Anti-HER2 binding molecules

Abstract

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

Description

[0001] All references or citations cited or referenced in this document, together with any manufacturer's instructions, descriptions, product specifications and product tables of any product mentioned in this document or in any document incorporated herein by reference, are incorporated herein in their entirety by reference.

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

[0003] This application claims priority to Australian provisional patent application AU 2019900973, filed on March 22, 2019. The entire contents of that document are incorporated herein by reference. Technical Field

[0004] This invention relates to a binding protein of the extracellular domain (ECD) of HER2 / ErbB2. More specifically, the binding protein binds to a conformational epitope exposed in cells in response to HER2 amplification or activation. Background Technology

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

[0006] HER2 overexpression and amplification have been identified in many cancer types, including breast cancer, biliary tract cancer, colon cancer, endometrial cancer, gastric 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 adverse outcomes in breast cancer and gastroesophageal junction (GEJ) cancers (Nagaraja et al., (2016) Eur J Surg Oncol 42(1):140-8); however, its role in other tumor types is not well defined.

[0007] The market for HER2 antibodies exceeded $10 billion in 2017, and sales are expected to increase in the future, with new clinical indications and markets emerging. Response to HER2-targeted therapy in patients with HER2 overexpression / amplification is influenced by several clinicopathological features, including tumor histology, degree of developmental abnormalities, disease stage, and the presence of potential risk factors. HER2 amplification or overexpression has also been used as a predictive biomarker for anti-HER therapy in various tumor types, including breast cancer, gastric cancer, 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 the review, see Parakh S et al., (2017) Cancer Treatment Reviews 59:1-21.

[0008] HER2 function

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

[0010] Anti-HER2 targeted therapy

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

[0012] (i) Monoclonal antibodies targeting domain IV of HER2: Earlier HER2 inhibition methods used monoclonal antibodies targeting the extracellular domain, the main example being trastuzumab (Albanell J et al., (1999) DrugsToday (Barc) 35(12):931-46). Other anti-HER2 antibodies have also been described, for example, Ko BK et al., (2015) MolOncol 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).

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

[0014] (iii) Small molecule inhibitors of HER2: Small molecule inhibitors targeting HER2 have been developed. Many target many receptors, including other members of the ErbB family, which may be advantageous because collateral signaling can be a mechanism of resistance to HER2 inhibitors (Ritter CA et al., (2007) Clin Cancer Res 13(16):4909-19). There are two approved tyrosine kinase inhibitors; lapatinib is an oral small molecule reversible inhibitor that inhibits both EGFR and HER2 kinases (Tevaarwerk AJ et al., (2009) Clin Ther 31:2332-48), and afatinib is an irreversible inhibitor of EGFR, HER2 and HER4 tyrosine kinase activity as well as the dimer containing EGFR and HER2 (Li D et al., (2008) Oncogene 27(34):4702-11). Neratinib is an irreversible pan-tyrosine kinase inhibitor of HER1, HER2, and HER4. It appears to affect downstream signal transduction in cells overexpressing HER2 and in cells with expanded EGFR, 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).

[0015] (iv) Antibody-drug conjugates targeting HER2: High expression of HER2 in HER2-positive cancer cells and low expression in normal tissues make them targets for antibody-drug conjugates (ADCs). Trastuzumab antibody-drug conjugate (T-DM1) was the first approved anti-HER2 ADC in solid tumors. It consists of trastuzumab linked to the potent cytotoxic drug DM1, a microtubule dimerization inhibitor. The antitumor effect of T-DM1 is associated with trastuzumab and DM1 metabolites (Juntilla T et al., (2011) Breast Cancer Res Treat 128(2):347-56). DM1 metabolites disrupt the microtubule network, leading to cell cycle arrest and apoptotic cell death. Although T-DM1 has shown the greatest benefit in tumors with high HER2 expression, it has also shown efficacy in different HER2 expression subgroups (Baselga J et al., (2016) Clin Cancer Res clincanres.2499.015). 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 comprising 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 comprising an anti-HER2 antibody with HT-19 linked to an auristatin-based cytotoxic payload (AF-HPA) (Bergstrom D et al., (2016) Can Res 76(4 Supplement) P4-14-28).

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

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

[0018] Despite various approaches, challenges remain. Major resistance to trastuzumab monotherapy occurs in 70% of breast cancers overexpressing HER2 (Vogel CL et al., (2002) 20(3):719-26), with most patients developing resistance during treatment. Many mechanisms have been proposed (see Parakh S et al., ibid.). The mechanisms of resistance to pertuzumab are less well understood compared to trastuzumab. Similar to other anti-HER2 therapies, both primary and acquired resistance to T-DM1 has occurred (Tan X et al., (2013) Can Res 73(8 Supplement):4629). The mechanisms of resistance to T-DM1 appear to depend on tumor size and duration of treatment; resistance has even been observed after long latency periods (Barok M et al., (2011) Breast Cancer Res 13(2):R46).

[0019] Despite the success of trastuzumab (Herceptin), pertuzumab, and T-DM1 conjugates, the toxicity of current HER2 antibodies is dose-limited, and resistance always develops as described above. Clearly, new strategies targeting HER2 without the associated toxicity characteristics are needed. Summary of the Invention

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

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

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

[0023] The inventors have discovered novel monoclonal antibodies, exemplified herein by an antibody called mAb104, which specifically recognizes aberrantly expressed HER2. In particular, the antibodies of the present invention recognize HER2 epitopes found in tumorigenic, hyperproliferative, or abnormal cells and undetectable in normal or wild-type cells. The antibodies of the present invention are further illustrated herein by examples of antibodies mAb105, mAb106, and mAb107.

[0024] This invention provides a HER2 / ErbB binding protein comprising an antigen-binding domain, wherein the antigen-binding domain specifically binds 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 conformatedly exposed in tumorigenic, hyperproliferative, or aberrant cells but not in normal or wild-type cells.

[0025] In another instance, the binding of the binding protein to its epitope does not block the binding of pertuzumab or trastuzumab / Herceptin. In one instance, the binding protein is not pertuzumab or trastuzumab. In one instance, the binding protein binding includes, for example, pertuzumab or trastuzumab. Figure 1 The region of residues 293 to 309 of the mature normal or wild-type human HER2 sequence shown in SEQ ID NO: 27. This region forms part of domain II in the HER2 extracellular domain (ECD). In one specific example, the epitope includes the amino acid sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1). This epitope is located in... Figure 1 The sequences are shown in bold and underlined. Although this epitope includes P294, L295, and H296, which are also present in pertuzumab-bound epitopes, the binding protein described here is non-pertuzumab-blocked and allows pertuzumab to bind to HER2 simultaneously. Epitopes can be determined using any conventional epitope mapping techniques known to those skilled in the art.

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

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

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

[0029] In another instance, the binding protein bound a smaller proportion of HER2 / ErbB2 on the cell surface of cancer cells compared to pertuzumab and / or trastuzumab. In yet another instance, when assessed by flow cytometry, the binding protein bound to HER2+-expressing cells by at least one, two, or three log-orders less than the binding of trastuzumab or pertuzumab. In some instances, the binding protein bound less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the total HER2 / ErbB2 expressed on the cancer cell surface. In one instance, the binding protein bound approximately 0.35–0.5% of the total HER2 / ErbB2 expressed on the cancer cell surface.

[0030] In one instance, the binding protein did not bind to normal gastric mucosa. In another instance, the binding protein did not bind to normal mammary cells.

[0031] In another instance, the binding protein can be internalized into tumor cells. In some instances, the binding protein has an anti-proliferative effect on tumor cells (e.g., gastric cells) in vivo. In other instances, the binding protein has an in vivo antitumor effect comparable to that of pertuzumab or trastuzumab. In yet another instance, the binding protein causes necrosis of tumor cells (e.g., breast tumor cells).

[0032] Exemplary HER2-binding proteins with this binding characteristic described herein include the variable region and / or CDR of an antibody called mAb104 or mAb106.

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

[0034] In another example, an antibody competitively inhibiting the binding of a HER2-binding protein, referred to as mAb104 or mAb106, to human HER2. In yet another example, an antibody competitively inhibiting the binding of a protein, referred to as mAb104 or mAb106, to a peptide consisting of the sequence shown in SEQ ID NO: 1.

[0035] In one example, the HER2 binding protein binds to a peptide comprising the sequence shown in SEQ ID NO: 1 or the sequence shown in SEQ ID NO: 1 at up to 75% of the amount of antibody binding, said antibody comprising VH comprising the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and VL comprising the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5.

[0036] In one example, the amount of bound protein or antibody is assessed by contacting a HER2-binding protein with a peptide comprising the sequence shown in SEQ ID NO: 1 and an amount of HER2-binding protein (e.g., 10 μg / ml) contacted with that peptide. The amount of HER2-binding protein bound to the peptide is then determined and compared with the amounts of antibodies comprising VH (including the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4) and VL (including the sequence shown 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 approximately 80%, 70%, 60%, or 40% of the antibody-bound amount.

[0037] This invention also provides a HER2-binding protein whose competitive inhibition is specified as the binding of antibodies to the following:

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

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

[0040] In one instance, HER2-binding proteins bind to the ECD of HER2 with an affinity dissociation constant (KD) of 2.90–3.20 nM, for example, those with… Figure 1 The sequence is shown. In another instance, KD is between approximately 2.90 and approximately 3 nM. In another instance, KD is approximately 3 nM.

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

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

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

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

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

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

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

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

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

[0050] GYX7FTX8YX9MX 10 (SEQ ID NO: 6)

[0051] Where X7 is S or T; X8 is G or D; X9 is F or G; X 10 It is H or N;

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

[0053] X 19 INX 20 YX 21 GX22 X 23 X 24 YX 25 X 26 X 27 FKX 28 (SEQ ID NO: 7)

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

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

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

[0057] Where X 50 It does not exist or is R;X 51 It does not exist or is F; X 52 It does not exist or is L; X 53 It does not exist or is N; X 54 It does not exist or is T; X 55 It does not exist or is V; X 56 It does not exist or is A; X 57 It does not exist or G;X 58 It does not exist or is R;X 59 It does not exist or is S; X 60 It is L or V; X 61 It is N or Y; and X 62 It is either A or D;

[0058] and / or

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

[0060] X14 X 15 SX 16 SX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 (SEQ ID NO: 9)

[0061] Where X 14 Is it K or S; X 15 Is it S or V; X 16 Is it Q or S; X 17 It is L or does not exist; X 18 It is L or does not exist; X 19 It is D or does not exist; X 20 It is S or does not exist; X 21 It is D or does not exist; X 22 It is G or does not exist; X 23 It is K or V; X 24 Is it T or G; X 25 Is it F or S; X 26 It is L or M; and X 27 It is either N or Y;

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

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

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

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

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

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

[0068] In one instance, the HER2-binding protein includes the following heavy chain variable region (VH) sequence:

[0069] X1X2QLX3QSGPELX4KPGX5X6VKISCKAS GYX7FTX8YX9MX 10 WVX 11 QX 12 X 13

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

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

[0072] in

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

[0074] In one instance, the HER2-binding protein also includes the following light chain variable region (VL) sequence:

[0075] X1IVX2TQSPX3X4X5SVX6X7GX8X9X 10 X 11 X 12 X 13 C X 14 X 15 SX 16 SX 17 X 18 X 19 X 20 X 21

[0076] X 22 X 23 X 24 X 25 X 26 X 27 WX 28 X 29 QX 30 PX 31 X 32 SPKX 33 X 34 IY LX 35 SX 36 LX 37 S GVPX 38 RFX 39 GSGSGTX40X 41 X 42 LX 43 ISX 44 X 45 EAEDX 46 X 47 X 48 YYC X 49 QX 50 X 51 X 52 X 53 PX 54 T FGX 55 GTKLEX 56 KR (SEQ ID NO: 13)

[0077] in

[0078] 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; X 10 Is it A or V; X 11 Is it S or T; X 12 Is it I or M; X 13 Is it S or T; X 14 Is it K or S; X 15 Is it S or V; X 16 Is it Q or S; X 17 It is L or does not exist; X 18 It is L or does not exist; X 19 It is D or does not exist; X 20 It is S or does not exist; X 21 It is D or does not exist; X 22 It is G or does not exist; X 23 It is K or V; X 24 Is it T or G; X 25 Is it F or S; X 26 Is it L or M; X27 Is it N or Y; X 28 Is it L or Y; X 29 Is it L or Q; X 30 It is R or K; X 31 Is it G or R; X 32 Is it Q or S; X 33 Is it R or P; X 34 Is it L or W; X 35 It is V or T; X 36 It is K or N; X 37 It is D or A; X 38 It is D or P; X 39是 T or S; X 40 Is it D or S; X 41 Is it F or Y; X 42 Is it T or S; X 43 Is it K or T; X 44 Is it R or S; X 45 It is V or M; X 46 It is L or A; X 47 It is G or A; X 48 It is V or T; X 49 Is it W or Q; X 50 Is it G or W; X 51 Is it T or S; X 52 It is H or S; X 53 It is F or N; X 54 It is W or P; X 55 It is G or A; and X 56 It is either I or L.

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

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

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

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

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

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

[0085] This invention also provides a HER2-binding protein comprising a heavy chain variable region (VH) sequence, wherein the heavy chain variable region sequence has CDR1, CDR2, and CDR3 sequences comprising or composed of the following:

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

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

[0088] This invention also provides a HER2-binding protein comprising a light chain variable region (VL) sequence, wherein the light chain variable region sequence has CDR1, CDR2, and CDR3 sequences comprising or composed of the following:

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

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

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

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

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

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

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

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

[0097] This invention also provides a HER2-binding protein comprising or composed of the following:

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

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

[0100] In one instance, the HER2-binding protein is selected from the following antigen-binding fragments:

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

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

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

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

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

[0106] (i) Double-chain antibody;

[0107] (ii) Triple-chain antibodies;

[0108] (iii) Quadruplex antibody;

[0109] (iv)Fab;

[0110] (v)F(ab′)2;

[0111] (vi)Fv; or

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

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

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

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

[0116] Based on the disclosure herein, the HER2-binding proteins of this invention include human, humanized, humanoid, chimeric, and primate proteins, which will be apparent to those skilled in the art.

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

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

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

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

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

[0122] The present invention also provides isolated nucleic acids encoding the HER2-binding protein or antibody of the present invention.

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

[0124] In instances of disclosures involving a single polypeptide HER2-binding protein, the expression construct may include a promoter linked to a nucleic acid encoding the polypeptide chain.

[0125] In examples involving multiple polypeptides that form HER2-binding proteins, the expression construct of the present invention includes a nucleic acid (e.g., including VH) operably linked to one of the coding polypeptides of a promoter and another nucleic acid (e.g., including VL) operably linked to another promoter.

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

[0127] (i) promoter

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

[0129] (iii) Internal ribosome entry sites; and

[0130] (iv) Nucleic acid encoding the second polypeptide.

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

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

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

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

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

[0136] The present invention further provides isolated cells expressing the HER2-binding protein or antibody of the present invention, or recombinant cells genetically modified to express the HER2-binding protein or antibody of the present invention. In one example, the cells are isolated hybridoma cells. In another example, the cells comprise the nucleic acid or expression construct of the present invention or:

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

[0138] (ii) a second expression construct comprising a nucleic acid encoding a promoter polypeptide (e.g., including VL),

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

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

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

[0142] The compositions of the present invention can be administered alone or in combination with other treatments, therapeutic agents, or pharmaceutical agents, simultaneously / in parallel, or sequentially. In one example, the HER2-binding protein or composition of the present invention is administered in combination with pertuzumab or trastuzumab. In another example, the HER2-binding protein or composition of the present invention is administered in combination with a tyrosine kinase inhibitor (e.g., lapatinib). It is also contemplated that the HER2-binding protein or composition described herein be administered simultaneously or sequentially with anticancer therapies (e.g., chemotherapy or radiotherapy). In yet another example, the HER2-binding protein or composition described herein may be administered simultaneously or sequentially with an immunotherapeutic agent or immunomodulatory agent.

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

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

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

[0146] This invention also provides HER2-binding proteins or diagnostic agents as described herein for histological examination of biological samples. Methods for preparing histological samples are well known to those skilled in the art.

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

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

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

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

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

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

[0153] In one instance, the HER2-binding protein was administered to the subject at a therapeutically effective dose.

[0154] Preferably, the test subjects are humans.

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

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

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

[0158] The present invention also provides a method for generating a HER2 / ErbB2 binding protein, comprising immunizing rodents with a cyclic peptide comprising the sequence H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 26); obtaining a hybridoma cell line from B cells of the immunized rodents and purifying an antibody from the hybridoma cell line. In one example, the peptide is cyclized via a disulfide bond. In another example, the peptide is cyclized via a disulfide bond between Cys2 and Cys18 side chains. In yet another example, the peptide is linked to a keyhole hemlock blue (KLH) protein. Attached Figure Description

[0159] Figure 1 The full-length protein sequence of the human receptor tyrosine-protein kinase HER2, including a 22-amino acid leader sequence. Peptide epitopes bound by mAb104 within cysteine-rich domain II are shown in bold underlined text.

[0160] Figure 2 The binding of 10 μg / mL mAb104, (B) mAb105, (C) mAb106, and (D) mAb107 to the extracellular domain of HER2, to cyclized and linear polypeptide immunogens linked to antibody-producing keyhole hemocyanin (KLH), or to control-irrelevant peptides linked to KLH was compared using ELISA. Binding activity of triplicate samples was measured using a Versamax microplate reader (molecular device) at 405 nm using Softmax Pro 4.8 software and mean ± SD. mAb104 (A) and mAb106 (C) showed the strongest binding activity to all immobilized HER2 forms, while mAb105 (B) showed the weakest binding. Specificity was confirmed by the lack of binding to the control peptide. The results represent two independent experiments.

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

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

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

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

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

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

[0167] Figure 8-2 ELISA analysis. Specificity of mAb104 (3–10,000 ng / mL) bound to the ELISA plate was assessed using ECDs coated with the recombinant sEGFR extracellular domain or HER2, HER3, or HER4. Controls included individual pNPP substrates and secondary anti-mouse antibody-alkaline phosphatase conjugates. (Data; mean ± SE; n = 3).

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

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

[0170] Figure 11-1 and 11-2 When used as monotherapy (A and B) or in combination with trastuzumab or pertuzumab (C and D), mAb104 did not affect ligand-dependent phosphorylation of the MAPK pathway and Akt in (A) SK-BR-3 and (B) BT474 cells in vitro. Cells were incubated in serum-free medium and treated with 100 μg / mL mAb104, trastuzumab, and pertuzumab alone or in combination for 24 h, followed by the addition of 100 ng EGF for 10 min. After whole-cell lysis, equal volumes of the lysate were loaded and separated on a 4–12% gel, then transferred to a nitrocellulose membrane. Immunoblots were performed on the membrane as shown in the figure. Results represent two experiments.

[0171] Figures 12-1 to 12-4 The effects of treatment on breast cancer cell viability and apoptosis were assessed by flow cytometry analysis with annexin-V and propidium iodide (PI) staining after 4 h of treatment with trastuzumab, pertuzumab, and mAb104 as monotherapy and in combination (total antibody 0.1 mg / mL in all groups) BT474 cells (A to H) and SK-BR-3 cells (I to P).

[0172] Figure 13 Antitumor effect of mAb104 in BT-474 breast cancer xenografts. Mice (n=5) were treated with 1 mg mAb104, trastuzumab, pertuzumab, and an allotype control. Tumor volume at the start of treatment was 100-120 mmHg. 3 The data shown in the growth curves indicate that the tumor volume ± SE in each treatment group was significantly smaller than that in the control group. * p<0.001; control group: mAb104; ** p<0.0001. Comparison of trastuzumab and pertuzumab.

[0173] Figure 14 Antitumor activity of mAb104 in BT-474 xenografts. Mice were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, and an allotype control (n=5 / group). Tumor volume at the start of treatment was 120-150 mmHg. 3 The data shown in the growth curve represent mean tumor volume ± SE. * p<0.01, compared with the mAb104 treatment group.

[0174] Figures 15-1 to 15-3 (A) Antitumor effect of mAb104 in a HER2-positive breast PDX model. Mice were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, and an allotype control (n=5 / group). The tumor volume at the start of treatment was 100-120 mm. 3 (B) Antitumor effect of mAb104 combined with trastuzumab in BT-474 xenografts. Mice (n=5) were treated with a total dose of 0.5 mg mAb104 + trastuzumab, trastuzumab + pertuzumab, or an allotype control. The tumor volume at the start of treatment was 120-150 mm. 3 . * p<0.0001 Control group vs. trastuzumab / mAb104. (c) Antitumor effect of mAb104 combined with trastuzumab in a HER2-positive breast PDX model. Mice (n=5) were treated with a total dose of 0.5 mg mAb104 + trastuzumab, trastuzumab + pertuzumab, or an allotype control. Tumor volume at the start of treatment was 120-150 mm. 3 . * p<0.0001, control group vs. mAb104; ** p<0.001, trastuzumab vs. trastuzumab / mAb104. The data shown in the growth curves for group AC represent mean tumor volume ± SE.

[0175] Figure 16-1 and 16-2Immunohistochemical evaluation of the following effects of BT-474 xenograft tumors: anti-HER2 monotherapy (0.5 mg dose) on (A) Ki67 proliferation, (B) downstream signal transduction of phosphorylated-Akt, and (C) the effect of scleroderma glycoprotein on the vascular system; or the combination of mAb104 and trastuzumab (0.5 mg total protein dose) on (D) Ki67 proliferation, (E) downstream signal transduction of phosphorylated-Akt, and (F) the effect of scleroderma glycoprotein on the vascular system. * p<0.001 Control group compared to trastuzumab.

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

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

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

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

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

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

[0182] Figure 23 Antitumor activity of mAb104 in NCI-N87 xenografts. Mice were treated with 1 mg mAb104, trastuzumab, pertuzumab, or an allotype control (n=5) or untreated. Tumor volume at the start of treatment was 100-120 mmHg. 3 The data shown in the growth curve represent mean tumor volume ± SE. * p≤0.01, control group: mAb104.

[0183] Figure 24 Antitumor activity of mAb104 in NCI-N87 xenografts. Mice (n=5) were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, or an allotype control, or without treatment. Tumor volume at the start of treatment was 100-120 mmHg. 3 Growth curves (A) and survival curves (B) are shown. The data shown in the growth curves represent mean tumor volume ± SE. The endpoint of the survival analysis was a tumor volume >1000 mm. 3 Or in a dying state. * p<0.001, control group: mAb104; ** p<0.0001 Control group vs. treatment group.

[0184] Figure 25 Antitumor activity of mAb104 in NCI-N87 xenografts. Mice (n=5) were treated with 0.1 mg mAb104, trastuzumab, pertuzumab, or an allotype control, or without treatment. Tumor volume at the start of treatment was 100-120 mmHg. 3 Growth curves (A) and survival curves (B) are shown. The data shown in the growth curves represent mean tumor volume ± SE. The endpoint of the survival analysis was a tumor volume >1000 mm. 3 Or in a dying state. * p≤0.001, control group: mAb104; ** p<0.0002 Control group vs. treatment group.

[0185] Figure 26 Antitumor activity of mAb104 in OE-19 xenografts. Mice (n=5) were treated with 1 mg mAb104, trastuzumab, pertuzumab, or an allotype control, or without treatment. Tumor volume at the start of treatment was 100-120 mmHg. 3 The data shown in the growth curve represent mean tumor volume ± SE. * p≤0.0001, control group: mAb104.

[0186] Figure 27 Antitumor activity of mAb104 in OE-19 xenografts. Mice (n=5) were treated with 0.5 mg mAb104, trastuzumab, pertuzumab, and an allotype control. Tumor volume at the start of treatment was 100-120 mmHg. 3 Growth curves (A) and survival curves (B) are shown. The data shown in the growth curves represent mean tumor volume ± SE. The endpoint of the survival analysis was a tumor volume >1000 mm. 3 Or in a dying state. * p<0.001, compared to trastuzumab. ** p<0.006, control group compared to treatment group.

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

[0188] Figure 29 Antitumor effects of mAb104 combined with trastuzumab in OE-19 xenografts. Mice (n=5) were treated with a total dose of 0.5 mg mAb104 + trastuzumab, trastuzumab + pertuzumab, or an allotype control. Tumor volume at the start of treatment was 100-120 mm. 3 Growth curves (A) and survival curves (B) are shown. The data shown in the growth curves represent mean tumor volume ± SE. The endpoint of the survival analysis was a tumor volume >1000 mm. 3 Or in a dying state. * p<0.0001 Control group vs. trastuzumab / mAb 104; ** p<0.0001 (Comparison of trastuzumab / pertuzumab) *** p<0.0001 Trastuzumab vs Trastuzumab / mAb 104; ± p<0.0005, control group compared to treatment group.

[0189] Figure 30-1 and 30-2 Immunohistochemistry was used to evaluate the effects of anti-HER2 monotherapy (0.5 mg dose) (A to C) or in combination with trastuzumab (D to F) on (A and D) Ki67 proliferation; (B and E) downstream signal transduction via phosphorylated-Akt staining; and (C and F) the effects on the vascular system via calyx glycoprotein staining.

