13(2):R46).

[0017] Despite the success of trastuzumab (Herceptin), pertuzumab, and the T-DM1 conjugate, the toxicity of current HER2 antibodies is dose-limiting, and resistance invariably develops, as discussed above. New strategies that target HER2 without the associated toxicity profile are clearly needed. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] Australian Provisional Patent Application No. AU2019900973 [Non-patent literature]

[0019] [Non-Patent Document 1] Nagaraja V.et al.,(2016) [Non-patent document 2] Eur.J.Surg.Oncol.42(1):140-8 [Non-patent document 3] Slamon DJ.et al.,(1987),Science,235{4785):177-82

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Summary of the Invention

[0020] The present disclosure provides isolated specific binding proteins that bind to the extracellular domain (ECD) of HER2 in a conformationally flexible region of domain II. In particular, the binding proteins exhibit no amino acid sequence changes or substitutions from the wild-type HER2 sequence and recognize a HER2 epitope that is exposed in cells in response to HER2 amplification or activation. The conformationally exposed epitope is found only in tumorigenic, hyperproliferative, or abnormal cells and is undetectable in normal or wild-type cells. "Wild-type" contemplates cells that express endogenous HER2, but specifically excludes cells that overexpress the HER2 gene; the term "wild-type" refers to a genotype or phenotype or other characteristic present in normal cells but not in abnormal or tumorigenic cells. [Means for solving the problem]

[0021] Interestingly, the binding proteins of the present disclosure do not block the binding of pertuzumab or trastuzumab / herceptin to HER2 on cancer cells, suggesting that this epitope region in domain II, when conformationally exposed, may allow the binding of the present binding molecules without blocking the binding of these antibodies, potentially enabling a dual therapy approach.

[0022] More specifically, the inventors have found that the present binding molecules bind to a smaller fraction of HER2 on the surface of cancer cells (e.g., when compared to pertuzumab or trastuzumab), while being just as potent in vivo at concentrations comparable to pertuzumab or trastuzumab, despite binding to fewer receptors. Because the binding molecules of the present disclosure are internalized and tumor cell specific, they are ideally suited as drug conjugates or agents in dual therapy approaches with other HER2 antibodies.

[0023] The specific binding proteins of the present disclosure, which may be antibodies or fragments thereof, such as immunogenic fragments thereof, do not bind to or recognize normal or wild-type cells that contain a normal or wild-type HER2 epitope in the absence of aberrant expression and in the presence of normal HER2 post-translational modifications. More specifically, the specific binding proteins of the present invention may be antibodies or fragments thereof that recognize a HER2 epitope that is present in cells that overexpress HER2 (e.g., the HER2 gene is amplified), particularly in the presence of aberrant post-translational modifications, and that is not detectable in cells that express HER2 under normal conditions, particularly in the presence of normal post-translational modifications.

[0024] The present inventors have discovered novel monoclonal antibodies, exemplified herein by the antibody designated mAb104, that specifically recognize aberrantly expressed HER2. In particular, the antibodies of the present disclosure recognize a HER2 epitope found in tumorigenic, hyperproliferative, or abnormal cells that is undetectable in normal or wild-type cells. The antibodies of the present disclosure are further exemplified by the antibodies mAb105, mAb106, and mAb107 described herein.

[0025] The present disclosure provides HER2 / ErbB binding proteins 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 example, the HER2 binding protein binds to a region of HER2 that is conformationally exposed in tumorigenic, hyperproliferative, or abnormal cells, but not in normal or wild-type cells.

[0026] In another example, binding of the binding protein to its epitope does not block pertuzumab or trastuzumab / Herceptin binding. In one example, the binding protein is not pertuzumab or trastuzumab. In one example, 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 HER2 extracellular domain (ECD). In a particular example, the epitope comprises the amino acid sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1). This epitope is shown in Figure 1 as the bold, underlined sequence. This epitope includes P294, L295, and H296, which are also present in the epitope to which pertuzumab binds; however, the currently described binding protein does not block pertuzumab but allows pertuzumab to simultaneously bind HER2. Epitopes may be determined by any conventional epitope mapping technique known to those skilled in the art.

[0027] In one example, the binding protein does not bind or does not substantially bind to human EGFR (HER1) or HER3 or HER4.

[0028] In one example, the binding protein has the characteristics of the antibodies identified and characterized by the inventors, and in particular recognizes aberrantly expressed HER2, such as that found in amplified HER2. In another example, the binding protein binds to tumor cell lines expressing high levels of HER2. In one example, HER2 overexpression is determined using immunohistochemical analysis. In a specific example, the staining pattern is evaluated and scored using the American Society of Clinical Oncology and College of American Pathologists (ASCO / CAP) recommendations for HER2 testing in breast cancer (Wolff AC. et al. (2013), Journal of Clinical Oncology, 31(31):3997-4013).

[0029] In another example, the binding protein binds to a cancerous cell selected from the group consisting of breast cancer, gastric cancer, squamous cell carcinoma, and colon cancer. In another example, the binding protein binds to a cell line selected from the group consisting of breast cancer (e.g., BT474, SK-BR3, SUM159PT, MDA-MB-453), gastric cancer (e.g., NCI-N87, MK N7), squamous cell carcinoma (e.g., A431), and colon cancer (COLO205, LIM1215).

[0030] In another example, the binding protein binds a smaller proportion of HER2 / ErbB on the cell surface of cancer cells compared to pertuzumab and / or trastuzumab. In another example, the binding protein binds to HER2+ expressing cells at least 1 log lower, at least 2 log lower, or at least 3 log lower orders of magnitude than trastuzumab or pertuzumab, as assessed by flow cytometry. In particular 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 binding protein binds to a proportion of HER2 / ErB2 between about 0.35% and 0.5% of the total HER2 / ErB2 expressed on the surface of cancer cells.

[0031] In one example, the binding protein does not bind to normal stomach mucosa, in another example, the binding protein does not bind to normal breast cells.

[0032] In another example, the binding protein can be internalized by tumor cells. In a particular example, the binding protein has an in vivo anti-proliferative effect on tumor cells (e.g., gastric cells). In another example, the binding protein has an in vivo anti-tumor effect comparable to that of pertuzumab or trastuzumab. In yet another example, the binding protein causes necrosis of tumor cells (e.g., breast tumor cells).

[0033] An exemplary HER2 binding protein described herein having such binding properties comprises the variable regions and / or CDRs of the antibody designated mAb104 or mAb106.

[0034] In one example, the binding protein binds to a peptide comprising or consisting of the sequence set forth in SEQ ID NO: 1, or binds to a sequence in the human HER2 ECD at a similar or substantially the same level or with similar or substantially the same affinity as the antibodies designated mAb 104 or mAb 106. In one example, the binding protein binds to a contiguous sequence of amino acids comprising or consisting of residues 293-309 of the mature, normal or wild-type human HER2 sequence as shown in Figure 1.

[0035] In another example, the HER2 binding protein competitively inhibits the binding of an antibody designated mAb104 or mAb106 to human HER2. In a further example, the protein competitively inhibits the binding of an antibody designated mAb104 or mAb106 to a peptide consisting of the sequence set forth in SEQ ID NO:1.

[0036] In one example, the HER2 binding protein binds to a peptide comprising or consisting of the sequence set forth in SEQ ID NO: 1 in an amount that is within 75% of the amount bound by an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.

[0037] In one example, the amount of bound protein or antibody is assessed by contacting the HER2-binding protein with a peptide consisting of the sequence set forth in SEQ ID NO: 1, and the amount of HER2-binding protein contacted with the peptide (e.g., 10 μg / ml). The amount of HER2-binding protein bound to the peptide is then determined and compared to the amount of antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, respectively, bound to the peptide. In one example, the amount of HER2-binding protein bound to the peptide is within about 80%, or 70%, or 60%, or 40% of the amount of bound antibody.

[0038] The present disclosure also provides an antibody named: (i) mAb104, the antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 3; or (ii) A peptide comprising or consisting of the sequence set forth in SEQ ID NO: 1 of mAb106, which comprises a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 5, or a HER2 binding protein that competitively inhibits binding of human HER2 to the ECD (e.g., Figure 1).

[0039] In one example, a HER2 binding protein binds to the ECD of HER2, e.g., having a sequence as shown in Figure 1, with an affinity dissociation constant (KD) of between 2.90 and 3.20 nM. In another example, the KD is between about 2.90 and about 3 nM. In another example, the KD is about 3 nM.

[0040] In one example, KD is assessed using surface plasmon resonance on a biosensor equipped with a streptavidin (SA) chip by capturing a biotin-conjugated human HER2 peptide (e.g., the peptide according to SEQ ID NO: 1) on the surface of the chip and passing a HER2-binding protein over it.

[0041] Exemplary HER2 binding proteins of the present disclosure have a K of about 3 nM (e.g., ±0.2 nM) as assessed by SA chip biotin peptide SPR. In one example, the HER2 binding protein has a K as shown in Table 7 for mAb104 or mAb106.

[0042] In one example, the HER2 binding protein of the present disclosure specifically binds to human HER2. In one example, binding of the protein is assessed by ELISA.

[0043] The human HER2 binding protein of the present disclosure may be an anti-HER2 recombinant antibody or synthetic antibody or monoclonal antibody or antigen-binding fragment thereof.

[0044] In one example, the HER2 binding protein is a chimeric antibody comprising human heavy and light chain constant region sequences, hi another example, the HER2 binding protein is a humanized or fully human antibody.

[0045] In one example, the HER2 binding protein comprises a heavy chain variable region sequence (VH) that has at least 55% identity to the heavy chain variable region sequence of mAb104 (SEQ ID NO:2).

[0046] In one example, the HER2 binding protein comprises a light chain variable region sequence (VL) that has at least 50% identity to the light chain variable region sequence of mAb104 (SEQ ID NO:3).

[0047] In one example, the binding protein comprises: (i) a VH CDR1 having the sequence shown as follows: [ka] In the sequence, X7 is S or T; X8 is G or D; X9 is F or G; 10 is H or N; (ii) a VH CDR2 having the sequence shown as follows: [ka] In the sequence, 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; (iii) a VH CDR3 having the sequence shown as follows: [ka] In the sequence, 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; and / or (iv) a VL CDR1 having the sequence shown as follows: [ka] In the sequence, X 14 is K or S;X 15 is S or V; X 16 is Q or S;X 17 is L or does not exist; X 18 is L or does not exist; X 19 is D or does not exist; X 20 is S or does not exist; X 21 is D or does not exist; X 22 is G or does not exist; 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; (v) a VL CDR2 having the sequence shown as follows: [ka] X 35 is D or E;X 36is K or T;X 37 is S or A; and (vi) a VL CDR3 having the sequence shown as follows: [ka] In the sequence, 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.

[0048] In one example, the HER2 binding protein comprises the heavy chain variable region sequence (VH) sequence shown below: [ka] In the array, X1 is E or Q; X2 is V or I; X3 is Q or V; X4 is V or K; X5 is A or E; X6 is S or T; X7 is S or T; X8 is G or D; X9 is F or G; 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; X28 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 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; X 65 is A or S.

[0049] In one example, the HER2 binding protein further comprises a light chain variable region sequence (VL) sequence shown below: [ka] In the array, X1 is D or Q; X2 is I or L; X3 is L or A; X4 is T or L; X5 is L or M; X6 is T or S; X7 is F or P; X8 is Q or E; X9 is P or K; 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 does not exist; X 18 is L or does not exist; X 19 is D or does not exist; X 20 is S or does not exist; X 21 is D or does not exist; X 22 is G or does not exist; 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 is T or S;X 40 is D or S;X 41is 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 54 is W or P;X 55 is G or A;X 56 is I or L.

[0050] In one example, the VH comprises or consists of a CDR1 sequence selected from GYSFTGYFMH (SEQ ID NO: 14) or GYTFTDYGMN (SEQ ID NO: 15).

[0051] In one example, the VH comprises or consists of a CDR2 sequence selected from RINPYNGDIRYNQNFKD (SEQ ID NO: 16) or WINTYTGKPTYDDDFKG (SEQ ID NO: 17).

[0052] In one example, the VH comprises or consists of a CDR3 sequence selected from LNFAY (SEQ ID NO: 18) or RFLNTVAGRSVYFDY (SEQ ID NO: 19).

[0053] In one example, the VL comprises or consists of a CDR1 sequence selected from KSSQSLLDSDGKTFLN (SEQ ID NO: 20) or SVSSSVGSMY (SEQ ID NO: 21).

[0054] In one example, the VL comprises or consists of a CDR2 sequence selected from LVSKLDS (SEQ ID NO: 22) or LTSNLAS (SEQ ID NO: 23).

[0055] In one example, the VL comprises or consists of a CDR3 sequence selected from WQGTHFPWT (SEQ ID NO: 24) or QQWSSNPPT (SEQ ID NO: 25).

[0056] The present disclosure provides: (i) SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18; or (ii) Also provided is a HER2 binding protein comprising a heavy chain variable region sequence (VH) having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19.

[0057] The present disclosure provides: (i) SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24; or (ii) Also provided is a HER2 binding protein comprising a light chain variable region sequence (VL) having CDR1, CDR2, and CDR3 sequences comprising or consisting of SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25.

[0058] In one example, the HER2 binding protein comprises CDRs having sequences 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.

[0059] In one example, the HER2 binding protein comprises CDRs having sequences 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.

[0060] In one example, the HER2 binding protein comprises a VH comprising a sequence that is at least 55% identical to the sequence set forth in SEQ ID NO:2, and / or a VL comprising a sequence that is at least 50% identical to the sequence set forth in SEQ ID NO:3, or a humanized, chimeric, or deimmunized form thereof.

[0061] In one example, the HER2 binding protein comprises a VH comprising a sequence that is at least 55% identical to the sequence set forth in SEQ ID NO: 4, and / or a VL comprising a sequence that is at least 50% identical to the sequence set forth in SEQ ID NO: 5, or a humanized, chimeric, or deimmunized version thereof.

[0062] In one example, the VH comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:2 or SEQ ID NO:4.

[0063] In one example, the VL comprises a sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:3 or SEQ ID NO:5.

[0064] This disclosure: (i) a VH set forth in SEQ ID NO: 2 and a VL set forth in SEQ ID NO: 3; or (ii) A HER2 binding protein comprising or consisting of a VH as set forth in SEQ ID NO:4 and a VL as set forth in SEQ ID NO:5 is also provided.

[0065] In one example, the HER2 binding protein is (i) Single chain Fv fragment (scFv); (ii) 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 (CH)2 and / or CH3; or (iv) An antigen-binding fragment selected from at least one of (i) and / or (ii) linked to a protein that extends the half-life of the antibody (e.g., human serum albumin (HSA)).

[0066] In another example of the present disclosure, the VL and VH are on separate polypeptide chains. For example, a HER2 binding protein may comprise: (i) diabody; (ii) triabodies; (iii) tetrabodies; (iv)Fab; (v) F(ab')2; (vi) Fv; or (vii) at least one of (i)-(vi) linked to a heavy chain constant region or Fc or heavy chain constant domain (CH)2 and / or CH3; or (viii) At least one of (i) to (vi) linked to a protein that extends the half-life of the antibody (e.g., human serum albumin (HSA)).

[0067] The present disclosure also provides chimeric antibodies comprising a VH and VL described herein, wherein the VH is linked to a heavy chain constant region and the VL is linked to a light chain constant region.

[0068] The present disclosure also provides chimeric antibodies comprising a VH and VL described herein, wherein the VH is linked to a human heavy chain constant region and the VL is linked to a human light chain constant region.

[0069] Based on the present disclosure herein, it will be apparent to one of skill in the art that the HER2 binding proteins of the present disclosure encompass human, humanized, synhumanized, chimeric, and primatized proteins.

[0070] The antibodies of the present disclosure may belong to any class, including IgM, IgG, IgE, IgA, IgD, or subclass. Exemplary subclasses of IgG are IgG1, IgG2, IgG3, and IgG4.

[0071] In one example, the HER2 binding protein is recombinant. In one example, the HER2 binding protein is synthetic.

[0072] The present disclosure also provides an anti-idiotype antibody or antigen-binding fragment thereof capable of binding to mAb104 or mAb106.

[0073] In one example, the HER2-binding protein or HER2-binding antibody of the present disclosure is conjugated to a moiety. The moiety may be a detectable moiety or a functional moiety. For example, the moiety may be selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a drug, a compound that increases the half-life of the HER2-binding protein in a subject, and mixtures thereof. In a specific example, the moiety may be selected from an immunoglobulin, a fragment or portion of an immunoglobulin, a therapeutic compound (e.g., a chemotherapy), a drug or bioactive agent, a toxin, or a radionuclide. Alternatively, the moiety may comprise an siRNA, a deoxyribozyme, or a ribozyme. Combinations of any of the foregoing moieties are also encompassed by the present disclosure. In one example, the HER2-binding protein is an antibody-drug conjugate. In another example, the antibody-drug conjugate comprises a HER2-binding protein of the present disclosure linked to monoethylauristatin E (MMAE), monoethylauristatin F (MMAF), a pyrrolobenzodiazepine (PBD), or emtansine (DM1). In certain examples, the conjugation is achieved via conjugation chemistry. In one example, the drug is conjugated to the HER2 binding protein via a cysteine ​​or lysine residue present in the HER2 binding protein. In another example, the conjugation is via a linker (e.g., a GS linker) as known in the art. In another example, the antibody-drug conjugate can be internalized upon binding to the HER2 receptor on tumor cells. The present disclosure also encompasses compositions comprising the conjugates described herein.

[0074] The serum half-life of a binding protein or antibody may be increased by incorporating a salvage receptor binding epitope into the antibody, such as those 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, half-life is increased by PEGylation.

[0075] The present disclosure also provides isolated nucleic acids encoding the HER2 binding proteins or HER2 binding antibodies of the present disclosure.

[0076] The present disclosure additionally provides an expression construct comprising a nucleic acid of the present disclosure operably linked to a promoter. Such an expression construct can be in a vector, such as a plasmid.

[0077] In examples of the present disclosure directed to a single polypeptide HER2 binding protein, the expression construct may include a promoter linked to a nucleic acid encoding that polypeptide chain.

[0078] In examples directed to multiple polypeptides that form a HER2 binding protein, the expression construct of the present disclosure includes a nucleic acid encoding one of the polypeptides (e.g., comprising a VH) operably linked to a promoter and a nucleic acid encoding another of the polypeptides (e.g., comprising a VL) operably linked to a separate promoter.

[0079] In another example, the expression construct may be, for example: (i) promoters; (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site; and (iv) A bicistronic expression construct comprising a nucleic acid encoding a second polypeptide operably linked in 5' to 3' order.

[0080] For example, the first polypeptide comprises a VH and the second polypeptide comprises a VL, or the first polypeptide comprises a VL and the second polypeptide comprises a VH.

[0081] The present disclosure also contemplates separate expression constructs, one encoding a first polypeptide (e.g., comprising a VH and, optionally, a heavy chain constant region or portion thereof) and the other encoding a second polypeptide (e.g., comprising a VL and, optionally, a light chain constant region). For example, the present disclosure contemplates (i) a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VH operably linked to a promoter); and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VL operably linked to a promoter); and wherein the first and second polypeptides associate to form a HER2 binding protein of the disclosure.

[0082] The present disclosure additionally provides isolated cells expressing the HER2-binding proteins or HER2-binding antibodies of the present disclosure, or recombinant cells genetically engineered to express the HER2-binding proteins or HER2-binding antibodies of the present disclosure. In one example, the cells are isolated hybridomas. In another example, the cells comprise a nucleic acid or expression construct of the present disclosure, or: (i) a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VH) operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VL) operably linked to a promoter; The first and second polypeptides then associate to form the HER2 binding protein or HER2 binding antibody of the present disclosure.

[0083] The present disclosure additionally provides a composition comprising a HER2 binding protein or nucleic acid or expression construct or cell of the present disclosure and a suitable carrier. In one example, the composition comprises a HER2 binding protein of the present disclosure.

[0084] In one example, the carrier is pharmaceutically acceptable.

[0085] The compositions of the present disclosure may be administered alone or in combination with other treatments, therapeutic agents, or drugs, either simultaneously / concurrently or sequentially. In one example, a HER2-binding protein or composition of the present disclosure is administered in combination with pertuzumab or trastuzumab. In one example, a HER2-binding protein or composition of the present disclosure is administered in combination with a tyrosine kinase inhibitor (e.g., lapatinib). It is also contemplated that a HER2-binding protein or composition as described herein may be administered simultaneously or sequentially with an anti-cancer therapy, such as chemotherapy or radiation therapy. In a further example, a HER2-binding protein or composition as described herein may be administered simultaneously or sequentially with an immunotherapeutic or immunomodulatory agent.

[0086] The HER2 binding proteins of the present disclosure may be used for treatment, diagnosis, or detection. In some examples, the HER2 binding proteins are linked to chemotherapeutic agents for use as theranostics.

[0087] The present disclosure also provides a diagnostic agent comprising a HER2 binding protein as described herein linked to a detectable label. In one example, the diagnostic agent is used to detect HER2-expressing tumor cells in vivo or in vitro.

[0088] In one example, the diagnostic agent can be used to detect the presence of HER2-expressing tumor cells 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, prosthetic groups, fluorescent materials, luminescent materials, electron-dense labels, labels for MRI, and radioactive materials.

[0089] The present disclosure also provides a HER2 binding protein or diagnostic agent as described herein for use in histological examination of a biological sample. Methods for preparing a histological sample will be familiar to those skilled in the art.

[0090] The present disclosure additionally provides a method for treating or preventing a HER2-expressing cancer in a subject, the method comprising administering to the subject a protein, nucleic acid, expression construct, cell, or composition that binds to HER2 of the present disclosure. In one example, the subject has cancer, such as breast cancer.

[0091] In one example, the method includes administering to a subject an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 2 and / or a VL comprising the sequence set forth in SEQ ID NO: 3, or a humanized or deimmunized version thereof.

[0092] In one example, the method includes administering to a subject an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and / or a VL comprising the sequence set forth in SEQ ID NO: 5, or a humanized or deimmunized version thereof.

[0093] The present disclosure additionally provides a protein or nucleic acid or expression construct or cell or composition that binds to HER2 of the present disclosure for use in medicine.

[0094] The present disclosure additionally provides a protein or nucleic acid or expression construct or cell or composition that binds to HER2 of the present disclosure for use in treating a proliferative disorder of cells expressing HER2.

[0095] In one example, the disclosure provides a method of treating a HER2-expressing proliferative disorder comprising administering to a subject in need thereof a HER2-binding protein or nucleic acid or expression construct or cell or composition of the disclosure. In one example, the cancer is selected from the group consisting of breast cancer, gastric cancer, gastroesophageal cancer, colon cancer, and squamous cell carcinoma.

[0096] In one example, the HER2 binding protein is administered to the subject in a therapeutically effective amount.

[0097] Preferably, the subject is a human.

[0098] The present disclosure additionally provides use of a HER2-binding protein or nucleic acid or expression construct or cell of the present disclosure in the manufacture of a medicament for the treatment of a HER2-expressing cancer.

[0099] The present disclosure additionally provides a method for detecting HER2 in a biological sample, the method comprising contacting the sample with a HER2-binding protein or a HER2-binding antibody of the present disclosure to form an antigen-protein complex, and detecting the complex, wherein detecting the complex indicates HER2 expression in the sample.

[0100] The present disclosure also provides a vaccine antigen comprising or consisting of a sequence according to SEQ ID NO: 1 together with a pharmaceutically acceptable carrier for generating antibodies to human HER2.