[0190] Figure 31-1 and 31-2 Immunohistochemistry was used to evaluate the effects of anti-HER2 monotherapy (0.5 mg dose) (A to C) or in combination with trastuzumab (D to F) on (A and D) Ki67 proliferation; (B and E) downstream signal transduction by phosphorylated-Akt staining; and (C and F) the effects on the vascular system by calyx glycoprotein staining.

[0191] Figure 32 Used for determination 89 Binding assay of the immunoreactive fraction of Zr-labeled anti-HER antibodies. A) shows a standard plot of specific binding against total applied radioactivity based on increased cell concentration. B) and C) are double inverse plots of the same data as in A, allowing determination of the immunoreactive fraction for conditions representing unlimited antigen overload.

[0192] Figure 33 A) of the combination of Scatcherd diagram 89Zr-labeled mAb104 and B) 89 Zr-labeled Herceptin / Trastuzumab binding to NCI-N87 gastric carcinoma cells. The x-axis represents the concentration of specifically bound antibody, and the y-axis is the ratio of the concentration of specifically bound antibody to the concentration of reactive free antibody. The binding capacity of each cell is determined by the intercept value at the x-axis, and the association constant is determined by the slope of the line.

[0193] Figure 34 A) Biodistribution of zirconium-89 labeled mAb104 in mice carrying NCI-N87 gastric cancer xenografts overexpressing HER2. B) Biodistribution of zirconium-89 labeled mAb104 and isotype controls in the blood and tumors of mice carrying NCI-N87 xenografts. High tumor-specific uptake was demonstrated using mAb104. (Data mean ± SEM, n = 5).

[0194] sequence list keywords

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

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

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

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

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

[0200] SEQ ID NO:6: VH CDR1 Common Sequence

[0201] SEQ ID NO:7: VH CDR2 common sequence

[0202] SEQ ID NO:8: VH CDR3 Common Sequence

[0203] SEQ ID NO:9: VL CDR1 common sequence

[0204] SEQ ID NO:10: VL CDR2 common sequence

[0205] SEQ ID NO:11: VL CDR3 common sequence

[0206] SEQ ID NO:12: VH common sequence

[0207] SEQ ID NO:13: VL co-occurrence sequence

[0208] SEQ ID NO:14: VH CDR1 of mAb104

[0209] SEQ ID NO:15: VH CDR1 of mAb106

[0210] SEQ ID NO:16: VH CDR2 of mAb104

[0211] SEQ ID NO:17: VH CDR2 of mAb106

[0212] SEQ ID NO:18: VH CDR3 of mAb104

[0213] SEQ ID NO:19: VH CDR3 of mAb106

[0214] SEQ ID NO:20: VL CDR1 of mAb104

[0215] SEQ ID NO:21: VL CDR1 of mAb106

[0216] SEQ ID NO:22: VL CDR2 of mAb104

[0217] SEQ ID NO:23: VL CDR2 of mAb106

[0218] SEQ ID NO:24: VL CDR3 of mAb104

[0219] SEQ ID NO:25: VL CDR3 of mAb106

[0220] SEQ ID NO:26: Cyclic peptide sequence for immunization

[0221] SEQ ID NO:27: Sequence of HER2 / ErbB2

[0222] SEQ ID NO:28: Light chain primer sequence

[0223] SEQ ID NO:29: Light chain primer sequence

[0224] SEQ ID NO:30: Light chain primer sequence

[0225] SEQ ID NO:31: Light chain primer sequence

[0226] SEQ ID NO:32: Light chain primer sequence

[0227] SEQ ID NO:33: Light chain primer sequence

[0228] SEQ ID NO:34: Light chain primer sequence

[0229] SEQ ID NO:35: Light chain primer sequence

[0230] SEQ ID NO:36: Light chain primer sequence

[0231] SEQ ID NO:37: Light chain primer sequence

[0232] SEQ ID NO:38 Light chain primer sequence

[0233] SEQ ID NO:39: Light chain primer sequence

[0234] SEQ ID NO:40: Light chain primer sequence

[0235] SEQ ID NO:41: Heavy chain primer sequence

[0236] SEQ ID NO:42: Heavy chain primer sequence

[0237] SEQ ID NO:43: Heavy chain primer sequence

[0238] SEQ ID NO:44: Heavy chain primer sequence

[0239] SEQ ID NO:45: Heavy chain primer sequence

[0240] SEQ ID NO:46: Heavy chain primer sequence

[0241] SEQ ID NO:47: Heavy chain primer sequence

[0242] SEQ ID NO:48: Heavy chain primer sequence

[0243] SEQ ID NO:49: Heavy chain primer sequence

[0244] SEQ ID NO:50: Heavy chain primer sequence

[0245] SEQ ID NO:51: Heavy chain primer sequence

[0246] SEQ ID NO:52: Heavy chain primer sequence

[0247] SEQ ID NO:53: Heavy chain primer sequence

[0248] SEQ ID NO:54: Light chain primer sequence

[0249] SEQ ID NO:55: Light chain primer sequence Detailed Implementation

[0250] General terms

[0251] Throughout this specification, unless otherwise specified or required by context, references to a single step, composition of matter, group of steps, or group of composition of matter shall be considered to include one or more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of composition of matter.

[0252] Those skilled in the art will understand that, in addition to those specifically described, the invention is susceptible to variations and modifications. It should be understood that the invention includes all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0253] This invention is not limited to the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this invention.

[0254] Unless otherwise specifically stated, any other instances of the invention should be adapted to the necessary modifications.

[0255] Unless otherwise expressly defined, all technical and scientific terms used herein should be regarded as having 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).

[0256] Unless otherwise stated, the recombinant protein, recombinant DNA technologies, molecular biology, microbiology, cell culture and immunology techniques used in this disclosure are standard methods well known to those skilled in the art. These techniques are described and explained in the following sources: J. Perbal, *A Practical Guide to Molecular Cloning*, John Wiley and Sons (1984); J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press (1989); T.A. Brown (ed.), *Essential Molecular Biology: A Practical Approach*, Volumes 1 and 2, IRL Press (1991); D.M. Glover and B.B. Hames (eds.), *DNA Cloning: A Practical Approach*, Volumes 1–4, IRL Press (1995 and 1996); and F.M. Ausubel et al. (eds.), *Current Protocols in Molecular Biology*, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0257] The descriptions and definitions of variable regions and their portions, immunoglobulins, antibodies and their fragments in this article can be further clarified by the 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.

[0258] Throughout this specification, the word “comprise” or variations thereof such as “comprises” or “comprising” shall be understood to mean including the stated elements, integers or steps, or groups of elements, integers or steps, but not excluding any other elements, integers or steps, or groups of elements, integers or steps.

[0259] As used herein, the term “derived from” should be used to indicate that the specified integer can be obtained from a particular source, although it does not necessarily have to be obtained directly from that source.

[0260] In the context of peptide sequences, the terms “composed of” or “essentially composed of” refer to a peptide sequence having a defined number of residues that are not covalently linked to a larger product.

[0261] Unless otherwise specifically stated, any embodiments herein should be understood to be applicable in accordance with any other embodiments.

[0262] Select definition

[0263] As used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. The terms “a” (or “an”) and “one or more” and “at least one” are used interchangeably herein.

[0264] Furthermore, when used herein, "and / or" should be considered as each of the two specified features or components being, or not being, a specific disclosure of the other. Therefore, the term "and / or" as used herein 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" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0265] The term "about" is used in this document to mean approximately, roughly, around, or in a region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the value. Typically, the term "about" is used in this document to indicate that the value above or below has changed by 10% (%).

[0266] It should be understood that the HER2-binding proteins, antibodies, nucleic acids, cells, and vectors in this article are in isolated forms. "Isolated" means a polypeptide, antibody, polynucleotide, vector, or cell that is not naturally found in its original form. Isolated polypeptides, antibodies, polynucleotides, vectors, or cells include those that have been purified to the point that they are no longer in their naturally found form. In some respects, isolated antibodies, polynucleotides, vectors, or cells are substantially pure. In some respects, isolated antibodies, polynucleotides, vectors, or cells are "recombinant."

[0267] The term "HER2" as used in this article should be understood as referring to Figure 1 The human HER2 receptor shown, particularly domain II of the HER2 receptor represented by amino acid residues 190-269 of the wild-type HER2 sequence (Coussens L et al., (1985) Science 230(4730):1132-9). The term HER2 is used interchangeably with ErbB2.

[0268] The term “abnormal expression” or “abnormally expressed” is intended to include a state in which an abnormal (usually increased) amount / level of protein is present, regardless of the direct cause of the abnormal amount or level. Abnormal expression includes and considers any situation or alteration in which protein expression or post-translational modification mechanisms in a cell are affected or otherwise disrupted due to enhanced protein expression or increased levels or amounts, including altered proteins, such as mutated proteins or variants expressed due to sequence changes, deletions or insertions, or altered folding. In this paper, abnormal expression is associated with HER2 expression observed in tumorigenic, hyperproliferating, or abnormal cells rather than wild-type or normal cells.

[0269] As used herein, the term "affinity" refers to the strength of binding between a single molecule and its ligand, and is generally expressed as the equilibrium dissociation constant (KD) of the reversible binding of two reagents. It is determined by the Koff / Kon ratio between the HER2-binding protein and HER2. KD is inversely proportional to affinity. The KD value is related to the concentration of the HER2-binding protein, therefore the lower the KD value (the lower the concentration), the higher the affinity of the binding protein. The affinity of the HER2-binding protein of the present invention for HER2 can be, for example, from about 100 nanomolars (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or higher.

[0270] As used herein, the term "binding" in relation to the interaction between HER2-binding proteins and their targets means that the interaction depends on the presence of a specific structure on the target (e.g., an antigenic determinant or epitope). For example, HER2-binding proteins recognize and bind to specific protein structures, rather than proteins in general.

[0271] As used herein, the term "binding protein" is intended to describe members of a pair of molecules that have specific binding properties to each other. Members of a specific binding pair can be of natural origin or wholly or partially synthetically produced. One member of the pair has a region or cavity on its surface that specifically binds to and is complementary to the specific spatial and polar organization of the other member of the pair. Thus, the members of the pair possess the property of specifically binding to each other. Examples of specific binding pair types are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, and enzyme-substrate. This application relates to antigen-antibody type reactions.

[0272] The term "antibody" describes a naturally occurring or partially or wholly synthetically produced immunoglobulin. The term also includes any polypeptide or protein having a binding domain homologous to an antibody-binding domain. The term also includes CDR transplantation antibodies. An "antibody" is any immunoglobulin that binds to a specific epitope, including antibodies and fragments thereof. The term includes polyclonal, monoclonal, and chimeric antibodies, the last mentioned antibodies being described in further detail in U.S. Patents 4,816,397 and 4,816,567. The term "antibody" includes wild-type immunoglobulin (Ig) molecules, which typically consist of four full-length polypeptide chains, two heavy (H) chains, and two light (L) chains, or equivalent Ig homologs (e.g., camelid nanobodies, which consist of only heavy chains); it includes full-length functional mutants, variants, or derivatives thereof that retain the essential epitope binding characteristics of Ig molecules, and includes bispecific, bispecific, multispecific, and bivariate domain antibodies; immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). 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 desired specificity. Therefore, the term includes antibody fragments, derivatives, functional equivalents, and homologs of antibodies, including any polypeptide that includes an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Therefore, this includes chimeric molecules that include an immunoglobulin-binding domain or equivalent fused to another polypeptide. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, as well as U.S. Patents 4,816,397 and 4,816,567. The term "antibody" also includes any "antibody fragment".

[0273] "Antibody fragment" refers to a molecule comprising at least one non-full-length polypeptide chain, including (i) Fab fragments, which are monovalent fragments composed of variable light chain (VL), variable heavy chain (VH), constant light chain (CL), and constant heavy chain 1 (CH1) domains; (ii) F(ab')2 fragments, which are divalent fragments comprising two Fab fragments linked by disulfide bonds through a hinge region; (iii) the heavy chain portion of Fab(Fd) fragments, which consist of VH and CH1 domains; (iv) variable fragment (Fv) fragments, which consist of the VL and VH domains of a single arm of the antibody; and (v) domain antibody (dAb) fragments, which include a single variable domain (Ward, ES et al., Nature). 341, 544-546 (1989)); (vi) Cameloid antibody; (vii) Separated complementarity-determining region (CDR); (viii) Single-chain Fv fragment in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); (ix) Biantibody, which is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to pair between the two domains on the same chain is used, thus forcing the domains to pair with the complementary domain of another chain and generate two antigen-binding sites (WO94 / 13804; P. Holliger et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci.) USA 90 6444-6448, (1993)); and (x) linear antibodies comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), the tandem Fv fragments forming 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 the heavy chain and / or light chain, or mutants, variants or derivatives thereof, alone or in any combination.

[0274] As used herein, the term "antigen-binding fragment" includes Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), VL and VH domain fragments, domain antibodies, trispecific (Fab3), bispecific (Fab2), biantibodies ((VL-VH)2 or (VH-VL)2), trivalent antibodies, tetravalent antibodies, small antibodies ((scFv-CH3)2), bispecific single-chain Fv (Bis-scFv), IgGδCH2, scFv-Fc, and (scFv)2-Fc. A "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody molecule and can be generated by cleaving the entire antibody molecule with papain to produce a fragment consisting of a complete light chain and a portion of a heavy chain. A "Fab' fragment" of an antibody molecule can be obtained by treating the complete antibody molecule with pepsin and then reducing it to produce a molecule consisting of a complete light chain and a portion of a heavy chain. Each antibody molecule treated in this way yields two Fab' fragments. The antibody's "F(ab')2 fragment" consists of a dimer of two Fab' fragments linked together by two disulfide bonds and is obtained by treating the intact antibody with pepsin without subsequent reduction. The "Fv fragment" is a genetically engineered fragment containing both light and heavy chain variable regions expressed as two chains. A "single-chain antibody" (SCA) is a genetically engineered single-chain molecule containing light and heavy chain variable regions linked by suitable flexible polypeptide linkers.

[0275] As used herein, “antibody variable region” refers to a portion of the light and heavy chains and the framework region (FR) of an antibody molecule that includes the amino acid sequence of the complementarity-determining regions (CDRs, i.e., CDR1, CDR2, and CDR3). 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 this invention, the amino acid positions assigned to the CDRs and FRs may be based on 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 the antibody or antigen-binding fragment is also based on Kabat numbering.

[0276] As used herein, the term "constant region" (CR) refers to the portion of an antibody molecule that confers effector function. The constant region of a humanized antibody for a test subject is derived from human immunoglobulins. The heavy chain constant region can be selected from any of five isotypes: α, δ, ε, γ, or μ. Furthermore, various subclasses of the heavy chain (e.g., the IgG subclass of the heavy chain) are responsible for different effector functions; therefore, by selecting the desired heavy chain constant region, antibodies with the desired effector function can be generated. Exemplary heavy chain constant regions are γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4). The light chain constant region can be of the κ or λ type, preferably the κ type.

[0277] The “frame region” (hereinafter referred to as FR) consists of the 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.

[0278] As used in this article, the term "syn CDR" (i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable domain that are essential for antigen binding. Each variable domain typically has three CDR regions, designated CDR1, CDR2, and CDR3. Each syn CDR may include amino acid residues from CDR regions as defined by Kabat (i.e., approximately residues 24-34 or 24-39 (L1)), 50-56 or 55-61 (L2) and 89-97 or 93-102 (L3) in the light chain variable domain, and 31-35 or 26-35 (H1), 50-65 or 50-66 (H2), and 95-102 or 97-108 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health. Health, Bethesda, MD. (1991) and / or those residues from the “hypervariant ring,” namely approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol Biol. 196: 901-917). In some cases, the complementarity-determining region may include amino acids from the CDR region and the hypervariant ring as defined by Kabat. Those skilled in the art will recognize that some variations in the positioning of the FR, such as as a result of mutations (e.g., deletions and / or insertions), may involve up to 5 residue changes, or 4 residue changes, or 2 residue changes, or 1 residue change (e.g., as in the exemplary antibody described herein).

[0279] As used herein, the term "monoclonal antibody" refers to a formulation of antibody molecules that are single-molecule compositions. Monoclonal antibodies exhibit single-binding specificity and affinity for a specific epitope. Monoclonal antibodies can be produced from any animal, such as mice, rats, rabbits, pigs, etc., or can be synthesized and contain partial or complete human sequences.

[0280] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species (e.g., mouse) or belonging to a specific antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species (e.g., primate) or belonging to a specific antibody class or subclass, as well as to the corresponding sequence in fragments of these antibodies, provided they exhibit the desired biological activity.

[0281] The term "humanized antibody" should be understood to refer to a chimeric molecule typically prepared using recombinant technology, which has an epitope binding site derived from a non-human immunoglobulin and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen binding site preferably includes a complementarity-determining region (CDR) from the non-human antibody, which is transplanted into an appropriate frame region within the variable domain of the human antibody and the remaining region from the human antibody.

[0282] As used in this article, the term "human antibody" in relation to antibody molecules and binding proteins refers to an antibody having variable (e.g., VH, VL, CDR, and FR regions) and constant antibody regions that are derived from or correspond to sequences found in humans (e.g., human germline or somatic cells).

[0283] As used herein, the term "specific binding" refers to a reaction or binding of a binding protein or antibody with a specific cell or substance that is more frequent, faster, longer-lasting, and / or has a greater affinity than its reaction or binding with other cells or substances. Reading this definition also allows us to understand, for example, that an antibody that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require exclusive or undetectable binding to another molecule, which is included in the term "selective binding." Generally, but not necessarily, binding refers to specific binding.

[0284] As used herein, the term "cellular proliferative disorder" and its grammatical variations, when used to refer to cells, tissues, or organs, refers to any unwanted, excessive, or abnormal growth, proliferation, differentiation, or survival of cells, tissues, or organs. Undesirable cellular proliferative disorders include both disease and physiological disorders, including benign proliferative disorders characterized by an unwanted, excessive, or abnormal number of cells, cell growth, cell proliferation, cell survival, or differentiation in the subject. Specific examples of such disorders include metastatic and non-metastatic tumor formation, tumors, and cancers (malignant tumors).

[0285] The term "identity" and its grammatical variations refer to two or more reference entities being identical. Thus, when two antibody sequences are identical, they have the same amino acid sequence at least within a reference region or portion. When two nucleic acid sequences are identical, they have the same polynucleotide sequence at least within a reference region or portion. Identity can be defined on a region (region or domain) of the sequence. % identity of polynucleotides is analyzed by GAP (Needleman and Wunsch, J. Mol Biol. 48:444-453. 1970) analysis (GCG procedure), where the nick creation penalty = 5 and the nick extension penalty = 0.3. Unless otherwise stated, the query sequence length is at least 45 nucleotides, and GAP analysis compares the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence length is at least 100 nucleotides, and GAP analysis compares the two sequences over a region of at least 100 nucleotides. Most preferably, the two sequences are compared over their entire length.

[0286] The term "isolated," including DNA, RNA, or protein, refers to a polynucleotide / peptide that is at least partially isolated from a polynucleotide / peptide sequence associated with or linked to its native state. Preferably, the isolated polynucleotide / peptide is at least 60% free, more preferably at least 75% free, and most preferably at least 90% free of other components naturally associated with it.

[0287] The term “nucleic acid” as used in this article may be used interchangeably with the term “polynucleotide”.

[0288] As used herein, the term "pharmaceutical composition" means any composition containing at least one therapeutic or bioactive agent and suitable for administration to a patient. Any of these formulations may be prepared by methods well-known and accepted in the art. See, for example, Gennaro, AR, ed., Remington: The Science and Practice of Pharmacy, 20th edition, Mack Publishing Co., Easton, Pa. (2000).

[0289] As used herein, the phrase “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, and / or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0290] "Test subject" refers to any subject requiring diagnosis, prognosis, or treatment, particularly mammalian subjects. As used herein, the term "test subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, bears, chickens, amphibians, reptiles, etc., and may be used interchangeably with the term "patient" where appropriate. Preferably, the test subject is a primate. In particular, the test subject is a human.

[0291] As used herein, the reference to “similar” binding level should be understood to mean that an antibody binds to an antigen at a level approximately 30%, 25%, or 20% of the level at which it binds to another antigen. The term can also refer to an antibody binding to an antigen at a level approximately 30%, 25%, or 20% of the level at which another antibody binds to the same antigen.

[0292] As used herein, the reference to “substantially the same level” should be understood to mean that an antibody binds to an antigen at a level of approximately 15%, 10%, or 5% of the level at which it binds to another antigen. The term can also refer to an antibody binding to an antigen at a level of approximately 5%, 4%, or 3% of the level at which another antibody binds to the same antigen.

[0293] The term "competitive inhibition" should be understood as the protein of the present invention reducing or preventing the binding of the generated antibody (e.g., mAb104) to domain II or a fragment thereof of human HER2. It will be apparent from the foregoing that the protein does not need to completely inhibit antibody binding, but only needs to reduce binding by a statistically significant amount, such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, the antibody is exposed to HER2 or a fragment thereof in the presence or absence of the protein. If antibody binding in the presence of the protein is less than antibody binding in the absence of the protein, the protein is considered to competitively inhibit antibody binding. In one instance, the protein and antibody are substantially simultaneously exposed to HER2. Other methods for determining competitive inhibition of binding are apparent to those skilled in the art and / or described herein. In one instance, the antigen-binding domain of the protein competitively inhibits antibody binding.

[0294] In the context of two epitopes, "overlap" means that the two epitopes share a sufficient number of amino acid residues to allow an antibody binding to one epitope to competitively inhibit the binding of an antibody binding to the other epitope. For example, the epitopes may share at least one, two, three, four, five, six, seven, eight, nine, or ten amino acids.

[0295] As used herein, the term "substantially non-binding" should be understood to mean that the protein (e.g., antibody) binds to the candidate antigen at a level 10%, 8%, 6%, or 5% above background. Background can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an allotype control antibody) and / or the level of binding detected in the presence of a negative control antigen. Binding levels are detected using biosensor analysis (e.g., Biacore), in which the protein is immobilized and contacted with the antigen.

[0296] The term "therapeuticly effective amount" should be understood to mean a sufficient amount of antibody or antigen-binding fragment to reduce or suppress one or more symptoms of a cell proliferation disorder to below the level observed and accepted as clinically characteristic of the disorder. Those skilled in the art will recognize that such an amount will vary depending on the antibody, fragment, and / or the specific subject and / or the type, severity, or level of the disease. Therefore, this term should not be construed as limiting the invention to a specific amount.

[0297] As used herein, the term "treat" (treating, treatment) and its grammatical variations refer to a protocol, course, procedure, or therapy in which an individual patient is subjected to treatment in which a physiological response or outcome is expected to be achieved. Because each treated patient may not respond to a particular treatment protocol, course, procedure, or therapy, treatment is not required to achieve the desired physiological response or outcome in every patient or patient group. Therefore, a given patient or patient group may not respond to treatment or may respond inadequately.

[0298] The terms "tumor" or "cancer" are used interchangeably to refer to a group of cells or cells that grow, proliferate, or survive in a manner greater than that of their normal counterparts (e.g., proliferative or differentiation disorders). Typically, the growth is uncontrolled.

[0299] The terms “104 antibody” or “mAb104” and any variants not specifically listed herein are used interchangeably and, as used throughout this application and claims, refer to protein material comprising one or more proteins, and extend to those proteins having the amino acid sequence data described herein and the activity profile set forth herein and in the claims. Therefore, proteins exhibiting substantially equivalent or altered activities are also considered. These modifications may be intentional, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as modifications obtained through mutation in a host that is a producer of the complex or its designated subunit. Furthermore, the terms “104 antibody” or “mAb104” are intended to include within their scope the proteins specifically listed herein, as well as all substantially homologous analogs and allele variants. Detailed Implementation

[0301] This invention is not limited to the specific embodiments described herein, which are for illustrative purposes only.

[0302] Antibody production

[0303] The general method for preparing monoclonal antibodies via hybridomas is well known. Antibody-producing cell lines that proliferate indefinitely can also be generated using techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. See, for example, M. Schreier et al., “Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies and T-cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980); and also see U.S. Patents 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. A group of monoclonal antibodies against HER2 can be screened for various properties, including isotype, epitope, affinity, etc., as described herein. Of particular interest are monoclonal antibodies that bind to domain II of aberrantly expressed HER2. Such monoclonal antibodies can be readily identified in specific binding member activity assays. High-affinity antibodies are also useful when immunoaffinity purification of natural or recombinant specific binding members is possible. Monoclonal antibodies that can be used to carry out the present invention can be generated by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules with appropriate antigen specificity. The culture is maintained under conditions and for a time sufficient to allow the hybridoma to secrete antibody molecules into the medium. The medium containing the antibody is then collected. The antibody molecules can then be further isolated using well-known techniques.

[0304] The antibodies of the present invention can also be produced by immunizing animals with purified antigens corresponding to cyclic peptides comprising residues 277 to 312 or residues 293 to 309 of mature normal or wild-type human HER2.

[0305] The HER2-binding protein of the present invention can also be synthesized using standard techniques such as solid-phase peptide synthesis and / or natural protein linking.

[0306] Other suitable techniques for 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.

[0307] Recombinant antibody production

[0308] The antibody and antigen-binding fragments of the present invention can also be recombined and produced using techniques and materials readily available to those skilled in the art.

[0309] The variable domain can be derived from any germline or rearranged human variable domain, or it can be a synthetic variable domain based on a common sequence of known human variable domains. Using recombinant DNA technology, the CDR-derived sequence of the present invention can be introduced into all components of a variable domain lacking a CDR region. For example, Marks et al. (Bio / Technology, 1992, 10:779-783) described a method for generating all components of an antibody variable domain, wherein a common primer targeting or adjacent to the 5' end of the variable domain region is used in conjunction with a common primer targeting the third frame region of the human VH gene to provide all components of a VH variable domain lacking one or more CDRs. Marks et al. further described how all components can be combined with the CDR of a specific antibody. Using similar techniques, the CDR-derived sequence of the present invention can be shuffled with all components of a VH or VL domain lacking one or more CDRs, and the shuffled complete VH or VL domain can be combined with a homologous VL or VH domain to provide the antibody of the present invention. All components can then be displayed in a suitable host system, such as the phage display system WO92 / 01047, to allow for the selection of appropriate specific binding members. All components can be displayed from 10 4 Any composition from 10 upwards from a single member 6 Up to 10 8 Or 10 10 Stemmer (Nature, 1994, 370:389-391) also disclosed similar shuffling or combinatorial techniques, describing techniques related to β-lactamase genes, but observed that the method could be used to produce antibodies.

[0310] Antibody affinity maturation can also be achieved using selection and / or mutagenesis methods known in the art.

[0311] The recombinant antibody of the present invention can also be produced by the phage display method disclosed in US 5,969,108.

[0312] The antibodies of the present invention may also include an antibody constant region or a portion thereof. For example, antibodies based on SEQ ID NO: 3 or 5 may have an antibody light chain constant domain comprising a human CK or Cλ chain linked to their C-terminus. Similarly, antibodies based on SEQ ID NO: 2 or 4 may have all or part of an immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE, IgD, and IgM) and any isotype subclass (particularly IgG1, IgG2b, and IgG4) linked to their C-terminus.

[0313] For recombinant production, it is preferable to isolate the nucleic acid encoding the antibody of the present invention and insert it into a reproducible vector for further cloning (DNA amplification) or for expression. The DNA encoding the antibody can be readily isolated or synthesized using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to the heavy and light chains of the DNA encoding the antibody). Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding the antibody of the present invention or a fragment thereof (e.g., from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence.

[0314] (i) Signal Sequence Components. The antibodies of the present invention can be generated not only through direct recombinant production but also as fusion peptides with heterologous polypeptides, preferably signal sequences or other polypeptides having specific cleavage sites at the N-terminus of mature proteins or polypeptides. Preferably, the heterologous signal sequence is a sequence recognized and processed by host cells (i.e., cleaved by signal peptidases). For prokaryotic host cells that do not recognize and process the natural antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leader sequences. For yeast secretion, the natural signal sequence can be replaced by, for example, yeast invertase leader sequences, α-factor leader sequences, or acid phosphatase leader sequences, Candida albicans glucoamylase leader sequences, or signals described in WO 90 / 13646. In mammalian cell expression, mammalian signal sequences and viral secretion leader sequences, such as the herpes simplex gD signal, are available. The DNA of this precursor region is linked to the DNA encoding the antibody within the reading frame.

[0315] (ii) Promoter components. Expression and cloning vectors typically contain promoters that are recognized by the host organism and can be efficiently linked to the antibody nucleic acid. Promoters suitable for prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and heterozygous promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters used in bacterial systems will also contain a Shine-Dalgarno (SD) sequence that is operatively linked to the DNA encoding the antibody.

[0316] Promoters are known to be used in eukaryotes. In fact, all eukaryotic genes have an AT-rich region approximately 25-30 bases upstream of the transcription start site. Another sequence found 70-80 bases upstream of the transcription start site in many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal to add a poly-A tail to the 3' end of the coding sequence. All of these sequences are appropriately inserted into eukaryotic expression vectors. Examples of suitable promoter sequences for yeast hosts include promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucoskinase. Other yeast promoters are inducible promoters with the added advantage of transcription controlled by growth conditions. These are promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, nitrogen metabolism-related degrading enzymes, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for yeast expression are further described in EP73,657. Yeast enhancers are also advantageously used in conjunction with yeast promoters.

[0317] Antibody transcription from vectors in mammalian host cells is controlled by promoters derived from viral genomes, such as polyomaviruses, fowlpox viruses, adenoviruses (e.g., adenovirus 2), CMV, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably simian virus 40 (SV40), from heterologous mammalian promoters, such as actin promoters or immunoglobulin promoters, or from heat shock promoters, provided that these promoters are compatible with the host cell system.

[0318] (iii) Enhancer element components. Transcription of DNA encoding the antibodies of this invention in higher eukaryotes is typically enhanced by inserting enhancer sequences into a vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are now known. However, enhancers from eukaryotic viruses are commonly used. Examples include the SV40 enhancer (bp 100-270) located late at the origin of replication, early promoter enhancers for cytomegalovirus, polyomavirus enhancers located late at the origin of replication, and adenovirus enhancers. See also Yaniv (1982) Nature 297:17-18 for enhancing elements used to activate eukaryotic promoters. The enhancer can be spliced ​​into the vector at the 5' or 3' position of the antibody coding sequence, but is preferably located at the 5' site of the promoter.

[0319] (iv) Transcription termination components. Expression vectors used for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences required to terminate transcription and stabilize mRNA. These sequences are typically obtained from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide fragments that are transcribed into polyadenylated fragments in the untranslated portion of mRNA encoding antibodies. A useful transcription termination component is the polyadenylated region of bovine growth hormone. See WO94 / 11026 and the expression vectors disclosed therein.

[0320] (v) Selection and transformation of host cells. Suitable host cells for cloning or expressing DNA in the vector described herein are the aforementioned prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens and Shigella, and Bacillus, such as Bacillus subtilis and Bacillus licheniformis, and Pseudomonas, such as Pseudomonas aeruginosa and Streptomyces. A preferred Escherichia coli cloning host is Escherichia coli 294 (ATCC 31, 446), although other strains such as Escherichia coli B, Escherichia coli X 1776 (ATCC 31, 537), and Escherichia coli W3110 (ATCC 27, 325) are suitable. These examples are illustrative and not limiting.

[0321] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or Bacillus baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and useful in this paper, such as *Schizosaccharomyces cerevisiae*; hosts of the *Kluyveromyces* genus, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces marxianus*, and *Kluyveromyces marxianus*; *Yersinia* genus (EP 402,226); *Pichia pastoris* (EP 183,070); *Candida* genus; *Trichoderma reesia* (EP 402,226). 244,234); Neurospora crassa; Schwannium genus such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Crested Yeast and Aspergillus hosts such as Aspergillus nidus and Aspergillus niger.

[0322] Suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants, along with corresponding permitted insect host cells, have been identified from hosts such as the grass armyworm (Spodoptera frugiperda), the mosquito (Aedes aegypti), the mosquito (Aedes albopictus), the fruit fly (Drosophila melanogaster), and the silkworm (Bombyx mori). Many viral strains for transfection are publicly available, such as the LI variant of the alfalfa silver-striped armyworm NPV and the Bm-5 strain of the silkworm NPV, and these viruses can be used as the viruses of this invention, particularly for transfecting grass armyworm cells.

[0323] Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); the human embryonic kidney cell line (293 or 293 cell subclones for growth in suspension culture, Graham et al. (1977) Gen Virol. 36:59); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO, Urlaub et al. (1980) Proc. Natl. Acad. Sci. USA 77:4216); mouse podocytes (TM4, Mather (1980) Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); and canine kidney cells (MDCK, ATCC CCL 10). 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al. (1982) Annals N.Y. Acad. Sci. 383: 44-68); MRC 5 cells; FS4 cells; and PER.C6 TM (Crucell NV).

[0324] Functionally equivalent antibody

[0325] The present invention also contemplates antibodies or antigen-binding fragments thereof having one or more amino acid additions, deletions, or substitutions of the heavy and light chain variable region sequences of the antibodies of the present invention, while still retaining the function of the antibodies of the present invention. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as those obtained by mutations in the host expressing the antibody.

[0326] Mutant (altered) peptides can be prepared using any technique known in the art. For example, the polynucleotides of the present invention can be mutagenized in vitro. Such in vitro mutagenization techniques involve subcloning the polynucleotide into a suitable vector, transforming the vector into a "mutant" strain such as *Escherichia coli* XL-1 Red (Stratagene), and multiplying the transformed bacteria for an appropriate number of generations. Products derived from mutated / altered DNA can be readily screened using the techniques described herein to determine whether they possess receptor-binding and / or inhibitory activity.

[0327] When designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the characteristics to be modified. The mutation site can be modified alone or in series, for example by (1) first replacing with a conserved amino acid and then replacing with more radicals depending on the results, (2) deleting the target residue or (3) inserting other residues near the localization site.

[0328] The amino acid sequence deletion is generally about 1-15 residues, more preferably about 1-10 residues, and usually about 1-5 consecutive residues.

[0329] Substitution mutants have at least one amino acid residue removed from the antibody and / or immunoglobulin chain molecule (including in the variable region) and a different residue inserted at its position. Sites of most interest in substitution mutagenesis include those identified as important for antigen binding. These sites, particularly those belonging to sequences of at least three other identical conserved sites in the human antibody and / or immunoglobulin chain, are preferably substituted in a relatively conserved manner. Such conserved substitutions are shown under the heading “Exemplary Substitutions” in the table below.

[0330] This invention also includes conserved amino acid substitutions. These refer to the amino acid substitutions listed in the table below.

[0331] Exemplary replacement

[0332]

[0333]

[0334] The amino acids used in this study are preferably in the "L" isomer form. However, residues in the D isomer form can replace any L-amino acid residue, provided that the polypeptide retains the desired functional properties for immunoglobulin binding. Modifications also include structural and functional analogs, such as peptide mimics having synthetic or non-natural amino acids or amino acid analogs and derived forms.

[0335] chimeric antibodies

[0336] Chimeric antibodies are prepared via recombinant methods by combining variable light and heavy chain regions (VL and VH) 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 produce antibodies that primarily have human domains. For example, chimeric antibodies include the variable region of a mouse antibody fused with a human constant region according to any embodiment herein. The production of such chimeric antibodies is known in the art and can be achieved by standard means (as described, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patents 5,807,715; 4,816,567 and 4,816,397). It is also considered that the human constant region of the chimeric antibody of the present invention may be selected from the constant regions of IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19.

[0337] Humanization and human antibodies

[0338] The antibodies of this invention can be humanized antibodies or human antibodies. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins (receptor antibodies) in which residues of the receptor's complementarity-determining region (CDR) are replaced by residues of the CDR from a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues not present in the receptor antibody or in the introduced CDR or framework sequence. Generally, humanized antibodies will include at least one, typically two, variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of the human immunoglobulin common sequence. Humanized antibodies will preferably also include at least a portion of the immunoglobulin constant region (Fc), typically a portion of the constant region (Fc) of human immunoglobulins (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).

[0339] Methods for humanizing nonhuman antibodies are known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are generally referred to as “input” residues, which are typically derived from the “input” variable domain. Humanization can be performed essentially as described by Jones et al. (ibid.); Riechmann et al. (ibid.); Verhoeyen et al. (1988) Science, 239:1534-1536), replacing the corresponding sequence of the human antibody with a rodent CDR or CDR sequence. Thus, such “humanized” antibodies are chimeric antibodies (US Patent 4,816,567), in which essentially no intact human variable domain is replaced by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are often human antibodies in which some CDR residues and possibly some FR residues are replaced by residues at similar sites in rodent antibodies.

[0340] Human antibodies can also be generated 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 applicable to the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al. (1991) J Immunol, 147:86-95). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production was observed, which was remarkably similar to that seen in humans in all respects, including gene rearrangements, assembly, and all antibody components. The method is described, for example, in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0341] A technique called “guided selection” can also be used to generate fully human antibodies that recognize selected epitopes. In this method, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., Bio / technology 12:899-903 (1988)).

[0342] Antibody affinity maturation can also be achieved using selection and / or mutagenesis methods known in the art. Preferably, the affinity of the maturation antibody is 5 times, more preferably 10 times, or even more preferably 20 or 30 times, that of the starting antibody (typically mouse, humanized, or human) used to prepare the maturation antibody.

[0343] Humanized and primate-like proteins

[0344] The HER2-binding protein of the present invention can be a human-like protein. The term "human-like protein" refers to a protein prepared by the method described in WO2007 / 019620. A human-like HER2-binding protein includes a variable region of an antibody, wherein the variable region includes a FR from a variable region of a New World primate antibody and a CDR from a variable region of a non-New World primate antibody. For example, a human-like HER2-binding protein includes a variable region of an antibody, wherein the variable region includes a FWR from a variable region of a New World primate antibody and a CDR from a mouse antibody, as described herein, for example. In one instance, the homohumanized HER2-binding protein is a HER2-binding antibody in which one or both variable regions are human-like.

[0345] The HER2-binding protein of the present invention may be a primate-like protein. A “primate-like protein” includes a variable region derived from an antibody produced after immunization of a non-human primate (e.g., a cynomolgus monkey). Optionally, the variable region of a non-human primate antibody is linked to a human constant region to generate a primate-like antibody. Exemplary methods for generating primate-like antibodies are described in US 6113898.

[0346] Deimmunizing antibodies and proteins

[0347] This invention also relates to deimmunizing antibodies or HER2-binding proteins. Deimmunizing antibodies have one or more epitopes, such as removed (i.e., mutated) B-cell epitopes or T-cell epitopes, thereby reducing the likelihood that a subject will generate an immune response against the antibody or protein. Methods for generating deimmunizing antibodies and proteins are known in the art and described, for example, in WO00 / 34317, WO 2004 / 108158, and WO 2004 / 064724.

[0348] Based on the description herein, methods for introducing appropriate mutations and expressing and measuring the resulting proteins will be apparent to those skilled in the art.

[0349] Antibody variable regions containing proteins.

[0350] Single domain antibodies

[0351] In some embodiments, the HER2-binding protein of the present invention is a single-domain antibody (which may be used interchangeably with the terms "domain antibody" or "dAb"). A single-domain antibody is a single polypeptide chain comprising all or part of the variable region of the antibody heavy chain. In some instances, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, US6248516; WO 90 / 05144 and / or WO 2004 / 058820).

[0352] Bivalent, Trivalent, and Quadrivalent Antibodies

[0353] Exemplary HER2-binding proteins that include an antibody-antigen binding domain are biantibodies, triantibodies, tetraantibodies, and more advanced protein complexes, such as those described in WO 98 / 044001 and WO 94 / 007921.

[0354] For example, a biantibody is a protein comprising two associated polypeptide chains, each polypeptide chain including the structure VL-X-VH or VH-X-VL, where VL is the variable region of the antibody light chain, VH is the variable region of the antibody heavy chain, and X is a linker including residues insufficient to allow association between VH and VL in a single polypeptide chain or which are absent. The VH of one polypeptide chain binds to the VL of the other polypeptide chain to form an antigen-binding site, i.e., forming an Fv molecule capable of specifically binding to one or more antigens. VL and VH can be the same in each polypeptide chain, or VL and VH can be different in each polypeptide chain, in order to form a bispecific dimer antibody (i.e., comprising two Fvs with different specificities).

[0355] Single-stranded Fv (scFv) fragment

[0356] Those skilled in the art will know that scFv comprises VH and VL regions within a single polypeptide chain. Between the VH and VL regions, the polypeptide chain also includes a polypeptide linker that enables the scFv to form the structure required for antigen binding (i.e., to associate the VH and VL regions of the single polypeptide chain with each other to form an Fv). For example, the linker comprises more than 12 amino acid residues, among which (Gly4Ser)3 is one of the more favorable linkers for scFv.

[0357] The present invention also considers disulfide-stabilized Fv (or diFv or dsFv), wherein a single cysteine ​​residue is introduced into the FR of VH and the FR of VL, and the cysteine ​​residue is linked by a disulfide bond to produce a stable Fv (see, for example, Brinkmann et al., (1993) Proc Natl Acad Sci USA 90:547-551).

[0358] Alternatively or additionally, the present invention provides a dimer scFv, i.e., a protein comprising two scFv molecules linked by non-covalent or covalent connections, such as by leucine zipper domains (e.g., derived from Fos or Jun) (see, for example, Kruif and Logtenberg, 1996). Alternatively, two scFvs may be linked by a peptide linker of sufficient length to allow the two scFvs to form and bind to an antigen, for example, as described in US20060263367.

[0359] For a review of scFv, see Ahmad ZA et al., (2012) Clinical and Developmental Immunology doi:10.1155 / 2012 / 980250.

[0360] microantibodies

[0361] Those skilled in the art will recognize that microbodies comprise the VH and VL domains of an antibody fused to the (CH2) and / or (CH3) domains of the antibody. Optionally, a microbody includes a hinge region between the VH and VL domains; this conformation is sometimes referred to as a flexible microbody. Microbodies do not include CH1 or CL. In one instance, the VH and VL domains are fused to the hinge region and the CH3 domain of the antibody. At least one variable region of the microbody binds HER2 in a manner consistent with the present invention. Exemplary microbodies and methods for their preparation are described, for example, in WO 94 / 09817.

[0362] Other protein-containing antibody variable regions

[0363] This invention also considers other variable regions containing HER2-binding proteins, such as:

[0364] (i) A protein with dual specificity for both bonds and pores, as described in US 5,731,168;

[0365] (ii) Heteroconjugated proteins, such as those described in US 4,676,980;

[0366] (iii) Heteroconjugated proteins produced using chemical cross-linking agents, such as those described in US 4,676,980;

[0367] (iv) Fab'-SH fragments, for example as described in Shalaby (1992)j Exp Med 1;175(1):217-25;

[0368] (v) Single-chain Fab; or

[0369] (vi)Fab3 (e.g., as described in EP 19930302894).

[0370] Protein-based, non-antibody antigen-binding domain

[0371] Immunoglobulins and immunoglobulin fragments

[0372] Examples of the compounds of the present invention are proteins comprising variable regions of immunoglobulins, such as T-cell receptors or heavy chain immunoglobulins (e.g., IgNA, camel antibodies).

[0373] The term "immunoglobulin" should be understood to include antigen-binding proteins that include immunoglobulin domains. An exemplary immunoglobulin is an antibody. Other proteins included in the term "immunoglobulin" include domain antibodies, camelid antibodies, and antibodies from cartilaginous fish (i.e., immunoglobulin neoantigen receptors (IgNARs)). Typically, camelid antibodies and IgNARs include the VH domain, however, they lack the VL domain and are often referred to as heavy chain immunoglobulins. Other "immunoglobulins" include T-cell receptors.

[0374] Heavy chain immunoglobulins

[0375] Heavy chain immunoglobulins differ structurally from many other forms of immunoglobulins (such as antibodies) because they include heavy chains but not light chains. Therefore, these immunoglobulins are also called "heavy chain-only antibodies." Heavy chain immunoglobulins are found in animals such as camels and cartilaginous fish (also known as IgNAR).

[0376] The variable regions present in naturally occurring heavy chain immunoglobulins are commonly referred to as the “VHH domain” in camel Ig and the V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions (called the “VH domain”) present in conventional 4-chain antibodies and the light chain variable regions (called the “VL domain”) present in conventional 4-chain antibodies.

[0377] Heavy chain immunoglobulins do not require the presence of a light chain that binds to the associated antigen with high affinity and high specificity. This means that single-domain binding fragments can be derived from heavy chain immunoglobulins, which are readily expressed and are generally stable and soluble. A general description of heavy chain immunoglobulins from camelids and their variable regions, as well as methods of their production and / or isolation and / or use, is found in references WO 94 / 04678, WO 97 / 49805, and WO 97 / 49805.

[0378] In particular, WO2005 / 118629 provides a general description of heavy chain immunoglobulins from cartilaginous fish and their variable regions, as well as methods for their production and / or isolation and / or use.

[0379] V-like protein

[0380] An example of the HER2-binding protein of the present invention is a T-cell receptor. The T-cell receptor has two V domains that bind to a structure similar to an antibody Fv module. Novotny et al., Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad SciUSA) 88:8646-8650, 1991, describe how to fuse the two V domains (designated α and β) of the T-cell receptor and express them as a single-chain polypeptide, and how to modify surface residues to reduce the direct hydrophobicity similar to the antibody scFv. Other disclosures describing the production of single-chain or multimeric T-cell receptors comprising two V-α and V-β domains include WO 1999 / 045110 or WO 2011 / 107595.

[0381] Other non-antibody proteins that include antigen-binding domains include proteins with V-like domains, which are typically monomeric. Examples of proteins including such V-like domains include CTLA-4, CD28, and ICOS. Further disclosure of proteins including such V-like domains is included in WO 1999 / 045110.

[0382] Adnectins

[0383] In one example, the HER2-binding protein of the present invention is adnectin.

[0384] Adnectins are based on the tenth type III fibronectin (10Fn3) domain of human fibronectin, in which the loop regions are modified to confer antigen binding. For example, the three loops at one end of the β-sandwich of the 10Fn3 domain can be engineered to enable Adnectin to specifically recognize antigens. See US 20080139791 or WO2005 / 056764 for more details.

[0385] Anticalins

[0386] In another example, the HER2-binding protein of this invention is anticicalin. Anticicalins are derived from lipid carrier proteins, a family of extracellular proteins that transport small hydrophobic molecules such as steroids, phenotypes, retinoids, and lipids. Lipid carrier proteins have a rigid β-sheet secondary structure with multiple loops at the open end of a conical structure, which can be engineered to bind antigens. Such engineered lipid carrier proteins are called anticalins. For a further description of anticalins, see US 7250297B1 or US 20070224633.

[0387] Affibody

[0388] In another embodiment, the HER2-binding protein of the present invention is an affinity compound. The affinity compound is a scaffold of the Z-domain (antigen-binding domain) of protein A derived from Staphylococcus aureus, which can be engineered to bind antigens. The Z-domain consists of a triple-helix bundle of approximately 58 amino acids. A library is generated by randomization of surface residues. See EP1641818 for further details.

[0389] High-affinity polymers (Avimers)

[0390] In another embodiment, the HER2-binding protein of the present invention is a high-affinity multimer. The high-affinity multimer is a multidomain protein derived from the A-domain scaffold family. The native domain of approximately 35 amino acids employs a defined disulfide-bonded structure. Diversity arises from natural variations exhibited by reorganizing the A-domain family. See WO 2002088171 for further details.

[0391] Ankylosing dermal repeats (DARPins)

[0392] In another example, the HER2-binding protein of this invention is a designed ankyrin repeat protein (DARPin). DARPin is derived from Ankyrin, a family of proteins that mediate the attachment of integrated membrane proteins to the cytoskeleton. A single ankyrin repeat sequence is a 33-residue motif consisting of two α-helices and one β-turn. They can be engineered to bind different target antigens by randomizing the residues in the first α-helix and β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (an affinity maturation method). See US 20040132028 for further details.

[0393] Other non-antibody peptides

[0394] Other non-antibody proteins that include binding domains include those based on human γ-lens proteins and human ubiquitin, the kunitz domain of human protease inhibitors, the PDZ domain of Ras-binding protein AF-6, scorpion venom (charybdotoxin), and those with C-type lectin domains (tetranectins).

[0395] constant region

[0396] This invention covers HER2-binding proteins that include variable regions and constant regions or their domains (e.g., Fc, CH2, and / or CH3 domains). Those skilled in the art will recognize the meaning of the terms constant region and constant domain from the disclosures and references discussed herein.

[0397] The constant region sequence that can be used to generate the HER2-binding protein of the present invention can be obtained from many different sources. In some instances, 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, can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one instance, human isotype IgG1 is used.

[0398] Various constant region gene sequences can be obtained in the form of publicly available deposits, or their sequences can be obtained from publicly available databases. Constant regions with specific effector functions (or lack thereof) or with specific modifications to reduce immunogenicity can be selected.

[0399] In one instance, the protein of the present invention has or exhibits effector functions that promote or achieve at least partial exhaustion, substantial exhaustion, or elimination of HER2-expressing cells. Such effector functions may enhance binding affinity to the Fc receptor, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0400] In one instance, the HER2-binding protein was able to induce enhanced levels of effector function.

[0401] In one instance, the level of effector function induced by the constant region was enhanced relative to the wild-type Fc region of either the IgG1 antibody or the IgG3 antibody.

[0402] In another example, the constant region is modified to increase the level of effector functions it can induce, compared to the unmodified constant region. Such modifications can be made at the amino acid level and / or the secondary structure level and / or the tertiary structure level and / or through glycosylation in the Fc region.

[0403] Those skilled in the art will understand that greater effector function can manifest itself in any of a variety of ways, such as higher levels of effect, more sustained effect, or faster rate of effect. Exemplary constant region modifications include amino acid substitutions, such as S239D / I332E, according to Kabat's EU index number, or S239D / A330L / I332E, according to Kabat's EU index number.

[0404] Other amino acid substitutions that enhance the ability of Fc region-inducible effector function are known in the art and / or described, for example, in US 6737056 or US 7317091.