[0101] The present disclosure also provides a method for producing a HER2 / ErbB2 binding protein, comprising immunizing a rodent with a cyclic peptide comprising the sequence H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 26), deriving a hybridoma cell line from B cells of the immunized rodent, and purifying antibodies from the hybridoma cell line. In one example, the peptide is cyclized via a disulfide bond. In one example, the peptide is cyclized via a disulfide bond between Cys2 and Cys18 in the side chain. In another example, the peptide is linked to keyhole limpet hemocyanin (KLH) protein. [Brief explanation of the drawings]

[0102] [Figure 1] Figure 1 shows the full-length protein sequence of human receptor tyrosine protein kinase HER2, including the 22-amino acid leader sequence. The peptide epitope bound by mAb104 within cysteine-rich domain II is underlined and in bold. [Figure 2]Figure 1 shows a comparison of binding of 10 μg / ml (A) mAb104, (B) mAb105, (C) mAb106, and (D) mAb107 to the HER2 extracellular domain, cyclic and linear peptide immunogens conjugated to keyhole limpet hemocyanin (KLH) on which the antibodies were generated, or an irrelevant control peptide conjugated to KLH using an ELISA-based assay. Binding activity of triplicate samples was measured by optical density absorbance readings at 405 nm using a VERsamax microplate reader (Molecular Devices) with Softmax Pro 4.8 software, and the mean ± SD was determined. mAb104 (A) and mAb106 (C) showed the strongest binding activity across all immobilized HER2 formats, while mAb105 (B) showed the weakest binding. Specificity was confirmed by the lack of binding to the control peptide. Results are representative of two independent experiments. [Figure 3] Figure 1 shows antibody binding 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 (commercially available positive control antibody 2242, Cell Signaling Technology, Beverly, MA), mAb104, mAb105, mAb106, and mAb107. Results are representative of two independent experiments. [Figure 4] Figure 1 shows the CDR designations (shown in bold and underlined) of the heavy and light chains of mAb104 (A and B) and mAb106 (C and D), respectively. CDR designations according to Kabat and Chothia numbering. [Figure 5] Comparison of binding of HER2 antibodies (A) trastuzumab, (B) mAb106, and (C) mAb104 to HER2 ECD was investigated by surface plasmon resonance using a BIAcore biosensor over antibody concentrations ranging from 320 μg / ml to 10 μg / ml (2133 to 66 nM). Traces represent binding and dissociation of antibodies in solution to immobilized recombinant HER2 ECD. Results are representative of two or more experiments. [Figure 6]Figure 1 shows an ELISA-based HER2-ECD binding competition assay. (A) Trastuzumab and pertuzumab do not affect the binding of mAb104 to ELISA plate-bound recombinant HER2 ECD; (B) mAb104 does not affect the binding of trastuzumab; and (C) mAb104 partially affects the binding of pertuzumab. (Data; mean ± SE; n=3) Results are representative of two experiments. [Figure 7-1] Figure 1 shows a FACS-based competition assay. Preincubation with a 10-fold excess of mAb104 (100 μg / ml) did not affect the binding of 10 μg / ml trastuzumab or pertuzumab to cancer cell surface HER2 on 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 are representative of two or more experiments. [Figure 7-2] Figure 1 shows a FACS-based competition assay. Preincubation with a 10-fold excess of mAb104 (100 μg / ml) did not affect the binding of 10 μg / ml trastuzumab or pertuzumab to cancer cell surface HER2 on 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 are representative of two or more experiments. [Figure 7-3] Figure 1 shows a FACS-based competition assay. Preincubation with a 10-fold excess of mAb104 (100 μg / ml) did not affect the binding of 10 μg / ml trastuzumab or pertuzumab to cancer cell surface HER2 on 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 are representative of two or more experiments. [Figure 7-4]Figure 1 shows a FACS-based competition assay. Preincubation with a 10-fold excess of mAb104 (100 μg / ml) did not affect the binding of 10 μg / ml trastuzumab or pertuzumab to cancer cell surface HER2 on 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 are representative of two or more experiments. [Figure 8-1] Lysates from cancer cell lines were separated by 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. Data are representative of three experiments. [Figure 8-2] ELISA analysis. Specificity of mAb104 (3-10,000 ng / mL) binding to ELISA plates coated with recombinant sEGFR ectodomain or ECD of HER2, HER3, or HER4. Controls with only pNPP substrate and secondary anti-mouse antibody alkaline phosphatase conjugate were included. (Data; mean ± SE; n=3). [Figure 9-1] Figure 1 shows the effect of mAb104 alone (A and B) or in combination with trastuzumab (C and D) or pertuzumab (E and F) on the proliferation of SK-BR-3 cells (A, C, and E) or BT-474 cells (B, D, and F) in vitro as measured by MTS assay. Cells were incubated for 5-7 days in serum-depleted medium with mAb104, trastuzumab, pertuzumab, or an isotype control as monotherapy, or with the combinations trastuzumab + pertuzumab, trastuzumab + mAb104, or pertuzumab + mAb104. Numbers of viable cells were measured at baseline and the end of the experiment. Results are expressed as mean ± SD; n = 3. Data are representative of two or more independent experiments. *mAb104, pertuzumab, and the isotype control antibody had no antiproliferative effect and overlapped with each other. [Figure 9-2]Figure 1 shows the effect of mAb104 alone (A and B) or in combination with trastuzumab (C and D) or pertuzumab (E and F) on the proliferation of SK-BR-3 cells (A, C, and E) or BT-474 cells (B, D, and F) in vitro as measured by MTS assay. Cells were incubated for 5-7 days in serum-depleted medium with mAb104, trastuzumab, pertuzumab, or an isotype control as monotherapy, or with the combinations trastuzumab + pertuzumab, trastuzumab + mAb104, or pertuzumab + mAb104. Numbers of viable cells were measured at baseline and the end of the experiment. Results are expressed as mean ± SD; n = 3. Data are representative of two or more independent experiments. *mAb104, pertuzumab, and the isotype control antibody had no antiproliferative effect and overlapped with each other. [Figure 9-3] Figure 1 shows the effect of mAb104 alone (A and B) or in combination with trastuzumab (C and D) or pertuzumab (E and F) on the proliferation of SK-BR-3 cells (A, C, and E) or BT-474 cells (B, D, and F) in vitro as measured by MTS assay. Cells were incubated for 5-7 days in serum-depleted medium with mAb104, trastuzumab, pertuzumab, or an isotype control as monotherapy, or with the combinations trastuzumab + pertuzumab, trastuzumab + mAb104, or pertuzumab + mAb104. Viable cell counts were measured at baseline and the end of the experiment. Results are expressed as mean ± SD; n = 3. Data are representative of two or more independent experiments. *mAb104, pertuzumab, and the isotype control antibody had no antiproliferative effect and overlapped with each other. [Figure 10-1]mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and downstream signaling of Akt in vitro in (A) SK-BR-3 cells and (B) BT474 cells. Cells were incubated in serum-depleted medium and treated with 100 μg / ml of mAb104, trastuzumab, or pertuzumab, alone or in combination, for 24 hours, after which whole cells were lysed. Equal volumes of lysates were then loaded and resolved on a 4-12% gel before being transferred to a nitrocellulose membrane. Membranes were immunoblotted as indicated. Data are representative of two experiments. [Figure 10-2] mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and downstream signaling of Akt in vitro in (A) SK-BR-3 cells and (B) BT474 cells. Cells were incubated in serum-depleted medium and treated with 100 μg / ml of mAb104, trastuzumab, or pertuzumab, alone or in combination, for 24 hours, after which whole cells were lysed. Equal volumes of lysates were then loaded and resolved on a 4-12% gel before being transferred to a nitrocellulose membrane. Membranes were immunoblotted as indicated. Data are representative of two experiments. [Figure 11-1] mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and ligand-dependent phosphorylation of Akt in vitro in (A) SK-BR-3 cells and (B) BT474 cells. Cells were incubated in serum-depleted medium and treated with 100 μg / ml mAb104, trastuzumab, or pertuzumab, alone or in combination, for 24 hours, after which 100 ng of EGF was added for 10 minutes. After total cell lysis, equal volumes of lysates were loaded and resolved on a 4-12% gel before transfer to nitrocellulose membranes. Membranes were immunoblotted as indicated. Data are representative of two experiments. [Figure 11-2]mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and ligand-dependent phosphorylation of Akt in vitro in (A) SK-BR-3 cells and (B) BT474 cells. Cells were incubated in serum-depleted medium and treated with 100 μg / ml mAb104, trastuzumab, or pertuzumab, alone or in combination, for 24 hours, after which 100 ng of EGF was added for 10 minutes. After total cell lysis, equal volumes of lysates were loaded and resolved on a 4-12% gel before transfer to nitrocellulose membranes. Membranes were immunoblotted as indicated. Data are representative of two experiments. [Figure 12-1] Figure 1 shows the effect of treatment on breast cancer cell viability and apoptosis assessed by flow cytometry analysis of Annexin V and propidium iodide (PI) staining after 4 hours of treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy and in combination (total antibody 0.1 mg / mL in all groups) in (A-H) BT474 cells and (I-P) SK-BR-3 cells. [Figure 12-2] Figure 1 shows the effect of treatment on breast cancer cell viability and apoptosis assessed by flow cytometry analysis of Annexin V and propidium iodide (PI) staining after 4 hours of treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy and in combination (total antibody 0.1 mg / mL in all groups) in (A-H) BT474 cells and (I-P) SK-BR-3 cells. [Figure 12-3] Figure 1 shows the effect of treatment on breast cancer cell viability and apoptosis assessed by flow cytometry analysis of Annexin V and propidium iodide (PI) staining after 4 hours of treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy and in combination (total antibody 0.1 mg / mL in all groups) in (A-H) BT474 cells and (I-P) SK-BR-3 cells. [Figure 12-4]Figure 1 shows the effect of treatment on breast cancer cell viability and apoptosis assessed by flow cytometry analysis of Annexin V and propidium iodide (PI) staining after 4 hours of treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy and in combination (total antibody 0.1 mg / mL in all groups) in (A-H) BT474 cells and (I-P) SK-BR-3 cells. [Figure 13] Figure 1 shows the antitumor effect of mAb104 in BT-474 breast cancer xenografts. Mice (n=5) were treated with 1 mg of mAb104, trastuzumab, pertuzumab, or isotype control. Tumor volumes were 100-120 mm3 at the start of treatment. Data shown in growth curves represent mean tumor volume ± SE. Tumors in individual treatment groups were significantly smaller than the control group. *p<0.001 control vs. mAb104; **p<0.0001 control vs. trastuzumab and pertuzumab. [Figure 14] Figure 1 shows the antitumor effect of mAb104 in BT-474 xenografts. Mice (n=5 / group) were treated with 0.5 mg of mAb104, trastuzumab, pertuzumab, or isotype control. Tumor volumes were 120-150 mm3 at the start of treatment. Data shown in the growth curves represent mean tumor volume ± SE. *p<0.01, control vs. mAb104-treated group. [Figure 15-1] (A) Antitumor effect of mAb104 in a HER2-positive breast PDX model. Mice (n = 5 / group) were treated with 0.5 mg of mAb104, trastuzumab, pertuzumab, or an isotype control. Tumor volumes were 100–120 mm3 at the start of treatment. [Figure 15-2] (B) Antitumor effect of mAb104 in combination with 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. Tumor volumes at the start of treatment were 100-120 mm3. *p<0.0001 control group vs. trastuzumab / mAb104. [Figure 15-3](C) Antitumor effect of mAb104 in combination with trastuzumab in a HER2-positive breast PDX model. Mice (n=5) were treated with a total dose of 0.5 mg of mAb104 + trastuzumab, trastuzumab + pertuzumab, or isotype control. Tumor volumes at the start of treatment were 120-150 mm3. *p<0.0001 control vs. mAb104; **p<0.001 trastuzumab vs. trastuzumab / mAb104. Data shown for growth curves in panels A-C represent mean tumor volume ± SE. [Figure 16-1] BT-474 xenograft tumors were evaluated by immunohistochemistry for: (A) the effect of anti-HER2 monotherapy (0.5 mg dose) on proliferation by Ki67, (B) downstream signaling by phospho-Akt staining, and (C) effects on vasculature by podocalyxin staining; or the effect of mAb104 (0.5 mg total protein dose) in combination with trastuzumab on (D) proliferation by Ki67, (E) downstream signaling by phospho-Akt staining, and (F) effects on vasculature by podocalyxin staining. *p<0.001 control vs. trastuzumab. [Figure 16-2] BT-474 xenograft tumors were evaluated by immunohistochemistry for: (A) the effect of anti-HER2 monotherapy (0.5 mg dose) on proliferation by Ki67, (B) downstream signaling by phospho-Akt staining, and (C) effects on vasculature by podocalyxin staining; or the effect of mAb104 (0.5 mg total protein dose) in combination with trastuzumab on (D) proliferation by Ki67, (E) downstream signaling by phospho-Akt staining, and (F) effects on vasculature by podocalyxin staining. *p<0.001 control vs. trastuzumab. [Figure 17]mAb104 does not inhibit the growth of (A) NCI-N87 gastric cancer cells and (B) OE19 gastric cancer cells in vitro, as measured by the MTS assay. Cells were incubated with mAb104, trastuzumab, pertuzumab, or an isotype control as monotherapy (A and B) in serum-depleted medium for 5–7 days. Viable cell counts were measured at baseline and the end of the experiment. Results are expressed as mean ± SD; n = 3. Data are representative of two or more independent experiments. *p<0.0001, control vs. trastuzumab. [Figure 18] mAb104 does not inhibit the growth of (A) NCI-N87 gastric cancer cells and (B) OE19 gastric cancer cells in vitro, as measured by the MTS assay. Cells were incubated with mAb104 in combination with trastuzumab + pertuzumab, trastuzumab + mAb104, or pertuzumab + mAb104 in serum-depleted medium for 5–7 days. Numbers of viable cells were measured at baseline and the end of the experiment. Results are expressed as mean ± SD; n = 3. Data are representative of two or more independent experiments. *p ≤ 0.005 compared to control. [Figure 19-1] mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and downstream signaling of Akt in (A) NCI-N87 cells and (B) OE-19 cells in vitro. Cells were incubated in serum-depleted medium and treated with 100 μg / ml of mAb104, trastuzumab, or pertuzumab alone or in combination with trastuzumab or pertuzumab for 24 hours, after which whole cells were lysed. Equal volumes of lysates were then loaded and resolved on a 4-12% gel before being transferred to a nitrocellulose membrane. Membranes were immunoblotted as indicated. Results are representative of two experiments. [Figure 19-2]mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and downstream signaling of Akt in (A) NCI-N87 cells and (B) OE-19 cells in vitro. Cells were incubated in serum-depleted medium and treated with 100 μg / ml of mAb104, trastuzumab, or pertuzumab alone or in combination with trastuzumab or pertuzumab for 24 hours, after which whole cells were lysed. Equal volumes of lysates were then loaded and resolved on a 4-12% gel before being transferred to a nitrocellulose membrane. Membranes were immunoblotted as indicated. Results are representative of two experiments. [Figure 20-1] mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and ligand-dependent phosphorylation of Akt in vitro in (A) NC-N87 cells and (B) OE-19 cells. Cells were incubated in serum-depleted medium and treated with 100 μg / ml mAb104, trastuzumab, or pertuzumab, alone or in combination, for 24 hours, after which 100 ng of EGF was added for 10 minutes. After total cell lysis, equal volumes of lysates were loaded and resolved on a 4-12% gel before transfer to nitrocellulose membranes. Membranes were immunoblotted as indicated. Results are representative of two experiments. [Figure 20-2] mAb104, when used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), does not affect the MAPK pathway and ligand-dependent phosphorylation of Akt in vitro in (A) NC-N87 cells and (B) OE-19 cells. Cells were incubated in serum-depleted medium and treated with 100 μg / ml mAb104, trastuzumab, or pertuzumab, alone or in combination, for 24 hours, after which 100 ng of EGF was added for 10 minutes. After total cell lysis, equal volumes of lysates were loaded and resolved on a 4-12% gel before transfer to nitrocellulose membranes. Membranes were immunoblotted as indicated. Results are representative of two experiments. [Figure 21-1]Figure 1 shows the effect of treatment on cancer cell viability and apoptosis assessed by propidium iodide (PI) staining after 4 hours of treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy or in combination in (A and B) NCI-N87 gastric cancer cells and (C) OE-19 esophageal cancer cells. [Figure 21-2] Figure 1 shows the effect of treatment on cancer cell viability and apoptosis assessed by propidium iodide (PI) staining after 4 hours of treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy or in combination in (A and B) NCI-N87 gastric cancer cells and (C) OE-19 esophageal cancer cells. [Figure 22] Confluent OE-19 cells utilized in the migration assay are shown. Images collected at 0 and 90 hours of antibody incubation at 0 or 100 μg / ml. The antibody did not retard OE-19 cell migration compared to the control antibody at a dose of 100 μg / mL at 90 hours post-treatment. [Figure 23] Figure 1 shows the antitumor effect of mAb104 in NCI-N87 xenografts. Mice (n=5) were treated with 1 mg of mAb104, trastuzumab, pertuzumab, isotype control, or no treatment. Tumor volumes were 100-120 mm3 at the start of treatment. Data shown in the growth curves represent mean tumor volume ± SE. *p≦0.01, control vs. mAb104. [Figure 24] Figure 1 shows the antitumor effect of mAb104 in NCI-N87 xenografts. Mice (n=5) were treated with 0.5 mg of mAb104, trastuzumab, pertuzumab, isotype control, or no treatment. Tumor volumes were 100-120 mm3 at the start of treatment. Growth curves (A) and survival curves (B) are shown. Data shown for growth curves represent mean tumor volume ± SE. The endpoints for survival analysis were tumor volume >1000 mm3 or moribund status. *p<0.001, control vs. mAb104; **p<0.0001, control vs. treated group. [Figure 25]Figure 1 shows the antitumor effect of mAb104 in NCI-N87 xenografts. Mice (n=5) were treated with 0.1 mg of mAb104, trastuzumab, pertuzumab, isotype control, or no treatment. Tumor volumes were 100-120 mm3 at the start of treatment. Growth curves (A) and survival curves (B) are shown. Data shown for growth curves represent mean tumor volume ± SE. The endpoints for survival analysis were tumor volume >1000 mm3 or moribund status. *p≦0.001, control vs. mAb104; **p<0.0002, control vs. treated group. [Figure 26] Figure 1 shows the antitumor effect of mAb104 in OE-19 xenografts. Mice (n=5) were treated with 1 mg of mAb104, trastuzumab, pertuzumab, isotype control, or no treatment. Tumor volumes were 100-120 mm3 at the start of treatment. Data shown in the growth curves represent mean tumor volumes ± SE. *p≦0.0001, control vs. mAb104. [Figure 27] Figure 1 shows the antitumor effect of mAb104 in OE-19 xenografts. Mice (n=5) were treated with 0.5 mg of mAb104, trastuzumab, pertuzumab, or an isotype control. Tumor volumes were 100-120 mm3 at the start of treatment. Growth curves (A) and survival curves (B) are shown. Data shown for growth curves represent mean tumor volume ± SE. The endpoints for survival analysis were tumor volume >1000 mm3 or moribund status. *p<0.001, control vs. trastuzumab; **p<0.006, control vs. treatment group. [Figure 28] Figure 1 shows the antitumor effect of mAb104 in combination with 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 volumes were 100-120 mm3 at the start of treatment. Data shown in growth curves represent mean tumor volume ± SE. *p<0.0001, control group vs. mAb104; **p<0.001, trastuzumab vs. trastuzumab / mAb104. [Figure 29]Figure 1 shows the antitumor effect of mAb104 in combination with 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 volumes were 100-120 mm3 at the start of treatment. Growth curves (A) and survival curves (B) are shown. Data shown for growth curves represent mean tumor volume ± SE. The endpoint for survival analysis was tumor volume >1000 mm3 or moribundity. *p<0.0001, control group vs. trastuzumab / mAb104; **p<0.0001, control vs. trastuzumab / pertuzumab; ***p<0.0001, trastuzumab vs. trastuzumab / mAb104; ±p<0.0005, control vs. treatment group. [Figure 30-1] NCI-N87 xenograft tumors were evaluated by immunohistochemistry for the effect of anti-HER2 monotherapy (0.5 mg dose) (A-C) or in combination with trastuzumab (D-F) on (A and D) proliferation by Ki67; (B and E) downstream signaling by staining for phosphor-Akt; and (C and F) vasculature by staining for podocalyxin. [Figure 30-2] NCI-N87 xenograft tumors were evaluated by immunohistochemistry for the effect of anti-HER2 monotherapy (0.5 mg dose) (A-C) or in combination with trastuzumab (D-F) on (A and D) proliferation by Ki67; (B and E) downstream signaling by staining for phosphor-Akt; and (C and F) vasculature by staining for podocalyxin. [Figure 31-1] OE-19 xenograft tumors were evaluated by immunohistochemistry for the effect of anti-HER2 monotherapy (0.5 mg dose) (A-C) or in combination with trastuzumab (D-F) on (A and D) proliferation by Ki67; (B and E) downstream signaling by staining for phosphor-Akt; and (C and F) vasculature by staining for podocalyxin. [Figure 31-2]OE-19 xenograft tumors were evaluated by immunohistochemistry for the effect of anti-HER2 monotherapy (0.5 mg dose) (A-C) or in combination with trastuzumab (D-F) on (A and D) proliferation by Ki67; (B and E) downstream signaling by staining for phosphor-Akt; and (C and F) vasculature by staining for podocalyxin. [Figure 32] Figure 1 shows a binding assay for the determination of the immunoreactive fraction of 89Zr-labeled anti-HER antibodies. A) shows a conventional plot of specific binding versus total radioactivity applied as a function of increasing cell concentration. B) and C) are double inverse plots of the same data as A, allowing the immunoreactive fraction to be determined for conditions representing infinite antigen excess. [Figure 33] Scatchard plots of A) 89Zr-labeled mAb104 binding and B) 89Zr-labeled Herceptin / Trastuzumab binding to NCI-N87 gastric cancer cells. The horizontal axis shows the concentration of specifically bound antibody, and the vertical axis shows the ratio of specifically bound to reactive free antibody. The binding capacity per cell was determined from the intercept on the horizontal axis, and the binding constant was determined from the slope of the line. [Figure 34] A) Biodistribution of zirconium-89 labeled mAb104 in mice bearing HER2-overexpressing NCI-N87 gastric cancer xenografts. B) Biodistribution of zirconium-89 labeled mAb104 and isotype control in the blood and tumors of mice bearing NCI-N87 xenografts. mAb104 showed high specific tumor uptake. (Data: Mean ± SEM, n=5).

[0103] Explanation of the sequence listing SEQ ID NO: 1: HER2 / ErbB2 epitope sequence SEQ ID NO: 2: VH of mAb104 SEQ ID NO: 3: VL of mAb104 SEQ ID NO: 4: VH of mAb106 SEQ ID NO: 5: VL of mAb106 SEQ ID NO: 6: Consensus sequence of VH CDR1 SEQ ID NO: 7: Consensus sequence of VH CDR2 SEQ ID NO: 8: Consensus sequence of VH CDR3 SEQ ID NO: 9: Consensus sequence of VL CDR1 SEQ ID NO: 10: Consensus sequence of VL CDR2 SEQ ID NO: 11: Consensus sequence of VL CDR3 SEQ ID NO: 12: Consensus sequence of VH SEQ ID NO: 13: Consensus sequence of VL 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 ID NO: 19: VH CDR3 of mAb106 SEQ ID NO: 20: VL CDR1 of mAb104 SEQ ID NO: 21: VL CDR1 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: Sequence of the cyclized peptide used for immunization SEQ ID NO: 27: HER2 / ErbB2 sequence SEQ ID NO: 28: Light chain primer sequence SEQ ID NO: 29: Light chain primer sequence SEQ ID NO: 30: Light chain primer sequence SEQ ID NO: 31: Light chain primer sequence SEQ ID NO: 32: Light chain primer sequence SEQ ID NO: 33: Light chain primer sequence SEQ ID NO: 34: Light chain primer sequence SEQ ID NO: 35: Light chain primer sequence SEQ ID NO: 36: Light chain primer sequence SEQ ID NO: 37: Light chain primer sequence SEQ ID NO: 38: Light chain primer sequence SEQ ID NO: 39: Light chain primer sequence SEQ ID NO: 40: Light chain primer sequence SEQ ID NO: 41: Heavy chain primer sequence SEQ ID NO: 42: Heavy chain primer sequence SEQ ID NO: 43: Heavy chain primer sequence SEQ ID NO: 44: Heavy chain primer sequence SEQ ID NO: 45: Heavy chain primer sequence SEQ ID NO: 46: Heavy chain primer sequence SEQ ID NO: 47: Heavy chain primer sequence SEQ ID NO: 48: Heavy chain primer sequence SEQ ID NO: 49: Heavy chain primer sequence SEQ ID NO: 50: Heavy chain primer sequence SEQ ID NO: 51: Heavy chain primer sequence SEQ ID NO: 52: Heavy chain primer sequence SEQ ID NO: 53: Heavy chain primer sequence SEQ ID NO: 54: Light chain primer sequence SEQ ID NO: 55: Light chain primer sequence DETAILED DESCRIPTION OF THE INVENTION

[0104] overview Throughout this specification, unless expressly stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0105] Those skilled in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described. The disclosure should be understood to include all such variations and modifications. The disclosure also includes all steps, features, compositions, and compounds referenced or indicated herein, individually or collectively, and all combinations or any two or more of such steps or features.

[0106] The present disclosure is not intended to be limited in scope by the specific examples described herein. Such specific examples are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0107] Any example of this disclosure shall be construed to apply mutatis mutandis to any other example of this disclosure, unless expressly stated otherwise.

[0108] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed 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).

[0109] Unless otherwise indicated, the recombinant protein, recombinant DNA techniques, molecular biology, microbiology, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H.Means (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and J.E. Coligan et al. (editors), Current Protocols in They are described and explained throughout the literature in sources such as Immunology, John Wiley & Sons (including all current editions).

[0110] The descriptions and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by a discussion 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, 5, Today, 18, 509.

[0111] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to include a stated element, integer, or step, or group of elements, integers, or steps, but not to exclude any other element, integer, or step, or group of elements, integers, or steps.

[0112] As used herein, the term "derived from" shall be construed to indicate that the specified integer may be obtained from a particular source, but is not necessarily obtained directly from that source.

[0113] The terms "consisting of" or "consisting essentially of" in the context of a peptide sequence refer to a peptide sequence of a defined number of residues that is not covalently attached to a larger product.

[0114] Any example herein shall be construed to apply mutatis mutandis to any other example unless expressly stated otherwise.

[0115] Selected Definitions As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more" and "at least one," can be used interchangeably herein.

[0116] Furthermore, as used herein, "and / or" shall be construed as specifically disclosing the two specified features or components with or without the other. Thus, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to encompass 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).

[0117] The term "about" is used herein to mean approximately, roughly, around, or within the region of. When the term "about" is used in conjunction with a numerical range, "about" modifies that range by extending the boundaries above and below the stated value. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent (%) above or below (high or low).

[0118] It will be understood that the HER2 binding proteins and antibodies, nucleic acids, cells, and vectors described herein are in isolated form. "Isolated" refers to 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 a form found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, or cell is substantially pure. In some embodiments, an isolated antibody, polynucleotide, vector, or cell is "recombinant."

[0119] As used herein, the term "HER2" is understood to refer to the human HER2 receptor as shown in Figure 1, particularly domain II of the HER2 receptor as 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 can be used interchangeably with ErbB2.

[0120] The term "aberrant expression" or "aberrantly expressed" is intended to encompass a condition in which there is an abnormal (usually increased) amount / level of a protein, regardless of the efficient cause of that abnormal amount or level. Abnormal expression includes and contemplates situations or changes in which the protein expression or post-translational modification machinery within a cell is overburdened or otherwise disrupted due to increased expression or increased level or amount of protein, including when an altered protein is expressed, such as in a mutated or variant protein due to sequence changes, deletions or insertions, or altered folding. In the present context, aberrant expression is associated with HER2 expression found in tumorigenic, hyperproliferative, or abnormal cells, but not in wild-type or normal cells.