[0405] In one instance, the glycosylation of the constant region is altered to increase its ability to induce enhanced effector function. In some instances, the Fc region according to the invention comprises a carbohydrate structure lacking (directly or indirectly) fucosylated fucose, i.e., the Fc region is “fucosylated.” These variants may have an improved ability to induce ADCC. Methods for generating fucosylated antibodies include expressing HER2-binding proteins (e.g., as described in Yumane-Ohnuki et al., 2004) in cell lines that cannot express α-1,6-fucosyltransferase (FUT8). Other methods include using cell lines that inherently produce antibodies capable of inducing enhanced effector function (e.g., duck embryo-derived stem cells for the production of viral vaccines, WO2008 / 129058; poultry). Recombinant protein production in cells (WO 2008 / 142124).

[0406] HER2-binding proteins may also include Fc regions capable of inducing enhanced CDC levels. For example, hybrids of IgG1 and IgG3 produce antibodies with enhanced CDC activity (Natsume et al., 2008).

[0407] Methods for determining the ability of an antibody or its antigen-binding fragment to induce effector function are known in the art and / or described herein.

[0408] In another example, the protein includes one or more amino acid substitutions that increase the half-life of the HER2-binding protein. For instance, the HER2-binding protein includes a constant region comprising one or more amino acid substitutions that increase the affinity of the constant region for the neonatal Fc region (FcRn). For example, the constant region has an increased affinity for FcRn at lower pH (e.g., about pH 6.0) to promote Fc / FcRn binding in endosomes. In one example, the constant region has an increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which promotes the re-release of Fc into the bloodstream after cellular recirculation. These amino acid substitutions can be used to prolong the protein's half-life by reducing clearance from the blood.

[0409] According to the EU numbering system, exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US 7083784.

[0410] The HER2-binding protein of this invention may include an IgG4 constant region or a stable IgG4 constant region. The term "stable IgG4 constant region" should be understood as referring to an IgG4 constant region that has been modified to reduce or undergo Fab arm exchange, or to form a hapten or a hapten. "Fab arm exchange" refers to a type of protein modification in human IgG4 in which the IgG4 heavy chain and its linked light chain (half-molecule) exchange with another IgG4 molecule to form a heavy-light chain pair. Thus, the IgG4 molecule can acquire two different Fab arms that recognize two different antigens (producing a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro using purified blood cells or reducing agents such as reduced glutathione. A "hapten" is formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0411] In one instance, according to the Kabat system, the stable IgG4 constant region includes proline at position 241 of the hinge region. This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, this residue is typically serine. When serine replaces proline, the IgG4 hinge region includes the sequence CPPC. At this point, those skilled in the art will recognize that the “hinge region” is the proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, conferring mobility on the two Fab arms of the antibody. The hinge region includes cysteine ​​residues involved in the inter-heavy chain disulfide bond. According to the Kabat numbering system, it is generally defined as the extension of human IgG11 from Glu226 to Pro243. By placing the first and last cysteine ​​residues forming the inter-heavy chain disulfide bond (SS) at the same position, the hinge regions of other IgG isotypes can be aligned with the IgG11 sequence (see, for example, WO 2010 / 080538).

[0412] Modified proteins

[0413] This invention provides a HER2-binding protein that has at least 80% sequence identity with the present invention and has the same functional properties described or claimed herein.

[0414] In one instance, the HER2 binding protein of the present invention comprises a sequence having at least 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% identity with the VL sequence disclosed herein, for example, SEQ ID NO: 3.

[0415] In another instance, the HER2 binding protein of the present invention comprises a sequence having at least 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% identity with the VH of the present invention herein, for example SEQ ID NO: 2.

[0416] The present invention also provides nucleic acids encoding the aforementioned proteins or nucleic acids that hybridize with them under moderate to highly stringent conditions.

[0417] The present invention also includes nucleic acids encoding proteins comprising the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, which differ from the exemplary sequences herein due to the degeneracy of the genetic code.

[0418] The present invention also includes nucleic acids encoding proteins comprising the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, which differ from the exemplary sequences herein due to the degeneracy of the genetic code.

[0419] The % identity of nucleic acids or peptides is determined by GAP (Needleman and Wunsch 1970) analysis (GCG procedure), with a nick penalty of 5 and a nick extension penalty of 0.3. The query sequence is preferably at least 50 residues long, and the GAP analysis compares the two sequences over a region of at least 50 residues. For example, if the query sequence is at least 100 residues long, the GAP analysis compares the two sequences over a region of at least 100 residues. In one instance, the two sequences are compared over their entire length.

[0420] Modified glycosylation

[0421] The glycosylation pattern of an antibody can be altered from the original glycosylation pattern of a reference antibody. This alteration involves 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. Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the attachment of a carbohydrate moieties to an asparagine residue side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of carbohydrate moieties to asparagine side chains. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation involves the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Adding glycosylation sites to an antibody is conveniently achieved by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). It can also be altered by adding or substituting one or more serine or threonine residues into the original antibody sequence (for O-linked glycosylation sites).

[0422] The modified glycoforms of the antibodies of the present invention can be used for a variety of purposes, including but not limited to enhancing or reducing effector function and / or modifying the half-life of antibodies (see, for example, WO / 2007 / 010401). Such alterations can result in a decrease or increase in C1q and CDC binding or FcγR and ADCC binding. For example, substitutions can be made in one or more amino acid residues in the heavy chain constant region, thereby resulting in alterations in effector function while maintaining the ability to bind antigens compared to modified antibodies, see US5,624,821 and US5,648,260. The engineered glycoforms can be produced by any method known to those skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, such as β(1,4)-N-acetylglucosamine transferase III (GnTIl 1), by expressing antibodies or fragments thereof in various organisms or cell lines derived from various organisms, or by modifying carbohydrates after expressing antibodies or fragments. Methods for producing engineered sugar forms are known in the art, including but not limited to 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 Nos. 6,602,684; 10 / 277,370; 10 / 113,929; PCT WO 00 / 61739A1; PCT WO 01 / 292246A1; PCT WO 02 / 311140A1; PCT WO 02 / 30954A1; Technology (Biowa, Inc., Princeton, NJ); GlycoMAb TM Glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, for example, WO 00061739; EA01229125; US 20030115614; Cong et al. (2004) JMB, 336: 1239-49.

[0423] Effector Function

[0424] The antibodies of the present invention may need to be modified in terms of effector function, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or additionally, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in that region. The resulting homodimeric antibodies may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). See Caron et al., J. J. Exp Med. 176:1 191-1 195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinkers described in Wolff et al., Research 53:2560-2565 (1993). Alternatively, antibodies with dual Fc regions can be modified to enhance complement cleavage and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0425] half life

[0426] To increase the serum half-life of an antibody, a salvage receptor-binding epitope can be incorporated into the antibody (especially an antibody fragment), for example, as described in U.S. Patent 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 can be increased by PEGylation.

[0427] Determining the activity of the binding protein of the present invention

[0428] Combined measurement

[0429] One form of this assay is the antigen-binding assay, as described, for example, in Scopes (1994) *Protein Purification: Principles and Practice*, Springer-Verlag. This method typically involves labeling the HER2-binding protein and contacting it with an immobilized antigen or a fragment thereof, for example, as... Figure 1The protein shown includes residues 293 to 309 of mature normal or wild-type human HER2. After washing to remove non-specific binding proteins, the amount of the marker is detected, and the result indicates the amount of binding protein. Alternatively, HER2 binding proteins can be immobilized and labeled with antigens. Panning assays can also be used. The examples in this article describe a flow cytometry-based binding assay.

[0430] HER2-binding proteins that competitively inhibit the binding of the HER2 antibody of the present invention to the epitope can be screened and identified using conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0431] Competitive binding assay

[0432] An assay for determining the binding of a HER2-binding protein that competitively inhibits the binding of antibodies (e.g., mAb104) of the present invention will be readily apparent to those skilled in the art. For example, the antibody of the present invention is conjugated to a detectable label, such as a fluorescent or radioactive label. The labeled antibody and the tested HER2-binding protein are then mixed and contacted with HER2 or a peptide including its epitope (e.g., residues 293 to 309 corresponding to 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 including its epitope in the absence of the HER2-binding protein. If the level of the labeled antibody is lower in the presence of the tested HER2-binding protein compared to the absence of the HER2-binding protein, the HER2-binding protein competitively inhibits antibody binding.

[0433] Optionally, the HER2-binding protein to be tested is conjugated to a label that differs from the antibody. This allows for the detection of the binding level of the HER2-binding protein to a protein or epitope.

[0434] In another example, HER2-binding protein binding to HER2 or a peptide including its epitope is allowed to be tested before contacting HER2 or a peptide including its epitope with the antibody described herein. The reduced amount of antibody bound in the presence of HER2-binding protein compared to the absence of HER2-binding protein indicates that HER2-binding protein competitively inhibits antibody binding to HER2. An interactive assay can also be performed using labeled HER2-binding protein, and antibody binding to HER2 or a peptide including its epitope is first allowed. In this case, the reduced amount of labeled HER2-binding protein binding to HER2 or a peptide including its epitope in the presence of antibody compared to the absence of antibody indicates that HER2-binding protein competitively inhibits antibody binding to HER2.

[0435] Affinity Measurement

[0436] Optionally, the dissociation constant (Kd), association constant (Ka), or binding constant (KD, i.e., Ka / Kd) of the HER2-binding protein to HER2 or its epitope-containing peptide is determined. In one embodiment, these constants of the HER2-binding protein are measured by a radiolabeled or fluorescently labeled HER2 binding assay. This assay equilibrates the HER2-binding protein with a minimum 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 determined. According to another embodiment, the constants are measured by using a surface plasmon resonance assay, for example, using a BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized HER2 or its regions.

[0437] Protein detection assay

[0438] One example of this invention is the detection of the presence of HER2 or HER2-expressing cells (e.g., breast cancer cells). The quantity, level, or presence of the protein or cells is determined using any of a variety of techniques known to those skilled in the art, such as those selected from the group consisting of flow cytometry, immunohistochemistry, immunofluorescence, Western blotting, Western blot, dot blot hybridization, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g., LC MS / MS), biosensor technology, evanescent fiber optic technology, or protein chip technology.

[0439] In one example, the assay used to determine the amount or level of protein is a semi-quantitative assay. In another example, the assay used to determine the amount or level of protein is a quantitative assay.

[0440] For example, proteins can be detected using immunoassays, such as those selected from the group consisting of immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), Western blotting, radioimmunoassay (RIA), biosensor assays, protein chip assays, and immunostaining assays (e.g., immunofluorescence).

[0441] Standard solid-phase ELISA or FLISA forms are particularly useful for determining protein concentrations from a variety of samples.

[0442] In one form, an ELISA or FLISA involves immobilizing the HER2-binding protein of the present invention, or a protein that binds to a different epitope of HER2, onto a solid matrix, such as a membrane, polystyrene or polycarbonate micropores, polystyrene or polycarbonate impregnated sheets, or a glass support. The sample is then physically associated with the immobilized protein, and HER2 is bound or “captured.” The bound HER2 is then detected using a second labeling compound that binds to a different epitope of HER2. Alternatively, a third labeling antibody that binds to a second (detection) antibody may be used. It will be apparent to those skilled in the art that the assay forms described herein are suitable for high-throughput applications, such as automated screening processes or microarray applications. Furthermore, variations of the above assays will be apparent to those skilled in the art, such as competitive ELISAs.

[0443] In another instance, peptides are detected intracellularly or on cells using methods known in the art, such as immunohistochemistry or immunofluorescence. Methods using immunofluorescence are exemplary because they are quantitative or at least semi-quantitative. Methods for quantifying the fluorescence intensity of stained cells are known in the art and described, for example, Cuello, 1984.

[0444] Biosensor devices typically employ electrode surfaces combined with current or impedance measurement elements, which are integrated into a device that binds to an assay matrix (e.g., as described in US 5567301). The HER2-binding protein of the present invention is incorporated into the surface of the biosensor device, and a biological sample is brought into contact with the device. Changes in current or impedance detected by the biosensor device indicate the binding of the protein to the HER2-binding protein. Some forms of biosensors known in the art also rely on surface plasmon resonance (SPR) to detect protein interactions, whereby changes in the reflected SPR surface indicate protein binding to a ligand or antibody (US 5485277 and US 5492840).

[0445] Because such systems are easily adaptable to the micrometer or nanometer scale, biosensors are particularly suitable for high-throughput analysis. Furthermore, such systems are conveniently suited for combining several detection reagents, allowing for the multiplexing of diagnostic reagents within a single biosensor unit. This enables the simultaneous detection of several proteins or peptides in small amounts of bodily fluids.

[0446] The binding of the protein to HER2 can also be detected by flow cytometry as described in the examples in this article.

[0447] Epitopes bound by the binding protein of the present invention

[0448] The inventors have developed binding molecules that specifically bind to conformationally exposed epitopes in domain II of human HER2, present in tumorigenic, overproliferating, or abnormal cells rather than wild-type or normal cells. These conformationally exposed epitopes are located at the distal portion of domain II, side-attached with disulfide bonds, allowing for flexible variation in this region and the exposure of the epitope, which has been bound to mAb104. In particular, the epitope appears to be exposed in cells responding to HER2 amplification or activation. Particularly surprising about the antibodies of this invention is that they do not block the binding of pertuzumab or trastuzumab to the extracellular domain of HER2, indicating that this epitope region of domain II allows antibody binding without blocking it when conformationally exposed, potentially allowing for dual therapeutic approaches.

[0449] The crystal structure of pertuzumab binding to HER2 has been determined (see Franklin MC et al., (2004) Cancer Cell, Vol. 5: 317-328). Pertuzumab binds to HER2 near the center of domain II, spatially blocking the binding pocket necessary for receptor dimerization and signal transduction. It is understood that the CDR H3 of pertuzumab contacts the HER2 residues Lys311 and His296 via hydrophobic and hydrogen bonds. His296 is completely embedded in the pertuzumab binding. Since pertuzumab specifically inhibits HER2 heterodimerization by blocking the pocket of the dimerization ring on HER2 that accepts the heterodimer receptor's partner, and not wishing to be bound by theory, the inventors hypothesize that the binding molecule of the present invention (e.g., mAb104) may spatially bind to different faces of the epitope ring in HER2. The inventors hypothesize that the epitope may be revealed in a subset of receptors on the surface of cancer cells, making it more readily available, when the receptor undergoes a conformational change due to redox disulfide bond transitions or aberrant expression of HER2 in cancer under conditions of overexpression or hypoxia, or when HER2 binds to a dimerized partner and undergoes a conformational change to reveal the ring bound by the currently binding molecule. Furthermore, it is known that antibodies do not bind to every amino acid in the epitope region, and this can be explained by the conformational properties of the mAb104 epitope, why closely opposed epitopes do not affect the binding of the two antibodies.

[0450] In one instance, the HER2-binding protein inhibited HER2 heterodimerization.

[0451] Antibody conjugates

[0452] This invention also provides a HER2-binding protein as described herein, conjugated to a portion. The portion may include, but is not limited to, a detectable or functional marker. In some embodiments, the portion is selected from the group consisting of radioisotopes, detectable markers, therapeutic compounds, colloids, toxins, nucleic acids, peptides, proteins, compounds that increase the half-life of HER2-binding proteins in a test subject, and mixtures thereof. As will be understood by those skilled in the art, the portion may be classified as one or more of the above lists. For example, the portion may be classified as therapeutic compounds and toxins.

[0453] In some implementations, the component is a radioactive isotope. Suitable radioactive isotopes include isotopes. 3 H, 14 C 32 P, 33 P, 35 S, 36 Cl、 47 Sc、 51 Cr 57 Co、 58 Co、 59 Fe、 67 Ga、 68 Ga、 89 Zr、 90 Y、 121 I, 124 I, 125 I, 131 I, 111 In、 177 Lu、 211 At、 198 Au、 67 Cu、 223 Ra、 225 Ac、 213 Bi、 99 Tc and 186 Re, they can be linked to the antibodies of the present invention using conventional chemical methods known in the field of antibody imaging.

[0454] In some implementations, the part is a detectable marker. Suitable detectable markers include, but are not limited to, radioactive markers, such as isotopes. 3 H, 14 C 32 P, 33 P, 35 S, 36 Cl、 47 Sc、 51 Cr 57 Co、 58 Co、 59 Fe、 67 Ga、 68 Ga、89 Zr、 90 Y、 121 I, 124 I, 125 I, 131 I, 111 In、 177 Lu、 211 At、 198 Au、 67 Cu、 223 Ra、 225 Ac、 213 Bi、 99 Tc and 186 The labeling can be linked to the antibody of the present invention using conventional chemical methods known in the field of antibody imaging. Labeling also includes fluorescent labels (e.g., fluorescein, rhodamine, Texas red, phycoerythrin) and labels conventionally used in MRI-CT imaging. These also include enzyme labels such as horseradish peroxidase, β-glucuronidase, β-galactosidase, urease, catalase, alkaline phosphatase, and chloramphenicol transferase. Labeling also includes peptide tags such as T7-, his-, myc-, HA-, and FLAG- tags. Labeling also includes chemical moieties, such as biotin, which can be detected by binding to specific homologous detectable moieties such as labeled avidin. Labeling also includes electron-dense reagents; energy transfer molecules; paramagnetic labels; chemiluminescence (imidazole, luciferase); and bioluminescent agents.

[0455] In some implementations, the component is a nucleic acid. Suitable nucleic acids include double-stranded DNA, single-stranded DNA, siRNA, DNase, or ribozyme.

[0456] In some embodiments, a portion is a therapeutic compound. Suitable therapeutic compounds include compounds capable of altering biological responses (e.g., but not limited to inhibiting or preventing cellular expression activity, causing cellular damage, or otherwise affecting cellular function). Such therapeutic compounds include, for example, but not limited to, chemical ablation agents, toxins, immunomodulators, cytokines, cytotoxic drugs, chemotherapeutic agents and / or drugs, and include, but are not limited to, the following: 4-deacetylated vincristine-3-carbohydrate; 5-fluoro-2′-deoxyuridine; 5-fluorouracil; 5-fluorouracil decarbonylation; 6-mercaptopurine; 6-thioguanine; abrin; abrin A chain; actinomycin D; 1-dehydrotestosterone; doxorubicin; tung oil protein; alkylation. Agents; alkylphosphocholine; aminopterin; angiopoietin; angioinhibinin; anthracyclines; aminopicrine; anti-angiogenic drugs; antifolate agents; antimetabolites; antimitotic substances; antibiotics; cytarabine; asparaginase-containing aurestatin derivatives (see, for example, but not limited to, U.S. Patent Publications 2008 / 0300192, 2009 / 0018086, 2009 / 0018086 and 2009 / 0111756, each of which is incorporated herein by reference in its entirety) ; Aurometrine E (see, for example, but not limited to, U.S. Patent 5,635,483, which is incorporated herein by reference in its entirety); Aurometrine E valerate benzylhydrazone; Aurometrine F phenylene diamine; Aurometrine; auromycins; diiodophenol mustard; bismuth; bleomycin; busulfan; chalcone; carboplatin; erythromycin; carmustine; cc-1065 compound (see, for example, but not limited to, U.S. Patents 5,475,092, 5,585,499) U.S. Patent Publications 2007 / 0135346, 2008 / 0260685, 2009 / 0281158, and 2009 / 0318668, and PCT Publication No. 5,846,545, 6,534,660, 6,586,618, 6,756,397, 7,049,316, 7,329,760, 7,388,026, 7,655,660, and 7,655,661; U.S. Patent Publications 2007 / 0135346, 2008 / 0260685, 2009 / 0281158, and 2009 / 0318668; and PCT Publication No. WO (2009 / 017394, each of which is incorporated herein by reference in its entirety); chlorambucil; cis-dichlorodiamineplatin (cisplatin); cladribine; colchicine; carbetatin; crotonin; curicin; cyclophosphamide; cytarabine; cytochalasin B; cytarabine; cytotoxin; dacarbazine; actinomycin; daunorubicin; carnation toxin; dibromomannitol; dihydroxyanthracin dione; diphtheria toxin; sulphurin-10; docetaxel; doxorubicin; doxorubicin hydrazide;Betamethasone (see, for example, but not limited to, U.S. Patent 7,214,685, which is incorporated herein by reference in its entirety); emetine; endostatin; enediyenes; enoxacin; epirubicin; esperamycin compounds (see, for example, but not limited to, U.S. Patent 4,675,187, which is incorporated herein by reference in its entirety); ethidium bromide; etoposide; fludarabine leukotoxin; gefitinib, gemcitabine; glucocorticoids; bacitracin D; granulocyte colony-stimulating factor; granulocyte-macrophage colony-stimulating factor; idarubicin; interleukin-1; interleukin-2; interleukin-6; lidocaine; lomustine; lymphokines; maytansine (See, for example but not limited to, U.S. Patents 4,137,230, 4,151,042, 4,162,940, 4,190,580, 4,225,494, 4,228,239, 4,248,870, 4,256,746, 4,260,608, 4,263,294, 4,264,596, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,317,821, 4,320,200, 4,322,348, 4,331,598, 4,360,462, 4,361,6) 50, 4,362,663, 4,364,866, 4,371,533, 4,424,219, 4,450,234, 5,141,736 and 5,217,713 (each incorporated herein by reference in its entirety); nitrogen mustard; melphalan (and other related nitrogen mustards); methotrexate; minor groove conjugates; photomycin; mitogellin; mitomycin C; mitomycin; mitoxantrone; MMAF-dimethylaminoethylamine; MMAF-Nt-butyl; MMAF-tetraethylene glycol; modeccin A chain; bitter melon inhibitor; monomethylaurestatin E (MMAE) (see, for example, but not limited to, U.S. Patent 6,884,800). 69, 7,098,308, 7,256,257 and 7,423,116, and U.S. Patent Publications 2003 / 0083263, 2004 / 0157782, 2005 / 0009751, 2005 / 0113308 and 2006 / 0229253, each of which is incorporated herein by reference in its entirety; monomethylaurestatin F (MMAF) (see, for example, but not limited to, U.S. Patent 7,498,298, and U.S. Patent Publications 2008 / 0226657, 2008 / 0248051, 2008 / 0248053 and 2009 / 0047296, each of which is incorporated herein by reference in its entirety); morpholino doxorubicin;N2′-Deacetylated-N2′-(c-mercapto-1-oxopropyl)-Maytansine (DM1) (see, for example, but not limited to, U.S. Patent 5,208,020, which is incorporated herein by reference in its entirety); N2′-Deacetylated-N2′-(4-mercapto-4-methyl-1-oxopentyl)-Maytansine (DM4) (see, for example, but not limited to, U.S. Patent 7,276,497, which is incorporated herein by reference in its entirety); neomycin; nerve growth factor (and other growth factors); onanaxone; paclitaxel; PE40; phenolmycin; pokeweed protein (PAPI, PAPII, and PAP-S); platelet-derived growth factor; procaine; prednisone; procaine; procarbazine; propranolol; Pseudomonas exotoxin A; puromycin; pyrrolobenzodiazepines; radioisotopes (e.g., such as but not limited to At); 211 Bi 212 Bi 213 、Cf 252 I 125 I 131 In 111 Ir 192 Lu 177 P 32 Re 186 Re 188 、Sm 153 Y 90 and W 188 ); retstrictocin; retstrictocin; ricin A; ricin; fussaponin; saponin; streptozotocin; suramin; tamoxifen; taxanes; taxanes; tacriazole; teniposide; tetracaine; thioepa chlorambucil; thiotepa; thrombolytic agents; tissue plasminogen activators; topoisomerase I inhibitors; topoisomerase II inhibitors; toxotere; trichothecenes; tumor necrosis factor; vincristine; vinca alkaloids; vinca genus; vincristine; vinorelbine; vinorelbine; yttrium; α-interferon; α-diaceticococcus; and β-interferon, and their analogues, homologs, fragments, variants and derivatives (see also Garnett (2001) Advanced Drug Delivery Review). (Reviews 53:171-216, which is incorporated herein by reference in its entirety).

[0457] In a preferred embodiment, the therapeutic compound is selected from the group consisting of aurestatin or a derivative thereof, maytansine or a derivative thereof (also known as maytanol), or pyrrolobenzodiazepine or a derivative thereof. In one example, the therapeutic agent is N2′-deacetylated-N2′-(c-mercapto-1-oxopropyl)-matansine (DM1). In another example, the therapeutic agent is monomethyl aurestatin E (MMAE). In yet another example, the therapeutic agent is pyrrolobenzodiazepine.

[0458] The present invention also considers, for example, immunotoxin conjugates described in WO 93 / 21232.

[0459] Suitable colloids include colloidal gold and gold nanoparticles. HER2-binding proteins can be conjugated to colloids using techniques known to those skilled in the art (see Jazayeri et al. (2016), Sensing and Bio-Sensing Research, 9:17-22).

[0460] In some implementations, the component 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 used include diphtheria A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain (from *Pseudomonas aeruginosa*), ricin A chain, abrin A chain, modeccin A chain, α-salicylicin, *Aleurites fordii* protein, caryophyllin protein, *Phytolacca americana* protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, *Jatropha curcas* toxin, croton toxin, *Euphorbia humifusa* inhibitor, *Euphorbia pekinensis* toxin, mitogellin, localized aspergillin, phenolmycin, enoxacin, and trichothecene compounds.

[0461] In some implementations, this involves compounds that increase the half-life of HER2-binding proteins in the test subjects. Suitable compounds for increasing the half-life of HER2-binding proteins in test subjects include PEG, recombinant PEG mimics (including flexible peptides such as XTEN, elastin-like peptides, gelatin-like peptides, and (Pro-Ala-Ser)). n ), carbohydrates (such as dextran, hydroxyethyl starch, polysialic acid and hyaluronic acid) and peptides / polypeptides (such as albumin and Fc protein of IgG).

[0462] Furthermore, the HER2 binding protein of the present invention, particularly the antibody and fragment thereof, can be conjugated to a secondary antibody to form an antibody heteroconjugate (see, for example, but not limited to, U.S. Patent 4,676,980, which is incorporated herein by reference in its entirety), can be administered alone or in combination with another agent (for example, but not limited to, the agents described above) (with or without the agent linked to or conjugated thereto), and / or can be conjugated to an anticancer prodrug activating enzyme capable of converting a prodrug into its active form.

[0463] As those skilled in the art will understand, the foregoing portions and other suitable portions can be conjugated or linked in any suitable manner to the HER2-binding protein of the present invention, particularly antibodies and fragments thereof, to produce antibody conjugates. For example, but not limited to, in various embodiments of the present invention, the HER2-binding protein and portions can be covalently linked and / or conjugated using linkers, spacer regions, and / or extension compounds, which in various embodiments of the present invention are cleavable, non-cleavable, and result in the internalization of the reagent into the target cells.

[0464] For example, such connectors, spacers, and / or extension compounds include, but are not limited to, the following: aminobenzoic acid spacer (see, for example, but not limited to, U.S. Patents 7,091,186 and 7,553,816, each of which is incorporated herein by reference in its entirety); maleimide hexanoyl; p-aminobenzylcarbamoyl (PAB); lysosomal enzyme cleavage connector (see, for example, but not limited to, U.S. Patent 6,214,345, which is incorporated herein by reference in its entirety); maleimide hexanoyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valine citrulline; 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) (see, for example, but not limited to, Yoshitake et al., (1979) Eur. J. Biochem., 101, 395-399, which is incorporated herein by reference in its entirety); 4 -(2-pyridyldithio)butyric acid N-succinimide ester (SPDB) (see, for example, but not limited to, U.S. Patent 4,563,304, which is incorporated herein by reference in its entirety); 4-(2-pyridylthio)valerate N-succinimide ester (SPP); valine-citrulline; and other linker, spacer, and / or extension compounds (see, for example, but not limited to, U.S. Patents 7,090,843, 7,223,837). U.S. Patent Publications 7,659,241, and U.S. Patent Publications 2004 / 0018194, 2004 / 0121940, 2006 / 0116422, 2007 / 0258987, 2008 / 0213289, 2008 / 0241128, 2008 / 0311136, 2008 / 0317747 and 2009 / 0010945, each of which is incorporated herein by reference in its entirety.

[0465] Generally, techniques for linking and / or conjugating the above-described portions and other portions to the HER2-binding protein of the present invention, particularly antibodies and fragments thereof, are known in the art. In various embodiments of the present invention, the HER2-binding protein and portions can be covalently linked and / or conjugated via lysine or cysteine ​​residues present in the HER2-binding protein. In one embodiment, the MMAE portion is linked by conjugation to a cysteine ​​residue. In one embodiment, the DM1 portion is linked by conjugation to a lysine residue. In one embodiment, the PBD (pyrrolobenzodiazepine) portion is linked by conjugation to a cysteine ​​residue. Suitable conjugation chemistry is reviewed in Jain et al. (2015) Pharmaceutical Research, 32:3526.See also, for example, but not limited to, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Second Edition), 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. In Applications), Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982), Parslow et al. (2016) Biomedicines, 4, 14, which are all incorporated herein by reference in their entirety.

[0466] In embodiments where the linked portion is a peptide or polypeptide, the conjugate can be a fusion protein in which the HER2-binding protein and the peptide or polypeptide form a single continuous polypeptide chain. These fusion proteins can be produced using techniques known in the art, including recombinant or synthetic techniques.

[0467] Furthermore, antibodies (including fragments thereof) and drugs that regulate the production or activity of specific binding members, antibodies, and / or their subunits may have certain diagnostic applications and may be used, for example, to detect and / or measure conditions such as cancer, precancerous lesions, conditions associated with or caused by excessive proliferative cell growth, etc. For example, specific binding members, antibodies, or their subunits may be used to generate polyclonal and monoclonal antibodies against themselves in various cell culture media using known techniques such as hybridoma techniques, for example, fused mouse splenic lymphocytes and myeloma cells. Similarly, small molecules that mimic or antagonize the activity of the specific binding members of the present invention may be discovered or synthesized and may be used in diagnostic and / or therapeutic regimens.

[0468] Specific binding members of radiolabeled molecules, particularly antibodies and fragments thereof, can be used in in vitro diagnostic techniques and in vivo radioimaging techniques, as well as radioimmunotherapy. In the case of in vivo imaging, the specific binding members of the present invention can be conjugated to imaging agents rather than radioisotopes, including but not limited to magnetic resonance imaging enhancers, wherein, for example, antibody molecules are loaded with a large number of 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 another aspect of the invention, specific binding members of radiolabeled molecules, particularly antibodies and fragments thereof, especially radioimmunoconjugates, can be used in radioimmunotherapy, particularly as radiolabeled antibodies for cancer treatment. In a further aspect, specific binding members of radiolabeled molecules, particularly antibodies and fragments thereof, can be used in radioimmunoguided surgical techniques, wherein they can identify and indicate the presence and / or location of cancer cells, precancerous cells, tumor cells, and hyperproliferating cells before, during, or after surgical removal.

[0469] Competitive inhibition

[0470] Antibodies that competitively inhibit the binding of the HER2 antibody of the present invention to the epitope can be screened and identified using conventional competitive binding assays known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0471] The composition of the present invention

[0472] The HER2-binding protein comprising its conjugate according to the present invention is typically administered in the form of a pharmaceutical composition, which may include at least one component other than the HER2-binding protein, HER2 antibody, or its antigen-binding fragment. Therefore, pharmaceutical compositions according to the present invention, and for use according to the present invention, may include, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. These substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, such as intravenous.

[0473] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder, or liquid form. Tablets may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may include physiological saline solutions, dextran or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0474] For intravenous, injectable, or site-specific administration, the active ingredient will be in the form of a parenteral-acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0475] In some embodiments, liposomes and / or nanoparticles may also be used with the active ingredient. The formation and use of liposomes are generally known to those skilled in the art. Liposomes can be formed from phospholipids dispersed in an aqueous medium and spontaneously forming multilayered concentric bilayer vesicles (also known as multilayered vesicles (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayered vesicles (SUVs) with diameters ranging from 200 to 500 angstroms, containing an aqueous solution in the core. When dispersed in water, phospholipids can form a variety of structures different from liposomes, depending on the molar ratio of lipid to water. At low ratios, liposomes are the preferred structure. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can exhibit low permeability to ions and polar substances, but undergo a phase transition at high temperatures, which significantly alters their permeability. The phase transition involves a change from a tightly packed, ordered structure called a gel state to a loosely packed, disordered structure called a fluid state.

[0476] HER2-binding proteins or compositions comprising them may be administered alone or in combination with other treatments, therapeutic agents, or pharmaceutical agents, either simultaneously or sequentially, depending on the condition to be treated. Furthermore, the present invention contemplates and includes compositions comprising the HER2-binding protein described herein and other agents or therapeutic agents (e.g., anticancer agents or therapeutic agents, hormones, other antiHER2 agents or antibodies, or antiEGFR agents or antibodies). More generally, these anticancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational regulators, cell growth or division inhibitors (e.g., antimitotic agents), or signal transduction inhibitors. Other treatments or therapies may include administration of appropriate doses of pain-relieving medications, such as nonsteroidal anti-inflammatory drugs (e.g., aspirin, acetaminophen, ibuprofen, or ketoprofen) or opioid preparations, such as morphine or antiemetics. The composition can be administered in combination with tyrosine kinase inhibitors (including but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosourea, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytarabine, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine and / or other chemotherapeutic agents (sequentially (i.e., before or after) or simultaneously). Therefore, these agents can be HER2-specific agents or tyrosine kinase inhibitors, such as lapatinib, afatinib, AG1478, ZD1 839, STI571, OSI-774, or SU-6668, or more common anticancer and antitumor agents, such as doxorubicin, cisplatin, temozolomide, nitrosourea, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytarabine, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. Furthermore, the composition can be used in conjunction with hormones (such as dexamethasone), immunomodulators (such as interleukins), tumor necrosis factor (TNF), or other cytokines or angiogenesis inhibitors that stimulate immune responses and reduce or eliminate cancer cells or tumors.

[0477] In some instances, the HER2-binding protein, or a composition including it, is combined with a chemotherapy agent, a radioimmunotherapy agent, or an immunotherapy agent. In one instance, the immunotherapy agent is a checkpoint inhibitor. In another instance, the checkpoint inhibitor is selected from ipilimumab (CTLA4), nivolumab (PD-1), pembrolizumab (PD-1), atezolizumab (PD-L1), avelumab (PD-L1), durvalumab (PD-L1), and cemiplimab (PD-1).

[0478] In some instances, HER2-binding proteins, or compositions including them, are administered together with immunosuppressive drugs.

[0479] In some instances, HER2-binding proteins or compositions including them are administered together with immunomodulators. Examples of suitable immunomodulators include interleukins (e.g., IL-2, IL-7, IL-12), cytokines (e.g., interferon, G-CSF), chemokines (e.g., CCL3, CCL26, and CXCL7), and immunomodulatory imide drugs (e.g., thalidomide).

[0480] The HER2-binding protein of this invention can be administered to patients in need of treatment via any suitable route, typically by injection into the bloodstream or CSF, or directly into the tumor site. The precise dosage will depend on many factors, including whether the antibody is for diagnostic or therapeutic purposes, the size and location of the tumor, the precise nature of the HER2-binding protein (whether it is a whole antibody, fragment, biantibody, etc.), and the nature of the detectable or functional marker attached to the antibody. When a radionuclide is used for treatment, a suitable minimum single dose is approximately 45 mCi / m 2 The maximum is approximately 250 mCi / m 2 The preferred dose range is 15-40 mCi, with a further preferred range of 20-30 mCi or 10-30 mCi. This treatment may require bone marrow or stem cell replacement. Typical antibody doses for tumor imaging or treatment are 0.5-40 mg, preferably 1-4 mg in the F(ab')2 form. Naked antibodies are preferably administered at doses of 20-1000 mg protein per dose, or 20-500 mg protein per dose, or 20-100 mg protein per dose. This is the dose for a single treatment in adult patients and can be adjusted proportionally for children and infants, as well as for other antibody forms proportional to molecular weight. Treatment may be repeated daily, twice weekly, weekly, or monthly at the physician's discretion.

[0481] Examples of suitable angiogenesis inhibitors (anti-angiogenic agents) include, but are not limited to, urokinase inhibitors, matrix metalloproteinase inhibitors (such as marimastat, neovastat, BAY 129566, AG3340, BMS 275291 and similar agents), inhibitors of endothelial cell migration and proliferation (such as TNP470, squalamine, 2-methoxyestradiol, compretastatin, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp., Madison, NJ), R115777 (Janssen Pharmaceutica, Inc., Titusville, NJ) and similar agents), antagonists of angiogenic growth factors (such as ZD6474, SU6668, antibodies against anti-angiogenic agents and / or their receptors (such as VEGF, bFGF and angiopoietin 1), thalidomide, thalidomide analogs (such as CC5013), and Sugeng. 5416, SU5402, anti-angiogenic ribozymes (e.g., angiozyme), interferon α (e.g., interferon α2a), suramin and similar drugs), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (e.g., SU011248), endothelial-specific integrin / survival signal transduction inhibitors (e.g., vitaxin and similar drugs), copper antagonists / chelators (e.g., tetrathiomolybdate, captopril and similar drugs), carboxymethyltriazole (CAI), ABT627, CM101, interleukin-12 (IL12), IM862, PNU145156E, and angiogenic nucleotide molecules (e.g., antisense-VEGF-cDNA, cDNA encoding angiostatin, cDNA encoding p53, and cDNA encoding defective VEGF receptor 2) and similar agents. Other examples of inhibitors of angiogenesis, neovascularization, and / or other angiogenesis include heparin derivatives and related molecules (e.g., heparinase III), temozolomide, NK4, macrophage migration inhibitory factor (MIF), cyclooxygenase 2 inhibitors, hypoxia-inducible factor 1 inhibitors, anti-angiogenic soy isoflavones, otepralatrexate, nicotinic acid and its analogues, somatostatin analogues, pentosan polysulfate, tecogallan sodium, dalteparin, tumor suppressor, thromboretin, NM3, carbetatin, angiostatin, avastatin, antibodies against other related targets (e.g., anti-α-v / β-3 integrin and anti-kininostatin mAb) and analogues.

[0482] Measuring cell viability and proliferation

[0483] Cytotoxicity and viability (apoptosis, lysis, growth and proliferation, etc.) can be measured based on calorimetry, luminescence, radiation, or fluorescence assays known in the art and in a variety of ways as described in the embodiments herein. Colorimetric techniques for determining cell viability include, for example, the trypan blue exclusion method. In short, cells are stained with trypan blue and counted using a hemocytometer. Live cells exclude the dye, while dead and dying cells absorb the blue dye and are easily distinguishable under an optical microscope. Neutral red is adsorbed by live cells and concentrated in lysosomes; live cells can be determined by quantitatively counting the number of neutral red-stained cells using an optical microscope.

[0484] Fluorescent techniques used to determine cell viability include, for example, propidium iodide, a fluorescent DNA intercalator. Propidium iodide is excluded from living cells but stains the nuclei of dead cells. Flow cytometry of propidium-labeled cells can then be used to quantify living and dead cells. The release of lactate dehydrogenase (LDH) indicates structural damage and death of cells and can be measured by spectrophotometric enzymatic assay. Bromodeoxyuridine (BrdU) is incorporated into newly synthesized DNA and detected with an antibody labeled with a fluorescent dye. The fluorescent dye Hoechst 33258 labels DNA and can be used to quantify cell proliferation (e.g., flow cytometry). Quantitative incorporation of the fluorescent dye carboxyfluorescein diacetate succinimide (CFSE or CFDA-SE) provides cell division analysis (e.g., flow cytometry). This technique can be used in vitro or in vivo. 7-Aminoactinomycin D (7-AAD) is a fluorescent intercalator that undergoes a spectral shift upon binding to DNA and can provide cell division analysis (e.g., flow cytometry).

[0485] Radiometric measurement techniques used to determine cell proliferation include, for example, [ 3 [H]-thymidine, which is incorporated into newly synthesized DNA in living cells and is frequently used to measure cell proliferation, can be quantified by scintillation counting of chromium released from dying cells. 51 Cr) to quantify cell viability.

[0486] Luminescence techniques used to determine cell viability include, for example, the CellTiter-Glo luminescence cell viability assay (Promega Madison Wis.). This technique quantifies the amount of ATP present to determine the number of live cells.

[0487] 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, which provides rapid cell counting and viability assays based on differential uptake of fluorescent reagents (Guava Technologies, Hayward, Calif.); 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 fluorescence assays. Quantitative cell proliferation assays are fluorescence-based assays that measure the fluorescence of DNA-dye complexes from lysed cells (Stratagene, La Jolla, Calif.). The CellTiter-Glo Cell Viability Assay is a luminescent assay used to measure cell viability (Promega, Madison Wis.).

[0488] application

[0489] (i) Diagnostic and therapeutic uses

[0490] The unique specificity of the HER2-binding protein of the present invention, particularly the antibody or fragment thereof, allows the binding protein to recognize the HER2 / ErbB2 epitope, which is found in tumorigenic, hyperproliferating, or abnormal cells and is undetectable in normal or wild-type cells. Furthermore, the protein binds to amplified EGFR but not to wild-type HER2, providing diagnostic and therapeutic uses for the identification, characterization, targeting, and treatment of, reducing, or eliminating various tumor cell types and tumor types (e.g., head and neck, breast, lung, bladder, or prostate tumors and gliomas), without the problems associated with normal tissue uptake that may occur with previously known HER2 antibodies.

[0491] Therefore, by utilizing the binding proteins of the present invention, particularly antibodies or fragments thereof, cells overexpressing HER2 can be identified, isolated, characterized, targeted, and treated or eliminated (e.g., by amplification).

[0492] Therefore, the HER2-binding proteins (e.g., antibodies) of the present invention can be used to specifically classify HER2-related tumors or tumorigenic cells by staining or otherwise identifying those tumors or cells in which HER2 overexpression is present. Furthermore, the antibodies of the present invention, such as mAb104, have demonstrated significant in vivo antitumor activity against tumors containing amplified HER2 and HER2-positive xenografts.

[0493] As described above, the inventors have discovered that, when expressed in normal cells, the HER2-binding protein of this invention recognizes the tumor-associated form of HER2 but not the normal wild-type receptor. Antibody recognition is believed to depend on a conformational response to HER2 amplification or activation, which opens the conformational epitopes exposed for binding.

[0494] mAb104 was shown to inhibit the growth of overexpressed (e.g., amplified) HER2 xenografts in human tumors and induce necrosis within such tumors.

[0495] (ii) Therapeutic HER2-binding proteins and their uses

[0496] In vivo properties, particularly concerning tumors: The blood ratio and clearance of the HER2 binding protein of the present invention will be at least comparable to mAb104. Following administration to human or animal test subjects, this specific binding member will exhibit a peak tumor-to-blood ratio >1:1. Preferably, at such a ratio, the specific binding member will also have a tumor-to-organ ratio greater than 1:1, preferably greater than 2:1, more preferably greater than 5:1. Preferably, at such a ratio, the binding protein will also have an organ-to-blood ratio <1:1 in organs distant from the tumor site. These ratios exclude organs from which the administered binding protein is catabolized and secreted. Thus, in the case of scFv and Fab, the binding member will be secreted via the kidneys. In the case of whole IgG, clearance will be at least partially via the liver. The peak localization ratio of intact antibodies is typically reached between 10 and 200 h after administration of the HER2 binding protein. More specifically, this ratio can be measured in approximately 0.2 to 1.0 g of tumor xenografts formed subcutaneously on one side of athymic nude mice.

[0497] The HER2-binding protein (e.g., an antibody) of the present invention can be labeled with a detectable or functional tag, or a partial tag. As will be understood by those skilled in the art, the tag can be defined under more than one category. Detectable tags include, but are not limited to, radioactive tags, such as isotopes. 3 H, 14 C 32 P, 35 S, 36 C1, 51 Cr 57 Co、 58 Co、59 Fe、 90 Y、 121 1. 124 1. 125 1. 131 I, πι In、 211 At、 198 Au、 67 Cu、 225 Ac、 213 Bi、 99 Tc and 186 Re, which can be linked to the antibody of the present invention using conventional chemical methods known in the field of antibody imaging. The labeling also includes fluorescent labels and labels conventionally used in the art for MRI-CT imaging. They also include enzyme labels such as horseradish peroxidase. The labeling also includes chemical moieties, such as biotin, which can be detected by binding to specific homologous detectable moieties such as labeled avidin.

[0498] Functional markers include substances designed to target tumor sites to cause tumor tissue destruction. These functional markers include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which can convert prodrugs into active drugs at the tumor site.

[0499] Furthermore, antibodies, including polyclonal and monoclonal antibodies, and drugs that regulate the production or activity of binding proteins, antibodies, and / or their subunits, may have certain diagnostic applications and can be used, for example, to detect and / or measure conditions such as cancer, precancerous lesions, conditions associated with or caused by excessive proliferative cell growth. For example, HER2 binding proteins, antibodies, or their subunits can be used to generate polyclonal and monoclonal antibodies against themselves in various cell culture media using known techniques such as hybridoma techniques, for example, fused mouse spleen lymphocytes and myeloma cells. Similarly, small molecules that mimic or antagonize the activity of the HER2 binding proteins of the present invention can be discovered or synthesized and can be used in diagnostic and / or therapeutic regimens.

[0500] Radiolabeled HER2-binding proteins, particularly antibodies and fragments thereof, can be used in in vitro diagnostic techniques and in vivo radiographic imaging techniques, as well as radioimmunotherapy. In the case of in vivo imaging, the HER2-binding proteins of the present invention can be conjugated to imaging agents rather than radioisotopes, including but not limited to magnetic resonance imaging enhancers, wherein, for example, antibody molecules are loaded with a large number of 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 another embodiment of the invention, radiolabeled HER2-binding proteins, particularly antibodies and fragments thereof, especially radioimmunoconjugates, can be used in radioimmunotherapy, particularly as radiolabeled antibodies for cancer treatment. In a further embodiment, radiolabeled HER2-binding proteins, particularly antibodies and fragments thereof, can be used in radioimmunoguided surgical techniques, wherein they can identify and indicate the presence and / or location of cancer cells, precancerous cells, tumor cells, and hyperproliferating cells before, during, or after surgical removal.

[0501] The immunoconjugates or antibody fusion proteins of the present invention, wherein the HER2 binding proteins of the present invention, particularly antibodies and their fragments, are conjugated or linked to other molecules or reagents, including but not limited to binding proteins conjugated to chemical ablation agents, toxins, immunomodulators, cytokines, cytotoxic drugs, chemotherapeutic agents, or drugs.

[0502] The efficacy of radioimmunotherapy (RAFT) has been demonstrated using various antibody-immunoconjugates. It has been evaluated in colorectal cancer. 131 I-labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 (Behr TM et al. (2002) Cancer94 (Supplement 4): 1373-81) and evaluated in medullary thyroid carcinoma. 90 The same Y-labeled antibody (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). Radioimmunotherapy using specific antibodies is also described in U.S. Patents 6,306,393 and 6,331,175.

[0503] Radioimmunoguided surgery (RIGS) has demonstrated efficacy and usefulness, including the use of anti-CEA antibodies and antibodies against tumor-associated antigens (Kim JC et al. (2002) Int J Cancer 97(4):542-7; Schneebaum S et al. (2001) World J Surg 25(12):1495-8; Avital S et al. (2000) Cancer 89(8):1092-8; McLosh DG et al. (1997) Cancer Biothcr Radiopharai 12(4):2S7-94).

[0504] The HER2-binding protein (e.g., an antibody) of this invention can be administered to patients in need of treatment via any suitable route, typically by injection into the bloodstream or CSF, or directly into the tumor site. The precise dosage will depend on many factors, including whether the antibody is for diagnostic or therapeutic purposes, the size and location of the tumor, the precise nature of the antibody (whether whole antibody, fragment, biantibody, etc.), and the nature of the detectable or functional marker attached to the antibody. When a radionuclide is used for treatment, a suitable maximum single dose is approximately 45 mCi / m 2 The maximum is approximately 250 mCi / m 2 The preferred dose range is 15-40 mCi, with a further preferred range of 20-30 mCi or 10-30 mCi. This treatment may require bone marrow or stem cell replacement. Typical antibody doses for tumor imaging or treatment are 0.5-40 mg, preferably 1-4 mg in the F(ab')2 form. Naked antibodies are preferably administered at doses of 20-1000 mg protein per dose, or 20-500 mg protein per dose, or 20-100 mg protein per dose. This is the dose for a single treatment in adult patients and can be adjusted proportionally for children and infants, as well as for other antibody forms proportional to molecular weight. Treatment may be repeated daily, twice weekly, weekly, or monthly at the physician's discretion.

[0505] These formulations may include a second binding protein, such as the EGFR-binding protein or HER2-binding protein described herein. In a particularly preferred form, the second binding protein is trastuzumab.

[0506] (iii) Anticancer treatment

[0507] The HER2-binding protein (e.g., an antibody) of this invention can be used in a variety of applications, including research, diagnostic, and therapeutic applications. In one example, this invention provides a method for treating or preventing a condition in a subject. As used herein, a "condition" is a disruption or interference with normal function.

[0508] (iv) Diagnostic testing

[0509] This invention also relates to a variety of in vitro or in vivo diagnostic applications, including methods for detecting the presence of aberrantly expressed HER2 by referencing the ability of aberrantly expressed HER2 to be recognized by the HER2-binding protein of this invention (e.g., an antibody). Diagnostic applications of the antibodies of this invention include in vitro and in vivo applications well-known and standard to those skilled in the art, and based on this specification. Diagnostic assays and kits for in vitro assessment and evaluation of HER2 status, particularly regarding aberrant HER2 expression, can be used to diagnose, evaluate, and monitor patient samples, including those known to have or suspected of having cancer, precancerous conditions, conditions associated with excessive proliferative cell growth, or samples from tumors. Assessment and evaluation of HER2 status can also be used to determine a patient's suitability for a clinical trial of a drug or for the administration of a specific chemotherapeutic agent or specific binding member (particularly an antibody) (including combinations thereof) of this invention with different agents or antibodies. This type of diagnostic monitoring and evaluation has been used in practice with antibodies against the HER2 protein in breast cancer (Hercep Test, Dako Corporation), where this assay is also used to evaluate patients using antibody therapy with Herceptin. In vivo applications include tumor imaging or assessment of an individual's cancer status, including radiographic imaging.