[0121] As used herein, the term "affinity" refers to the strength of binding of a single molecule to its ligand and is usually expressed as the equilibrium dissociation constant (KD) for the reversible binding of the two agents. It is determined by the ratio of Koff / Kon between the HER2-binding protein and HER2. KD and affinity are inversely proportional. The KD value is related to the concentration of the HER2-binding protein, so 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 disclosure for HER2 can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM) or greater.

[0122] As used herein, the term "binding," with respect to the interaction of a HER2 binding protein with a target, means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the target. For example, a HER2 binding protein recognizes and binds to a specific protein structure, rather than to proteins in general.

[0123] The term "binding protein" as used herein is intended to describe members of a pair of molecules that have binding specificity for one another. Members of a specific binding pair may be naturally occurring or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface or cavity that specifically binds to, and is complementary to, a particular spatial and polar organization of the other member of the pair. Thus, the members of the pair have the property of specifically binding to one another. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions.

[0124] The term "antibody" describes a natural or partially or wholly synthetically produced immunoglobulin. The term also covers any polypeptide or protein having a binding domain that is, or is homologous to, an antibody binding domain. CDR-grafted antibodies are also contemplated by the term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that bind a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the most recently mentioned of which are described in more detail in U.S. Pat. Nos. 4,816,397 and 4,816,567. The term "antibody(s)" includes wild-type immunoglobulin (Ig) molecules, generally comprising four full-length polypeptide chains, two heavy (H) chains and two light (L) chains, or their equivalent Ig homologs (e.g., camelid nanobodies containing only heavy chains); full-length functional mutants, variants, or derivatives thereof that retain the essential epitope-binding properties of Ig molecules; and dual-specific, bispecific, multispecific, and dual-variable domain antibodies; immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Because antibodies can be modified in many ways, the term "antibody" should be interpreted to encompass any specific-binding member or substance having a binding domain with the required specificity. Thus, the term encompasses antibody fragments, derivatives, functional equivalents, and homologs of antibodies, including any polypeptide, whether natural or wholly or partially synthetic, that contains an immunoglobulin binding domain. Chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, and U.S. Pat. Nos. 4,816,397 and 4,816,567. Any "antigen-binding fragment" is also included within the meaning of the term "antibody."

[0125] "Antibody fragment" means a molecule comprising at least one polypeptide chain that is not full-length, and includes: (i) a Fab fragment, which is a monovalent fragment consisting of the variable light chain (VL), variable heavy chain (VH), constant light chain (CL), and constant heavy chain 1 (CH1) domains; (ii) an F(ab')2 bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) the heavy chain portion of an Fab (Fd) fragment consisting of the VH and CH1 domains; (iv) a variable fragment (Fv) fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a domain antibody (dAb) fragment comprising a single variable domain (Ward, ES et al., Nature, 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity-determining region (CDR); (viii) a single-chain Fv fragment in which the VH and VL domains are linked by a peptide linker that allows the two domains to combine to form an antigen-binding site (Bird et al., Nature, 341, 544-546 (1989)). al, Science, 242, 423-426, 1988; Huston et al, PNAS, USA, 85, 5879-5883, 1988); (ix) diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (WO 94 / 13804; P. Holliger et al. Proc. Natl. Acad. Sci. USA, 906444-6448, (1993)); and (x) linear antibodies comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with complementary light chain polypeptides; (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 heavy and / or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

[0126] As used herein, the term "antigen-binding fragment" is intended to include Fab, Fab', F(ab'), Fv, Fd, single-chain Fv (scFv), disulfide-linked Fv (sdFv), VL and VH domain fragments, domain antibodies, trispecific (Fab), bispecific (Fab), diabodies ((VL-VH) or (VH-VL)), triabodies (trivalent), tetrabodies (tetravalent), minibodies ((scFv-CH), bispecific single-chain Fv (Bis-scFv), IgG delta CH, scFv-Fc, and (scFv)-Fc. A "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody molecule and can be generated by digestion of a whole antibody molecule with the enzyme papain to generate fragments consisting of an intact light chain and a portion of the heavy chain. A "Fab" fragment of an antibody molecule can be obtained by treating whole antibody molecules with pepsin, followed by reduction, to generate a molecule consisting of an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained for each antibody molecule treated in this manner. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked by two disulfide bonds and can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction. An "Fv fragment" is a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain, expressed as two chains. A "single-chain antibody" (SCA) is a genetically engineered single-chain molecule containing the variable region of a light chain and the variable region of a heavy chain linked by a suitable, flexible polypeptide linker.

[0127] As used herein, "antibody variable region" refers to a portion of the light and heavy chains of an antibody molecule comprising the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and 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 method used in the present invention, the amino acid positions assigned to the CDRs and FRs may be defined 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 also follows the amino acid numbering of Kabat.

[0128] As used herein, the term "constant region" (CR) refers to the portion of an antibody molecule that confers effector function. The constant region of the subject humanized antibody is derived from a human immunoglobulin. The heavy chain constant region can be selected from any of five isotypes: alpha, delta, epsilon, gamma, or mu. Furthermore, various subclasses of heavy chains (e.g., IgG heavy chain subclasses) are involved in various effector functions, and therefore, antibodies with desired effector functions can be produced by selecting the desired heavy chain constant region. Preferred heavy chain constant regions are gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4). The light chain constant region can be kappa or lambda type, preferably kappa type.

[0129] "Framework regions" (hereinafter FR) are those variable domain residues other than the CDR residues. Each variable domain of a naturally occurring antibody typically has four FRs, identified as FR1, FR2, FR3, and FR4.

[0130] As used herein, the term "complementarity determining region" (synonymous CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3. Each CDR consists of amino acid residues derived 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 approximately residues 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). (Health, Bethesda, MD. (1991)), and / or the "hypervariable loops," i.e., about residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and about residues 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 complementarity determining regions may comprise amino acids from both the CDR regions and the hypervariable loops as defined according to Kabat. Those skilled in the art will recognize some variation in the configuration of the FRs, for example, as a result of mutations (e.g., deletions and / or insertions), e.g., up to five residues, or four residues, or two residues, or one residue (e.g., as in the antibodies exemplified herein).

[0131] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays 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 synthetically produced and can be partially or fully human in sequence.

[0132] The term "chimeric antibody" refers to antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species (e.g., mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species (e.g., primate) or belonging to another antibody class or subclass, and similarly refers to fragments of such antibodies, so long as they exhibit the desired biological activity.

[0133] The term "humanized antibody" shall be understood to refer to chimeric molecules, generally prepared using recombinant techniques, which have an epitope-binding site derived from an immunoglobulin from a non-human species, while the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site preferably comprises the complementarity-determining regions (CDRs) from the non-human antibody grafted into appropriate framework regions within the variable domains of a human antibody, with the remaining regions derived from the human antibody.

[0134] The term "human antibody," as used herein in connection with antibody molecules and binding proteins, refers to an antibody having variable antibody regions (e.g., VH, VL, CDR, and FR regions) and constant antibody regions that are derived from or correspond to sequences found in a human, e.g., in a human germline or somatic cell.

[0135] As used herein, the term "specifically binds" shall be interpreted to mean that a binding protein or antibody reacts with or associates with a particular cell or substance more frequently, rapidly, for longer duration, and / or with greater affinity than with alternative cells or substances. It is also understood by 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, "specific binding" does not necessarily require exclusive or undetectable binding of another molecule, which is encompassed by the term "selective binding." Generally, although not necessarily, reference to binding refers to specific binding.

[0136] As used herein, "cell proliferative disorder" and grammatical variations thereof, when used in reference to a cell, tissue, or organ, refers to any unwanted, excessive, or abnormal cell, tissue, or organ growth, proliferation, differentiation, or survival. Unwanted cell proliferative disorders include diseases and physiological conditions that are both benign hyperplastic conditions characterized by unwanted, excessive, or abnormal cell numbers, cell growth, cell proliferation, cell survival, or differentiation in a subject. Specific examples of such disorders include metastatic and non-metastatic neoplasias, tumors, and cancers (malignancies).

[0137] The term "identity" and its grammatical variations mean that two or more referenced entities are the same. Thus, if two antibody sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. If two nucleic acid sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. Identity can be over a defined region (region or domain) of the sequence. The percent identity of polynucleotides is measured by GAP (Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. Unless otherwise specified, the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 100 nucleotides in length, 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.

[0138] The term "isolated," including DNA, RNA, or protein, refers to a polynucleotide / polypeptide that is at least partially separated from the polynucleotide / polypeptide sequence with which it is associated or linked in its native state. Preferably, isolated polynucleotides / polypeptides are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.

[0139] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide."

[0140] As used herein, the term "pharmaceutical composition" refers to any composition containing at least one therapeutically or biologically active agent and suitable for administration to a patient. All such formulations can be prepared by methods well known and accepted in the art. See, e.g., Gennaro, A.R., ed., Remington: The Science and Practice of Pharmacy, 20th Edition, Mack Publishing Co., Easton, Pa. (2000).

[0141] The phrase "pharmaceutically acceptable" is employed herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, and / or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0142] "Subject" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., 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.

[0143] As used herein, reference to a "similar" level of binding will be understood to mean that an antibody binds to an antigen at a level that is within about 30%, 25%, or 20% of the level at which it binds to another antigen. The term can also mean that an antibody binds to an antigen at a level that is within about 30%, 25%, or 20% of the level at which another antibody binds to the same antigen.

[0144] As used herein, reference to "substantially the same level" of binding will be understood to mean that an antibody binds to an antigen at a level that is within about 15%, 10%, or 5% of the level at which it binds to another antigen. The term can also mean that an antibody binds to an antigen at a level that is within about 5%, 4%, or 3% of the level at which another antibody binds to the same antigen.

[0145] The term "competitively inhibit" is to be understood to mean that the protein of the present disclosure reduces or prevents the binding of the recited produced antibody (e.g., mAb104) to domain II of HER2 or a fragment thereof. From the above, it will be apparent that the protein need not completely inhibit antibody binding, but rather only reduce binding by a statistically significant amount, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Methods for measuring 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 either in the presence or absence of the protein. If antibody binding is less in the presence of the protein than in the absence of the protein, the protein is considered to competitively inhibit antibody binding. In one example, the protein and antibody are exposed to HER2 substantially simultaneously. Additional methods for measuring 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 competes to inhibit antibody binding.

[0146] "Overlapping," in the context of two epitopes, shall be interpreted to mean that the two epitopes share a sufficient number of amino acid residues to allow an antibody that binds to one epitope to compete and inhibit the binding of an antibody that binds to the other epitope, e.g., the epitopes share at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acids.

[0147] As used herein, the term "does not substantially bind" shall be understood to mean that a protein, e.g., an antibody, binds to a candidate antigen at a level that is less than 10%, or less than 8%, or less than 6%, or less than 5% above background. Background can be the level of binding signal detected in the absence of protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using a biosensor assay (e.g., Biacore) in which the protein is immobilized and contacted with the antigen.

[0148] The term "therapeutically effective amount" shall be construed 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 tolerated as clinically characteristic of that disorder. One of skill in the art will recognize that such amounts will vary depending on the particular antibody, fragment, and / or the particular subject, and / or the type or severity or level of disease. Thus, this term should not be construed to limit the invention to a specific amount.

[0149] As used herein, the terms "treat," "treating," "treatment," and grammatical variations thereof refer to subjecting an individual patient to a protocol, regimen, process, or therapy, wherein it is desired to obtain a physiological response or result in the patient. Treating does not require that a desired physiological response or result be achieved in every patient or patient population, as not every patient treated may respond to a particular treatment protocol, regimen, process, or therapy. Thus, a given patient or patient population may fail to respond, or may respond poorly, to treatment.

[0150] The terms "tumor" and "cancer" are used interchangeably and refer to a cell or population of cells whose growth, proliferation, or survival exceeds that of normal counterparts, e.g., a cell proliferative or differentiative disorder. Growth is usually uncontrolled.

[0151] The terms "104 antibody" or "mAb104," and any variants not specifically recited, may be used interchangeably herein and, as used throughout this application and claims, refer to proteinaceous material, including single or multiple proteins, and extend to those proteins having the amino acid sequence data set forth herein and the activity profile described herein and in the claims. Thus, proteins exhibiting substantially equivalent or altered activity are also contemplated. These modifications may be deliberate, such as those obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in the host that is the producer of the complex or its designated subunit. The terms "104 antibody" or "mAb104" are also intended to include within their scope the proteins specifically recited herein, as well as all substantially homologous analogs and allelic variations.

[0152] Detailed Description of the Embodiments The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only.

[0153] antibody generation The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created 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. Patent 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; and 4,493,890. Panels of monoclonal antibodies raised 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 using specific binding member activity assays. High-affinity antibodies are also useful when immunoaffinity purification of native or recombinant specific binding members is possible. Monoclonal antibodies useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing hybridomas secreting antibody molecules of appropriate antigen specificity. The culture is maintained under conditions and for a period of time sufficient for the hybridomas to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques.

[0154] The antibodies of the present disclosure can also be generated by immunizing animals with purified antigen corresponding to a cyclic peptide comprising residues 277-312 or residues 293-309 of normal or wild-type mature human HER2.

[0155] The HER2 binding proteins of the present disclosure may also be synthesized by standard techniques such as, for example, solid phase peptide synthesis and / or native protein ligation.

[0156] Suitable techniques that may further be employed 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.

[0157] Recombinant antibody production The antibodies and antigen-binding fragments of the present invention can also be produced recombinantly using techniques and materials readily available to those of skill in the art.

[0158] The variable domains may be derived from any germline or rearranged human variable domain, or may be synthetic variable domains based on consensus sequences of known human variable domains. Sequences derived from the CDRs of the present invention may be introduced into a repertoire of variable domains lacking CDR regions using recombinant DNA technology. For example, Marks et al. (Bio / Technology, 1992, 10:779-783) describe a method for generating a repertoire of antibody variable domains in which a consensus primer directed to or adjacent to the 5' end of the variable domain region is used in conjunction with a consensus primer to the third framework region of a human VH gene to provide a repertoire of VH variable domains lacking one or more CDRs. Marks et al. further describe how to combine this repertoire with the CDRs of a particular antibody. Using similar techniques, the CDR-derived sequences of the invention may be combined with a repertoire of VH or VL domains lacking one or more CDRs, and the combined complete VH or VL domains may be combined with cognate VL or VH domains to provide antibodies of the invention. The repertoire may then be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, so that suitable specific binding members may be selected. The repertoire may then be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, so that suitable specific binding members may be selected. 4 From the individual members upwards, e.g., 10 6 ~10 8 or 10 10 A similar shuffle or combinatorial technique has also been described by Stemmer (Nature, 1994, 370:389-391), who describes techniques involving β-lactamase genes, but observes that this approach may also be used to generate antibodies.

[0159] Antibodies may also be affinity matured using known selection and / or mutagenesis methods as known in the art.

[0160] The recombinant antibodies of the present invention can also be produced by phage display methods, such as those disclosed in US Pat. No. 5,969,108.

[0161] The antibodies of the present invention may further comprise 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 Cκ chain 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 from any antibody isotype, e.g., IgG, IgA, IgE, IgD, and IgM, and any isotype subclass, particularly IgG1, IgG2b, and IgG4.

[0162] For recombinant production, nucleic acids encoding an antibody of the invention are preferably isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to DNA encoding the antibody heavy and light chains). Many vectors are available. Generally, vector components include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding an antibody of the invention or a fragment thereof (e.g., from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence.

[0163] (i) Signal sequence component: The antibodies of the present invention can be recombinantly produced not only directly but also as a fusion polypeptide with a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The selected heterologous signal sequence is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is substituted with a prokaryotic signal sequence selected from the group of, for example, the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the native signal sequence may be substituted with, for example, the yeast invertase leader, α-factor leader, or acid phosphatase leader, the C. albicans glucoamylase leader, or a signal described in WO 90 / 13646. For expression in mammalian cells, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, are available. The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.

[0164] (ii) Promoter component: Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody nucleic acid. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the antibody-encoding DNA.

[0165] Promoters for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence, which may signal addition of a poly(A) tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors. Examples of promoter sequences suitable for use with yeast hosts include promoters for 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, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters that are inducible promoters with the added advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

[0166] Transcription of the antibody from the vector in mammalian host cells is controlled by a promoter derived from the genome of a virus such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), CMV, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40), from a heterologous mammalian promoter, such as the actin promoter or an immunoglobulin promoter, or from a heat shock promoter, provided that such a promoter is compatible with the host cell system.

[0167] (iii) Enhancer Element Component: Transcription of a DNA encoding an antibody of the present invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, an enhancer from a eukaryotic virus will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, (1982), Nature, 297:17-18, for a review of enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced ​​into the vector at a position 5' or 3' to the antibody-coding sequence, but is preferably located 5' from the promoter.

[0168] (iv) Transcription termination component: Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are commonly available from the 5' and, occasionally, the 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94 / 11026 and the expression vector disclosed therein.

[0169] (v) Selection and transformation of host cells: Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotes, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, e.g., gram-negative or gram-positive organisms, such as Enterobacteriaceae, e.g., Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, e.g., B. subtilis and B. licheniformis, Pseudomonas, e.g., P. aeruginosa, and Streptomyces. One 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 also suitable. These examples are illustrative rather than limiting.

[0170] In addition to prokaryotes, eukaryotic microbes 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 among lower eukaryotic host microorganisms. However, for example, Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger, are generally available and useful herein.

[0171] Suitable host cells for the expression of glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0172] Examples of useful mammalian host cell lines are monkey kidney CV1 transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al. (1977), Gen. Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL10); 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 CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al. (1982), Annals NYAcad. Sci. 383:44-68); MRC5 cells; FS4 cells; and PER.C6™ (Crucell NV).

[0173] Functionally equivalent antibodies The present disclosure also contemplates antibodies or antigen-binding fragments thereof with 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 which still retain the function of the antibodies of the invention. These modifications may be deliberate, for example, through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts expressing the antibodies.

[0174] Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, polynucleotides of the invention can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques involve subcloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain, e.g., E. coli XL-1 red (Stratagene), and growing the transformed bacteria for a suitable number of generations. Products derived from mutated / altered DNA can be readily screened using the techniques described herein to determine whether they have receptor-binding and / or -inhibitory activity.

[0175] In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the feature(s) to be modified. The sites for mutation can be modified individually or sequentially, for example, by (1) substituting initially with conservative amino acid choices, followed by more radical choices depending on the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the positioned site.

[0176] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues, and usually about 1 to 5 contiguous residues.

[0177] Substitutional mutations involve removing at least one amino acid residue in an antibody and / or immunoglobulin chain molecule, including the variable regions, and inserting a different residue in its place. Sites of greatest interest for substitutional mutagenesis include those identified as important for antigen binding. Such sites, particularly those that fall within the sequence of at least three other identically conserved sites in human antibodies and / or immunoglobulin chains, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 below under the heading of "exemplary substitutions."

[0178] Conservative amino acid substitutions are also contemplated in the present invention and are intended to mean the amino acid substitutions shown in the table below. [Table A]

[0179] The amino acids described herein are preferably in the "L" isomeric form. However, residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin binding is retained by the polypeptide. Modifications also include structural and functional analogs, such as synthetic or non-natural amino acids or amino acid analogs and peptidomimetics having derivatized forms.

[0180] chimeric antibodies Chimeric antibodies are produced by recombinant means by combining variable light and heavy chain regions (VL and VH) obtained from antibody-producing cells of one species with constant light and heavy chain regions from another species. Typically, chimeric antibodies utilize rodent or rabbit variable regions and human constant regions to create an antibody with primarily human domains. For example, a chimeric antibody may contain a variable region from a murine antibody, such as those described herein, according to any embodiment, fused to a human constant region. The production of such chimeric antibodies is known in the art and may be accomplished by standard means (e.g., as described 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). Furthermore, it is contemplated that the human constant region of the chimeric antibody of the invention may be selected from the constant region of IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, or IgG19.

[0181] Humanized and human antibodies The antibodies of the present disclosure may be humanized or human. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that 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).

[0182] Methods for humanizing non-human antibodies are known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, usually taken from an "import" variable domain. Humanization can essentially be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the methods of Jones et al., supra; Riechmann et al., supra; Verhoeyen et al., Science, 239:1534-1536 (1988). Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0183] Human antibodies can also be produced using various techniques known in the art, 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 suitable for preparing 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 produced by introducing human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigen challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0184] Fully human antibodies that recognize a selected epitope can also be generated using a technique called "guided selection," in which a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., Bio / technology, 12:899-903 (1988)).

[0185] Antibodies may be affinity matured using known selection and / or mutagenesis techniques as known in the art. Preferred affinity-matured antibodies have 5-fold, more preferably 10-fold, and even more preferably 20 or 30-fold greater affinity than the starting antibody (generally murine, humanized, or human) from which the mature antibody is prepared.

[0186] Synthetic Humanized and Primatized Proteins The HER2 binding protein of the present disclosure may be a synthetic humanized protein. The term "synthetic humanized protein" refers to a protein prepared by the method described in WO2007 / 019620. The synthetic humanized HER2 binding protein comprises a variable region of an antibody, wherein the variable region comprises FRs derived from a New World primate antibody variable region and CDRs derived from a non-New World primate antibody variable region. For example, the synthetic humanized HER2 binding protein comprises a variable region of an antibody, wherein the variable region comprises FRs derived from a New World primate antibody variable region and CDRs derived from, for example, a mouse antibody as described herein. In one example, the synthetic humanized HER2 binding protein is a HER2 binding antibody in which one or both of the variable regions are synthetically humanized.

[0187] The HER2 binding protein of the present disclosure may be a primatized protein. A "primatized protein" comprises a variable region(s) from an antibody generated after immunization of a non-human primate (e.g., a cynomolgus monkey). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to create a primatized antibody. An exemplary method for producing a primatized antibody is described in US6113898.

[0188] Deimmunized antibodies and proteins The present disclosure also contemplates deimmunized antibodies or HER2 binding proteins. Deimmunized antibodies have one or more epitopes, such as B cell epitopes or T cell epitopes, removed (i.e., mutated), thereby reducing the likelihood that a subject will develop an immune response to the antibody or protein. Methods for producing deimmunized antibodies and proteins are known in the art and are described, for example, in WO00 / 34317, WO2004 / 108158, and WO2004 / 064724.

[0189] Methods for introducing suitable mutations and expressing and assaying the resulting proteins will be apparent to those skilled in the art based on the disclosure herein.

[0190] A protein containing an antibody variable region. Single Domain Antibodies In some examples, the HER2 binding proteins of the present disclosure are single domain antibodies (used interchangeably with the terms "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain that comprises all or a portion of the heavy chain variable region of an antibody. In particular examples, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US6248516; WO90 / 05144; and / or WO2004 / 058820).

[0191] Diabodies, triabodies, and tetrabodies Exemplary HER2 binding proteins that comprise an antibody antigen-binding domain are diabodies, triabodies, tetrabodies, and higher order protein complexes such as those described in, for example, WO98 / 044001 and WO94 / 007921.

[0192] For example, a diabody is a protein comprising two associated polypeptide chains, each comprising the structure VL-X-VH or VH-X-VL, where VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, and X is a linker containing insufficient residues to allow the VH and VL in a single polypeptide chain to associate (or form an Fv), or is absent; 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 to 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, forming a bispecific diabody (i.e., comprising two Fvs with different specificities).

[0193] Single chain Fv (scFv) fragment Those skilled in the art will recognize that an scFv comprises a VH domain and a VL domain in a single polypeptide chain. The polypeptide chain further comprises a polypeptide linker between the VH and VL that enables the scFv to form the desired structure for antigen binding (i.e., when the VH and VL of the single polypeptide chain associate with each other to form an Fv). For example, the linker may comprise more than 12 amino acid residues of (Gly4Ser)3, which is one of the more preferred linkers for scFvs.

[0194] The present disclosure also contemplates disulfide-stabilized Fvs (or diFvs or dsFvs), in which a single cysteine ​​residue is introduced into the VH FR and the VL FR, and the cysteine ​​residues are linked by a disulfide bond to create a stable Fv (see, e.g., Brinkmann et al. (1993), Proc. Natl. Acad. Sci. USA, 90:547-551).

[0195] Alternatively or additionally, the present disclosure provides dimeric scFvs, i.e., proteins comprising two scFv molecules linked non-covalently or covalently, for example, by a leucine zipper domain (e.g., from Fos or Jun) (see, e.g., Kruif and Logtenborg, 1996). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to allow both scFvs to form and bind to antigen, e.g., as described in US20060263367.

[0196] For a review of scFv, see Ahmad ZA. et al., (2012), Clinical and Developmental Immunology doi:10.1155 / 2012 / 980250.

[0197] Mini Body Those skilled in the art will be aware that a minibody comprises the VH and VL domains of an antibody fused to the CH2 and / or CH3 domains of the antibody. Optionally, the minibody comprises a hinge region between the VH and VL; this configuration is sometimes referred to as a Flex minibody. Minibodies do not comprise a 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 of the variable regions of the minibody binds to HER2 in the manner of the present disclosure. Exemplary minibodies and methods for their production are described, for example, in WO 94 / 09817.

[0198] Other antibody variable region-containing proteins The present disclosure provides, for example: (i) the "lock and key" bispecific proteins described in US 5,731,168; (ii) heterocomplex proteins, such as those described in US Pat. No. 4,676,980; (iii) heterocomplex proteins created using chemical cross-linkers, such as those described in US 4,676,980; (iv) Fab'-SH fragments, as described, for example, in Shalaby, (1992), J. Exp. Med. 1;175(1):217-25; (v) a single-chain Fab; or (vi) HER2 binding proteins containing other variable regions, such as Fab3 (eg, as described in EP19930302894), are also contemplated.