[0510] The presence of HER2 in cells can be determined using in vitro or in vivo immunological methods known to those skilled in the art. For example, the HER2 receptor forms a complex with one or more antibodies, and one member of this complex is labeled with a detectable marker. The most commonly used markers for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, etc. Many fluorescent materials are known and can be used as markers. These include, for example, fluorescein, rhodamine, auramine, Texas red, AMCA blue, and fluorescein yellow. Anti-HER2 antibodies can also be labeled with radioactive elements or enzymes. Radioactive labeling can be detected by any currently available counting procedure. Preferred isotopes may be selected from... 3 H, 14 C 32 P, 35 S, 36 Cl、 51 Cr 57 Co、 58 Co、 59 Fe、 90 Y、 121 I, 124 1. 125 1. 131 I5 111 In、 211 At、 198 Au、 67 Cu、 225 Ac、 213 Bi、99 Tc and 186 Re. Enzyme labels are also useful and can be detected by any currently used colorimetric, spectrophotometric, fluorescence spectrophotometric, galvanometry, or gasometry technique. The enzyme is conjugated to the selected particle by reacting with bridging molecules such as carbodiimide, diisocyanate, glutaraldehyde, etc. Many enzymes that can be used in these methods are known and usable. Preferred enzymes include peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase, and alkaline phosphatase. U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043 are mentioned as examples of alternative labeling materials and methods disclosed therein.

[0511] Reagent test kit

[0512] The present invention also contemplates the use of therapeutic or diagnostic kits comprising the HER2-binding protein of the present invention for use in this therapeutic approach. Such kits typically contain a pharmaceutically acceptable formulation of at least one HER2-binding protein of the present invention (such as an antibody or fragment) in a suitable container. The kit can be used to detect the presence of the HER2 receptor in a biological sample. The antibody compositions of the present invention can be provided in liquid or lyophilized form, alone or in combination with other antibodies specific to other epitopes. Labeled or unlabeled antibodies can be included in the kit along with auxiliary components (e.g., buffers such as Tris, phosphates and carbonates, stabilizers, excipients, antimicrobial agents, and / or inert proteins such as bovine serum albumin). For example, antibodies can be provided as a lyophilized mixture with auxiliary components, or auxiliary components can be provided alone for the user to combine. These auxiliary substances are typically present in amounts less than about 5 wt% based on the amount of active antibody and typically in total amounts of at least about 0.001 wt% based on antibody concentration. When using secondary antibodies capable of binding antibodies, such antibodies can be provided in the kit, for example, in separate vials or containers. Secondary antibodies (if present) are typically labeled and can be formulated in a manner similar to the antibody formulations described in this article.

[0513] Commercially available test kits suitable for use by medical professionals can be prepared to determine the presence or absence of aberrant HER2 expression, including but not limited to amplified HER2, in suspected target cells. Based on the testing techniques discussed above, one category of such kits will contain at least a labeled HER2 or its binding partner, such as an antibody specific to it, and of course, depending on the guidelines of the chosen methodology, such as “competitive,” “sandwich,” “DASP,” etc. The kits may also contain peripheral reagents, such as buffers, stabilizers, etc.

[0514] Therefore, a test kit can be prepared to demonstrate the presence or abnormal expression of HER2 in cells, comprising:

[0515] (a) A predetermined amount of at least one labeled immunochemically reactive component, obtained by directly or indirectly linking the HER2 binding protein or its specific binding partner as described herein to a detectable label;

[0516] (b) Other reagents; and

[0517] (c) Instructions for use of the kit.

[0518] More specifically, diagnostic test kits may include:

[0519] (a) A known amount of the HER2-binding protein (or binding partner) as described above, which typically binds to a solid phase to form an immunoadsorbent, or, in an alternative, binds to a suitable tag, or a variety of such end products (or their binding partners), one of each.

[0520] (b) Other reagents, if necessary; and

[0521] (c) Instructions for use of the test kit.

[0522] In another example, a test kit can be prepared and used for the above-mentioned purposes, which operates according to a predetermined protocol (e.g., "competitive," "sandwich," "double antibody," etc.) and includes:

[0523] (a) Labeled components obtained by conjugating HER2-binding proteins with detectable labels;

[0524] (b) One or more other immunochemical reagents, wherein at least one reagent is a ligand or an immobilized ligand, the ligand being selected from the group consisting of:

[0525] (i) a ligand capable of binding to the labeled component (a);

[0526] (ii) A ligand capable of binding to the binding partner of the labeled component (a);

[0527] (iii) a ligand capable of binding to at least one analyte; and

[0528] (iv) a ligand capable of binding to at least one binding pair of at least one analyte; and

[0529] (c) Guidance on implementing protocols for detecting and / or measuring one or more components of the immunochemical reaction between HER2, HER2-binding protein and its specific binding partner.

[0530] Based on the above, an assay system can be prepared for screening potential drugs that effectively regulate HER2 activity, HER2 aberrant expression, and / or HER2-binding protein activity or binding. The receptor or binding protein can be introduced into the assay system, or the expected drug can be introduced into the resulting cell culture, and the culture can then be examined to observe any changes in cell S-phase activity, whether due to the addition of the expected drug alone or due to the effect of a known amount of reagent added.

[0531] Those skilled in the art will understand that many changes and / or modifications can be made to the invention as illustrated in the specific embodiments without departing from the scope of the invention as broadly described. Therefore, these embodiments are to be considered illustrative rather than restrictive in all respects.

[0532] The invention is further described in the following non-limiting embodiments.

[0533] Example

[0534] Those skilled in the art will understand that various changes and / or modifications can be made to the above embodiments without departing from the broad scope of this disclosure. Therefore, this embodiment is to be considered illustrative rather than restrictive in all respects.

[0535] Materials and methods

[0536] Cell lines and culture conditions

[0537] Parental lines were obtained from the American Type Culture Collection Center (ATCC, USA), Asterand Bioscience (USA), Ludwig Cancer Institute, or Australian Cell Bank (Australia). Cells were cultured in the supplier-recommended medium at 37°C in an incubator with 5% CO2. All media were supplemented with 10% fetal bovine 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 changed when 80% confluence was reached. Cells were used for experiments during the exponential growth phase. To passage adherent cells, the medium was removed and an appropriate volume (based on growth surface area) of 2 mM EDTA and trypsin (Life Technologies) was added. TM The cell lines were prepared using PBS solutions from Australia. The cell lines are described in Table 1.

[0538] Table 1 Cell lines

[0539]

[0540] Antibodies and antigens

[0541] Primary antibodies were purchased from commercially available sources listed in Table 2 below or purified from hybridoma supernatant using protein-g affinity chromatography.

[0542] Table 2. Antibodies used in this application

[0543]

[0544] antigen

[0545] Linear and cyclic peptide immunogens and unrelated control peptides were chemically synthesized by Mimitopes Pty Ltd (Clayton, Australia) and conjugated with keyhole hemocyanin (KLH). The linear peptide sequence is H-CPLHNQEVTAEDGTQR-NH2, and the cyclic peptide sequence is H-GCPLHNQEVTAEDGTQRC-NH2, where the N-terminal H- indicates a free amine. A control-independent peptide, H-LEEKKGNYVVTDHC-NH2, conjugated with KLH, was also prepared.

[0546] Based on previously published methods, the HER2 extracellular domain (HER2-ECD) was generated in the laboratory of academic collaborator Professor AW Burgess (The Walter and Eliza Hall Institute of Medical Research) (Xu Y, Soo P, Walker F, Zhang HH, Redpath N, Tan CW et al., LRIG1 extracellular domain: Structure and function analysis. Journal of molecular biology. 2015; 427(10):1934-48.). In short, the synthetic DNA corresponding to the human HER2-ECD was... The protein was cloned into an expression vector, expressed in Hi5 insect cells, and purified using anti-FLAG M2 beads (Sigma-Aldrich). Further purification was achieved by gel filtration in 20 mM Tris–HCl (pH 8.5) and 100 mM NaCl.

[0547] Cell biology reagents and sources

[0548] Details of the reagents are provided in the table below.

[0549] Reagents used (Table 3)

[0550]

[0551] Culture medium and solution

[0552] Agarose (1-1.5%): Dissolve agarose in 1×TAE to a final concentration of 1-1.5% w / v.

[0553] Agarose buffer 1x: 10mM BisTris HCl pH6.5; 0.2mM EDTA; 100mM NaCl.

[0554] Blocking buffer: 5% w / v skim milk powder (Fonterra, Mount Waverly, Australia), 0.1% v / v Tween 20 (ICN Biomedicals) in Tris buffered saline (TBS; 20 mM Tris-HCl, 150 mM NaCl).

[0555] Diethanolamine-HCl buffer: Diethanolamine (10% or 0.1M), MgCl2·6H2O (1mM), NaN3

[0556] DMEM-10: Dulbecco's modified Eagles medium supplemented with 10% FCS and 2mM L-alanyl-L-glutamine Glutamax. TM ), 100 U / ml penicillin and 100 mg / ml streptomycin.

[0557] EDTA-PBS: A PBS solution of 2 mM EDTA, without Ca2+. 2+ or Mg 2+ .

[0558] EDTA-PBS-3% FCS: EDTA-PBS supplemented with 3% FCS.

[0559] MATRIGEL TM Matrix:

[0560] Composition MATRIGEL TM Matrix (Product Information)

[0561]

[0562] RIPA buffer: 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 10 mM NaF and protease inhibitor (pH 7.5).

[0563] RPMI-10 medium: RPMI-1640 contains 10% fetal bovine serum (FCS) and 2 mM L-alanyl-L-glutamine (Glutamax). TM ), 100 U / ml penicillin and 100 mg / ml streptomycin.

[0564] Run buffer (Western blot): SDS run buffer (Invitrogen) was diluted to 1× run buffer in milliq H2O.

[0565] TBS (10×): 24.2g Alkali; 80g sodium chloride; 970ml H2O, adjust pH to 7.5 using HCl, total volume 1000ml.

[0566] TBS-T: Supplement with 0.05% 20 of 1×TBS.

[0567] Cell biology instruments

[0568] The table below provides detailed information on cell biology instruments.

[0569] Table 4 Instrument Details

[0570]

[0571] Immunity and monoclonal antibody production in mice

[0572] Female BALB / c mice were immunized with a 30 μg peptide containing the conformationally exposed region of the HER2 extracellular domain. The cyclic peptide, as determined by structural modeling, was synthesized from the sequence H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 26) and conjugated to keyhole hemocyanin (KLH) as a carrier protein. The peptides were injected intraperitoneally at four-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) (Flies DB, Chen L. A simple and rapid vortex method for preparing antigen / adjuvant emulsions for immunization. Journal of immunological methods. 2003; 276(1):239-42) for the first injection and with Freund's incomplete adjuvant for the second injection. Two booster injections of the peptide immunogen were then performed separately. Three days after the final immunization, mice were sacrificed, and spleen cells from hyperimmunized mice were harvested and fused with the mouse myeloma cell line SP2 / 0 at a ratio of 1:50 to generate hybridomas (Yokoyama WM, Christensen M, Santos GD, Miller D, Ho J, Wu T et al., Production of monoclonal antibodies. Current protocols in immunology. 2006:2.5.1-2.5.29). The fused cells were grown in complete RPMI medium supplemented with 10% FCS and additives. The supernatant of the growing cells was screened using an indirect ELISA.

[0573] Enzyme-linked immunosorbent assay (ELISA)

[0574] Polystyrene 96-well plates were coated overnight at 40°C with 3 μg / mL HER2-ECD peptide-KLH antigen as a linear or cyclic peptide, a negative control-KLH conjugated peptide, or recombinant HER2 ECD in PBS. The plates were blocked for 1 h at room temperature (RT) with PBS containing 3% FCS. The plates were then incubated for 1 h with serially diluted hybridoma supernatant starting at a 1:50 dilution and appropriate controls. After three washes, the plates were incubated for 1 h at room temperature with an anti-mouse IgG HRP conjugate (1:2000 dilution). After three more washes, phosphatase activity was measured using pNPP substrate, and absorbance (OD) was read at 405 nm using a Versamax microplate reader (molecular device) with Softmax Pro 4.8 software.

[0575] After identifying positive hybridomas from four candidate clones (referred to herein as mAb104, mAb105, mAb106, or mAb107), purified antibodies were evaluated using ELISA. Polystyrene 96-well plates were coated with 3% FCS in PBS at room temperature for 1 h. The peptide was diluted to a final peptide concentration of 1 mg / mL in 0.1% acetic acid. This peptide solution was further diluted to 30 μg / mL in 3% FCS-PBS. The plates were incubated with linear or cyclic peptide immunogens conjugated with KLH, HER2-ECD, or the negative control peptide-KLH in dilution buffer (3% FCS-PBS) at RT for 1 h. After washing the plates three times with 0.05% Tween 20-PBS, the wells were incubated again with 10 μg / mL mAb104, mAb105, mAb106, or mAb107 for 1 h. After washing, the plate was incubated with anti-mouse Ig-alkaline phosphatase (SigmaA-3688) (1:3000 dilution) at room temperature for 1 h. After washing three more times, phosphatase activity was measured using pNPP substrate, and the absorbance was read at 405 nm using a Versamax microplate reader (molecular device) with Softmax Pro 4.8 software.

[0576] Coat polystyrene 96-well plates overnight at 40°C with 50 mL / well of PBS solution containing 3 μg / mL recombinant ErbB2 ECD, ErbB3 ECD, ErbB4 ECD, or EGFR501. Block the plates with PBS containing 3% FCS for 1 h at room temperature (RT). Incubate the plates with serially diluted 10 μg / mL purified antibody and appropriate controls for 1 h at room temperature. After washing three times, incubate the plates with anti-mouse IgG AP conjugate (1:2000 dilution) for 1 h at room temperature. After washing three more times, measure phosphatase activity using pNPP substrate and read the absorbance (OD) at 405 nm using a SPECTROstar microplate reader (BMG LABTECH, Victoria, Australia).

[0577] FACS analysis

[0578] Cells (1×10) 4Inoculate HER2 antibody or IgG1 isotype control antibody into 96-well plates, add 10 μg / ml of anti-HER2 antibody or IgG1 isotype control antibody, and incubate at 4°C for 1 h. Humanized antibodies were detected using Alexa-488-conjugated anti-human IgG antibody for trastuzumab and pertuzumab. Conjugated mAb104, mAb105, mAb106, or mAb107 or mouse isotype control LMH-3 were detected using Alexa-488-conjugated anti-mouse IgG antibody, and fluorescence was read on a Becton Dickinson FACScan (CellQuestPro version 4.0.2). Negative controls included separate secondary antibody and separate cell background fluorescence.

[0579] Biosensor Analysis

[0580] At BIAcore TM Surface plasmon resonance (SPR) kinetics analysis was performed using a carboxymethyl dextran-coated sensor chip (CM5-S, GE LifeSciences) in the T200 system. The test channels were derived into HER2-ECDs with 200 response units (RU) using standard amine coupling chemistry (0.05M NHS / 0.2M EDC). A blank control channel was derived using ethanolamine to correct for refractive index effects.

[0581] Samples of mAb104, mAb106, pertuzumab, or trastuzumab anti-HER2 antibodies were diluted to concentrations from 320 μg / mL to 0 μg / mL in PBS / 0.005% Tween 20 buffer, a two-fold dilution (2133 to 0 nM). Samples were injected at 45 μL / min for 200 s (30 μL, 30 μL, 10 μL / min) with PBS buffer containing 0.005% Tween-20 on the immobilized HER2-ECD, using PBS / 0.005% Tween 20 as the run buffer. Dissociation was monitored after the injection phase by allowing the run buffer to flow over the chip surface for 600 s. The bound antibody was eluted and the chip surface was regenerated between samples by injecting 30 μL of 50 mM NaOH at 30 μL / min for 30 s.

[0582] Western Blot analysis

[0583] The responsiveness of anti-HER2 monoclonal antibodies to native HER2 was determined using Western blot. Trypsin-digested cells were lysed with RIPA buffer [50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 200 mM Na3VO4, 0.5% deoxycholate, 0.05% SDS, 10 mM NaF and a mixture of protease inhibitors (1 mM, CA, USA)] for 20 min and centrifuged at 17,000 rpm for 15 min. 10 μg of cell lysate was run on a 4–12% gradient Nu-PAGE gel and... 2. Electrotransfer was performed using a ThermoFisher gel transfer apparatus onto nitrocellulose membranes. The presence of EGFR and HER2 was assessed by detecting the blot with the corresponding antibody using commercially available antibodies and mAb104 bound to HER2. The blot was observed on a Storm 804 Phosphoimager (Amersham Bioscience) and analyzed using ImageQuant TL image analysis software (2005 version).

[0584] Immunohistochemistry

[0585] To confirm the tumor selectivity of mAb104, an immunohistochemical method was developed and used to screen mAb104 reactivity in a range of normal and tumor tissue types. Changes in antigen retrieval, primary antibody concentration, and incubation time were evaluated before optimizing the conditions as described above. The final protocol is briefly discussed below. Slides were placed in a 60°C oven for 30 min and then transferred to a xylene bath, changing the bath after 10 min. The slides were then rehydrated in 100% ethanol (replaced twice) for 10 min each time, followed by rehydration in a 70% ethanol bath for 10 min. The slides were rinsed three times in double-distilled (dd) H2O for approximately 2 min each time. The slides were then quenched in 3% H2O2 for 20 min. Antigen retrieval was achieved by treating the slides in a 10% (v / v) EDTA buffer bath at 100°C for 30 min. After cooling and rinsing with phosphate-buffered saline (PBS), the slides were placed in a protein blocking agent (SuperBlock). TM T20, Pre-incubate in the solution for 60 min. Then incubate the slide with mAb104 primary antibody (2.5 μg / mL) at room temperature for 60 min. After staining with the primary antibody, detect the bound antibody with streptavidin-horseradish peroxidase (HRP)-labeled anti-mouse secondary antibody (Dakocytomation, Carpinteria, CA, USA). The bound antibody is detected with 3,3′-diaminobenzidine (DAB) substrate and counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated and fixed in ethanol and xylene.

[0586] Using this method, tissue microarrays (TMA) were employed to examine the expression of these proteins in 11 normal human tissues and 10 common tumor types (ductal carcinoma in situ of the breast, mesothelioma, colorectal and gastric adenocarcinoma, renal cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, prostate adenocarcinoma, and glioblastoma) from 9–27 different human donors. The tumor and normal tissues were not from the same patient (i.e., mismatched). 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.

[0587] Ki-67: The 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", Cancer Biology Symposium (1990)). The antigen was recovered for 20 min at 100°C in 10% (v / v) citrate buffer (pH 6.0) in water. After cooling, it was reacted with a protein blocking agent (SuperBlock) at room temperature. TM T20, Block nonspecific binding sites for 20 min. Rabbit anti-human Ki-67 primary antibody (RM-9106-S1, The antibody was diluted 1:100 in blocking buffer and incubated at room temperature for 2 hours. After washing away excess antibody, the bound antibody was detected for 30 minutes at room temperature using a suitable secondary antibody (Dakocytomation, Carpinteria, CA, USA). The bound antibody was detected using 3,3'-diaminobenzidine (DAB) substrate. The slides were then counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated and fixed in ethanol and xylene.

[0588] Apoptosis Apoptotic cells were detected using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay via an in situ cell death detection kit and fluorescein (11684795910 Roche, USA). Slides prepared from paraffin-embedded tissue were dewaxed and rehydrated as described above, then washed three times in ddH2O for approximately 2 min each time. Tissue sections were incubated in proteinase K working solution at room temperature for 20 min, followed by two washes in PBS. Positive and negative controls were prepared according to product specifications. 100 μl of the TUNEL reaction mixture or 100 μl of the control labeling solution for the negative control was added to each slide and incubated in a humidified chamber at 37°C for 60 min. After incubation, the slides were washed three times with PBS. After application to avoid evaporation loss, 50 μl of Covertor-POD was applied to slides with coverslips and incubated in a humidified chamber at 37°C for 30 min. After washing three times with PBS, apply 50–100 μl of DAB substrate to a glass slide and incubate at room temperature for 10 min. Wash the slide with PBS and analyze under an optical microscope.

[0589] Foot calyx glycoprotein: Slides from paraffin-embedded tissue were dewaxed and rehydrated as before. After quenching the slides in 3% H₂O₂ for 20 min at room temperature, the antigen was recovered by incubating the slides in 10% (v / v) citrate buffer (pH 6.0) at 100°C for 20 min. 15 μg / mL of goat anti-mouse foot calyx glycoprotein primary antibody (catalog number AF1556, R&D) was used. Add the antibody to each slide and incubate at room temperature for 2 hours. Then wash the slides and detect the bound antibody using anti-goat HRP, counterstain with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrate and fix in ethanol and xylene.

[0590] p-AktSlides derived from paraffin-embedded tissue were dewaxed by heating to 60°C and rehydrated in xylene and fractionating alcohol. After washing the slides three times in ddH2O, antigen retrieval was performed by incubating the slides in 0.01M citrate buffer (pH 6.0) in a 95% water bath for 20 min. Once the slides were cooled, endogenous peroxidase activity was quenched by incubation in PBS and 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, Tween 20 (0.1%; TBS-T). The slides were then incubated overnight at 4°C with primary antibody (rabbit polyclonal phosphate-Akt (Ser473; Cell Signaling Technology, Beverly, MA, catalog number 9277, IHC specific) at a 1:100 dilution in TBS-T. After washing the slides three times in TBS-T for approximately two minutes each time, the slides were incubated for 1 hour with rabbit biotinylated secondary antibody at a 1:200 dilution. The bound antibodies were detected using DAB substrate and counterstained with hematoxylin and eosin (H&E) (BDH Laboratory, Poole, UK), dehydrated and fixed in ethanol and xylene.

[0591] Cell proliferation assay

[0592] Cells in serum-deserium-free culture medium (1×10) 4 Cells were seeded in 96-well microtiter plates and allowed to adhere overnight. The next day, serially diluted antibodies with appropriate controls were added, and measurements were taken at time 0 (T=0). The remaining plates were incubated for 3 to 5 days. Cell viability was assessed using the MTS colorimetric activity assay with 3-(4,5-dimethylthiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS) as a substrate (Promega, Australia). Absorbance was assessed at 490 nm using a VersaMax microplate reader (Molecular Devices, USA) and SoftMax Pro 5.4.1 software (MolecularDevices, USA). Absorbance at 630 nm was also measured as background and subtracted from the 490 nm reading. Experiments were performed in triplicate and repeated 2 to 3 independent runs. All data were normalized to the signal at the time of compound addition (T=0). Dose-response curves were analyzed using GraphPad Prism 4.03 (Graphpad Software Inc., La Jolla, CA, USA).

[0593] Downstream signal transduction

[0594] Cells (1×10)6 Two copies of the culture medium were inoculated into 6-well plates and allowed to establish overnight. The medium in each well was discarded and replaced with serum-free medium containing the desired antibody at a total concentration of 10 mg / mL. At the specified time point (24 h), half of the wells were treated with 100 ng EGF for 10 min at room temperature. The reaction was terminated by washing with ice-cold PBS and lysed for 30 min with 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 a mixture of protease inhibitors (1 mM, CA, USA)]. The plates were then centrifuged at 17,000 rpm for 15 min. The total protein concentration was determined using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hemel Hempstead, UK). MAPK activation was assessed by Western blotting using commercial antibodies purchased from Cell Signaling Technology targeting HER2 (#4290), pHER2 (#2243), HER3 (#12708), pHER3 (#4791), EGFR (#4267), pEGFR (#3777), AKT (#4691), pAKT (#4060), ERK (#4695), and pERK (#4370). Anti-GAPDH (AbC-1001) antibody was purchased from AbClon. Bands were visualized using AbSignal (AbClon, AbC-3001).

[0595] Cell death detection by enzyme-linked immunosorbent assay (ELISA)

[0596] Cell death and apoptosis were assessed using ELISA analysis (ELISA Plus kit for cell death detection; Roche Molecular Biochemicals) (Holdenrieder S, Stieber P, Bodenmüller H, Fertig G, Fürst H, Schmeller N et al., Nucleosomes in serum as a marker for cell death. Clinical Chemistry and Laboratory Medicine. 2001; 39(7):596-605). In short, cells were cultured in 96-well plates and allowed to establish overnight. Cells were treated with trastuzumab, pertuzumab, and mAb104 as single and combined therapies in serum-free (1%) growth medium for 24 h. The plates were centrifuged at 200 × g for 10 min at 4 °C. The supernatant was carefully removed, 200 μl of the manufacturer's lysis buffer was added, and the plates were incubated at room temperature for 30 min. After incubation, the plates were centrifuged, and 20 μl of supernatant and three aliquots of cell lysate solution were placed into streptavidin-coated microplates. An additional 80 μl of immunoassay reagent containing a mixture of anti-histone-biotin and anti-DNA-POD was added to the supernatant. The plates were incubated at room temperature in a shaking incubator for 2 h. Using ABTS (2,2'-hydrazine bis-3-ethyl-benzothiazoline-6-sulfonic acid) as a substrate, the degree of apoptosis was quantitatively determined photometrically using a Versamax microplate reader (Molecular Devices) running Softmax Pro 4.8 software at a wavelength of 405 nm and a reference wavelength of 490 nm.