[0199] Proteins containing non-antibody-based antigen-binding domains Immunoglobulins and immunoglobulin fragments An example of a compound of the present disclosure is a protein comprising the variable region of an immunoglobulin, such as a T cell receptor or a heavy chain immunoglobulin (eg, IgNA, camelid antibody).

[0200] The term "immunoglobulin" will be understood to include any antigen-binding protein that contains an immunoglobulin domain. An exemplary immunoglobulin is an antibody. Additional proteins encompassed by the term "immunoglobulin" include domain antibodies, camelid antibodies, and antibodies from cartilaginous fish (i.e., immunoglobulin novel antigen receptors (IgNARs)). Camelid antibodies and IgNARs generally contain a VH but lack a VL and are often referred to as heavy chain immunoglobulins. Other "immunoglobulins" include T cell receptors.

[0201] Heavy chain immunoglobulins Heavy chain immunoglobulins are structurally distinct from many other immunoglobulin forms (e.g., antibodies) in that they contain heavy chains but no light chains. Thus, such immunoglobulins are also referred to as "heavy chain-only antibodies." Heavy chain immunoglobulins are found, for example, in camelids and cartilaginous fish (also called IgNARs).

[0202] The variable regions present in naturally occurring heavy chain immunoglobulins are commonly referred to as "VHH domains" in camelid Igs and V-NARs in IgNARs to distinguish them from the heavy chain variable regions (called "VH domains") present in conventional four-chain antibodies and the light chain variable regions (called "VL domains") present in conventional four-chain antibodies.

[0203] Heavy chain immunoglobulins do not require the presence of light chains to bind relevant antigens with high affinity and specificity. This means that single domain binding fragments can be derived from heavy chain immunoglobulins, which are easily expressed and generally stable and soluble. A general description of camelid-derived heavy chain immunoglobulins and their variable regions and methods for their production and / or isolation and / or use can be found, inter alia, in the following references: WO94 / 04678, WO97 / 49805, and WO97 / 49805.

[0204] A general description of cartilaginous fish-derived heavy chain immunoglobulins and their variable regions and methods for making and / or isolating and / or using them can be found, inter alia, in WO2005 / 118629.

[0205] V-like protein An example of a HER2-binding protein of the present disclosure is a T cell receptor. T cell receptors have two V domains that integrate into a structure similar to the Fv module of an antibody. Novotny et al., Proc. Natl. Acad. Sci. USA, 88:8646-8650, 1991, describe how the two V domains of a T cell receptor (designated alpha and beta) can be fused and expressed as a single polypeptide chain, and how surface residues can be modified to reduce hydrophobicity, similar to antibody scFvs. Other publications describing the construction of single-chain or multimeric T cell receptors containing two V-alpha and V-beta domains include WO1999 / 045110 or WO2011 / 107595.

[0206] Other non-antibody proteins containing antigen-binding domains include proteins with V-like domains, which are generally monomeric. Examples of such V-like domain-containing proteins include CTLA-4, CD28, and ICOS. Further disclosure of such V-like domain-containing proteins is contained in WO1999 / 045110.

[0207] Adnectins In one example, the HER2 binding protein of the present disclosure is an adnectin.

[0208] Adnectins are based on the tenth fibronectin type III (10Fn3) domain of human fibronectin, in which the loop regions have been modified to confer antigen binding. For example, the three loops at one end of the β-sandwich of 10Fn3 can be engineered to enable Adnectins to specifically recognize antigens. For further details, see US20080139791 or WO2005 / 056764.

[0209] Anticalin In a further example, the HER2-binding protein of the present disclosure is an anticalin. Anticalins are derived from lipocalins, 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 cone-like structure that can be engineered to bind to antigens. Such engineered lipocalins are known as anticalins. For further description of anticalins, see US7250297B1 or US20070224633.

[0210] Affibody In a further example, the HER2 binding protein of the present disclosure is an affibody. Affibodies are scaffolds derived from the Z domain (antigen-binding domain) of Staphylococcus aureus protein A that can be engineered to bind to antigens. The Z domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see EP1641818.

[0211] Abima In a further example, the HER2 binding protein of the present disclosure is an avimer. Avimers are multi-domain proteins derived from the A-domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide bond structure. Diversity is generated by the assortment of natural mutations exhibited by the A-domain family. For further details, see WO2002088171.

[0212] DARPins In a further example, the HER2-binding protein of the present disclosure is a designed ankyrin repeat protein (DARPin). DARPins are derived from ankyrin, a family of proteins that mediate the attachment of complex membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha helices and one beta turn. DARPins can be engineered to bind different target antigens by randomizing residues within the first alpha helix and beta turn of each repeat. The binding interface can be increased by increasing the number of modules (affinity maturation). For further details, see US20040132028.

[0213] Other non-antibody polypeptides Other non-antibody proteins that contain binding domains include those based on human gamma-crystallin and human ubiquitin (affilin), the Kunitz-type domains of human protease inhibitors, the PDZ domain of the Ras-binding protein AF-6, scorpion toxin (charybdotoxin), and C-type lectin domains (tetranectin).

[0214] constant region The present disclosure encompasses HER2 binding proteins comprising a variable region and a constant region or domain(s) thereof, e.g., Fc, CH2, and / or CH3 domains. One of skill in the art would know the meaning of the terms constant region and constant domain based on the disclosure herein and the references discussed herein.

[0215] Constant region sequences useful for producing the HER2 binding proteins of the present disclosure may be obtained from a number of different sources. In some examples, the constant region of the HER2 binding protein, or a portion thereof, is derived from a human antibody. Furthermore, the constant domain, or a portion thereof, may 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 example, the human isotype IgG1 is used.

[0216] Various constant region gene sequences are available in the form of publicly accessible deposits, or the sequences are available from publicly available databases. Constant regions can be selected that have particular effector functions (or lack particular effector functions) or with particular modifications to reduce immunogenicity.

[0217] In one example, a protein of the present disclosure has or exhibits an effector function that promotes or enables at least partial depletion, substantial depletion, or elimination of cells expressing HER2. Such effector function may be enhanced binding affinity to an Fc receptor, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0218] In one example, a HER2 binding protein can induce enhanced levels of effector function.

[0219] In one example, the level of effector function induced by the constant region is enhanced compared to the wild-type Fc region of an IgG1 antibody or compared to the wild-type Fc region of an IgG3 antibody.

[0220] In another example, the constant region is modified to increase the level of effector function it can induce compared to a constant region without such modifications. Such modifications can be at the amino acid level, and / or at the secondary structure level, and / or at the tertiary structure level, and / or to the glycosylation of the Fc region.

[0221] Those skilled in the art will appreciate that greater effector function may be manifested in multiple ways, for example, as a greater level of effect, a more persistent effect, or a more rapid effect. Exemplary constant region modifications include amino acid substitutions, for example, S239D / I332E as numbered according to the EU index of Kabat, or S239D / A330L / I332E as numbered according to the EU index of Kabat.

[0222] Further amino acid substitutions that enhance the ability of an Fc region to induce effector function are known in the art and / or are described, for example, in US Pat. No. 6,737,056 or US Pat. No. 7,317,091.

[0223] In one example, glycosylation of the constant region is modified to enhance its ability to induce enhanced effector function. In some examples, the Fc region of the present disclosure comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region, i.e., the Fc region is "non-fucosylated." Such variants may have improved ability to induce ADCC. Methods for producing non-fucosylated antibodies include expressing HER2-binding proteins in cell lines that cannot express α-1,6-fucosyltransferase (FUT8) (e.g., as described in Yumane-Ohnuki et al., 2004). Other methods include the use of cell lines that originally produce antibodies capable of inducing enhanced effector function (e.g., duck embryonic stem cells for viral vaccine production (WO 2008 / 129058); production of recombinant proteins in avian EBX® cells (WO 2008 / 142124)).

[0224] HER2-binding proteins can also contain Fc regions that can induce enhanced levels of CDC. For example, hybrids of IgG1 and IgG3 produce antibodies with enhanced CDC activity (Natsume et al., 2008).

[0225] Methods for determining the ability of an antibody or antigen-binding fragment thereof to induce effector function are known in the art and / or described herein.

[0226] In another example, the protein contains one or more amino acid substitutions that increase the half-life of the HER2-binding protein. For example, the HER2-binding protein contains a constant region containing 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 a higher affinity for FcRn at lower pH, e.g., about pH 6.0, facilitating Fc / FcRn binding in endosomes. In one example, the constant region has a higher affinity for FcRn at about pH 6 compared to about pH 7.4, which promotes Fc re-release into the blood after cellular recycling. These amino acid substitutions are useful for extending the half-life of the protein by reducing clearance from the blood.

[0227] Exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.

[0228] The HER2-binding protein of the present disclosure can comprise an IgG4 constant region or a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange or the formation of half antibodies or the formation of half antibodies. "Fab arm exchange" refers to a type of protein modification of human IgG4 in which one IgG4 heavy chain and associated light chain (half molecule) are exchanged with a heavy chain / light chain pair from another IgG4 molecule. Thus, an IgG4 molecule may 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 with purified blood cells or a reducing agent such as reduced glutathione. "Half antibodies" occur when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0229] In one example, a stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat system. This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, this residue is generally serine. Following the substitution of serine for proline, the IgG4 hinge region contains 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 region and Fab region, conferring mobility to the two Fab arms of the antibody. The hinge region contains cysteine ​​residues involved in inter-heavy chain disulfide bonds. The hinge region is generally defined as the section from Glu226 to Pro243 of human IgG1 according to the Kabat numbering system. Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions (see, for example, WO2010 / 080538).

[0230] modified proteins The present disclosure provides HER2 binding proteins that have at least 80% identity to the sequences of the present disclosure and have the same functional characteristics as described or claimed herein.

[0231] In one example, a HER2 binding protein of the present disclosure 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 a VL sequence disclosed herein, e.g., SEQ ID NO:3.

[0232] In another example, a HER2 binding protein of the disclosure 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 a VH disclosed herein, e.g., SEQ ID NO:2.

[0233] The present disclosure also provides nucleic acids encoding the aforementioned proteins or nucleic acids that hybridize thereto under moderate to high stringency conditions.

[0234] The present disclosure also encompasses nucleic acids that encode proteins comprising the sequences shown in SEQ ID NO:2 and SEQ ID NO:3, but which differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0235] The present disclosure also encompasses nucleic acids that encode proteins comprising the sequences shown in SEQ ID NO:4 and SEQ ID NO:5, which differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0236] The percent identity of nucleic acids or polypeptides is measured by GAP (Needleman and Wunsch. 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is preferably at least 50 residues in length, and 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 GAP analysis aligns the two sequences over a region of at least 100 residues. In one example, the two sequences are aligned over their entire length.

[0237] Modified glycosylation The glycosylation pattern of an antibody may be altered from the original glycosylation pattern of a reference antibody. Alteration means 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 antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the 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 except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either 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 hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0238] Modified glycoforms of the antibodies of the present disclosure may be useful for a variety of purposes, including, but not limited to, increasing or decreasing effector function and / or altering the half-life of the antibody (see, e.g., WO / 2007 / 010401). Such changes may result in decreased or increased C1q binding and CDC, or decreased or increased FcγR binding and ADCC. Substitutions can be made, for example, at one or more amino acid residues in the heavy chain constant region, thereby resulting in altered effector function while retaining the ability to bind antigen compared to the modified antibody; see U.S. Pat. Nos. 5,624,821 and 5,648,260. Engineered glycoforms may be produced by any method known to those of skill in the art, for example, by using engineered or mutant expression strains, by co-expression with one or more enzymes, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTII 1), by expressing the antibody or fragment thereof in different organisms or cell lines derived from different organisms, or by expressing the antibody or fragment and then modifying the carbohydrate(s). Methods for purifying engineered glycoforms are known in the art, including those described in 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. Application No. 10 / 277,370; U.S. Application No. 10 / 113,929; PCT WO00 / 61739A1; PCT WO01 / 292246A1; PCT WO02 / 311140A1; PCT These include, but are not limited to, those described in WO 02 / 30954 A1; Potelligent® technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland).See, e.g., WO00061739; EA01229125; US20030115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0239] Effector function It may be desirable to modify the antibody of the disclosure with respect to effector function, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or additionally, cysteine ​​residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176:1-195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may be prepared using heterobifunctional cross-linkers such as those described in Wolff et al. Cancer Research, 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design, 3:219-230 (1989).

[0240] Half-life To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into the antibody (particularly an antibody fragment), as described, for example, 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, antibody half-life may be increased by pegylation.

[0241] Assaying the activity of the binding proteins of the present disclosure Binding assay One form of such an assay is an antigen-binding assay, as described, for example, in Scopes (1994), Protein Purification: Principles and Practice, Springer-Verlag. Such methods generally involve labeling a HER2-binding protein and contacting it with an immobilized antigen or a fragment thereof, e.g., a protein containing residues 293-309 of mature, normal, or wild-type human HER2, as shown in Figure 1. Following washing to remove nonspecifically bound proteins, the amount of label and resulting bound proteins are detected. Of course, the HER2-binding protein can be immobilized and the antigen can be labeled. Panning-type assays can also be used. The Examples herein describe flow cytometry-based binding assays.

[0242] HER2 binding proteins that competitively inhibit the HER2 antibodies of the invention for binding to the epitope can be screened and identified using conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assays (ELISAs).

[0243] Competitive binding assay Assays for determining HER2-binding proteins that competitively inhibit the binding of an antibody of the present disclosure (e.g., mAb104) will be clear to those of skill in the art. For example, an antibody of the present disclosure is conjugated to a detectable label, such as a fluorescent or radioactive label. The labeled antibody and the test HER2-binding protein are then mixed and contacted with HER2 or a peptide containing an epitope thereof (e.g., corresponding to residues 293 to 309 of domain II of human HER2). The level of the labeled antibody is then measured and compared to the level measured when the labeled antibody is contacted with HER2 or a peptide containing an epitope thereof in the absence of the HER2-binding protein. If the level of the labeled antibody is reduced in the presence of the test HER2-binding protein compared to the absence of the HER2-binding protein, the HER2-binding protein competitively inhibits the binding of that antibody.

[0244] Optionally, the test HER2 binding protein is conjugated to a label distinct from the antibody, which allows for the level of binding of the test HER2 binding protein to the protein or epitope to be detected.

[0245] In another example, a test HER2-binding protein may be bound to HER2 or a peptide containing an epitope thereof prior to contacting HER2 or a peptide containing an epitope thereof with an antibody 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. Alternatively, a reciprocal assay may be performed using a labeled HER2-binding protein to first allow the antibody to bind to HER2 or a peptide containing an epitope thereof. In this case, a decrease in the amount of labeled HER2-binding protein bound to HER2 or a peptide containing an epitope thereof 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.

[0246] Affinity assay Optionally, the dissociation constant (Kd) or association constant (Ka) or binding constant (KD, i.e., Ka / Kd) of the HER2-binding protein for HER2 or its epitope-containing peptide is measured. For example, these constants for the HER2-binding protein are measured by a radioactively or fluorescently labeled HER2 binding assay. This 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 to remove unbound HER2, the amount of label is measured. In another example, the constant is measured by using a surface plasmon resonance assay, for example, BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized HER2 or a region thereof.

[0247] Protein detection assays One example of the present disclosure detects the presence of HER2 or cells expressing HER2 (e.g., breast cancer cells). The amount, level, or presence of the protein or cells is measured using any of a variety of techniques known to those skilled in the art, such as flow cytometry, immunohistochemistry, immunofluorescence, immunoblot, Western blot, dot blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, LC MS / MS), biosensor technology, evanescent fiber optic technology, or protein chip technology.

[0248] In one example, the assay used to measure the amount or level of a protein is a semi-quantitative assay. In another example, the assay used to measure the amount or level of a protein is a quantitative assay.

[0249] For example, the protein is detected by 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).

[0250] Standard solid-phase ELISA or FLISA formats are particularly useful for determining protein concentrations from a variety of samples.

[0251] In one form, ELISA or FLISA involves immobilizing the HER2-binding protein of the present disclosure or a protein that binds to a different epitope of HER2 on a solid matrix, such as a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick, or a glass support. A sample is then brought into physical contact 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 the 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. Furthermore, variations of the above-described assays, such as competitive ELISA, will be apparent to those skilled in the art.

[0252] In an alternative example, the polypeptide is detected in or on a cell using methods known in the art, such as immunohistochemistry or immunofluorescence. Immunofluorescence methods 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.

[0253] Biosensor devices generally use an electrode surface in conjunction with a current or impedance measuring element, combined with an assay substrate (e.g., as described in US5567301) and integrated into the device. The HER2-binding proteins of the present disclosure are incorporated onto the surface of the biosensor device, and a biological sample is contacted with the device. A change in current or impedance detected by the biosensor device indicates protein binding 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 surface reflectance in surface plasmon resonance indicates protein binding to a ligand or antibody (US5485277 and US5492840).

[0254] Biosensors are particularly useful in high-throughput analysis because such systems can be easily adapted to the micro- or nanoscale. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This allows for the simultaneous detection of several proteins or peptides in small volumes of body fluids.

[0255] Binding of proteins to HER2 can also be detected using flow cytometry as described herein in the Examples.

[0256] Epitopes bound by the binding proteins of the present disclosure The present inventors have generated a binding molecule specific for a conformationally exposed epitope in domain II of human HER2, which is present in tumorigenic, hyperproliferative, or abnormal cells but not in wild-type or normal cells. This conformationally exposed epitope is located in the distal portion of domain II and is flanked by disulfide bonds, allowing flexibility in this region and exposing the epitope for mAb104 binding. In particular, the epitope appears to be exposed in cells in response to HER2 amplification or activation. What is particularly surprising about this antibody is that it does not block the binding of pertuzumab or trastuzumab to the extracellular domain of HER2, indicating that the conformational exposure of this epitope region in domain II may allow this antibody to bind without blocking the binding of these antibodies, potentially enabling a dual therapy approach.

[0257] The crystal structure of pertuzumab bound to HER2 has been determined (see Franklin M.C. et al., (2004), Cancer Cell, vol. 5:317-328). Pertuzumab binds to HER2 near the center of domain II, sterically blocking the binding pocket required for receptor dimerization and signal transduction. CDR H3 of pertuzumab is understood to make hydrophobic and hydrogen-bonding contacts with residues Lys311 and His296 of HER2. His296 is completely hidden upon pertuzumab binding. Without wishing to be bound by theory, the inventors hypothesize that because pertuzumab specifically inhibits HER2 heterodimerization by blocking the pocket of HER2 that accommodates the dimerization loop of the heterodimeric receptor partner, it is sterically possible for the binding molecule of the present invention (e.g., mAb104) to bind to another face of the epitope loop of HER2. We hypothesize that this epitope may appear on a subset of receptors on the surface of cancer cells when the receptor undergoes conformational changes due to redox disulfide bond switching or aberrant expression in conditions of HER2 overexpression or hypoxia in cancer, or when HER2 binds to a dimerization partner and undergoes a conformational change to expose the loop bound by the current binding molecule, making it more accessible. Furthermore, it is known that within the epitope region, antibodies do not bind to all amino acids within that region, and due to the conformational nature of the mAb104 epitope, this may explain why closely opposing epitopes do not affect the binding of both antibodies.

[0258] In one example, the HER2 binding protein inhibits the heterodimerization of HER2.

[0259] antibody complex The present invention also provides a HER2 binding protein described herein conjugated to a moiety. The moiety can include, but is not limited to, a detectable label or a functional label. In some embodiments, the moiety is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases 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 lists. For example, the moiety can be classified as both a therapeutic compound and a toxin.

[0260] In some embodiments, the moiety is a radioisotope. Suitable radioisotopes include the isotope 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 may be linked to the antibodies of the invention using conventional chemistries known in the art of antibody imaging.

[0261] In some embodiments, the moiety is a detectable label. Suitable detectable labels include, for example, isotopes 3 H, 14 C. 32P, 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 Labels include, but are not limited to, radioactive labels such as Re, which may be linked to the antibodies of the present invention using conventional chemistry known in the art of antibody imaging. Labels also include fluorescent labels (e.g., fluorescein, rhodamine, Texas Red, phycoerythrin) and labels conventionally used in the art for MRI-CT imaging. They also include enzyme labels such as horseradish peroxidase, β-glucoronidase, 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 specific cognate detectable moieties, such as biotin, which may be detected via binding to labeled avidin. Labels also include electron-dense reagents; energy transfer molecules; paramagnetic labels; chemiluminescent (imidazole, luciferase); and bioluminescent agents.

[0262] In some embodiments, the moiety is a nucleic acid. Suitable nucleic acids include double-stranded DNA, single-stranded DNA, siRNA, deoxyribozymes, or ribozymes.

[0263] In some embodiments, the moiety is a therapeutic compound. Suitable therapeutic compounds include compounds that can modify a biological response (e.g., without limitation, inhibiting or preventing the expression activity of a cell, causing destruction of a cell, or otherwise affecting the function of a cell). Such therapeutic compounds include, for example, but are not limited to, chemical ablative agents, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents, and / or drugs, including, but not limited to, the following: 4-desacetylvinblastine-3-carbazidizide; 5-fluoro-2'-deoxyuridine; 5-fluorouracil; 5-fluorouracil decarbonate; 6-mercaptopurine; 6-thioguanine; abrin; abrin A chain; actinomycin D; 1-dehydrotestosterone; adriamycin; Aleurites fordii proteins; alkylating agents; alkylphosphocholines; aminopterins; angiogenins; angiostatins; anthracyclines; anthramycins; antiangiogenic agents; antifolates; antimetabolites; antimitotics; antibiotics; ara-C; asparaginase auristatin derivatives (e.g., without limitation, U.S. Patent Publication Nos. 2008 / 0300192, 2009 / 0018086, 2009 / 0018087, 2009 / 0018089, 2009 / 0018090, 2009 / 0018091, 2009 / 0018092, 2009 / 0018093, 2009 / 0018094, 2009 / 0018095, 2009 / 0018096, 2009 / 0018097, 2009 / 0018098, 2009 / 0018099, 2009 / 00180997, 2009 / 00180998, 2009 / 001809 ... 86, and 2009 / 0111756); auristatin E (see, for example, but not limited to, U.S. Patent No. 5,635,483, incorporated herein by reference in its entirety); auristatin E valeryl benzylhydrazone; auristatin F phenylenediamine; auristatin; auromycin; bis-iodophenol mustard; bismuth; bleomycin; busulfan; calicheamicin; carboplatin; carminomycin; carmustine;CC-1065 compounds (such as, but not limited to, U.S. Pat. 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 Publication No. 2007 / 0135346, 2007 / 0135346, 2007 / 0135347, 2007 / 0135348, 2007 / 0135349 ... See PCT Publication Nos. 08 / 0260685, 2009 / 0281158, and 2009 / 0318668, and PCT Publication No. WO2009 / 017394); chlorambucil; cis-dichlorodiamineplatinum (cisplatin); cladribine; colchicine (colchicine); combrestatin; crotin; curicin; cyclophosphamide; cytarabine; cytochalasin B; cytosine arabinoside; cytoxin; dacarbazine; dactinomycin (actinomycin ); daunorubicin (daunomycin); dianthin protein; dibromomannitol; dihydroxyanthracenedione; diphtheria toxin; dolastatin-10; doxetaxel; doxorubicin; doxorubicin hydrazide; duocarmycins (see, for example, but not limited to, U.S. Pat. No. 7,214,685, incorporated herein by reference in its entirety); emetine; endostatin; enediyne; enomycin; epirubicin; esperamicin compounds (see, for example, but not limited to, U.S. Pat. No. 4,675,187, incorporated herein by reference in its entirety); ethidium bromide; etoposide; fludarabine, geronin; 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;Maytansinol (for example, but not limited to, U.S. Pat. Nos. 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, 4,361,650, 4,362,663, 4,364,866, 4,371,533, 4,424,219, 4,450,234, 5,141,736, and 5,217,713); mechlorethamine; melphalan (and other related nitrogen mustards); methotrexate; minor group bilirubin under; mithramycin; mitogellin; mitomycin C; mitomycin; mitoxantrone; MMAF-dimethylaminoethylamine; MMAF-Nt-butyl; MMAF-tetraethylene glycol; modeccin A chain; momordica charantia inhibitor; monomethyl auristatin E (MMAE) (for example, but not limited to, those disclosed in U.S. Pat. Nos. 6,884,869, 7,098,308, 7,256,257, and 7,423,116, and U.S. Patent Publication No. 2003 / 0111294, each of which is incorporated by reference in its entirety). / 0083263, 2004 / 0157782, 2005 / 0009751, 2005 / 0113308, and 2006 / 0229253); 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 by reference in its entirety); morpholinodoxorubicin;N2'-deacetyl-N2'-(c-mercapto-1-oxopropyl)-maytansine (DM1) (see, for example, but not limited to, U.S. Pat. No. 5,208,020, which is incorporated by reference in its entirety); N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) (see, for example, but not limited to, U.S. Pat. No. 7,276,497, which is incorporated by reference in its entirety); neocarzinostatin; nerve growth factor (and other growth factors); onapristone; paclitaxel; PE40; phenomycin; pHytolacca americana proteins (PAPI, PAPII, and PAP-S); platelet-derived growth factor; plicamycin; prednisone; procaine; procarbazine; propranolol; Pseudomonas exotoxin A; puromycin; pyrrolobenzodiazepines, radioisotopes (e.g., 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; restrictocin; ricin A; ricin; Sapaonaria officinalis inhibitors; saporin; streptozotocin; suramin; tamoxifen; taxanes; taxoids; taxol; teniposide; tetracaine; thioepa chlorambucil; thiotepa; thrombotic agents; tissue plasminogen activator; topoisomerase I inhibitors; topoisomerase II inhibitors; toxotere; trichothecenes; tumor necrosis factors; vinblastine; vinca alkaloids; vinca; vincristine; vindesine; vinorelbine; yttrium; alpha-interferon; alpha-sarcin; and beta-interferon, as well as analogs, homologs, fragments, variants, and derivatives thereof (see also Garnett, (2001), Advanced Drug Delivery Reviews, 53:171-216, which is incorporated herein by reference in its entirety).

[0264] In a preferred embodiment, the therapeutic compound is selected from the group consisting of an auristatin or a derivative thereof, maytansine or a derivative thereof (also called a maytansinoid), or a pyrrolobenzodiazepine or a derivative thereof. In one example, the therapeutic agent is N2'-deacetyl-N2'-(c-mercapto-1-oxopropyl)-maytansine (DM1). In another example, the therapeutic agent is monomethylauristatin E (MMAE). In another example, the therapeutic agent is a pyrrolobenzodiazepine.

[0265] The present disclosure also contemplates immunotoxin conjugates, for example, as described in WO93 / 21232.

[0266] Suitable colloids include gold colloids and gold nanoparticles. The HER2 binding protein may be bound 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).

[0267] 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. Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, diansin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0268] 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 (XTEN, elastin-like polypeptides, gelatin-like polypeptides, and (Pro-Ala-SER) n Examples of suitable polymers include flexible polypeptides such as PEG, PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-30, PEG-31, PEG-32, PEG-33, PEG-34, PEG-35, PEG-36, PEG-37, PEG-38, PEG-39, PEG-40, PEG-41, PEG-4

[0269] Additionally, the HER2 binding proteins, particularly antibodies and fragments thereof, of the present disclosure may be conjugated to a secondary antibody to form an antibody heteroconjugate (see, e.g., without limitation, U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety), may be administered alone or in combination with another agent (e.g., without limitation, those agents described above) (with or without an agent linked or conjugated thereto), and / or may be conjugated to an anti-cancer prodrug-activating enzyme capable of converting a prodrug to its active form.

[0270] As will be appreciated by those of skill in the art, the above moieties, as well as other suitable moieties, may be attached or linked to the HER2 binding proteins of the present disclosure, particularly antibodies and fragments thereof, in any suitable manner to create antibody conjugates. For example, and without limitation, in various embodiments of the present invention, the HER2 binding protein(s) and moieties may be covalently attached and / or conjugated using linkers, spacers, and / or stretcher compounds, which in various embodiments of the present invention are cleavable and non-cleavable, such that the agent(s) are internalized by the target cell.

[0271] For example, such linker, spacer and / or stretcher compounds include the following: aminobenzoic acid spacers (see, for example, but not limited to, U.S. Pat. Nos. 7,091,186 and 7,553,816, which are incorporated by reference in their entireties); maleimidocaproyl; p-aminobenzylcarbamoyl (PAB); lysosomal enzyme cleavable linkers (see, for example, but not limited to, U.S. Pat. No. 6,214,345, which is incorporated by reference in its entirety); maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valine citrulline; N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (see, for example, but not limited to, Yoshitake et al., U.S. Pat. Nos. 7,091,186 and 7,553,816, which are incorporated by reference in their entireties). al. (1979) Eur. J. Biochem., 101, 395-399); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, for example, but not limited to, U.S. Pat. No. 4,563,304, which is incorporated herein by reference in its entirety); N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP); valine-citrulline; and other linker, spacer, and / or stretcher compounds (see, for example, but not limited to, See U.S. Patent Nos. 7,090,843, 7,223,837, and 7,659,241, and U.S. Patent Publication Nos. 2004 / 0018194, 2004 / 0121940, 2006 / 0116422, 2007 / 0258987, 2008 / 0213289, 2008 / 0241128, 2008 / 0311136, 2008 / 0317747, and 2009 / 0010945, which are incorporated by reference in their entireties.

[0272] Generally speaking, techniques for attaching and / or conjugating the above moieties, as well as other moieties, to the HER2-binding proteins of the present disclosure, particularly antibodies and fragments thereof, are known in the art. In various embodiments of the present invention, the HER2-binding protein(s) and / or moieties may be covalently attached and / or conjugated via a lysine or cysteine ​​residue present on the HER2-binding protein. In one embodiment, the moiety MMAE is attached by conjugation to a cysteine ​​residue. In one embodiment, the moiety DM1 is attached by conjugation to a cysteine ​​residue. In one embodiment, the moiety PBD (pyrrolobenzodiazepine) is attached by conjugation to a cysteine ​​residue. Suitable conjugation chemistries are reviewed in Jain et al. (2015), Pharmaceutical Research, 32:3526.See, for example, but not by way of limitation, 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 Prospects Of The Therapeutic Use See also "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.

[0273] In embodiments where the binding moiety is a peptide or polypeptide, the conjugate may be a fusion protein in which the HER2 binding protein and the peptide or polypeptide form a single continuous polypeptide chain. Such fusion proteins may be made using techniques known in the art, including recombinant or synthetic techniques.

[0274] Antibodies, including fragments thereof, and agents which modulate the production or activity of specific binding members, antibodies and / or their subunits may also have particular diagnostic applications and may be used to detect and / or measure conditions such as cancers, precancerous lesions, conditions associated with or caused by hyperproliferative cell growth, etc. For example, the specific binding members, antibodies, or subunits thereof may be used to generate both polyclonal and monoclonal antibodies against themselves in a variety of cell cultures by known techniques, such as hybridoma technology, utilizing, for example, fused mouse splenic lymphocytes and myeloma cells. Similarly, small molecules may be discovered or synthesized which mimic or antagonize the activity(ies) of the specific binding members of the invention and may be used in diagnostic and / or therapeutic protocols.

[0275] Radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in in vitro diagnostic techniques and in in vivo radioimaging techniques and radioimmunotherapy. For in vivo imaging, the specific binding members of the present invention may be conjugated to imaging agents other than radioisotopes, including, but not limited to, magnetic resonance imaging-enhancing agents, in which, for example, antibody molecules are charged with multiple paramagnetic ions via 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 ferbium. In a further aspect of the present invention, radiolabeled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabeled antibodies for cancer treatment. In yet a further aspect, radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in radioimmuno-guided surgery techniques, in which they can identify and indicate the presence and / or location of cancer cells, pre-cancerous cells, tumor cells, and / or hyperproliferative cells before, during, or after surgery to remove such cells.

[0276] Competitive inhibition Antibodies that competitively inhibit the binding of the HER2 antibody of the invention to its epitope can be screened and identified using conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assays (ELISA).

[0277] Compositions of the Invention HER2-binding proteins, including conjugates thereof, according to the present disclosure are typically administered in the form of pharmaceutical compositions, which may contain at least one component in addition to the HER2-binding protein, HER2 antibody, or antigen-binding fragment thereof. Thus, pharmaceutical compositions according to the present disclosure and for use in accordance with the present invention may contain, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials will depend on the route of administration, which may be oral or by injection, e.g., intravenous injection.

[0278] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline, dextrose, or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may also be included.

[0279] For intravenous injection or injection at the site of pain, the active ingredient will be in the form of a pyrogen-free, parenterally acceptable aqueous solution having suitable pH, isotonicity, and stability. Those skilled in the art will be able to prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0280] In some embodiments, liposomes and / or nanoparticles may also be employed 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, which spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 angstroms, containing aqueous solutions at their cores. When dispersed in water, phospholipids can form a variety of structures other than liposomes, depending on the lipid to water molar ratio. At low ratios, liposomes are the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can exhibit low permeability to ionic and polar substances, but undergo a phase transition at elevated temperatures that significantly changes their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less ordered structure, known as the fluid state.

[0281] HER2 binding proteins or compositions comprising them may be administered alone or in combination with other treatments, therapeutic agents, or drugs, either simultaneously or sequentially depending on the condition being treated. Additionally, the present disclosure contemplates and includes compositions comprising the HER2 binding proteins described herein and other drugs or therapeutic agents, such as, for example, anti-cancer drugs 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 modulators, inhibitors of cell growth or division (e.g., antimitotic agents), or signal transduction inhibitors. Other treatments or therapeutic agents may include, for example, administration of a suitable dose of an analgesic, such as a nonsteroidal anti-inflammatory drug (e.g., aspirin, paracetamol, ibuprofen, or ketoprofen), or an opiate such as morphine, or an antiemetic. The compositions can be administered in combination (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, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, 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 general anti-cancer and anti-tumor agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine.Additionally, the compositions may be administered with hormones such as dexamethasone, immunomodulators such as interleukins, tumor necrosis factor (TNF) or other growth factors or cytokines that stimulate the immune response and the reduction or elimination of cancer cells or tumors, or angiogenesis inhibitors.

[0282] In some examples, the HER2 binding protein or a composition comprising the same is used in combination with a chemotherapeutic agent, a radioimmunotherapy agent, or an immunotherapy agent. In one example, the immunotherapy agent is a checkpoint inhibitor. In a further example, 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).

[0283] In some examples, the HER2 binding protein or composition comprising same is administered in conjunction with an immunosuppressant drug.

[0284] In some examples, the HER2 binding protein or a composition comprising the same is administered together with an immunomodulatory agent. Examples of suitable immunomodulatory agents include interleukins (e.g., IL-2, IL-7, IL-12), cytokines (e.g., interferons, G-CSF), chemokines (e.g., CCL3, CCL26, and CXCL7), and immunomodulatory agents (e.g., thalidomide).

[0285] The HER2 binding proteins of the present disclosure may be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream or CSF, or directly to the site of the tumor. The exact dose will depend on a number of factors, including whether the antibody is diagnostic or therapeutic, the size and location of the tumor, the exact nature of the HER2 binding protein (whether whole antibody, fragment, diabody, etc.), and the nature of any detectable or functional label attached to the antibody. When radionuclides are used in therapy, a suitable minimum single dose is about 45 mCi / m 2 , up to approximately 250 mCi / m 2The preferred dosage range is 15-40 mCi, with a more preferred range being 20-30 mCi, or 10-30 mCi. Such therapy may require bone marrow or stem cell replacement. Typical antibody doses for either tumor imaging or tumor therapy range from 0.5-40 mg, preferably 1-4 mg of antibody in F(ab')2 form. Naked antibodies are preferably administered at doses of 20-1000 mg of protein per dose, or 20-500 mg of protein per dose, or 20-100 mg of protein per dose. This is a single treatment dose for adult patients; it may be adjusted proportionally for children and infants, and for other antibody formats proportional to molecular weight. Treatment may be repeated daily, twice weekly, weekly, or monthly, at the physician's discretion.

[0286] Examples of suitable angiogenesis inhibitors (antiangiogenic agents) include urokinase inhibitors, matrix metalloproteinase inhibitors (e.g., marimastat, neovastat, BAY129566, AG3340, BMS275291, and similar agents), inhibitors of endothelial cell migration and proliferation (e.g., 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 (e.g., ZD6474, SU6668, angiogenic agents and / or antibodies to their receptors (e.g., VEGF, bFGF, and angiopoietin 1), thalidomide, thalidomide analogs (e.g., CC5013), Sugen5416, SU5402, antiangiogenic ribozymes (e.g., angiozyme), interferon alpha (e.g., interferon alpha 2a), suramin and similar agents), VEGF-R kinase inhibitors and other antiangiogenic tyrosine kinase inhibitors (e.g., SU011248). These include, but are not limited to, inhibitors of endothelial-specific integrin / survival signaling (e.g., vitaxin and similar agents), copper antagonists / chelators (e.g., tetrathiomolybdate, captopril and similar agents), carboxyamidotriazole (CAI), ABT627, CM101, interleukin 12 (IL12), IM862, PNU145156E, as well as nucleotide molecules that inhibit angiogenesis (e.g., antisense-VEGF-cDNA, cDNA encoding angiostatin, cDNA encoding p53, and cDNA encoding deletions of VEGF receptor 2) and similar agents.Other examples of inhibitors of angiogenesis, neovascularization, and / or other vascularization include anti-angiogenic heparin derivatives and related molecules (e.g., heperinase III), temozolomide, NK4, macrophage migration inhibitory factor (MIF), cyclooxygenase 2 inhibitors, inhibitors of hypoxia-inducible factor 1, anti-angiogenic soy isoflavones, oltipraz, fumagillin and its analogs, somatostatin analogs, pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin, thrombospondin, NM3, combrestatin, canstatin, avastatin, antibodies against other related targets (e.g., anti-alpha-v / beta-3 integrin and anti-kininostatin mAbs), and similar agents.

[0287] Measurement of cell viability and proliferation Cell damage and viability (such as cell apoptosis, lysis, and proliferation) can be measured in a variety of ways, including calorimetric, luminescent, radiometric, or fluorometric assays, as known in the art and described in the Examples 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, whereas dead and dying cells take up the blue dye and are easily distinguished under a light microscope. Neutral red is adsorbed by live cells and concentrated in cell lysosomes; viable cells can be determined under a light microscope by quantifying the number of cells stained with neutral red.

[0288] Fluorometric techniques for determining cell viability include, for example, propidium iodide, a fluorescent DNA intercalating agent. 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 cellular structural damage and death and can be measured with a spectrophotometric enzyme assay. Bromodeoxyuridine (BrdU) is incorporated into newly synthesized DNA and can be detected with a fluorochrome-conjugated antibody. 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) can provide cell division analysis (e.g., flow cytometry). This technique can be used either in vitro or in vivo. 7-aminoactinomycin D (7-AAD) is a fluorescent intercalator that undergoes a spectral shift upon binding to DNA, which can provide cell division analysis (e.g., flow cytometry).

[0289] Radioactive techniques for measuring cell proliferation include, for example, the incorporation of ribonucleotides into newly synthesized DNA in living cells, which are often used to measure cell proliferation. 3 3H]-thymidine from dead cells. 51 Cr) release can be quantified by scintillation counting to quantify cell viability.

[0290] Luminescent techniques for measuring cell viability include, for example, the CellTiter-Glo Luminescent Cell Viability Assay (Promega Madison Wis.), which quantifies the amount of ATP present to determine the number of viable cells.

[0291] Commercially available kits for measuring cell viability and cell proliferation include, for example, the Cell Proliferation Biotrak ELISA (Amersham Biosciences, Piscataway, NJ); the Guava ViaCount assay (Guava Technologies, Hayward, Calif.), which provides rapid cell number and viability determination based on differential uptake of a fluorescent reagent; the CyQUANT Cell Proliferation Assay Kit (Molecular Probes, Inc., Eugene, Oreg.); and the CytoLux Assay Kit (PerkinElmer Life Sciences Inc., Boston, Mass.). The DELFIA Assay Kit (PerkinElmer Life Sciences Inc., Boston, Mass.) can measure cell proliferation and viability using time-resolved fluorometry. The Quantos Cell Proliferation Assay is a fluorescence-based assay that measures the fluorescence of DNA-dye complexes from lysed cells (Stratagene, La Jolla, Calif.). The CellTiter-Glo Cell Viability Assay is a luminescent assay for measuring cell viability (Promega, Madison, Wis.).

[0292] use (i) Diagnostic and therapeutic applications The unique specificity of the HER2 binding proteins, particularly antibodies or fragments thereof, of the present disclosure, whereby the binding protein(s) recognize a HER2 / ErbB2 epitope found on tumorigenic, hyperproliferative or abnormal cells and not detectable on normal or wild-type cells, and where the protein(s) bind to amplified EGFR but not wild-type HER2, provides diagnostic and therapeutic applications for identifying, characterizing, targeting and treating, mitigating or eliminating numerous tumorigenic cell types and tumor types, for example, head and neck, breast, lung, bladder or prostate tumors and gliomas, but without the problems associated with normal tissue uptake that may be seen with previously known HER2 antibodies.

[0293] Thus, cells that overexpress HER2 (e.g., by amplification) may be recognized, isolated, characterized, targeted, and treated or eliminated utilizing the binding protein(s) of the present disclosure, particularly antibody(ies) or fragments thereof.

[0294] Thus, the HER2 binding proteins (e.g., antibodies) of the present disclosure can specifically characterize HER2 tumors or tumorigenic cells by staining or otherwise recognizing tumors or cells in which HER2 overexpression is present. Furthermore, antibodies of the present invention, such as those exemplified by mAb104, exhibit significant in vivo antitumor activity against tumors containing amplified HER2 and HER2-positive xenografts.

[0295] As outlined above, the inventors have found that the HER2 binding proteins of the present disclosure recognize tumor-associated forms of HER2, but not the normal wild-type receptor when expressed in normal cells. Antibody recognition is believed to depend on a conformation in response to HER2 amplification or activation that makes conformationally exposed epitopes available for binding.

[0296] mAb104 has been shown to inhibit the growth of overexpressed (eg, amplified) HER2 xenografts of human tumors and to induce necrosis within such tumors.

[0297] (ii) Therapeutic HER2-binding proteins and uses The in vivo properties of the HER2 binding proteins of the present disclosure, particularly with respect to tumor:blood ratio and rate of clearance, will be at least comparable to those of mAb104. Following administration to a human or animal subject, such specific binding members will exhibit a peak tumor:blood ratio of >1:1. Preferably, at such ratios, the specific binding members will also have a tumor:organ ratio of greater than 1:1, preferably greater than 2:1, and even more preferably greater than 5:1. Preferably, at such ratios, the binding proteins will also have an organ:blood ratio of <1:1 in organs distant from the tumor site. These ratios exclude organ catabolism and secretion of the administered binding protein. Thus, in the case of scFv and Fab, the binding members will be secreted via the kidney. In the case of whole IgG, clearance will be at least partially via the liver. The peak localization ratio of the intact antibody will typically be achieved between 10 and 200 hours after administration of the HER2 binding protein. More specifically, the ratio may be measured in tumor xenografts of approximately 0.2 to 1.0 g formed subcutaneously in one flank of athymic nude mice.

[0298] The HER2 binding proteins (e.g., antibodies) of the present disclosure may be labeled with a detectable or functional label or moiety. As will be appreciated by those of skill in the art, labels may be defined under more than one category. Detectable labels include, for example, isotopes 3 H, 14 C. 32 P, 35 P, 35 S, 36 Cl, 51 Cr, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I, 115 In, 211 At, 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc, 186Labels include, but are not limited to, radioactive labels such as Re, which may be linked to the antibodies of the present disclosure using conventional chemistry known in the art of antibody imaging. Labels also include fluorescent labels and labels conventionally used in the art for MRI-CT imaging. They also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin that can be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.

[0299] Functional labels include agents designed to target tumor sites and cause tumor tissue destruction. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase that can convert a prodrug to an active drug at the tumor site.

[0300] Antibodies, including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of binding proteins, antibodies, and / or their subunits may also have particular diagnostic applications and may be used to detect and / or measure conditions, such as cancers, precancerous lesions, and conditions associated with or caused by hyperproliferative cell proliferation, etc. For example, HER2 binding proteins, antibodies, or their subunits may be used to generate both polyclonal and monoclonal antibodies against themselves in various cell cultures by known techniques, such as hybridoma technology utilizing fused mouse splenic lymphocytes and myeloma cells. Similarly, small molecules that mimic or antagonize the activity(ies) of the HER2 binding proteins of the present disclosure may be discovered or synthesized and may be used in diagnostic and / or therapeutic protocols.

[0301] Radiolabeled HER2-binding proteins, particularly antibodies and fragments thereof, are useful in in vitro diagnostic techniques, in vivo radioimaging techniques, and radioimmunotherapy. For in vivo imaging, the HER2-binding proteins of the present invention may be conjugated to an imaging agent other than a radioisotope(s), including, but not limited to, a magnetic resonance imaging-enhancing agent, in which, for example, an antibody molecule is charged with multiple paramagnetic ions via 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 ferbium. In a further example of the present disclosure, radiolabeled HER2-binding proteins, particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabeled antibodies for cancer treatment. In yet a further example, radiolabeled HER2 binding proteins, particularly antibodies and fragments thereof, are useful in radioimmuno-guided surgery techniques, where they can identify and indicate the presence and / or location of cancer cells, precancerous cells, tumor cells, and / or hyperproliferative cells before, during, or after surgery to remove such cells.

[0302] Immunoconjugates or antibody fusion proteins of the present disclosure in which the HER2 binding proteins, particularly antibodies and fragments thereof, of the present disclosure are conjugated or linked to other molecules or agents further include, but are not limited to, binding proteins conjugated to chemoablative agents, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents or drugs.

[0303] Radioimmunotherapy (RAFT) has already begun and has demonstrated efficacy using various antibody immunoconjugates. 131 The I-labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 has been evaluated in colorectal cancer (Behr TM. et al. (2002), Cancer, 94(4 Suppl):1373-81). 90The same Y-labeled antibody has been evaluated in medullary thyroid carcinoma (Stein R. et al. (2002), Cancer, 94(1):51-61). Radioimmunotherapy using monoclonal antibodies has also been evaluated and reported for non-Hodgkin's lymphoma and pancreatic cancer (Goldenberg DM. (2001), Crit. Rev. Oncol. Hematol. 39(1-2):195-201; Gold DV. et al. (2001), Crit. Rev. Oncol. Hematol. 39, (1-2) 147-54). Methods for radioimmunotherapy with specific antibodies are also described in U.S. Patent Nos. 6,306,393 and 6,331,175.

[0304] Radioimmuno-guided surgery (RIGS), which involves the use of anti-CEA antibodies and antibodies against tumor-associated antigens, has demonstrated efficacy and usefulness (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):1092-8; McLosh DGet al. (1997), Cancer Biothcr Radiopharai. 12(4):2S7-94).

[0305] The HER2 binding proteins (e.g., antibodies) of the present disclosure may be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream or CSF, or directly to the site of the tumor. The exact dose will depend on a number of factors, including whether the antibody is diagnostic or therapeutic, the size and location of the tumor, the exact nature of the antibody (whether whole antibody, fragment, diabody, etc.), and the nature of any detectable or functional label attached to the antibody. When radionuclides are used in therapy, a preferred maximum single dose is about 45 mCi / m 2 , up to approximately 250 mCi / m 2The preferred dosage ranges are 15-40 mCi, with 20-30 mCi or 10-30 mCi being more preferred. Such therapy may require bone marrow or stem cell replacement. Typical antibody doses for either tumor imaging or tumor therapy range from 0.5-40 mg, preferably 1-4 mg of antibody in F(ab')2 form. Naked antibodies are preferably administered at doses of 20-1000 mg of protein per dose, or 20-500 mg of protein per dose, or 20-100 mg of protein per dose. This is the dose for a single treatment in adult patients; it may be adjusted proportionally for children and infants, and for other antibody formats proportional to molecular weight. Treatment may be repeated daily, twice weekly, weekly, or monthly, at the physician's discretion.

[0306] These formulations may also include a second binding protein, such as an EGFR or HER2 binding protein described herein. In a particularly preferred form, the second binding protein is trastuzumab.

[0307] (iii) Anticancer therapy The HER2 binding proteins (e.g., antibodies) of the present disclosure are useful in a variety of applications, including research, diagnostic, and therapeutic applications. In one example, the present disclosure provides a method of treating or preventing a disorder in a subject. As used herein, a "disorder" is a disruption or interruption of normal function.

[0308] (iv) Diagnostic assays The present disclosure also relates to various in vitro or in vivo diagnostic applications, including methods for detecting the presence of aberrantly expressed HER2 by reference to its ability to be recognized by the HER2 binding proteins (e.g., antibodies) of the present invention. Diagnostic applications of the antibody(ies) of the present invention include in vitro and in vivo applications that are well known and standard to those of skill in the art and are based on the present description. Diagnostic assays and kits for assessing and evaluating HER2 status in vitro, particularly with respect to aberrant expression of HER2, may be used to diagnose, evaluate, and monitor patient samples, including those known to have or suspected of having cancer, precancerous conditions, conditions associated with hyperproliferative cell growth, or derived from tumor samples. Assessment and evaluation of HER2 status is also useful for determining a patient's suitability for drug clinical trials or for administration of a specific binding member, particularly an antibody, of the present disclosure, including a particular chemotherapeutic agent or combination thereof, versus a different agent or antibody. This type of diagnostic monitoring and assessment has already been practiced using antibodies against the HER2 protein in breast cancer (Hercep Test, Dako Corporation), and this assay is also used to assess patients for antibody therapy with Herceptin. In vivo applications include tumor imaging or assessment of an individual's cancer status, including radiological imaging.

[0309] The presence of HER2 in cells can be confirmed by in vitro or in vivo immunological procedures 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 label. The most commonly employed labels for these tests are radioactive elements, enzymes, or chemicals that fluoresce when exposed to ultraviolet light. Many fluorescent substances are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA Blue, and Lucifer Yellow. Anti-HER2 antibodies can also be labeled with radioactive elements or enzymes. Radioactive labels can be detected by any of the currently available counting procedures. Preferred isotopes are 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I5, 111 In, 211 At, 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc, and 186Re. Enzyme labels are similarly useful and can be detected by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gas-measuring techniques. The enzyme is attached to the selected particle by reaction with a bridging molecule such as, for example, carbodiimide, diisocyanate, glutaraldehyde, or the like. Many enzymes that can be used in these procedures are known and available. Preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase and peroxidase, and alkaline phosphatase. U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 are referenced by way of example for their disclosure of alternative labeling materials and methods.