[0597] Apoptosis analysis

[0598] Cell viability was determined using propidium iodide (PI) uptake and annexin V binding. In short, cells were cultured in 24-well plates (5 × 10⁻⁶ cells / well). 4 Cells were incubated overnight. Cells were treated with the antibody as a single therapy and in combination with appropriate controls or in culture medium for 24 h. Cells were transferred to 96-well plates, washed three times with cold PBS, and resuspended in binding buffer containing 2.5 μl FITC annexin V and 2.5 μl PI at room temperature in the dark with gentle agitation for 15 min. After incubation, an additional 150 μl of binding buffer was added, followed by flow cytometry analysis.

[0599] Migration (wound healing) test

[0600] To assess the effect of mAb014 on cell migration, OE-19 cells (1×10⁻⁶) were used.5 Cells were seeded in 6-well plates and allowed to grow 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 microscopy images were taken immediately after treatment (designated as T0) for 72 h.

[0601] The effect of mAb104 on ErbB receptor dimerization

[0602] Cells in serum-free medium were seeded into 12-well plates and allowed to adhere overnight. Cells were treated with two copies of 10 μg / mL relevant antibody or control for 1 h. At the specified time points, half of the wells were treated with 100 ng EGF for 10 min at room temperature. The reaction was terminated by washing with ice-cold PBS, and the cells were incubated with BS3 (bis(sulfosuccinimide) substrate ((BS3), Pierce, Rockford, IL, USA) at room temperature for 20 min with gentle shaking according to the manufacturer's instructions (Staros JV. N-hydroxysulfosuccinimide active esters: bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry. 1982; 21(17):3950-5). After quenching the cross-linking reaction mixture with a buffer containing 10 mM Tris-HCl, the cells were washed twice with cold PBS and then with 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 The mixture of NaF and protease inhibitors (1M, CA, USA) was used for lysation for 30 min. Cell lysates were immunoprecipitated with relevant antibodies, and EGFR and HER2 were immunoblotted.

[0603] DNA gel electrophoresis

[0604] DNA gel electrophoresis was performed on a gel containing 1% (w / v) DNA-grade agarose (Bioline). The agarose was diluted in 1×Tris-EDTA (TAE) buffer (Invitrogen) using... Preparation of safe DNA gel dye (Invitrogen). All DNA samples were diluted with 10×Orange G (Sigma) gel loading buffer stock solution before loading onto agarose gels. Electrophoresis was performed at 70–150 V, using a 1kb Plus DNA ladder (Invitrogen) as a reference for size estimation. DNA bands were visualized and photographed under UV light on a bio-Rad.

[0605] Hybridoma cDNA Synthesis

[0606] The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, California, USA) is used to reverse transcribe (RT) 10 μL (~1-5 μg) of RNA into cDNA using a 20 μL reaction volume. Follow the standard method using T100. TM (BIO-RAD, USA) or cDNA synthesis was performed in 200 μl thin-walled polypropylene PCR tubes (Eppendorf, USA) using a thermal cycler under the following reaction and cycling conditions.

[0607] cDNA synthesis conditions:

[0608] 25℃ for 10 minutes

[0609] 37℃ for 120 minutes

[0610] 85℃ for 5 minutes

[0611] Keep at 4℃

[0612] Mouse light chain variable region primers:

[0613] (i)ATG AAG TTG CCT GTT AGG CTG TTG GTG CTG (SEQ ID NO: 28)

[0614] (ii)ATG GAG WCA GAC ACA CTC CTG YTA TGG GT (SEQ ID NO: 29)

[0615] (iii)ATG AGT GTG CTC ACT CAG GTC CTG GSG TTG (SEQ ID NO: 30)

[0616] (iv)ATG AGG RCC CCT GCT CAG WTT YTT GGM WTC TTG (SEQ ID NO: 31)

[0617] (v)ATG GAT TTW CAG GTG CAG ATT WTC AGC TTC(SEQ ID NO:32)

[0618] (vi)ATG AGG TKC YYT GYT SAG YTY CTG RGG(SEQ ID NO: 33)

[0619] (vii)ATG GGC WTC AAG ATG GAG TCA CAK WYY CWG G(SEQ ID NO:34)

[0620] (viii)ATG TGG GGA YCT KTT TYC MMT TTT TCA ATT G(SEQ ID NO:35)

[0621] (ix)ATG GTR TCC WCA SCT CAG TTC CTT G(SEQ ID NO:36)

[0622] (x)ATG TAT ATA TGT TTG TTG TCT ATT TCT(SEQ ID NO:37)

[0623] (xi)ATG GAA GCC CCA GCT CAG CTT CTC TTC C(SEQ ID NO:38)

[0624] (xii)ATG AAG TTT CCT TCT CAA CTT CTG CTC(SEQ ID NO:39)

[0625] Reverse primer sequence for the variable region of the mouse light chain:

[0626] MKC: TGG ATG GTG GGA AGA TG (SEQ ID NO: 40)

[0627] Mouse heavy chain variable region primers:

[0628] (i)ATG AAA TGC AGC TGG GTC ATS TTC TTC(SEQ ID NO:41)

[0629] (ii)ATG GGA TGG AGC TRA TCA TSY TCT T(SEQ ID NO: 42)

[0630] (iii)ATG AAG WTG TGG TTA AAC TGG GTT TTT(SEQ ID NO:43)

[0631] (iv)ATG RAC TTT GWY TCA GCT TGR TTT(SEQ ID NO: 44)

[0632] (v)ATG GAC TCC AGG CTC AAM AGT TTT CCT T(SEQ ID NO:45)

[0633] (vi)ATG GCT GTC YTR GSG CTR CTC TTC TGC(SEQ ID NO:46)

[0634] (vii)ATG GRA TGG AGC KGG RTC TTT MTC TT(SEQ ID NO:47)

[0635] (viii)ATG AGA GTG CTG ATT CTT TTG TG(SEQ ID NO:48)

[0636] (ix)ATG GMT TGG GTG TGG AMC TTG CTA TTC CTG(SEQ ID NO:49)

[0637] (x)ATG GGC AGA CTT ACA TTC TCA TTC CTG(SEQ ID NO:50)

[0638] (xi)ATG GAT TTT GGG CTG ATT TTT TTT ATT G(SEQ ID NO:51)

[0639] (xii)ATG ATG GTG TTA AGT CTT CTG TAC CTG (SEQ ID NO: 52)

[0640] Reverse primer sequence for the variable region of the mouse heavy chain:

[0641] MHC:CCAGTGGATAGACAGATG(SEQ ID NO:53)

[0642] Degenerate forward and reverse primers for the variable region of the mouse light chain:

[0643] κF:GCC GAA TTC GAY ATT GTG MTS ACM CAR WCT MCA(SEQ ID NO:54)

[0644] κR:CCG GTC GAC GGA TAC AGT TGG TGC AGC ATC (SEQ ID NO: 55)

[0645] Symbol annotation:

[0646] 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

[0647] B = G, T, or C; D = G, A, or T; N = A, C, G, or T; V = G, A, or C

[0648] Polymerase chain reaction

[0649] To amplify the DNA fragment used for cloning, use according to the manufacturer's instructions. Pfx DNA polymerase (Invitrogen). Using T100 according to standard methods. TM (BIO-RAD, USA) or PCR reaction mixing was performed in 200 μl thin-walled polypropylene PCR tubes using an Eppendorf thermal cycler under the following reaction and cycling conditions.

[0650] PCR conditions:

[0651] 94℃ for 3 minutes

[0652] (94℃ 1min → x C) a 55-90s b →72℃ for 2 minutes) Continue for 15-25 cycles.

[0653] 72C / 10min

[0654] Keep at 4℃

[0655] In vivo studies

[0656] NOD-SCID-IL2R - / - Mice (4 to 6 weeks old, Animal Research Center, Perth, Australia) were treated with 5 × 10 6 NCI-N87 or 8×10 6 BT-474 cells were subcutaneously injected into the lateral region of a Matrigel (BD Biosciences) container. Mice injected with BT-474 cells were implanted with estrogen granules 24 hours prior. The formula (L×W) was used. 2The tumor volume is calculated as (W) / 2, where "W" represents the width of the tumor and "L" represents the length of the tumor. The tumor is then allowed to grow to approximately 100 mm. 3 Mice were then randomly assigned to different treatment groups based on size. Tumors that failed to transplant were excluded from further analysis. Treatment was administered three times weekly at the dose indicated for intraperitoneal injection for three weeks. Animals were observed post-treatment, and tumors were considered reached when the mean tumor volume was >1000 mm. 3 Animals were euthanized or exhibited prolonged stress symptoms. Postmortem tumors were excised and processed into formalin-fixed, paraffin-embedded sections. These sections were collected for reverse-phase protein array (RPPA), and excess tissue was 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}] × 100, where T = mean tumor volume of the treated animal at the endpoint, T0 = mean tumor volume of the treated animal at time 0, C = mean tumor volume of the control animal at the endpoint, and C0 = mean tumor volume of the vehicle control animal at time 0.

[0657] All animal research protocols were approved by the Austin Health Animal Ethics Committee (Protocol #A2015 / 05297) and were conducted in accordance with the Scientific Animal Management and Use Guidelines (8th edition, 2013).

[0658] Reverse protein array (RPPA)

[0659] Proteins were extracted from HER2-overexpressing PDX breast tumors and subjected to RPPA as previously described (Hennessy BT, Lu Y, Gonzalez-Angulo AM, Carey MS, Myhre S, Ju Z et al., A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers. Clinical proteomics. 2010; 6(4):129). Tumor samples were homogenized and lysed at the end of the treatment using lysis buffer (catalog number #9803, Cell Signaling Technology, Beverly, MA, USA), supplemented with protease and phosphatase inhibitors (Roche Applied Science catalog number #05056489001, Penzberg, Germany). TMThe BCA protein assay kit was used to determine protein concentration, which was 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.

[0660] The lysates were serially diluted 5-fold with lysis buffer to obtain a 1:16 dilution. The lysates were arranged in an 11×11 pattern on nitrocellulose-coated slides (Grace Biolab). Samples were probed with 297 confirmed primary antibodies using a tyramine-based signal amplification method and visualized by DAB colorimetric reaction. The slides were scanned, analyzed, and quantified using an Array-Pro analyzer (Meyer Instruments, INC., Houston, TX) to generate spot intensities.

[0661] Each dilution curve was fitted using a logistic model (“hypercurve fitting”, developed by the Department of Bioinformatics and Computational Biology at MD Anderson Cancer Center in Houston, Texas). All data were polished to median normality using a protein load correction factor and converted to linear values ​​using median expression levels from all antibody experiments. In the heatmap, “red” indicates above the median and “green” indicates below the median.

[0662] Statistical analysis

[0663] use The analysis was performed using version 5.04. All p-values ​​were two-tailed, and values ​​≤0.05 were considered significant.

[0664] To compare means, the Student t-test or the nonparametric Mann-Whitney U test is used, where only two groups are considered. To compare between three or more groups, parametric data are analyzed using ANOVA, and if p ≤ 0.05, a post-hoc test using the Bonferroni method is performed to determine which groups are significantly different. For multiple groups, the nonparametric test used is the Kruskal-Wallis test, and a post-hoc test is performed if p ≤ 0.05 to determine which groups are significantly different.

[0665] Survival rates were also compared among the groups. If the log-rank test results for all groups were significantly different (p≤0.05), a post-hoc test was performed using further log-rank tests to determine which group was significantly different.

[0666] Example 1 - In vitro antibody production and characterization

[0667] The immunoantigens and immunization protocols have been described above. Through a series of immunization and screening strategies involving HER2 peptides, recombinant proteins, and cell-based assays expressing HER2, the inventors have finally succeeded in generating tumor-specific monoclonal antibodies against the conformational flexible region of HER2 domain II.

[0668] The inventors employed an immunization strategy using a linear peptide linked to biotin, GST, MBP, and KLH carrier proteins, and Baf / 03 hematopoietic cells transfected to express erbB2 (which does not express HER members on its cell surface). erbB2 had a cysteine ​​mutation to expose the peptide ring, but this was unsuccessful in generating any clones. Immunization with cells expressing the mutant plus a recombinant mutant ECD of ErbB2 did not produce mAbs binding to the peptide, but instead bound to different sites within the ErbB2 ECD. After a single immunization with a KLH-linked cyclized peptide, the inventors were able to obtain monoclonal antibody clones that recognize the peptide and bind to erbB2-expressing cells. This is summarized in the table below (Table 5).

[0669] Table 5 Results of the immunization regimen

[0670]

[0671] Using an immunization protocol, the inventors generated hybridoma clones that produced novel monoclonal antibodies (mAbs) targeting the conformationally exposed region of the HER2 extracellular domain, which were thought to be available for binding only under conditions found in tumor cells. These monoclonal antibodies targeting the conformational epitope, H-GCPLHNQEVTAEDGTQRC-NH2 (SEQ ID NO: 1), were generated by immunizing mice with a peptide immunogen derived from the HER2 extracellular domain. This sequence is shown in [the original text]. Figure 1 The provided HER2 sequence number is underlined. This sequence was exported from the ncbi database at the following link: https: / / www.ncbi.nlm.nih.gov / protein / NP_004439.2 .

[0672] This region is within domain II, but far from the known epitopes 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).

[0673] Hybridoma screening

[0674] ELISA-based assays were used to screen hybridoma culture supernatants (designated mAb104, mAb105, mAb106, and mAb107) for specificity toward the HER2 extracellular domain (ECD) and the cyclic and linear peptides of the antigens targeted by the antibodies. 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 both reducing and non-reducing conditions. Immunoglobulin isotypes of the selected antibodies were detected using a monoclonal antibody isotype kit (Thermo Fisher Scientific, IL, USA), revealing that all antibodies were IgG1 with a K-light chain. ELISA analysis results of the purified mAbs are shown below. Figure 2 As shown.

[0675] Monoclonal antibodies mAb104 and mAb106 exhibited the strongest binding activity against all peptide conformations, while mAb105 showed the weakest binding (see [link to relevant documentation]). Figure 2 B). Clones that produce antibodies with the highest affinity were selected for further development, namely mAb104 and mAb106, and antibodies against them were selected for further analysis and characterization.

[0676] FACS detection of hybridoma cell binding affinity

[0677] The binding of 10 μg / ml purified antibodies (mAb104, mAb105, mAb106, and mAb107) to HER2-overexpressing breast cancer cell lines (BT474, SK-BR-3, and MDA-MB-453) and gastric cancer cell line (NCI-N87) was tested by flow cytometry.

[0678] The results are summarized in Table 6 below and represent two or more experiments.

[0679] Table 6: FACS analysis of antibodies

[0680] mAb104 mAb105 mAb106 mAb107 MDA-MB-453 + - - - BT 474 ++ - + - SK-BR-3 ++ - - - NCI-N87 +++ - +++ +++

[0681] Of all the cell lines evaluated, mAb104 showed the highest binding compared to other antibodies. Among the evaluated cell lines, mAb104 showed the highest logarithmic shift in the NCI-N87 and SK-BR-3 cell lines. No mAb105 binding was observed in any of the evaluated cell lines. Binding was lower than that of commercial HER2-binding antibodies for all antibodies. The inventors propose that these antibodies bind to a subset of receptors on the cell surface, with the epitope only exposed to a portion of the HER2 receptor population.

[0682] via Western Combined analysis of blotting

[0683] The ability of these novel antibodies to bind to the HER2 protein was further evaluated by Western blot analysis in human breast cancer cell lines (BT474, SK-BR-3, and MDA-MB-453) and gastric cancer cell line (NCI-N87). Trypsin-digested cells were washed, lysed, and immunoblotted with their respective purified antibodies. Anti-HER2 antibody 2242 (Cell Signaling Technology, Beverly, MA) was used as a positive control.

[0684] Consistent with previous ELISA and FACS data, mAb104 showed the strongest binding in all cell lines (mAb104>mAb106>mAb107>mAb105). Figure 3 ), and combined with cell lines for all four tests.

[0685] sequences of mAb104 and mAb106

[0686] The PCR reaction was purified using a BioLine Isolate II PCR and gel extraction kit (BIO-52059). The purified amplification products were sent to the Monash Micromon DNA sequencing facility for Sanger DNA sequencing using the same primers as those used for sequencing.

[0687] CDRs are defined according to the Chothian and Kabat numbering systems and the amino acids in CDRs falling into either system. The heavy and light chain variable region sequences are as follows: Figure 4 As shown.

[0688] The complementarity-determining region sequences for each antibody are provided below:

[0689] mAb104 VH chain

[0690] CDR1: GYSFTGYFMH (SEQ ID NO: 14)

[0691] CDR2: RINPYNGDIRYNQNFKD (SEQ ID NO: 16)

[0692] CDR3: LNFAY (SEQ ID NO: 18)

[0693] mAb104 VL chain

[0694] CDR1: KSSQSLLDSDGKTFLN (SEQ ID NO: 20)

[0695] CDR2: LVSKLDS (SEQ ID NO: 22)

[0696] CDR3: WQGTHFPWT (SEQ ID No: 24)

[0697] mAb106 VH chain

[0698] CDR1: GYTFTDYGMN (SEQ ID NO: 15)

[0699] CDR2: WINTYTGKPTYDDDFKG (SEQ ID NO: 17)

[0700] CDR3: RFLNTVAGRSVYFDY (SEQ ID NO: 19)

[0701] mAb106 VL chain

[0702] CDR1: SVSSSVGSMY (SEQ ID NO: 21)

[0703] CDR2: LTSNLAS (SEQ ID NO: 23)

[0704] CDR3: QQWSSNPPT (SEQ ID NO: 25)

[0705] BIAcore Analysis

[0706] The epitope of mAb104 is side-bonded with a disulfide bond, suggesting flexibility around the epitope site and exposure to mAb104-bound epitopes under certain conditions or environments.

[0707] The binding properties and epigenetic affinity of purified mAb104 and mAb106 to HER2 compared to trastuzumab were detected using a BIAcore T200 surface plasmon resonance (BIAcore) instrument. The extracellular domain of recombinant HER2 was immobilized on a CM5 sensor chip, and then various concentrations of mAb104, mAb106, and commercial anti-HER2 mAb were passed through the sensor to determine epigenetic binding affinity.

[0708] like Figure 5 As shown in C, mAb104 exhibits high binding affinity with a KD in the nanomolar range. The binding affinity of mAb104 is one log lower than that of trastuzumab and similar to that of pertuzumab (Table 7).

[0709] Table 7: Binding affinity of mAb104

[0710] Antibody <![CDATA[K D (nM)]]> <![CDATA[Chi 2 ]]> mAb104 2.91 0.03 mAb106 3.18 0.13 Trastuzumab 0.1 0.12 pertuzumab 1.9 14.3

[0711] In summary, mAb104, mAb106, and mAb107 bound to a spectrum of tumor cells overexpressing HER2. mAb104 consistently appeared to exhibit stronger in vitro binding than mAb106. Based on the results of these initial screening experiments, mAb104 was selected for further evaluation.

[0712] Example 2 Epitope Analysis and Competition Determination

[0713] Using the method described above, based on the homology modeling 3D structure of the antibody Fv domain and the known X-ray structure of human HER2, the variable domain of mAb104 antibody that binds to the antigenic epitope located on HER2 domain II was calculated and predicted (Zhang W, Zeng X, Zhang L, Peng H, Jiao Y, Zeng J et al., Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5) , Journal of Computer-Aided Molecular Design. 2013; 27(6):539-50).

[0714] The predicted HER2 binding of mAb104 was compared with the known crystal structures of pertuzumab and trastuzumab binding to HER2 (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). Unwilling to be bound by theory, it is believed that the binding of mAb104 to HER2 requires a conformational change that occurs upon receptor activation, as previously observed in antibodies specifically targeting a locally misfolded region of tumor-associated EGFR (Garrett TP, Burgess AW, GanHK, Luwor RB, Cartwright G, Walker F et al., Proceedings of the National Academy of Sciences. 2009; 106(13):5082-7). In this study, the disulfide bond in domain II of HER2 ECD can be dynamically formed and broken.

[0715] Compared to other known HER2-binding antibodies, the epitope recognized by mAb104...

[0716] The antibody H2-18 (Lu et al. (2016) Oncotarget 7(41)), Chinese patent CN 104447993, recognizes an epitope within domain I of HER2 / ErbB2. H2-18 inhibits the growth of trastuzumab-resistant breast cancer cells in vivo and in vitro, and induces programmed cell death in both trastuzumab-sensitive and resistant breast cancer cell lines.

[0717] Antibody A21 (Hu S et al. (2015) Oncotarget 6(3):1695-1706) appears to recognize a large conformational epitope comprising most of the region derived from ErbB2 EC domain I. A bivalent antibody for A21 was found to be essential for its inhibitory activity against tumor cells, as well as for ErbB2 phosphorylation and receptor downregulation.

[0718] Trastuzumab / Herceptin (4D5) binds to the juxtamembrane region of subdomain IV of ErbB2 and can interrupt ErbB2 activation by metalloproteinase cleavage and also block ErbB2 dimerization.

[0719] Pertuzumab (2C4) can directly disrupt the binding between ErbB2 and other ErbB receptors by epitopes in or near subdomain II, thereby inhibiting tumor cell growth.

[0720] The epitopes recognized by pertuzumab and mAb104 consist of only three amino acids: P294, L295, and H296. While it is understandable that H296 is completely masked upon pertuzumab binding (Franklin MC et al., (2004) Cancer Cell 5:317). The epitopes bound by pertuzumab consist of H245, Y252, F257, D285, V286, S288, T290, P294, L295, H296, K311, K314, and P315.

[0721] Conversely, mAb104 identifies the epitope of the sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1).

[0722] Although both antibodies recognize epitopes within domain II of HER2, the epitopes bound by pertuzumab and mAb104 are distinctly different.

[0723] To avoid being bound by theory, the inventors hypothesize that pertuzumab and mAb104 bind to opposite face / side of domain II of HER2 / ErbB2, thus explaining why mAb104 does not block pertuzumab binding, despite being closely opposed epitopes. Conformational changes in domain II, such as those occurring during activation, hypoxia, and / or aberrant expression, would allow the antibody to bind to CPLHNQEVTAEDGTQRC in domain II and mAb104 to bind alone to a small subset of the HER2 receptor, which has undergone such conformational changes. However, the desired conformational change is possible if, for example, the disulfide bond preceding the mAb104 epitope (C293 / C309, excluding the leader sequence) (C277 / C289, excluding the leader sequence) is transiently broken or undergoes a disulfide bond transition, allowing rearrangement to expose at least a portion of domain II of the mAb104 epitope. During rearrangement, the pertuzumab-binding epitope remains largely undisturbed. However, in computer simulations of adsorption of recombinant HER2-ECD for ELISA capture, the structural rearrangement of domain II is fixed, and the smaller steric hindrance of mAb104 binding can lead to reduced pertuzumab binding (e.g., as shown below). The potential loss of binding affinity can be balanced by synergistic interactions between the two antibodies.

[0724] Competition Measurement

[0725] To better define the epitopes of mAb104, ELISA was used to compare the binding ability of mAb104 to HER2-ECD and to the binding ability of pertuzumab domain II binding antibody to the spatially distant trastuzumab domain IV epitope. Figure 6 In these experiments, the inventors evaluated the effect of pre-incubation with mAb014 on the binding of trastuzumab and pertuzumab. Figure 6 B and C), and the effect of pre-incubation with trastuzumab and pertuzumab on mAb104 binding was also determined.

[0726] The inventors demonstrated that trastuzumab and mAb104 do not affect each other's binding to HER2-ECD. Figure 6 A and B). The inventors also demonstrated that prior incubation with pertuzumab does not affect mAb104 binding ( Figure 6 A). However, interestingly, prior to incubation with mAb104, the binding of pertuzumab to HER2-ECD was reduced ( Figure 6 C) indicates that the binding of mAb104 to its epitope may cause some steric hindrance to pertuzumab in certain situations.

[0727] Competition for endogenous HER2 between mAb104 and pertuzumab and trastuzumab was measured by flow cytometry in HER2-overexpressing mammary glands (BT474 and SK-BR-3). Figure 7-1 and 7-2 ) and gastric cell lines (NCI-N87 and OE19; Figure 7-3 and 7-4 Further investigations were conducted using the two sequential incubation methods described above. Experiments in these groups were pre-incubated with a high dose (100 μg / mL) of mAb104 to maximize the observed changes in the effect on trastuzumab and pertuzumab binding. Incubation with much higher doses of mAb104 did not affect the binding of trastuzumab or pertuzumab to HER2 on the cell surface. Inconsistencies in the results of mAb104 competing with pertuzumab using flow cytometry and ELISA can be explained by differences between the antigen preparations used in the assays, i.e., the presence of HER2 in its physiological conformation when analyzed by flow cytometry, while it is partially denatured in ELISA, and subsequent epitope presentation and availability.

[0728] Example 3: Binding of mAb104 to HER2 on the cell surface

[0729] The inventors used a group of cell lines with differential HER2 expression to examine the binding pattern and efficiency of mAb104 by FACS analysis.

[0730] The results are summarized in Table 8 below. The results are compared with those of secondary antibody binding only.