[0310] kit The present disclosure also contemplates the use of therapeutic or diagnostic kits comprising the HER2-binding proteins of the present disclosure for use in the therapeutic methods of the present invention. Such kits will generally contain a pharmaceutically acceptable formulation of at least one HER2-binding protein (e.g., an antibody or fragment) of the present disclosure in suitable container means. The kits can be used to detect the presence of HER2 receptors in biological samples. The antibody compositions of the present disclosure can be provided in liquid or lyophilized form, alone or in combination with additional antibodies specific for other epitopes. The antibodies, which can be labeled or unlabeled, can be included in the kit along with auxiliary components (e.g., buffers such as Tris, phosphate, and carbonate, stabilizers, excipients, biocides, 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 combination by the user. Generally, these auxiliary materials will be present in less than about 5% by weight based on the amount of active antibody, and usually in a total amount of at least about 0.001% by weight based on the antibody concentration. If a second antibody capable of binding to the antibody is employed, such antibody can be provided in the kit, for example, in a separate vial or container. The second antibody, if present, will usually be labeled and can be formulated in a manner similar to the antibody formulations described herein.

[0311] Commercially available test kits suitable for use by medical professionals may be prepared to determine the presence or absence of aberrant expression of HER2, including, but not limited to, amplified HER2, in suspected target cells. According to the testing techniques discussed above, one class of such kits will contain at least labeled HER2 or its binding partner, e.g., an antibody specific thereto, and, of course, instructions depending on the method selected, e.g., "competitive," "sandwich," "DASP," etc. The kit may also contain peripheral reagents such as, for example, buffers, stabilizers, etc.

[0312] therefore, (a) a predetermined amount of at least one labeled immunochemically reactive component obtained by direct or indirect linkage of a HER2 binding protein described herein or its specific binding partner to a detectable label; (b) with other reagents; A test kit for demonstrating the presence or potential of cells for aberrant expression of HER2 may be prepared, comprising (c) instructions for use of the kit.

[0313] More specifically, the diagnostic test kit comprises: (a) a known amount of a HER2-binding protein (or binding partner) as described above, or a plurality of such end products (or their binding partners), typically bound to a solid phase to form an immunoadsorbent, or alternatively, bound to a suitable tag, one of each; (b) with other reagents as needed; (c) Instructions for use of the test kit.

[0314] In a further example, test kits may be prepared and used for the above purposes, operating according to predetermined protocols (e.g., "competitive," "sandwich," "double antibody," etc.), which include: (a) a labeled component obtained by coupling a HER2 binding protein to a detectable label; (b) one or more additional immunochemical reagents, at least one of which is a ligand or an immobilized ligand, selected from the group consisting of: (i) a ligand capable of binding to the labeled component (a); (ii) a ligand capable of binding to the binding partner of labeled component (a); (iii) a ligand capable of binding to at least one of the determined component(s); and (iv) a ligand capable of binding to at least one of the binding partners of at least one of the components to be determined; and (c) Instructions for carrying out a protocol for detecting and / or determining one or more components of the immunochemical reaction between HER2, HER2 binding proteins, and their specific binding partners.

[0315] In accordance with the above, an assay system may be prepared to screen for potential drugs effective in modulating HER2 activity, aberrant HER2 expression, and / or HER2 binding protein activity or binding. The receptor or binding protein may be introduced into the test system, and a candidate drug may be introduced into the resulting cell culture, after which the culture may be examined to observe changes in the S-phase activity of the cells due to the addition of the candidate drug alone or due to the effect of additional amounts of a known drug(s).

[0316] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0317] The present invention is further illustrated in the following non-limiting examples. [Example]

[0318] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as shown in the above-described embodiments without departing from the scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0319] Materials and Methods Cell lines and culture conditions Parental lines were obtained from the American Type Culture Collection (ATCC, USA), Asterand Bioscience (USA), the Ludwig Institute for Cancer Research, or Cell Bank Australia (Australia). Cells were cultured in the supplier-recommended medium at 37°C in a 5% CO2 incubator. 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). Cells were passaged and the medium was replaced at 80% confluence. Cells were used for experiments in the exponential growth phase. To passage adherent cells, the medium was removed and an appropriate volume (based on growth surface area) of PBS solution containing 2 mM EDTA and trypsin (Life Technologies™, Australia) was added. The cell lines are listed in Table 1. [Table 1]

[0320] Antibodies and antigens Primary antibodies were purchased from commercial sources listed in Table 2 below or purified from hybridoma supernatants using protein-G affinity chromatography. [Table 2-1] [Table 2-2]

[0321] antigen Linear and cyclized peptide immunogens and an unrelated control peptide were chemically synthesized and conjugated to keyhole limpet hemocyanin (KLH) by Mimitopes Pty Ltd (Clayton, Australia). The linear peptide sequence was H-CPLHNQEVTAEDGTQR-NH2, and the cyclic peptide sequence was H-GCPLHNQEVTAEDGTQRC-NH2, where the N-terminal H represents a free amine. A control unrelated peptide conjugated to KLH, H-LEEKKGNYVVTDHC-NH2, was also prepared.

[0322] The HER2 extracellular domain (HER2-ECD) was generated in the laboratory of our collaborator, Professor A.W. Burgess (The Walter and Eliza Hall Institute of Medical Research), based on a previously published method (Xu Y, Soo P, Walker F, Zhang H.H., Redpath N, Tan C.W., et al. LRIG1 extracellular domain: Structure and function analysis. Journal of molecular biology. 2015;427(10):1934-48.). Briefly, synthetic DNA (GenScript®) corresponding to human HER2-ECD was cloned into an expression vector, expressed in Hi5 insect cells, and purified with 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.

[0323] Cell Biology Reagents and Suppliers Reagent details are provided in the table below. [Table 3]

[0324] Media and Solutions Agarose (1–1.5%): Agarose dissolved in 1x TAE to a final concentration of 1–1.5% w / v Agarose buffer 1x: 10 mM BisTris-HCl, pH 6.5; 0.2 mM EDTA; 100 mM NaCl Blocking buffer: Tris-buffered saline (TBS; 20 mM Tris-HCl, 150 mM NaCl) containing 5% w / v skim milk powder (Fonterra, Mount Waverly, Australia), 0.1% v / v Tween 20 (ICN Biomedicals). Diethanolamine-HCl buffer: diethanolamine (10% or 0.1 M), MgCl2.6H2O (1 mM), NaN3 DMEM-10: Dulbecco's modified Eagle's medium supplemented with 10% FCS, 2 mM L-alanyl-L-glutamine GlutaMAX™, 100 U / ml penicillin and 100 mg / ml streptomycin EDTA-PBS: 2 mM EDTA in PBS without Ca2+ or Mg2+ EDTA-PBS-3% FCS: EDTA-PBS supplemented with 3% FCS MATRIGEL™ Matrix: Composition MATRIGEL™ Matrix (Product Information) [Table B]

[0325] RIPA buffer: 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 10 mM NaF, and protease inhibitors (pH 7.5) RPMI-10 medium: RPMI-1640 containing 10% fetal calf serum (FCS), 2 mM L-alanyl-L-glutamine (GlutaMAX™), 100 U / ml penicillin, and 100 mg / ml streptomycin. Running buffer (Western blot): 20x NuPAGE® SDS running buffer (Invitrogen) diluted to 1x running buffer with milliq HO TBS (10x): 24.2 g Trizma® Base; 80 g Sodium Chloride; 970 ml H2O, adjust pH to 7.5 using HCl, total volume 1000 ml TBS-T: 1× TBS supplemented with 0.05% Tween® 20

[0326] cell biology equipment Details of the cell biology instrument are provided in the table below. [Table 4]

[0327] Immunization of mice and production of monoclonal antibodies Female BALB / c mice were immunized with 30 μg of a peptide containing the conformationally exposed region of the HER2 extracellular domain, synthesized as a cyclic peptide with the sequence H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 26), as determined by structural modeling, and conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. Injections were administered intraperitoneally at 4-week intervals. The antigen was prepared in phosphate-buffered saline (PBS, pH 7.2) and then mixed with Freund's complete adjuvant (Sigma, St. Louis, MO) for the first injection (Flies DB, Chen LA. Simple and rapid vortex method for preparing antigen / adjuvant emulsions for immunization. Journal of immunological methods. 2003;276(1):239-42) and Freund's incomplete adjuvant for the second injection. This was followed by two booster injections of the peptide immunogen alone. Three days after the final immunization, 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. Supernatants from growing cells were screened using indirect ELISA.

[0328] Enzyme-linked immunosorbent assay (ELISA) Polystyrene 96-well plates were coated overnight at 4°C in PBS with 3 μg / ml of HER2-ECD peptide-KLH antigen, negative control-KLH-conjugated peptide, or recombinant HER2-ECD as either linear or cyclic peptides. Plates were blocked with 3% FCS in PBS for 1 hour at room temperature (RT). Plates were incubated for 1 hour with serially diluted hybridoma supernatants starting at a 1:50 dilution along with appropriate controls. After three washes, plates were incubated for 1 hour at RT with HRP-conjugated anti-mouse IgG (1:2000 dilution). After three additional washes, phosphatase activity was measured using pNPP substrate reading absorbance (OD) at 405 nm using a Versamax microplate reader (Molecular Devices) with Softmax Pro 4.8 software.

[0329] Following identification of positive hybridomas, antibodies purified from four candidate clones (referred to herein as mAb104, mAb105, mAb106, or mAb107) were evaluated by ELISA. Polystyrene 96-well plates were coated with 3% FCS in PBS for 1 hour at room temperature. A final peptide concentration of 1 mg / ml was achieved by diluting the peptide with 0.1% acetic acid. This peptide solution was further diluted to 30 μg / ml in 3% FCS-PBS. Plates were incubated with linear or cyclic peptide immunogens conjugated to KLH, HER2-ECD, or negative control peptide-KLH in dilution buffer (3% FCS-PBS) for 1 hour at room temperature. After washing the plates three times with 0.05% Tween 20-PBS, wells were incubated with 10 μg / ml of mAb104, mAb105, mAb106, or mAb107 for an additional hour at room temperature. After washing, plates were then incubated with anti-mouse Ig-alkaline phosphatase (Sigma A-3688) (1:3000 dilution) for 1 h at RT. After three further washes, phosphatase activity was measured using pNPP substrate reading optical density absorbance at 405 nm using a Versamax microplate reader (Molecular Devices) with Softmax Pro 4.8 software.

[0330] Polystyrene 96-well plates were coated overnight at 4°C with 50 ml / well of recombinant ErbB2 ECD, ErbB3 ECD, ErbB4 ECD, or EGFR501 at 3 μg / ml in PBS. Plates were blocked with 3% FCS in PBS for 1 hour at room temperature (RT). Plates were incubated with serially diluted purified antibodies at 10 μg / ml along with appropriate controls for 1 hour at RT. After three washes, plates were incubated with AP-conjugated anti-mouse IgG (1:2000 dilution) for 1 hour at RT. After three additional washes, phosphatase activity was measured using pNPP substrate with absorbance (OD) reading at 405 nm using a SPECTROstar microplate reader (BMG LABTECH, Victoria, Australia).

[0331] FACS analysis Cells (1 × 10) seeded in a 96-well plate 4 ) were incubated with 10 μg / ml of anti-HER2 antibody or IgG1 isotype control antibody for 1 hour at 4°C. The humanized antibodies trastuzumab and pertuzumab were detected using an Alexa-488-conjugated anti-human IgG antibody. Bound mAb104, mAb105, mAb106, mAb107, or mouse isotype control LMH-3 was detected using an Alexa-488-conjugated anti-mouse IgG antibody, and fluorescence was read on a Becton Dickinson FACScan (CellQuestPro version 4.0.2). Negative controls included secondary antibody alone and background fluorescence of cells alone.

[0332] Biosensor analysis Surface plasmon resonance (SPR) kinetic analysis was performed on a BIAcore™ T200 system using a carboxymethyldextran-coated sensor chip (CM5-S, GE Life Sciences). The test channel was derivatized with HER2-ECD to 200 response units (RU) using standard amine coupling chemistry (0.05 M NHS / 0.2 M EDC). A blank control channel was derivatized with ethanolamine to correct for refractive index effects.

[0333] Samples of anti-HER2 antibodies, mAb104, mAb106, pertuzumab, or trastuzumab, were diluted 2-fold (2133 to 0 nM) in PBS / 0.005% Tween 20 buffer to concentrations ranging from 320 μg / ml to 0 μg / ml. Using PBS / 0.005% Tween 20 as the running buffer, samples were injected over the immobilized HER2-ECD at 45 μL / min for 200 s (30 μL at 10 μL / min). After the injection step, dissociation was monitored by flowing the running buffer over the chip surface for 600 s. Bound antibodies were eluted, and the chip surface was regenerated between samples by injecting 30 μL of 50 mM NaOH at 30 μL / min for 30 s.

[0334] Western blot analysis Western blot analysis was used to measure the reactivity of anti-HER2 monoclonal antibodies to native HER2. Trypsinized cells were lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 200 mM Na3VO4, 0.5% deoxycholate, 0.05% SDS, 10 mM NaF, and 1 mM protease inhibitor cocktail kit, Sigma-Aldrich, CA, USA) for 20 min and centrifuged at 17,000 rpm for 15 min. Ten μg of cell lysate protein was run on a 4-12% gradient Nu-PAGE gel and electrophoretically transferred to a nitrocellulose membrane using an iBlot® 2 gel transfer apparatus (ThermoFisher). The presence of EGFR and HER2 was assessed by probing blots with the respective antibodies, using commercially available antibodies and mAb104-bound HER2. Blots were viewed on a Storm 804 Phosphoimager (Amersham Bioscience) for analysis using ImageQuant TL image analysis software (version 2005).

[0335] immunohistochemistry To confirm the tumor selectivity of mAb104, an immunohistochemistry method was developed and used to screen various normal and tumor tissue types for mAb104 reactivity. Variations in antigen retrieval, primary antibody concentration, and incubation time were evaluated prior to optimizing the conditions as described. Below, only the final protocol is briefly described. Slides were placed in a 60°C oven for 30 minutes, followed by a 10-minute bath change and transfer to a xylene bath. Slides were then rehydrated in two changes of 100% ethanol for 10 minutes each, followed by a 70% ethanol bath for 10 minutes. Slides were rinsed three times with double-distilled (dd) H2O, each wash lasting approximately 2 minutes. Slides were then quenched in 3% H2O2 for 20 minutes. Antigen retrieval was achieved by treating the slides in a 10% (v / v) EDTA buffer bath at 100°C for 30 minutes. After cooling and washing with phosphate-buffered saline (PBS), the slides were preincubated with protein blocking reagent (SuperBlock™ T20, ThermoFisher®) for 60 minutes. Next, the slides were incubated with mAb104 primary antibody (2.5 μg / ml) for 60 minutes at room temperature. After staining with the primary antibody, bound antibody was detected using a streptavidin-horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Dakocytomation, Carpinteria, CA, USA). Bound antibody was detected with 3,3'-diaminobenzidine (DAB) substrate, counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated with ethanol and xylene, and mounted.

[0336] Using this method, the expression of these proteins was examined in 11 normal human tissues and 10 common tumor types (intraductal carcinoma of the breast, mesothelioma, colorectal and gastric adenocarcinoma, renal cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, prostate adenocarcinoma, and glioblastoma multiforme) from 9 to 27 different human donors using tissue microarrays (TMAs). Tumor and normal tissues were not derived from the same patient (i.e., unpaired). 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.

[0337] Ki-67: Expression of the human protein Ki-67 is strictly correlated with cell proliferation and is present in all active phases 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)). The antigen was retrieved for 20 minutes in 10% (v / v) aqueous citrate buffer (pH 6.0) at 100°C. After cooling, nonspecific binding sites were blocked with protein blocking reagent (SuperBlock™ T20, ThermoFisher®) for 20 minutes at room temperature. Rabbit anti-human Ki-67 primary antibody (RM-9106-S1, ThermoFisher®) diluted 1:100 in blocking buffer was incubated for 2 hours at room temperature. After washing away excess antibody, bound antibody was detected using a species-appropriate secondary antibody (Dakocytomation, Carpinteria, CA, USA) for 30 minutes at room temperature. Bound antibody was detected with 3,3'-diaminobenzidine (DAB) substrate. Slides were counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated in ethanol and xylene, and mounted.

[0338] Apoptosis: Apoptotic cells were detected using the terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay with an in situ cell death detection kit, fluorescein (11684795910, Roche, USA). Slides prepared from paraffin-embedded tissues were dewaxed and rehydrated as described above and rinsed three times with ddH2O, each wash lasting approximately 2 minutes. Tissue sections were incubated in proteinase K in working solution for 20 minutes at room temperature, followed by two PBS rinses. Positive and negative controls were prepared according to the manufacturer's specifications. 100 μl of the TUNEL reaction mixture or 100 μl of control labeling solution for the negative control was added to each slide and incubated for 60 minutes at 37°C in a humidified chamber. Following the incubation period, the slides were washed three times with PBS. After application, 50 μl of Covertor-POD was applied to the coverslipped slide to avoid evaporative loss and incubated for 30 minutes at 37°C in a humidified chamber. After three washes with PBS, 50–100 μl of DAB substrate was applied to the slide and incubated for 10 minutes at room temperature. Slides were washed with PBS and analyzed by light microscopy.

[0339] Podocalyxin: Slides generated from paraffin-embedded tissues were dewaxed and rehydrated as before. After quenching the slides with 3% H2O2 at room temperature for 20 minutes, antigen retrieval was performed by incubating the slides in 10% (v / v) citrate buffer (pH 6.0) at 100°C for 20 minutes. 15 μg / ml goat anti-mouse podocalyxin primary antibody (catalog no. AF1556, R&D Systems®) was added to each slide and incubated at room temperature for 2 hours. Sections were then washed, and bound antibody was detected using anti-goat HRP, counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated with ethanol and xylene, and mounted.

[0340] p-Akt: Slides generated from paraffin-embedded tissues were dewaxed by heating to 60°C and rehydrated in xylene and graded alcohols. After rinsing the slides three times with ddH2O, antigen retrieval was performed by incubating the slides in 0.01 M citrate buffer (pH 6.0) in a 95% water bath for 20 minutes. After cooling, the slides were rinsed sequentially with PBS and 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and Tween 20 (0.1%; TBS-T). Endogenous peroxidase activity was removed by incubation in TBS-T containing 3% hydrogen peroxide for 15 minutes at room temperature. Next, sections were incubated overnight at 4°C with primary antibody (rabbit polyclonal phospho-Akt (Ser473; Cell Signaling Technology, Beverly, MA, Cat. No. 9277, specific for IHC) diluted 1:100 in TBS-T. After washing the slides three times with TBS-T, each wash lasting approximately 2 minutes, the slides were incubated for 1 hour with rabbit biotinylated secondary antibody at a 1:200 dilution. Bound antibody was detected using DAB substrate, counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated in ethanol and xylene, and mounted.

[0341] Cell proliferation assay Cells (1 × 10) in serum-depleted medium 4) were seeded into 96-well microtiter plates and allowed to adhere overnight. The next day, antibodies were added in serial dilutions along with appropriate controls, and one plate was harvested for time 0 (T=0) measurements. The remaining cell plates were incubated for 3–5 days. Cell viability was assessed using a colorimetric MTS viability assay using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega, Australia) as the substrate. Absorbance was assessed 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 this value was subtracted from the 490 nm reading. Experiments were performed in triplicate and repeated for two to three 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).

[0342] Downstream signaling cells (1×10 6(1000 cells / well) were seeded in duplicate into 6-well plates and incubated overnight. The medium from each well was discarded and replaced with serum-free medium containing the desired antibody at a total concentration of 10 mg / ml. At the indicated time points (24 h), half of the wells were treated with 100 mg of EGF for 10 min at room temperature. The reaction was stopped by washing with ice-cold PBS, and the cells were lysed for 30 min in RIPA buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 200 mM Na3VO4, 0.5% deoxycholate, 0.05% SDS, 10 mM NaF, and 1 ml of protease inhibitor cocktail set, CA, USA]. This was followed by centrifugation at 17,000 rpm for 15 min. Total protein concentration was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hemel Hempstead, UK). MAPK activation was assessed by Western blot using commercially available 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. Anti-GAPDH (AbC-1001) antibody was purchased from AbClon. Bands were visualized using AbSignal (AbClon, AbC-3001).

[0343] Detection of cell death by enzyme-linked immunosorbent assay (ELISA) Cell death and apoptosis were assessed using an ELISA assay (Cell Death Detection ELISA Plus kit; Roche Molecular Biochemicals) according to the manufacturer's instructions (Holdenrieder S, Stieber P, Bodenmuller H, Fertig G, Furst H, Schmeller N, et al. Nucleosomes in serum as a marker for cell death. Clinical Chemistry and Laboratory Medicine. 2001;39(7):596-605). Briefly, cells were cultured in 96-well plates overnight. Cells were treated for 24 hours in serum-depleted (1%) growth medium with trastuzumab, pertuzumab, and mAb104 as monotherapy and in combination. Plates were centrifuged at 200 × g for 10 minutes at 4°C. The supernatant was carefully removed, and 200 μl of the manufacturer's lysis buffer was added and incubated at room temperature for 30 minutes. Following incubation, the plates were centrifuged, and 20 μl of the supernatant and cell lysis solution were transferred in triplicate to streptavidin-coated microplates. An additional 80 μL of immunoreagent containing a mixture of anti-histone-biotin and anti-DNA-POD was added to the supernatant. The plates were incubated for 2 hours at room temperature in a shaking incubator. The extent of apoptosis was quantitatively measured photometrically with ABTS (2,2'-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid) as a substrate, using a microplate reader at a wavelength of 405 nm and a reference wavelength of 490 nm using a Versamax microplate reader (Molecular Devices) running Softmax Pro 4.8 software.

[0344] Apoptosis assay Cell viability was measured using propidium iodide (PI) uptake and Annexin V binding. Briefly, cells (5 × 10 4(1000 cells / well) were cultured in 24-well plates overnight. Cells were treated with antibodies as monotherapy, in combination, or in medium along with appropriate controls for 24 hours. Cells were transferred to 96-well plates, washed three times with cold PBS, and then resuspended in binding buffer with 2.5 μl of FITC-annexin V and 2.5 μl of PI for 15 minutes in the dark at room temperature with gentle agitation. An additional 150 μl of binding buffer was added after incubation before flow cytometry analysis.

[0345] Migration (wound healing) assay To evaluate the effect of mAb 014 on cell migration, OE-19 cells (1 × 10 5 Cells (800 cells / well) were seeded into 6-well plates and grown to 80% confluence. Three parallel scratches were made in each well using a 100 μl pipette tip. Cells were treated with 100 μg / ml of the desired antibody or isotype control. Phase-controlled micrographs were taken starting immediately after 72 hours of treatment (designated TO).

[0346] Effect of mAb104 on ErbB receptor dimerization Cells were seeded in serum-depleted medium into 12-well plates and allowed to adhere overnight. In duplicate, cells were treated with 10 μg / ml of the relevant antibody or control for 1 hour. At the indicated time points, half of the wells were treated with 100 ng of EGF for 10 minutes at room temperature. The reaction was stopped by washing with ice-cold PBS, and the cells were incubated with BS3 (bis(sulfosuccinimidyl) substrate ((BS3), Pierce, Rockford, IL, USA) for 20 minutes at room temperature with gentle rocking 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, the cells were washed twice with cold PBS and lysed for 30 minutes in RIPA buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 200 mM Na3VO4, 0.5% deoxycholate, 0.05% SDS, 10 mM NaF, and 1 mM protease inhibitor cocktail (Biochem, CA, USA)]. Cell lysates were subjected to immunoprecipitation with the relevant antibodies and immunoblotted for EGFR and HER2.

[0347] DNA gel electrophoresis DNA gel electrophoresis was performed on gels containing 1% (w / v) DNA-grade agarose (Bioline) prepared in 1x Tris-acetate EDTA (TAE) buffer (Invitrogen) with SYBR® Safe DNA Gel Stain (Invitrogen). All DNA samples were diluted with a 10x stock of Orange G (Sigma) gel loading buffer before loading onto the agarose gel. Electrophoresis was performed at 70–150 V, and a 1 kb Plus DNA ladder (Invitrogen) was used as a reference for size estimation. DNA bands were visualized under UV light on a transilluminator (Bio-Rad) and photographed.

[0348] Hybridoma cDNA synthesis A High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, California, USA) was used for reverse transcription (RT) of 10 μL (approximately 1–5 μg) of RNA into cDNA using a 20 μL reaction size. cDNA synthesis was performed in 200 μL thin-walled polypropylene PCR tubes (Eppendorf, USA) using a T100™ (BIO-RAD, USA) or MasterCycler® (Eppendorf, USA) thermal cycler according to standard methods with the following reaction and cycling conditions: cDNA synthesis conditions: 25°C for 10 minutes 37°C for 120 minutes 85°C for 5 minutes Keep at 4°C

[0349] Mouse light chain variable region primers (i) ATG AAG TTG CCT GTT AGG CTG TTG GTG CTG (SEQ ID NO: 28) (ii) ATG GAG WCA GAC ACA CTC CTG YTA TGG GT (SEQ ID NO: 29) (iii) ATG AGT GTG CTC ACT CAG GTC CTG GSG TTG (SEQ ID NO: 30) (iv) ATG AGG RCC CCT GCT CAG WTT YTT GGM WTC TTG (SEQ ID NO: 31) (v) ATG GAT TTW CAG GTG CAG ATT WTC AGC TTC (SEQ ID NO: 32) (vi)ATG AGG TKC YYT GYT SAG YTY CTG RGG (SEQ ID NO: 33) (vii) ATG GGC WTC AAG ATG GAG TCA CAK WYY CWG G (SEQ ID NO: 34) (viii) ATG TGG GGA YCT KTT TYC MMT TTT TCA ATT G (SEQ ID NO: 35) (ix)ATG GTR TCC WCA SCT CAG TTC CTT G (SEQ ID NO: 36) (x) ATG TAT ATA TGT TTG TTG TCT ATT TCT (SEQ ID NO: 37) (xi)ATG GAA GCC CCA GCT CAG CTT CTC TTC C (SEQ ID NO: 38) (xii) ATG AAG TTT CCT TCT CAA CTT CTG CTC (SEQ ID NO: 39) Reverse primer sequence of mouse light chain variable region MKC: TGG ATG GTG GGA AGA TG (SEQ ID NO: 40) Mouse heavy chain variable region primers (i) ATG AAA TGC AGC TGG GTC ATS TTC TTC (SEQ ID NO: 41) (ii) ATG GGA TGG AGC TRA TCA TSY TCT T (SEQ ID NO: 42) (iii) ATG AAG WTG TGG TTA AAC TGG GTT TTT (SEQ ID NO: 43) (iv)ATG RAC TTT GWY TCA GCT TGR TTT (SEQ ID NO: 44) (v)ATG GAC TCC AGG CTC AAM AGT TTT CCT T (SEQ ID NO: 45) (vi)ATG GCT GTC YTR GSG CTR CTC TTC TGC (SEQ ID NO: 46) (vii) ATG GRA TGG AGC KGG RTC TTT MTC TT (SEQ ID NO: 47) (viii) ATG AGA GTG CTG ATT CTT TTG TG (SEQ ID NO: 48) (ix) ATG GMT TGG GTG TGG AMC TTG CTA TTC CTG (SEQ ID NO: 49) (x) ATG GGC AGA CTT ACA TTC TCA TTC CTG (SEQ ID NO: 50) (xi)ATG GAT TTT GGG CTG ATT TTT TTT ATT G (SEQ ID NO: 51) (xii) ATG ATG GTG TTA AGT CTT CTG TAC CTG (SEQ ID NO: 52) Reverse primer sequence of mouse heavy chain variable region MHC: CCAGTGGATAGACAGATG (SEQ ID NO: 53) Degenerate forward and reverse primers for the mouse light chain variable region Kappa F:GCC GAA TTC GAY ATT GTG MTS ACM CAR WCT MCA (SEQ ID NO: 54) Kappa R: CCG GTC GAC GGA TAC AGT TGG TGC AGC ATC (SEQ ID NO: 55) Symbol meaning: 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 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

[0350] polymerase chain reaction To amplify DNA fragments for cloning, Platinum® Pfx DNA polymerase (Invitrogen) was used according to the manufacturer's instructions. PCR reaction mixes were then run in 200 μL thin-walled polypropylene PCR tubes (Eppendorf) using a T100™ (BIO-RAD, USA) or MasterCycler® (Eppendorf) thermal cycler according to standard methods, with the following reaction and cycle conditions: PCR conditions 94°C for 3 minutes (94℃ for 1 minute → x℃ a for 55 to 90 seconds b → 72℃ for 2 minutes) for 15 to 25 cycles 72℃ / 10 minutes Keep at 4°C

[0351] In vivo testing NOD-SCID-IL2R - / - Mice (4–6 weeks old, Animal Research Centre, Perth, Australia) were inoculated with 5 × 10 6 NCI-N87 cells or 8 x 10 6 BT-474 cells were injected subcutaneously in the flank region in Matrigel (BD Biosciences). Mice injected with BT-474 cells were implanted with estrogen pellets 24 hours prior. 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. 3 Tumors were allowed to grow to a size of 1000 mm and mice were randomized into different treatment groups. Tumors that failed to engraft were excluded from further analysis. Treatment was administered via intraperitoneal injection at the indicated doses three times per week for 3 weeks. Animals were observed after treatment to determine if the mean tumor volume was >1000 mm. 3Mice were sacrificed when they reached the end of their normal range of risk or showed prolonged symptoms of stress. Postmortem tumors were excised, processed into formalin-fixed, paraffin-embedded sections, and collected for reverse-phase protein array (RPPA), with excess tissue stored at -80°C. At the end of treatment, the percentage of tumor growth inhibition (%TGI) was calculated as follows: %TGI = [1 - {T / T0 / C / C0} / 1 - {C0 / C}] x 100, where T = treated mean tumor volume at endpoint, T0 = treated mean tumor volume at time 0, C = control mean tumor volume at endpoint, and C0 = vehicle control mean tumor volume at time 0.

[0352] All animal study protocols were approved by the Austin Health Animal Ethics Committee (protocol number A2015 / 05297) and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th Edition 2013).

[0353] Reverse Phase Protein Array (RPPA) Proteins were extracted from HER2-overexpressing breast PDX tumors, and RPPA was performed 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). Tumor samples obtained at the end of treatment were lysed by homogenization using lysis buffer (catalog no. 9803, Cell Signaling Technology, Beverly, MA, USA) supplemented with protease and phosphatase inhibitors (Roche Applied Science catalog no. 05056489001, Penzberg, Germany). Protein concentration was determined using the Pierce™ BCA Protein Assay Kit and normalized to 1 mg / ml. Samples were boiled with 2-mercaptoethanol and SDS. The processed protein lysates were sent to MD Anderson Cancer Center, Houston, TX, USA for RPPA analysis as described below.

[0354] Lysates were serially diluted in lysis buffer in 5-fold serial dilutions to achieve a 1:16 dilution. Lysates were plated onto nitrocellulose-coated slides (Grace Biolab) in an 11 x 11 format. Samples were probed with 297 validated primary antibodies using a tyramide-based signal amplification approach and visualized by DAB colorimetric reaction. Slides were scanned, analyzed, and quantified using an Array-Pro Analyzer (Meyer Instruments, INC. Houston, TX) to generate spot intensities.

[0355] Each dilution curve was fitted to a logistic model ("Supercurve Fitting" developed by the Department of Bioinformatics and Computational Biology at MD Anderson Cancer Center, Houston, TX, USA). All data were normalized by median analysis of variance for protein loading correction factors and converted to linear values ​​using the median expression level of all antibody experiments. In the heatmap, "red" means above the median and "green" means below the median.

[0356] statistical analysis Analyses were performed using Prism® version 5.04. All p values ​​were two-sided, and values ​​of 0.05 or less were considered significant.

[0357] For comparison of means, Student's t-test or nonparametric Mann-Whitney U test was employed, and only two groups were considered. For comparisons between three or more groups, parametric data were analyzed by ANOVA, and if p ≤ 0.05, post-hoc tests using the Bonferroni method were performed to determine which group(s) were significantly different. The nonparametric test employed for multiple groups was the Kruskal-Wallis test, and if p ≤ 0.05, post-hoc tests were performed to determine which group(s) were significantly different.

[0358] Survival rates of the groups were also compared, and if the log-rank test was significantly different across all groups (p≦0.05), a further post-hoc log-rank test was performed to determine which group(s) were significantly different.

[0359] Example 1 - Generation and characterization of antibodies in vitro The immunization antigen and immunization protocol have been previously described. Through a series of immunization and screening strategies, including HER2 peptide, recombinant protein, and HER2-expressing cell-based assays, we finally succeeded in generating tumor-specific monoclonal antibodies against the conformationally flexible region of domain II of HER2.

[0360] We attempted an immunization strategy using linear peptides linked to biotin, GST, MBP, and KLH carrier proteins with Baf / 03 hematopoietic cells (which do not express HER members on the cell surface) transfected to express erbB2 with cysteine ​​mutations that exposed the peptide loop, but were unsuccessful in generating clones. Immunization with mutant-expressing cells and recombinant mutant ECD ErbB2 did not generate mAbs that bound the peptide but to different locations within the ECD of ErbB2. It was only once, when we immunized with cyclized peptides linked to KLH, that we were able to obtain monoclonal antibody clones that recognized the peptide and bound to erbB2-expressing cells. This is summarized in the following table (Table 5). [Table 5]

[0361] Using an immunization protocol, we generated hybridoma clones producing novel monoclonal antibodies (mAbs) directed against a conformationally exposed region of the HER2 extracellular domain that is believed to be available for binding only under conditions found on tumor cells. These monoclonal antibodies were generated against a conformational epitope by immunizing mice with a peptide immunogen derived from the HER2 extracellular domain, folded as a loop via a cysteine ​​(C) residue, and linked to KLH protein: H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 1). This sequence is underlined in the human HER2 sequence provided in Figure 1. This sequence was derived from the NCBI database at the following link: https: / / www.ncbi.nlm.nih.gov / protein / NP_004439.2.

[0362] 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).

[0363] Hybridoma screening Hybridoma culture supernatants designated mAb104, mAb105, mAb106, and mAb107 were screened for specificity using an ELISA-based assay for reactivity to loop (cyclic) and linear peptides of the HER2 extracellular domain (ECD), as well as the antigen against which the antibodies were raised. 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 Scientific Inc., IL, USA) and were all found to be IgG1 with a K-light chain. The results of the ELISA analysis of the purified mAbs are shown in Figure 2.

[0364] Monoclonal antibodies mAb104 and mAb106 showed the strongest binding activity for all peptide configurations, while mAb105 showed the lowest binding (see Figure 2B). Clones producing antibodies with the highest affinity, i.e., mAb104 and mAb106, were selected for further development, and these antibodies were selected for further analysis and characterization.

[0365] Hybridoma binding analysis by FACS The degree of binding of 10 μg / ml of purified antibodies (mAb104, mAb105, mAb106, and mAb107) to cellular HER2 was examined by flow cytometry in HER2-overexpressing breast cancer (BT474, SK-BR-3, and MDA-MB-453) and gastric cancer (NCI-N87) cell lines.

[0366] The results are summarized in Table 6 below and are representative of more than two experiments. [Table 6]

[0367] In all cell lines evaluated, mAb104 showed the highest binding compared to other antibodies. Of the cell lines evaluated, mAb104 showed the highest log shift in the NCI-N87 and SK-BR-3 cell lines. Binding of mAb105 was not observed in any of the cell lines evaluated. For all antibodies, binding was less than that of commercially available HER2-binding antibodies. The inventors' findings suggest that these antibodies bind to only a small fraction of the receptors on the cell surface, suggesting that the epitope is exposed on only a small portion of the HER2 receptor population.

[0368] Binding analysis by Western blot The ability of these novel antibodies to bind to HER2 protein was further evaluated in human breast cancer (BT474, SK-BR-3, and MDA-MB-453) and gastric cancer (NCI-N87) cell lines by Western blot analysis. Trypsinized cells were washed, lysed, and immunoblotted with each purified antibody. The anti-HER2 antibody, 2242 (Cell Signaling Technology, Beverly, MA), was used as a positive control.

[0369] Consistent with previous ELISA and FACS data, mAb104 showed the strongest binding across all cell lines (mAb104 > mAb106 > mAb107 > mAb105) (Fig. 3), binding to all four cell lines examined.

[0370] Sequences of mAb104 and mAb106 PCR reactions were purified using a BioLine Isolate II PCR and Gel Extraction Kit (BIO-52059), and the purified amplification products were sent for Sanger DNA sequencing at the Monash Micromon DNA sequencing facility, using the same primers as the sequencing primers to amplify the products.

[0371] The CDRs are defined according to both the Chothian and Kabat numbering systems and the amino acids of the CDRs that correspond to both. The variable region sequences of the heavy and light chains are shown in Figure 4.

[0372] The sequences of the complementarity determining regions of each antibody are provided below. VH chain of mAb104 CDR1: GYSFTGYFMH (SEQ ID NO: 14) CDR2: RINPYNGDIRYNQNFKD (SEQ ID NO: 16) CDR3: LNFAY (SEQ ID NO: 18) VL chain of mAb104 CDR1: KSSQSLLDSDGKTFLN (SEQ ID NO: 20) CDR2: LVSKLDS (SEQ ID NO: 22) CDR3: WQGTHFPWT (SEQ ID NO: 24) VH chain of mAb106 CDR1: GYTFTDYGMN (SEQ ID NO: 15) CDR2: WINTYTGKPTYDDDFKG (SEQ ID NO: 17) CDR3: RFLNTVAGRSVYFDY (SEQ ID NO: 19) VL chain of mAb106 CDR1: SVSSSVGSMY (SEQ ID NO: 21) CDR2: LTSNLAS (SEQ ID NO: 23) CDR3: QQWSSNPPT (SEQ ID NO: 25)

[0373] BIAcore analysis The fact that the epitope bound by mAb104 is adjacent to a disulfide bond suggests flexibility around the epitope site and exposure of the epitope for binding by mAb104 under certain conditions or circumstances.

[0374] The binding characteristics and apparent affinities of purified mAb104 and mAb106 for HER2 compared to trastuzumab were investigated by surface plasmon resonance (Biacore) using a BIAcore T200. After immobilizing recombinant HER2 extracellular domain onto a CM5 sensor chip, various concentrations of mAb104, mAb106, and a commercially available anti-HER2 mAb were passed over the sensor to measure apparent binding affinity.

[0375] As shown in Figure 5C, mAb104 exhibited high binding affinity, with a KD in the nanomolar range. The binding affinity of mAb104 is several logs lower than that reported for trastuzumab and similar to that of pertuzumab (Table 7). [Table 7]

[0376] In summary, mAb104, mAb106, and mAb107 bind to a variety of HER2-overexpressing tumor cells. mAb104 consistently appeared to exhibit stronger in vitro binding than mAb106. Based on these initial screening findings, mAb104 was selected for further evaluation.

[0377] Example 2: Epitope analysis and competition assays The mAb104 antibody variable domain, which binds an antigenic epitope located in domain II of HER2, was computationally predicted from a homology-modeled 3D structure of the antibody Fv domain and the known X-ray structure of human HER2 using a previously described method (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 (MAb4-5). Journal of Computer-Aided Molecular Design. 2013;27(6):539-50).

[0378] The predicted HER2 binding of mAb104 was compared with the known crystal structures of HER2 bound to 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 binding of mAb104 to HER2 requires a conformational change that occurs upon receptor activation, as previously described 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 tumor-associated EGFR. Proceedings of the National Academy of Sciences. 2009;106(13):5082-7), during which disulfide bonds in domain II of the HER2 ECD may be dynamically formed and broken.

[0379] Epitope recognized by mAb104 compared to other known HER2-binding antibodies The antibody H2-18 (Lu et al. (2016), Oncotarget, 7(41)) Chinese Patent CN104447993) recognizes an epitope within domain I of HER2 / ErbB2. H2-18 has been shown to inhibit the growth of trastuzumab-resistant breast cancer cells in vivo and in vitro, and induces programmed cell death in both trastuzumab-sensitive and -resistant breast cancer cell lines.

[0380] The antibody A21 (Hu S. et al. (2015), Oncotarget, 6(3):1695-1706) appears to recognize a conformational epitope primarily comprising a large region from the EC domain I of ErbB2. The antibody bivalency of A21 was found to be necessary for its inhibitory activity against tumor cells, as well as for ErbB2 phosphorylation and receptor downregulation.

[0381] Trastuzumab / Herceptin (4D5) binds to the juxtamembrane region of subdomain IV of ErbB2 and can prevent activation of ErbB2 by metalloproteinase cleavage and can also prevent ErbB2 dimerization.

[0382] Pertuzumab (2C4), which has an epitope within or near subdomain II, directly disrupts the association between ErbB2 and other ErbB receptors and can therefore inhibit tumor cell proliferation.

[0383] The epitopes recognized by pertuzumab and mAb104 share only three common amino acids, P294, L295, and H296, but H296 is understood to be completely hidden upon binding of pertuzumab (Franklin MC. et al. (2004), Cancer Cell, 5:317). The epitope to which pertuzumab binds consists of H245, Y252, F257, D285, V286, S288, T290, P294, L295, H296, K311, K314, and P315.

[0384] In contrast, the epitope recognized by mAb104 is the sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1).

[0385] While both antibodies recognize epitopes within domain II of HER2, the epitopes bound by pertuzumab and mAb104 are clearly different.

[0386] Without wishing to be bound by theory, the inventors hypothesize that Pertuzumab and mAb104 bind to opposite faces / sides of domain II of HER2 / ErbB2, explaining why mAb104 does not block Pertuzumab binding despite closely opposing epitopes. Conformational changes in domain II, which occur, for example, during activation, hypoxia, and / or abnormal expression, allow both antibodies to bind to a small subpopulation of HER2 receptors undergoing such conformational changes, allowing only mAb104 to bind to CPLHNQEVTAEDGTQRC in domain II. However, the required conformational change may be possible if, for example, the disulfide bond (C277 / C289 (numbering without the leader sequence)) preceding the mAb104 epitope (C293 / C309 (numbering without the leader sequence)) is temporarily broken or undergoes a disulfide bond switch that allows rearrangement of at least a portion of domain II exposing the mAb104 epitope. The epitope to which Pertuzumab binds may remain largely undisturbed during rearrangement. However, computational analysis using recombinant HER2-ECD adsorbed for ELISA capture shows that although the structural rearrangement of domain II is fixed, it is possible that slight steric hindrance due to the binding of mAb104 may result in reduced Pertuzumab binding (e.g., as shown below). The potential loss of binding affinity may be balanced by the synergistic interaction between the two antibodies.

[0387] Competition assay To further define the epitope of mAb104, we used ELISA to compare the binding of mAb104 to the HER2-ECD and its ability to interfere with the binding of the domain II-binding antibody pertuzumab and the binding of the spatially distant domain IV epitope of trastuzumab (Figure 6). In these experiments, we assessed the effect of preincubation with mAb014 on the binding of trastuzumab and pertuzumab (Figures 6B and C) and also determined the effect of preincubation with trastuzumab and pertuzumab on mAb104 binding.

[0388] We showed that trastuzumab and mAb104 did not affect each other's binding to HER2-ECD (Figures 6A and 6B). We also demonstrated that preincubation with pertuzumab did not affect the binding of mAb104 (Figure 6A). Interestingly, however, preincubation with mAb104 reduced the binding of pertuzumab to HER2-ECD (Figure 6C), indicating that the binding of mAb104 to the epitope may sterically hinder pertuzumab under certain circumstances.

[0389] Competition between mAb104 and pertuzumab and trastuzumab for endogenous HER2 was further examined by flow cytometry in HER2-overexpressing breast cancer cell lines (BT474 and SK-BR-3; Figures 7-1 and 7-2) and gastric cancer cell lines (NCI-N87 and OE19; Figures 7-3 and 7-4) using the two sequential incubation approaches discussed above. In these series of experiments, preincubation with a high dose (100 μg / ml) of mAb104 was utilized to maximize variability in determining its effect on trastuzumab and pertuzumab binding. Preincubation with much higher doses of mAb104 did not affect the binding of either trastuzumab or pertuzumab to cell surface HER2. The discrepancy in results for mAb104 competing with pertuzumab using flow cytometry and ELISA may be explained by differences between the antigenic preparations in the assays, i.e., the presence of HER2 partially denatured in ELISA versus in its physiological conformation when analyzed by flow cytometry, and the resulting epitope presentation and availability.

[0390] Example 3: Binding of mAb104 to cell surface HER2 We investigated the pattern and efficiency of mAb104 binding by FACS analysis using a panel of cell lines with differential HER2 expression.

[0391] These results are summarized below in Table 8. Results are compared to binding with the secondary antibody alone. [Table 8] FACS analysis of mAb104 binding to HER2-expressing cells: BT474, SK-BR-3, NCI-N87, OE-19, MDA-MB-231, and MCF7 cells were incubated with 10 μg / ml of trastuzumab, pertuzumab, mAb104, or secondary antibody alone, and the extent of binding was determined by FACS analysis. Results are representative of two or more experiments.

[0392] Among HER2-overexpressing cell lines, mAb104 showed the strongest binding to the HER2 subpopulation in the gastric cancer cell line NCI-N87, whereas only negligible HER2 binding was observed in the HER2-low expressing cell lines (MDA-MB-231 and MCF-7).

[0393] FACS demonstrated greater fluorescence for trastuzumab (Herceptin) and pertuzumab, indicating that they bound to more HER2 receptors in all cell lines evaluated compared to mAb104 (Table 8), with no difference in the extent of binding observed between the two humanized antibodies across the various cell lines. Our findings support the hypothesis that mAb104 binds to a subset of receptors on the cell surface and explain the observed differences in the extent of binding between the antibodies.

[0394] Specificity of mAb104 for HER2 To confirm the specificity of mAb104 for endogenously expressed HER2 and HER3, we performed Western blot assays using lysates from various HER2-positive and -negative cancer cell lines prepared under reducing conditions (Figure 8-1).

[0395] Antibody 2242, a commercially available antibody from Cell Signaling Technology raised against an intracellular HER2 epitope, was used as a global HER2 positive control. As demonstrated in Figure 8A, mAb104 showed strong reactivity across a variety of HER2-expressing cancer cell lines, comparable to the control antibody in providing a positive signal representative of the HER2 overexpression status of the various cell lines. The correlation between HER2 binding of mAb104 and control 2242 is a reflection of the HER2 epitope recognized by mAb104, as revealed under reducing conditions. HER3 expression levels were very low in the cancer cell lines examined, as confirmed by FACS analysis and in other previous 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-3 and BT474 breast carcinoma cells. Cytometry Part. A. 2001;44(4):338-48.).

[0396] HER2 / ErbB2 specificity was also assessed by ELISA assay. As shown in Figure 8-2, mAb104 was specific for ErbB2 / HER2 and did not bind the extracellular domain of EGFR / HER1 or the ECD of ErbB3 / HER3 or ErbB4 / HER4.

[0397] Efficacy of mAb104 in breast cancer in vitro Example 4: Antiproliferative effect of mAb104 The effect of mAb104 as monotherapy and in combination with trastuzumab or pertuzumab on the proliferation of HER2-overexpressing breast cancer cell lines was measured by MTS cell proliferation assay in serum-starved conditions (1% FCS) using increasing concentrations up to a maximum concentration of 100 μg / ml (Figure 9).

[0398] Trastuzumab significantly reduced proliferation of BT-474 (Figure 9B) and SK-BR-3 (Figure 9A) compared to an isotype control antibody (p=0.0006 and p=0.0005, respectively; two-sided), whereas pertuzumab monotherapy had no significant antiproliferative activity in the cell lines evaluated (p=0.22 and p=0.15, respectively; two-sided; see Figures 9A and 9B). These findings are supported by 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. 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 MC, 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 BK,Lee SY,Lee YH,Hwang IS,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). .

[0399] mAb104 did not show significant growth inhibition compared to the isotype control antibody in either the SK-BR-3 or BT-474 cell lines (p=0.33 and p=0.2, respectively; two-tailed) (FIGS. 9A and 9B).

[0400] As shown in Figures 9C and 9D, the combination of trastuzumab and pertuzumab significantly inhibited the proliferation of the evaluated cell lines (BT474, p=0.0008 and SK-BR-3, p=0.0007; two-sided); however, there was no statistically significant difference compared to trastuzumab monotherapy (BT-474, p=0.59 and SK-BR-3, p=0.51; two-sided, respectively).

[0401] The addition of mAb104 to trastuzumab or pertuzumab did not affect the individual antiproliferative activities of trastuzumab and pertuzumab (Figure 9C-F). The antiproliferative effect of mAb104 in combination with trastuzumab in the evaluated cell lines was not statistically different from that of trastuzumab in combination with pertuzumab (BT-474, p = 0.66; SK-BR-3, p = 0.47).

[0402] Thus, unlike trastuzumab and pertuzumab, mAb104 had no detectable antiproliferative effect against HER2-positive cell lines. The complex interplay between multiple receptor kinases and signaling pathways that occurs in vivo cannot always be fully recapitulated in vitro, and the effects of therapeutic agents that require activated receptors undergoing conformational changes may not be measurable in vitro. Antibodies targeting conformationally exposed epitopes on activated EGFR have shown significant antitumor activity in vivo despite failing to demonstrate any growth inhibition or altered signaling 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].

[0403] Example 5: Effects of mAb104 on ErbB receptors and downstream signaling pathways Given the functional differences observed with mAb104 in proliferation assays, we sought to examine the effects of mAb104 on MAPK and Akt ligand-independent pathways in serum-starved SK-BR-3 and BT-474 breast cancer cell lines after 24 hours of treatment. To assess the effects of mAb104 on ligand-dependent signaling pathways, cell lines were treated with anti-HER2 antibody for 24 hours, followed by the addition of 100 ng of EGF for 10 minutes.

[0404] The results of the ligand-independent and -dependent effects are shown in Figures 10 and 11, respectively. 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), we focused on EGFR-HER2 signaling because HER3 expression levels were very low in the cancer cell lines, as shown in Figure 8.

[0405] In this set of experiments (Figures 10 and 11), we demonstrated that 24-hour treatment with trastuzumab and pertuzumab did not significantly reduce overall 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).

[0406] The effects of anti-HER2 antibodies were also assessed on the Akt and MAPK pathways using phosphorylation-specific antibodies. In both breast cancer cell lines (BT-474 and SK-BR-3), trastuzumab treatment resulted in a decrease in Akt phosphorylation without altering total Akt protein levels, indicating a decrease in Akt phosphorylation activity rather than downregulation of Akt protein. These findings are consistent with other studies (Lu Q, et al. supra; Yakes FM, et al. (2002), Cancer Research. 62(14):4132-41).

[0407] In BT-474 cells, trastuzumab treatment resulted in a decrease in MAPK activity, as indicated by a decrease in phospho-MAPK (Figures 10C and 10D). In contrast, no change in MAPK activity was observed in SK-BR-3 cells (Figures 10A and 10B), consistent with other studies (Cuello M, et al. (2001), Cancer research. 61(12):4892-900).

[0408] As shown in Figures 10A and B, treatment with mAb104 as monotherapy did not produce detectable changes in the amount of total or phosphorylated protein in the cell lines evaluated.

[0409] In BT-474 cells, the combination of trastuzumab and pertuzumab significantly reduced the levels of phospho-Akt and phosphorylated p44 / p42 MAPK compared with either drug alone, but had no effect on total Akt or MAPK (Figure 10D). In contrast, in SK-BR-3 cells, signaling through the MAPK cascade was not inhibited by the drug combination, as indicated by unchanged 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 with the combination of trastuzumab and mAb104 and with trastuzumab and pertuzumab. There was no significant difference in the degree of downregulation between the two drug combinations (Figures 10C and D).

[0410] Under ligand-stimulated conditions (Figure 11), trastuzumab and pertuzumab did not affect the MAPK or Akt signaling pathways in BT-474 cells (Figures 11A and 11B). In the SK-BR-3 cell line, pretreatment with pertuzumab resulted in a decrease in Akt phosphorylation 10 min after EGF stimulation, but total Akt levels remained unchanged (Figure 11A). These findings are consistent with other reports (Henjes F, et al. (2012), Oncogenesis, 1(7):e16). Trastuzumab inhibits Akt-mediated signaling due to the abrogation of ligand-independent signaling, whereas pertuzumab blocks ligand-induced signaling. Addition of EGF to cells treated with mAb104 did not result in changes in the total or phosphorylated levels of MAPK and Akt pathway proteins in the breast cancer cell lines evaluated. In both cell lines, treatment with various combinations of mAb104 had no effect on MAPK or Akt signaling compared to control antibodies (FIGS. 11C and D).

[0411] Example 6: Effect of mAb104 on apoptosis in vitro The apoptosis-inducing activity of mAb104 was measured in BT-474 and SK-BR-3 cells using flow cytometry with the Dead Cell Apoptosis Kit (ThermoFisher Scientific, Catalog No. V13241). The fractions of early and late apoptotic cells were quantified by quadrant analysis. Compared with cells alone, treatment with trastuzumab or pertuzumab did not induce apoptosis (Figure 12), consistent with previous studies (Rockhoff G, et al. (2007), Cell proliferation. 40(4):488-507; Nahta Ret al. (2004), Cancer Research, 64(7):2343-6; Lu Q, et al. supra). Compared with BT-474 (Figure 12A-G), treatment of SK-BR-3 cells with trastuzumab resulted in a higher number of apoptotic cells, but this was not statistically significant (Figure 12I-O). Similarly, mAb104 did not induce significant apoptosis in the cell lines evaluated compared to cells alone (p=0.494). No differences in apoptotic activity were observed between the antibodies (p=0.726).

[0412] No increase in apoptotic activity was observed in cells exposed to the combination of trastuzumab and pertuzumab or mAb104 compared to single-agent treatment. In BT-474, 89.9% of cells survived after treatment with trastuzumab monotherapy, compared with 91.8% after treatment with trastuzumab and mAb104 (Figure 12A-G). No difference in apoptotic activity was observed between the combination of trastuzumab and pertuzumab or mAb104 (Figure 12A-F and G).

[0413] An interesting finding was the large number of necrotic cells seen after treatment with mAb104 compared to cells alone, as well as with trastuzumab and pertuzumab (Figures 12H and 12P), although this difference was not statistically significant. For BT-474 cells, 1.8% of cells were necrotic compared to <0.5% of cells in all other groups; similarly, after treatment with mAb104, more SK-BR-3 cells became necrotic than the other treatment groups (Figure 12P), although 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.

[0414] The complex interplay of multiple receptor kinases and signaling pathways that occurs in vivo cannot always be fully recapitulated in vitro and may reflect differences in HER2 processing and function. The influence of the tumor microenvironment on HER2 function or other in vivo factors is required to demonstrate the functional efficacy of mAb104, which targets a conformationally exposed epitope on HER2. Antibodies targeting a conformationally exposed epitope on activated EGFR have shown significant antitumor activity in vivo despite failing to demonstrate any growth inhibition or altered signaling 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).

[0415] Despite the lack of detectable in vitro activity seen with mAb104, we proceeded to investigate its efficacy in vivo in HER2-overexpressing cancer xenograft models.

[0416] In vivo efficacy of mAb104 Example 7: Efficacy of mAb104 monotherapy in HER2-overexpressing / amplified ER-positive breast cancer xenografts We evaluated the efficacy of mAb104 in mice bearing established tumor xenografts of the ER-positive, HER2-overexpressing breast cancer cell line, BT-474. 3 Once this was reached, a dose of 1 mg / antibody treatment of mAb104, trastuzumab, pertuzumab, or control antibody was administered three times weekly for three weeks.

[0417] The results are presented in Figure 13. At the end of treatment (day 32), all tumors in the treatment groups were significantly smaller than those in the control group (ANOVA, p<0.0006). Post-hoc testing using the Bonferroni method showed that all treatment groups were significantly smaller (p≦0.001) compared to the control group. The mean tumor volume at day 32 was 337.2 mm 3 (control group), 4.8 mm 3 (trastuzumab), 6.7 mm 3 (pertuzumab), and 48.7 mm 3 In the trastuzumab and pertuzumab treatment groups, significant antitumor responses persisted until the end of the study (day 39), one week after treatment was stopped. However, with mAb104, tumor growth resumed once treatment was stopped. At the end of the study (day 39), there was no significant difference in growth inhibition between the treatment groups (p=0.14).

[0418] We also performed a multicenter study of established BT-474 tumor xenografts (120–150 mm 3 The efficacy of low dose 0.5 mg / antibody treatment of mAb104, trastuzumab, pertuzumab and control IgG administered three times weekly for three weeks was evaluated in patients with rhesus malabsorption (rhesus malabsorption) and rhesus malabsorption (rhesus malabsorption).

[0419] The results are presented in Figure 14. Trastuzumab treatment prevented further tumor growth; pertuzumab and mAb104 reduced the tumor growth rate compared to the control group. At the end of the study (day 52), tumors in all treatment groups were significantly smaller than the control group (ANOVA, p<0.038). The mean tumor volume was 927.5 mm 3 (control), 182.4 mm 3 (trastuzumab), 415.0 mm 3 (Pertuzumab) and 469.1 mm 3 (mAb104). Post-hoc testing using the Bonferroni method showed that mice in the treated groups had significantly smaller tumors than the control group (trastuzumab, p = 0.0035; pertuzumab, p = 0.02; and mAb014, p = 0.008). At 0.5 mg / ml, mAb104 exhibited similar antitumor efficacy to pertuzumab in this model (p = 0.97, two-tailed). While treatment with trastuzumab resulted in numerically greater tumor growth inhibition, there were no significant differences between trastuzumab and pertuzumab (p = 0.22, two-tailed) or mAb104 (p = 0.15, two-tailed) at the end of the study (day 51).

[0420] Survival analysis by log-rank analysis showed that mice treated with anti-HER2 antibody were significantly lower than those in the control group due to ethical considerations (i.e., tumor size ≤1000 mm). 3 The mice in the treatment group had a significantly (p<0.002) higher survival rate than the control group at the time of culling for IVF. The median survival time for the control group mice was 41 days, while the median survival time had not been reached for the treated mice by the time the experiment was terminated (day 52).

[0421] Example 8: Efficacy of mAb104 monotherapy in HER2-overexpressing / amplified breast cancer PDX models We evaluated the efficacy of mAb104 in a patient-derived xenograft (PDX) model of breast cancer overexpressing / amplified HER2. Because the donor samples were untreated, tumor sensitivity to anti-HER2 therapy was assumed to be 100%. On day 64, established tumor volumes were 100-120 mm. 3If the tumor growth rate was between 0.5 and 100 mg / kg, mice were treated with mAb104, trastuzumab, pertuzumab, or control IgG at a total dose of 0.5 mg three times a week for three weeks. The results of the tumor growth curves are presented in Figure 15A.

[0422] Anti-HER2 therapy had an immediate effect on PDX growth rates. Following cessation of treatment on day 86, all anti-HER2 treatments demonstrated comparable anti-tumor efficacy, with tumor growth rate delay continuing until approximately day 125, when the tumor growth curve began to parallel that of the control group. At the end of the study, on day 145, the control group was terminated due to ethical considerations. At 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 group (trastuzumab, p=0.02; pertuzumab, p=0.02; mAb014, p=0.038). The mean tumor volume was 1099.2 mm. 3 (control), 761.2 mm 3 (mAb104), 632.8mm 3 (trastuzumab) and 691.3 mm 3 mAb104 demonstrated potent antitumor activity in this model comparable to approved HER2-targeted therapies, but there were no significant differences between the anti-HER2 therapies (p = 0.547 (two-sided) for trastuzumab vs. mAb104 and p = 0.754 (two-sided) for pertuzumab vs. mAb104).

[0423] Survival analysis showed that mice in the treated groups had significantly longer survival times than the control group (p<0.0005), and post-hoc tests showed that all groups treated with anti-HER2 antibodies survived significantly longer than control mice (p<0.001). The median survival time for control mice was 145 days, while the median survival time for treated mice had not been reached by the time the experiment was terminated.

[0424] Example 9: Efficacy of mAb104 in combination with trastuzumab in HER2-overexpressing / amplified ER-positive breast cancer xenografts The combination of trastuzumab and pertuzumab has documented more effective antitumor activity and prevention of metastatic tumor spread compared to either antibody alone, regardless of HER2 expression. Given the distinct domain II epitope binding site of mAb104 compared to trastuzumab and pertuzumab, and our observation of the potent antitumor activity of mAb104 as monotherapy in vivo, we proceeded to evaluate mAb104 in combination with trastuzumab compared to trastuzumab alone or in combination with pertuzumab.

[0425] We evaluated the effect of mAb104 in combination with trastuzumab in an established BT-474 breast cancer xenograft tumor model. Each mouse received 0.25 mg of trastuzumab and 0.25 mg of mAb104 or pertuzumab, three times per week for 3 weeks, to achieve a total dose of 0.5 mg / treatment or equivalent control antibody. Average tumor volumes were 100-120 mm. 3 Once this was achieved, processing began.

[0426] The results are presented in Figure 15B. The antitumor effect was evident within 10 days of treatment initiation and continued after treatment was stopped. At the end of the study period on day 50, the control group was terminated for ethical reasons due to tumor burden. The mean ± SD tumor volumes of the trastuzumab alone and combination groups were significantly smaller than the control group (ANOVA, p<0.0001). Post-hoc tests were then performed with the Bonferroni method. Tumors treated with the combination therapy were significantly smaller than the control group (p<0.0001), measuring 88.9 mm. 3 (mAb104 + trastuzumab) and 43.6 mm 3 (trastuzumab + pertuzumab). No complete tumor regression was observed in any of the treatment groups (Figure 15B). Combination treatment with mAb104 and trastuzumab resulted in greater tumor shrinkage compared to trastuzumab alone, but the difference in tumor size between the combination and monotherapy groups was not statistically significant (p = 0.09 by ANOVA).

[0427] Survival analysis showed that mice in both combination groups had significantly longer survival times than the control group (p<0.002). The median survival time for the control group was 44 days, while the median survival time was not reached for mice in the combination-treated group. Log-rank analysis showed no statistical difference between the two combination groups (p=0.21, two-tailed); treatment with mAb104 and trastuzumab significantly inhibited tumor growth compared to mAb104 monotherapy (p0.04, two-tailed) (Figure 15A).

[0428] This therefore suggests that mAb104 in combination with trastuzumab provides enhanced antitumor activity compared to either monotherapy alone.

[0429] Example 10: Efficacy of mAb104 in combination with trastuzumab in HER2-overexpressing / amplified ER-positive breast cancer PDX models We evaluated the effect of co-antibody treatment in HER2-overexpressing / amplified breast cancer PDX models. Because donor samples were anti-HER2 naive, tumor sensitivity to treatment was assumed to be 100%. Established tumor volumes of 100-120 mm were used. 3 If between 0.01 and 0.15, mice were treated with a total treatment dose of 0.5 mg trastuzumab or isotype control alone, or mAb104 and trastuzumab in combination, or trastuzumab plus pertuzumab, three times a week for three weeks.

[0430] At the completion of treatment on day 85, significant differences were observed between all treatment groups compared to the control group (p<0.0001) (Figure 15C). Furthermore, the combination group was more effective than trastuzumab alone (p=0.001). The greater antitumor effect of the combination group continued until the end of the study on day 145, when the control group was terminated for ethical reasons due to tumor burden. At day 145, tumors in all treatment groups remained significantly smaller than the control group (p<0.0001). The mean tumor volume was 164.4 mm 3 (mAb104 + trastuzumab) and 84.2 mm 3(trastuzumab + 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 observed in any of the treatment groups.

[0431] Compared with trastuzumab monotherapy, cotreatment with mAb104 and trastuzumab resulted in significantly greater tumor volume reduction (p<0.0001 by ANOVA), followed by post-hoc testing with the Bonferroni method. Tumors in the combination group were significantly smaller than those treated with trastuzumab alone (p<0.0049).

[0432] Survival analysis showed that mice in both combination groups had significantly longer survival times than the control group (p<0.0005) as well as the single-treatment groups (p<0.0014). The median survival time for the control group was 145 days, but this was not reached for mice in the combination treatment group.

[0433] The results in Figures 15A and C show that after 145 days, the mean tumor size for trastuzumab alone was 632.8 mm 3 and 761.2 mm for mAb104 3 When mAb104 was combined with trastuzumab, tumor size was reduced substantially to 164.4 mm 3 This reduction in tumor size suggests that the combination of mAb104 with trastuzumab resulted in enhanced antitumor activity compared with monotherapy with either trastuzumab or mAb104 alone.

[0434] Example 11: Reverse Phase Protein Array (RPPA) Analysis Lysates from HER2- breast PDX tumors (n=2 / group) were collected at the completion of treatment on day 85 and analyzed by RPPA. This RPPA analysis included a panel of over 300 antibodies detecting total proteins and / or their activated forms. Key proteins are involved in critical signaling pathways, including the phosphatidylinositol 3-kinase (PI3K) / Akt pathway, the extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) pathway, the Janus kinase (JAK) / signal transducer and activator of transcription (STAT) pathway, apoptosis pathways, and the cell cycle, including cell death and survival. Collected data were normalized to protein load and transformed into linear values ​​for analysis. Percent fold changes were calculated as the ratio of differences in protein expression between the control group and tumor samples treated with either trastuzumab, pertuzumab, mA104, or their combination.

[0435] Although mAb104 exhibited significant antitumor activity in the HER2 breast cancer PDX model, no significant changes in protein levels were observed compared to other antibodies evaluated (Table 9). [Table 9-1] [Table 9-2]

[0436] Example 12: Immunohistochemical analysis of established tumors Mice (n=2) bearing established BT-474 breast cancer xenografts from each of the monotherapy and combination treatment groups were sacrificed 1 day after the last treatment, and xenograft tissue samples were obtained and prepared for IHC analysis of tumor growth, downstream signaling, and angiogenesis.

[0437] The effect of mAb104 monotherapy on tumor growth was examined by Ki67 staining, and the results are presented in Figure 16A. In BT-474 xenograft tumors, treatment with anti-HER2 antibodies did not significantly reduce growth compared to the control group (p = 0.625 by ANOVA; post-hoc analysis showed no difference between the different treatment groups). The mean H-scores were 102.6 (control), 83.9 (trastuzumab), 91.1 (pertuzumab), and 99.7 (mAb104).

[0438] To determine whether the antiproliferative effect was mediated by downregulation of the Akt pathway, Akt was assessed by phosphoprotein assay (Figure 16B). For BT-474, the H-scores for phospho-Akt were not significantly different between the treatment and control groups (p = 0.958 by ANOVA, post-hoc analysis showed no differences between treatment groups). The mean H-scores were 129.6 (control), 124.3 (trastuzumab), 114.6 (pertuzumab), and 132.5 (mAb104).

[0439] Because trastuzumab has been shown to have antiangiogenic effects (Parakh S, (2017), Cancer Treatment Reviews. 59:1-21), we investigated the effect of mAb104 on the microvascular density of tumor tissue by staining for podocalyxin (Figure 16C). Immunohistochemical staining was performed as previously described. Microvascular density (%) was calculated as the ratio of the positively stained area in the viable region to the total observed area. In BT-474 xenograft tumors, significant antiangiogenic activity of trastuzumab was observed (p<0.001), whereas mAb104 and pertuzumab had no significant effect on tumor vasculature compared to control antibodies (p=0.987).

[0440] 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-tailed). The mean H-scores were 129.6 (control), 7.4 (trastuzumab + pertuzumab), and 97.6 (trastuzumab + mAb104) (Figure 16D). These findings are consistent with a previous report (Brockhoff G, (2007), Cell proliferation. 40(4):488-507).

[0441] No effect on pAkt (FIG. 16E) or angiogenesis (FIG. 16F) was observed in BT-474 tumor samples treated with trastuzumab plus pertuzumab or trastuzumab plus mAb104 when compared to control antibodies.

[0442] stomach cancer Example 13: Antiproliferative effect of mAb104 in vitro In vitro, trastuzumab significantly (p<0.0001) inhibited the proliferation of NCI-N87 and affected the proliferation of OE-19 gastric cancer cells (Figure 17), consistent with previous reports (Gravalos C, et al. (2008), Annals of Oncology. 19(9):1523-9). Pertuzumab as monotherapy did not have significant antiproliferative activity compared with the control antibody in the cell lines evaluated (NCI-N87, p=0.02; and OE19, p=0.96). These findings are consistent with other studies (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), Cancer research. 64(7):2343-6; Gong SJ, et al. (2004), Cancer letters. 214(2):215-24; Ko BK, et al. (2015), Molecular oncology. 9(2):398-408; Tomioka H, ​​et al. (2012), International journal of oncology. 41(2):551-8), despite differences in incubation time and dose.

[0443] Monoclonal antibody mAb104 also did not show significant growth inhibition in vitro compared to isotype control antibodies (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).

[0444] The combination of trastuzumab and pertuzumab significantly inhibited the growth of the evaluated gastric cancer / GEJ cell lines (Figure 18). However, there was no statistical difference compared to trastuzumab monotherapy. The effect of mAb104 in combination with trastuzumab or pertuzumab on growth was evaluated; the addition of mAb104 did not add any antiproliferative effect compared to the individual antibodies alone. Importantly, 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).

[0445] Example 14: Effects of mAb104 on ErbB receptors and downstream signaling pathways In the HER2-overexpressing gastric cancer cell lines, NCI-N87 and OE19, treatment with anti-HER2 antibodies as monotherapy did not affect total or phosphorylated proteins in the MAPK and Akt signaling pathways (Figures 19A and 19B), 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).

[0446] Combined treatment of trastuzumab with pertuzumab or mAb104 in the NCI-N87 gastric cancer cell line resulted in downregulation of phospho-Akt and phosphorylated p44 / p42 MAPK, while leaving total protein levels unchanged (Fig. 19C and D).

[0447] Under ligand-stimulated conditions (Figures 20A and B and 20C and D), mAb104 had no effect on 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).

[0448] The described changes in signaling following combined treatment with trastuzumab and pertuzumab in this cell line are consistent with other studies (Yamashita-Kashima Y, et al. (2011), Clinical Cancer Research. 17(15):5060-70; Ko BK, et al. (2015), Molecular oncology. 9(2):398-408; Tomioka H, ​​et al. (2012), International journal of oncology. 41(2):551-8) and are similar to those observed with trastuzumab and mAb104. These results suggest that mAb104 in combination with trastuzumab inhibits the activity of ErbB family proteins and suppresses downstream signaling. In contrast, combined treatment with trastuzumab and pertuzumab or mAb104 did not alter the levels of total or activated ErbB family proteins or the MAPK and Akt signaling cascades in OE19. In both gastric cancer cell lines, combined treatment with pertuzumab and mAb104 had no effect on the signaling cascades, nor on the levels of total or phosphorylated ErbB protein.

[0449] Example 15: Effect of mAb104 on apoptosis of gastric cancer cells Flow cytometry was used to measure the apoptosis-inducing activity of mAb104 in NCI-N87 (Figures 21A and B) and OE-19 cell lines (Figure 21C), and the early and late apoptotic cell fractions were quantified by quadrant analysis (Figure 21). Cells treated with trastuzumab did not induce apoptosis compared to the control antibody; in comparison, treatment with pertuzumab resulted in more cells in the late apoptotic stage. These findings are consistent with other published studies. mAb104 increased the late apoptotic cell population compared to single-agent treatment with trastuzumab or pertuzumab. In OE-19, none of the antibodies induced apoptosis as monotherapy or in combination therapy compared to the control antibody (Figure 21C).

[0450] Treatment with the combination of trastuzumab and pertuzumab resulted in significantly more apoptotic cells than the combination of trastuzumab and mAb104. After treatment with trastuzumab and pertuzumab, 61.4% of cells were viable, compared with 90.5% after trastuzumab with mAb104 (Figure 21A). The combination of trastuzumab and mAb104 did not induce apoptosis compared with control antibody or other monotherapy treatments.

[0451] A key finding was the presence of a greater number of necrotic cells after treatment with mAb104 compared to the control antibody and trastuzumab and pertuzumab (Figure 21C). In both cell lines evaluated, necrotic cells were increased twofold after treatment with mAb014 compared to the other treatment groups. Interestingly, treatment of cells with mAb104 in combination with trastuzumab did not result in an increase in the number of cells undergoing necrosis.

[0452] Example 16: Effect of mAb104 on migration (wound healing) assay Due to its morphology and growth pattern, the OE-19 cell line was used to evaluate the effect of mAb104 on migration. NCI-N87 cells were not used because they typically initially attach to small is...

Claims

1. 10. A HER2 binding protein comprising an antigen binding domain, said antigen binding domain specifically binding to an epitope within domain II of HER2 comprising residues 293-309 of the mature normal or wild-type human HER2 sequence according to FIG. 1 (SEQ ID NO:27), said epitope being exposed in response to HER2 amplification or activation, said epitope being expressed in tumorigenic, hyperproliferative, or abnormal cells but not in normal or wild-type cells, and further wherein said binding protein has the sequence H-GCPLHNQEVTAEDGTQRC-NH ２ (SEQ ID NO: 26).

2. the HER2 binding protein is each, (i) a heavy chain variable region sequence (VH) having CDR1, CDR2 and CDR3 sequences comprising or consisting of GYSFTGYFMH (SEQ ID NO: 14), RINPYNGDIRYNQNFKD (SEQ ID NO: 16) and LNFAY (SEQ ID NO: 18); and, each, 2. The HER2 binding protein of claim 1, comprising: (i) a light chain variable region sequence (VL) having CDR1, CDR2, and CDR3 sequences comprising or consisting of KSSQSLLDSDGKTFLN (SEQ ID NO: 20), LVSKLDS (SEQ ID NO: 22), and WQGTHFPWT (SEQ ID NO: 24).

3. the HER2 binding protein is each, (i) a heavy chain variable region sequence (VH) having CDR1, CDR2 and CDR3 sequences comprising or consisting of GYTFTDYGMN (SEQ ID NO: 15), WINTYTGKPTYDDDFKG (SEQ ID NO: 17), and RFLNTVAGRSVYFDY (SEQ ID NO: 19); and, each, (i) a light chain variable region sequence (VL) having CDR1, CDR2, and CDR3 sequences comprising or consisting of SVSSSSVGSMY (SEQ ID NO: 21), LTSNLAS (SEQ ID NO: 23), and QQWSSNPPT (SEQ ID NO: 25); 2. The HER2 binding protein of claim 1, comprising:

4. 3. The HER2 binding protein of claim 2, wherein the HER2 binding protein comprises a VH comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 2, and a VL comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 3, or a humanized, chimeric or deimmunized form thereof.

5. 4. The HER2 binding protein of claim 3, wherein the HER2 binding protein comprises a VH comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 4, and a VL comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 5, or a humanized, chimeric or deimmunized form thereof.

6. 3. The HER2 binding protein of claim 2, wherein the HER2 binding protein comprises a VH set forth in SEQ ID NO:2 and a VL set forth in SEQ ID NO:

3.

7. 4. The HER2 binding protein of claim 3, wherein the HER2 binding protein comprises a VH set forth in SEQ ID NO: 4 and a VL set forth in SEQ ID NO:

5.

8. the HER2 binding protein is (i) single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) at least one of (i) and / or at least one of (ii) linked to a heavy chain constant region or Fc or heavy chain constant domain CH2 and / or CH3; or (iv) a HER2 binding protein according to any one of claims 1 to 7, which is at least one of (i) and / or at least one of (ii) linked to a protein that extends the half-life of the antibody.

9. the HER2 binding protein is (i) diabodies; (ii) triabodies; (iii) tetrabodies; (iv) Fab; (v)F(ab')2; (vi) Fv; or (vii) at least one of (i)-(vi) linked to a heavy chain constant region or Fc or heavy chain constant domain CH2 and / or CH3; or (viii) A HER2 binding protein according to any one of claims 1 to 8, which is at least one of (i) to (vi) linked to a protein that extends the half-life of the antibody.

10. 10. The HER2 binding protein of any one of claims 1 to 9, conjugated to a detectable or functional moiety.

11. 10. The HER2 binding protein of any one of claims 1 to 9, conjugated to a drug.

12. A combination comprising (i) a HER2 binding protein according to any one of claims 1 to 9 and (ii) an anti-HER2 antibody, a chemotherapeutic agent, a radioimmunotherapeutic agent or an immunotherapeutic agent, or a combination thereof.

13. A composition comprising a HER2 binding protein according to any one of claims 1 to 11 and a suitable carrier, or comprising a combination according to claim 12.

14. A HER2 binding protein according to any one of claims 1 to 11, a combination according to claim 12, or a composition according to claim 13 for treating a HER2-expressing cancer in a subject.

15. 15. The composition of claim 14, wherein the HER2-expressing cancer is colorectal cancer, breast cancer, lung cancer, bladder cancer, gastric cancer, or gastroesophageal cancer.

16. 12. A method for detecting HER2 in a biological sample, the method comprising contacting the sample with a HER2 binding protein of any one of claims 1 to 11 and detecting a complex, wherein detecting the complex indicates HER2 expression in the sample.

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