[0731] Table 8. Binding of mAb104

[0732] Herceptin pertuzumab mAb104 BT-474 +++ +++ - SK-BR-3 +++ +++ - NCI-N87 +++ +++ ++ OE-19 +++ +++ + MDA-MB-231 ND ND - MCF-7 ND ND -

[0733] FACS analysis was performed on the binding of mAb104 to HER2-expressing cells BT474, SK-BR-3, NCI-N87, OE-19, MDA-MB-231, and MCF7. The cells were co-incubated with 10 μg / mL trastuzumab, pertuzumab, mAb104, or secondary antibody alone, and the degree of binding was analyzed by FACS. Results represent two or more experiments.

[0734] In HER2-overexpressing cell lines, mAb104 showed the strongest binding to the HER2 population in the gastric cell line NCI-N87, while negligible HER2 binding was observed in low HER2-expressing cell lines (MDA-MB-231 and MCF-7).

[0735] Trastuzumab (Herceptin) and pertuzumab showed greater fluorescence in FACS, indicating that they bound to a greater number of HER2 receptors on all evaluated cell lines compared to mAb104 (Table 8). No difference in binding intensity was observed between the two humanized antibodies across different cell lines. The inventors’ findings support the hypothesis that mAb104 binds to a subset of receptors on the cell surface and will explain the differences observed in binding intensity between the antibodies.

[0736] mAb104 specificity for HER2

[0737] To confirm the specificity of mAb104 for endogenously expressed HER2 and HER3, the inventors performed Western blot analysis using lysates from different HER2-positive and HER3-negative cancer cell lines prepared under reducing conditions. Figure 8-1 ).

[0738] Antibody 2242 was used as a positive control for total HER2. This commercially available antibody, derived from cell signal transduction technology, targets intracellular HER2 epitopes. As shown in Figure 8A, mAb104 exhibited strong reactivity against various HER2-expressing cancer cell lines and was comparable to control antibodies that provided positive signals representing HER2 overexpression states across multiple cell lines. The correlation between mAb104 and control 2242 HER2 binding reflects the HER2 epitopes recognized by mAb104 under reducing conditions. HER3 expression levels were very low in the cancer cell lines studied, as also observed by FACS analysis and in previous studies (Brockhoff G, Heiss P, Schlegel J, Hofstaedter F, Knuechel R. Interaction of epidermal growth factor receptor, c-erbB2 and c-erbB3 receptors, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells. Cytometry Part A. 2001; 44(4):338-48.).

[0739] The specificity of HER2 / ErbB2 was assessed using an ELISA method. Figure 8-2 As shown, mAb104 is specific to ErbB2 / HER2 and does not bind to the extracellular domain of EGFR / HER1, or the ECD of ErbB3 / HER3 or ErbB4 / HER4.

[0740] Efficacy of mAb104 in In Vitro Breast Cancer

[0741] Example 4: Antiproliferative effect of mAb104

[0742] The effect of mAb104 as a monotherapy or in combination with trastuzumab or pertuzumab on the proliferation of HER2-overexpressing breast cancer cell lines was determined by an MTS cell proliferation assay in serum-depleted conditions (1% FCS) using a maximum concentration increased to 100 μg / mL (Figure 9).

[0743] Compared with the isotype control antibody, trastuzumab significantly reduced the proliferation of BT-474 (Fig. 9B) and SK-BR-3 (Fig. 9A) (p = 0.0006 and p = 0.0005, respectively; bilateral), while pertuzumab monotherapy did not have any significant antiproliferative activity in the evaluated cell lines (p = 0.22 and p = 0.15, respectively; bilateral, see Figs. 9A and 9B). These findings are consistent with other studies (Brockhoff G, Heckel B, Schmidt-Bruecken E, Planter M, Hofstaedter F, Vollmann A et al., Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK-BR-3 breast cancer cell proliferation. Cell proliferation. 2007; 40(4):488-507; Tokuda Y, Ohnishi Y, Shimamura K, Iwasawa M, Yoshimura M, Ueyama Y et al., In vitro and in vivo antitumor 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 xenograftmodels. Clinical Cancer Research. 2011;17(15):5060-70; Nahta R, Hung MC, EstevaFJ. 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 andchemotherapeutic 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. Molecularoncology. 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). .

[0744] Compared with the isotype control antibody, mAb104 did not show any significant growth inhibition in either the SK-BR-3 and BT-474 cell lines (p = 0.33 and p = 0.2, respectively; two-sided) (Figs. 9A and 9B).

[0745] As shown in Figures 9C and 9D, the combination of trastuzumab and pertuzumab significantly inhibited proliferation in 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 with trastuzumab monotherapy (BT-474 p ​​= 0.59 and SK-BR-3 p = 0.51; two-sided).

[0746] Adding mAb104 to either trastuzumab or pertuzumab did not affect the individual antiproliferative activities of trastuzumab and pertuzumab (Figure 9C-F). In the evaluated cell lines, the antiproliferative effect of the mAb104 / trastuzumab combination was not statistically different from that of the trastuzumab / pertuzumab combination (BT-474p = 0.66; SK-BR-3p = 0.47).

[0747] Therefore, unlike trastuzumab and pertuzumab, mAb104 did not exhibit detectable antiproliferative activity against HER2-positive cell lines. The complex interactions of multiple receptor kinases and signal transduction pathways that occur in vivo cannot always be fully replicated in vitro, and the effects of therapeutic agents that capture activated receptors that require conformational changes may be unmeasurable in vitro. Antibodies targeting conformationally exposed epitopes on activated EGFR have shown significant antitumor activity in vivo, although they have failed to show any growth inhibition or altered signal transduction in vitro [Johns TG, Perera RM, Vernes SC, Vitali AA, Cao DX, Cavenee WK et al. The efficacy of epidermal growth factor receptor-specific antibodies against glioma xenografts is influenced by receptor levels, activation status, and heterodimerization. Clinical Cancer Research. 2007; 13(6):1911-25.]

[0748] Example 5: Effects of mAb104 on ErbB receptors and downstream signal transduction pathways

[0749] Considering the functional differences of mAb104 observed in proliferation assays, the inventors sought to investigate the effect of 24 h of mAb104 treatment on the MAPK and Akt ligand-independent pathways in SK-BR-3 and BT-474 breast cancer cell lines under serum starvation conditions. To evaluate the role of mAb104 in ligand-dependent signal transduction pathways, the cell lines were treated with anti-HER2 antibody for 24 h, followed by the addition of 100 ng EGF for 10 min.

[0750] The results of ligand-independent and ligand-dependent effects are presented in Figures 10 and 11, respectively. The inventors focused on EGFR-HER2 signal transduction because HER3 expression levels are very low in cancer cell lines, as demonstrated in Figure 8, which is consistent with other studies (Brockhoff G et al., Interaction of epidermal growth factor receptor, c-erbB2 and c-erbB3 receptors, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells. Cytometry Part A. 2001; 44(4):338-48).

[0751] In this series of experiments (Figures 10 and 11), the inventors found that treatment with trastuzumab and pertuzumab for 24 hours did not significantly reduce total HER2 expression in 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).

[0752] The effects of anti-HER2 antibodies on the Akt and MAPK pathways were also assessed using phosphate-specific antibodies. In two breast cancer cell lines (BT-474 and SK-BR-3), trastuzumab treatment resulted in decreased Akt phosphorylation without a change in total Akt protein levels, indicating a reduction in phosphorylation activity rather than a downregulation of Akt protein. These findings are consistent with other studies (Lu Q et al., ibid.; Yakes FM et al., (2002) Cancer research. 62(14): 4132-41).

[0753] In BT-474 cells, trastuzumab treatment led to a decrease in MAPK activity, as shown by a decrease in phosphate-MAPK (Fig. 10C and D). In contrast, no change in MAPK activity was observed in SK-BR-3 cells (Fig. 10A and B), consistent with other studies (Cuello M et al., (2001) Cancer research. 61(12): 4892-900).

[0754] As shown in Figures 10A and 10B, treatment with mAb104 as a monotherapy did not result in detectable changes in the amount of total protein or phosphorylated protein in the evaluated cell lines.

[0755] In BT-474 cells, the combination of trastuzumab and pertuzumab reduced phosphorylated Akt and phosphorylated p44 / p42 MAPK levels to a greater extent than either drug alone, without any effect on total Akt or MAPK (Fig. 10D). Conversely, in SK-BR-3, signal transduction via the MAPK cascade was not inhibited by the combination of drugs, as previously shown by the unchanged levels of phosphorylated p44 / p42 MAPK (Nahta R et al., (2004) Cancerresearch.64(7):2343-6). Similar changes in the Akt and MAPK signaling cascade were observed with trastuzumab and mAb104, and with the combination of trastuzumab and pertuzumab. There was no significant difference in the degree of downregulation between the two drug combinations (Fig. 10C and D).

[0756] Under ligand-stimulated conditions (Fig. 11), trastuzumab and pertuzumab did not affect the MAPK or Akt signaling pathways in BT-474 (Fig. 11A and B). In the SK-BR-3 cell line, pretreatment with pertuzumab 10 min after EGF stimulation resulted in reduced Akt phosphorylation, while total Akt remained unchanged (Fig. 11A). These findings are consistent with other reports (Henjes F et al., (2012) Oncogenesis 1(7):e16). Trastuzumab inhibited Akt-mediated signal transduction due to the abolition of ligand-independent signal transduction, while pertuzumab prevented ligand-induced signal transduction. Addition of EGF to mAb104-treated cells did not result in changes in the amount of total or phosphorylated MAPK and Akt pathway proteins in the evaluated breast cancer cell lines. In both cell lines, treatment with various mAb104 combinations had no effect on MAPK or Akt signaling compared to the control antibody (Fig. 11C and D).

[0757] Example 6: Effect of mAb104 on in vitro cell apoptosis

[0758] The apoptosis-inducing activity of mAb104 in BT-474 and SK-BR-3 cells was determined by flow cytometry using a dead cell apoptosis kit (Thermo Fisher Scientific, catalog number V13241). Early and late apoptotic cell fractions were quantified by quadrant analysis. Treatment with trastuzumab or pertuzumab did not induce any apoptosis compared to cells without apoptosis, consistent with previous studies (Rockhoff G et al., (2007) Cell proliferation. 40(4):488-507; Nahta R et al., (2004) Cancer Research 64(7):2343-6; Lu Q et al., ibid.) (Fig. 12). Treatment of SK-BR-3 cells with trastuzumab resulted in a greater number of apoptotic cells compared to BT-474 (Fig. 12A-G), however this was not statistically significant (Fig. 12I-O). Similarly, mAb104 did not induce any significant apoptosis in the evaluated cell lines compared to cells alone (p = 0.494). No difference in apoptotic activity was observed between the antibodies (p = 0.726).

[0759] Compared with single-agent treatment, no increase in apoptotic activity was observed in cells exposed to a combination of trastuzumab and pertuzumab or mAb104. In BT-474, 89.9% of cells survived after treatment with trastuzumab monotherapy, compared to 91.8% after treatment with trastuzumab and mAb104 (Fig. 12A-G). No difference in apoptotic activity was observed between combinations of trastuzumab and pertuzumab or mAb104 (Fig. 12A-F and G).

[0760] Interestingly, treatment with mAb104 resulted in a greater number of necrotic cells compared to treatment with cells alone, as well as treatment with trastuzumab and pertuzumab (Fig. 12H and P), although this difference was not statistically significant. In BT-474 cells, 1.8% of cells underwent necrosis, compared to <0.5% in all other groups; similarly, treatment with mAb104 resulted in a higher number of necrotic SK-BR-3 cells compared to other treatment groups (Fig. 12P), although this difference was not statistically significant. Interestingly, treatment with mAb104 in combination with trastuzumab did not lead to an increase in the number of cells undergoing necrosis.

[0761] The complex interactions of multiple receptor kinases and signal transduction pathways occurring in vivo cannot always be fully replicated in vitro and can reflect differences in HER2 processing and function. Furthermore, the influence of the tumor microenvironment on HER2 function or other in vivo factors is essential for the functional effect of mAb104 targeting conformationally exposed epitopes on HER2 to be confirmed. Antibodies targeting conformationally exposed epitopes on activated EGFR have shown significant antitumor activity in vivo, although they have failed to show any growth inhibition or altered signal transduction in vitro (Johns TG, Perera RM, Vernes SC, Vitali AA, Cao DX, Cavenee WK et al., The efficacy of epidermal growth factor receptor-specific antibodies against glioma xenografts is influenced by receptor levels, activation status, and heterodimerization. Clinical Cancer Research. 2007; 13(6):1911-25).

[0762] Despite the lack of detectable in vitro activity of mAb104, the inventors continued to investigate its in vivo role in cancer xenograft models that overexpress HER2.

[0763] Internal efficacy of mAb104

[0764] Example 7: mAb104 monotherapy in HER2-overexpressing / amplified ER-positive breast cancer xenografts effect

[0765] The inventors evaluated the efficacy of mAb104 in mice carrying established tumor xenografts of the ER-positive, HER2-overexpressing breast cancer cell line BT-474. Once the tumor volume reached 100-120 mm... 3 Administer mAb104, trastuzumab, pertuzumab, or control antibody at a dose of 1 mg / antibody three times a week for three weeks.

[0766] The results are shown in Figure 13 At the end of treatment (day 32), all tumors in the treatment group were significantly smaller than those in the control group (ANOVA p < 0.0006). Post-hoc tests using the Bonferroni method showed that all tumors in the treatment group were significantly smaller compared to the control group (p ≤ 0.001). The mean tumor volume on day 32 was 337.2 mm. 3(Control group), 4.8mm 3 (Trastuzumab), 6.7mm 3 (pertuzumab) and 48.7mm 3 (mAb104). Significant antitumor responses persisted until the end of the study (day 39) in both the trastuzumab and pertuzumab treatment groups, one week after treatment cessation. However, for mAb104, tumor growth resumed after treatment cessation. At the end of the study (day 39), no significant difference in growth inhibition was observed between the treatment groups (p = 0.14).

[0767] The inventors also evaluated the results of establishing BT-474 tumor xenografts (120-150 mm). 3 The efficacy of treatment with a lower dose of mAb104, trastuzumab, pertuzumab, and control IgG at 0.5 mg / mL for three weeks, administered three times a week, was evaluated.

[0768] The results are shown in Figure 14 Trastuzumab treatment halted further tumor growth; pertuzumab and mAb104 reduced 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 those in the control group (ANOVA p < 0.038). The mean tumor volume measured was 927.5 mm. 3 (Control) 182.4mm 3 (Trastuzumab), 415.0mm 3 (Pertuzumab) and 469.1mm 3 (mAb104). Post-hoc testing using the Bonferroni method showed that mice in the treatment group had significantly smaller tumors compared to the control group (trastuzumab p = 0.0035, pertuzumab p = 0.02 and mAb014 p = 0.008). At 0.5 mg / mL, mAb104 showed similar antitumor efficacy to pertuzumab in this model (p = 0.97, two-sided). Although treatment with trastuzumab resulted in numerically greater tumor growth inhibition, there was no significant difference between trastuzumab and pertuzumab (p = 0.22, two-sided) or mAb104 (p = 0.15, two-sided) at the end of the study (day 51).

[0769] Survival based on log-rank analysis was considered ethically (i.e., tumor size ≤1000mm). 3 When the control group was removed, the survival rate of mice treated with anti-HER2 antibody was significantly longer than that of the control group (p<0.002). The median survival time of mice in the control group was 41 days, while the mice in the treatment group did not reach the median survival time at the end of the experiment (day 52).

[0770] Example 8: Efficacy of mAb104 monotherapy in a HER2-overexpressing / amplified breast PDX model

[0771] The inventors evaluated the role of mAb104 in a patient-derived xenograft (PDX) model of breast cancer with HER2-overexpression / amplification. Donor samples were untreated, therefore tumor susceptibility to anti-HER2 therapy was assumed to be 100%. Once the tumor size of 100-120 mm measured on day 64 was determined... 3 The tumor volume was determined by treating mice with a total dose of 0.5 mg of mAb104, trastuzumab, pertuzumab, or control IgG three times a week for three weeks. The tumor growth curve results are shown in Figure 15A.

[0772] Anti-HER2 therapy directly affected the growth rate of PDX. After treatment was discontinued on day 86, all anti-HER2 treatments showed equivalent antitumor efficacy, and the delay in tumor growth rate persisted until day 125, at which point the tumor growth curve began to parallel the control group's growth rate. At the end of the study, on day 145, the control group was excluded for ethical reasons. On day 145, all treatment groups were significantly smaller than the control group (ANOVA p < 0.04) (Figure 15A). Post-hoc testing using the Bonferroni method showed that mice in the treatment groups had significantly smaller tumors compared to the control group (trastuzumab p = 0.02; pertuzumab = 0.02 and mAb104 p = 0.038). The mean tumor volume was 1099.2 mm. 3 (Control) 761.2mm 3 (mAb104), 632.8mm 3 (Trastuzumab) and 691.3mm 3 (Pertuzumab). mAb104 showed strong and equivalent antitumor activity to approved HER2-targeted therapies in this model, with no significant difference between anti-HER2 therapies (p = 0.547 (bilateral) trastuzumab vs. mAb104 and p = 0.754 (bilateral) pertuzumab vs. mAb104).

[0773] In survival analysis, mice in the treatment group had significantly longer survival than those in the control group (p<0.0005), and post-exposure testing showed that all groups treated with anti-HER2 antibody had significantly longer survival than control mice (p<0.001). The median survival of mice in the control group was 145 days, while mice in the treatment group did not reach the median survival at the end of the experiment.

[0774] Example 9: The combination of mAb104 and trastuzumab in ER-positive breast xenografts with overexpression / amplification of HER2 The efficacy of

[0775] The combination of trastuzumab and pertuzumab has been demonstrated in the literature to have more potent antitumor activity and prevent metastatic tumor spread compared to either antibody alone, independent of HER2 expression. Given the different domain II epitope binding sites of mAb104 compared to trastuzumab and pertuzumab, and the inventors' observations of the effective antitumor activity of mAb104 as a monotherapy in vivo, the inventors continue to evaluate the combination of mAb104 and trastuzumab, either alone or in combination with pertuzumab.

[0776] The inventors evaluated the efficacy of the combination of mAb104 and 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 to achieve a total dose of 0.5 mg / treatment or equivalent control antibody, three times a week for three weeks. Once the average tumor volume reached 100-120 mm, the combination was evaluated. 3 Then treatment began.

[0777] The results are shown in Figure 15B. Antitumor efficacy was evident within the first 10 days of treatment and continued after treatment cessation. At the end of day 50 of the study period, the control group was excluded for ethical reasons regarding tumor burden. The mean ± SD tumor volume in both the trastuzumab monotherapy group and the combination group was significantly smaller than that in the control group (ANOVA p < 0.0001). Post-hoc tests were then performed using the Bonferroni method. Tumors treated with the combination therapy were significantly smaller than those in the control group (p < 0.0001), measuring 88.9 mm. 3 (mAb104 plus trastuzumab) and 43.6mm 3 (Trastuzumab plus pertuzumab). Complete tumor regression was not observed in either treatment group (Figure 15B). Concomitant treatment with mAb104 and trastuzumab resulted in greater tumor reduction compared to trastuzumab alone; however, the difference in tumor size between the combination and monotherapy groups was not statistically significant (by ANOVA, p = 0.09).

[0778] Survival analysis showed that mice in both combination groups had significantly longer survival times compared to the control group (p<0.002). The median survival time in the control group was 44 days, while mice in the combination therapy groups did not reach the median survival time. Log rank analysis showed no statistically significant difference between the two combination groups (p=0.21, two-sided); treatment with mAb104 and trastuzumab significantly inhibited tumor growth compared to mAb104 monotherapy (p<0.04, two-sided) (Figure 15A).

[0779] Therefore, this suggests that the combination of mAb104 and trastuzumab provides enhanced antitumor activity compared to either monotherapy alone.

[0780] Example 10: The combination of mAb104 and trastuzumab in a HER2-overexpressing / amplified ER-positive breast PDX model. efficacy

[0781] The inventors evaluated the efficacy of concurrent antibody therapy in a HER2-overexpressing / amplified breast PDX model. Donor samples were untreated with anti-HER2, thus 100% tumor sensitivity to treatment was assumed. Once the tumor size of 100-120 mm was determined... 3 Tumor volume was measured between weeks, and mice were treated with a total therapeutic dose of 0.5 mg trastuzumab three times a week or an allotype control, either mAb104 or trastuzumab alone or in combination, or trastuzumab plus pertuzumab, for three weeks.

[0782] At day 85, when treatment was completed, significant differences were observed between all treatment groups and the control group (p<0.0001) (Figure 15C). Furthermore, the combination group was more effective than trastuzumab alone (p=0.001). The greater antitumor efficacy of the combination group persisted until the study ended on day 145, at which point the control group was excluded for ethical reasons regarding tumor burden. On day 145, tumors in all treatment groups remained significantly smaller than those in the control group (p<0.0001). The mean tumor volume measured was 164.4 mm. 3 (mAb104 plus trastuzumab) and 84.2mm 3 (Trastuzumab plus pertuzumab) (Figure 15C). The difference in tumor volume between the two combination groups did not reach statistical significance (p = 0.46, two-sided). Complete tumor regression was not observed in either treatment group.

[0783] Compared with trastumab monotherapy, concomitant treatment with mAb104 and trastumab resulted in significantly greater tumor volume shrinkage (by ANOVA, p<0.0001). Post-hoc tests were then performed using the Bonferroni method. Tumors in the combination group were significantly smaller compared with trastumab monotherapy (p<0.0049).

[0784] Survival analysis showed that mice in both combination groups had significantly longer survival times compared to the control group (p<0.0005) and the single-treatment group (p=0.0014). The median survival time in the control group was 145 days, while the median survival time in the combination treatment group was not reached.

[0785] The results in Figures 15A and 15C indicate that after day 145, the mean tumor size with trastuzumab alone was 632.8 mm. 3 mAb104 is 761.2 mm. 3 When mAb104 was combined with trastuzumab, the tumor size was significantly reduced to 164.4 mm. 3This reduction in tumor size suggests that the combination of mAb104 and trastuzumab results in enhanced antitumor activity compared to monotherapy with either trastuzumab or mAb104 alone.

[0786] Example 11: Reverse Phase Protein Array (RPPA) Analysis

[0787] Lysates were collected from HER2-breast PDX tumors (n=2 / group) at day 85 of treatment completion and analyzed by RPPA. A panel of over 300 antibodies, detecting total protein and / or its activated forms, were included in this RPPA analysis. Key proteins are involved in critical signal transduction pathways, including the phosphatidylinositol 3-kinase (PI3K) / AKT pathway, extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) pathway, Janus kinase (JAK) / signal transduction and activating factor (STAT) pathway, apoptosis pathway, and cell cycle (including cell death and survival). Collected data were normalized for protein loading and converted to linear values ​​for analysis. fold changes were calculated as the ratio of differences in protein expression between control and tumor samples treated with trastuzumab, pertuzumab, or mA104, or combinations thereof.

[0788] Although mAb104 showed significant antitumor activity in the HER2-breast PDX model, no significant changes in protein levels were observed compared to other antibodies evaluated (Table 9).

[0789] Table 9: Differential expression of key proteins treated with trastuzumab, pertuzumab, and mAb104 as assessed by RPPA

[0790]

[0791]

[0792]

[0793]

[0794]

[0795] *ns: Not significant. This list evaluates fold changes in key pathway kinases and their downstream effectors. Fold changes were calculated as the ratio of differences in protein expression between the control and treatment groups. p-values ​​were obtained using a t-test for the comparisons shown.

[0796] Immunohistochemical analysis of tumors established in Example 12

[0797] Mice (n=2) carrying established BT-474 breast tumor xenografts from each of the single-therapy and combination therapy groups were sacrificed one day after the last treatment, and xenograft tissue samples were obtained and prepared for IHC analysis for tumor proliferation, downstream signal transduction and angiogenesis.

[0798] The effect of mAb104 monotherapy on tumor proliferation was detected by Ki67 staining, and the results are shown in Figure 16A. In BT-474 xenograft tumors, anti-HER2 antibody treatment did not significantly reduce proliferation compared with the control group (ANOVA, p = 0.625; post-hoc analysis showed no difference between the treatment groups). The mean H-scores were 102.6 (control), 83.9 (trastuzumab), 91.1 (pertuzumab), and 99.7 (mAb104).

[0799] To determine whether the antiproliferative effect is mediated by downregulation of the Akt pathway, Akt was assessed by phosphoprotein assay (Figure 16B). In BT-474, no significant difference in H-scores of phosphate-Akt was observed between the treatment and control groups (by ANOVA, p = 0.958; no difference in the treatment group in post-hoc analysis). The mean H-scores were 129.6 (control), 124.3 (trastuzumab), 114.6 (pertuzumab), and 132.5 (mAb104).

[0800] Trastuzumab has been shown to have anti-angiogenic activity (Parakh S, (2017) Cancer treatment reviews. 59: 1-21). Therefore, the inventors examined the effect of mAb104 on microvessel density in tumor tissue by calyx glycoprotein staining (Figure 16C). Immunohistochemical staining was performed as described above. Microvessel 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, although significant anti-angiogenic activity of trastuzumab was observed (p < 0.001), mAb104 and pertuzumab had no significant effect on the tumor vascular system compared to the control antibody (p = 0.987).

[0801] Compared with the control (p<0.05) and with trastuzumab and mAb104 (p=0.017, two-sided), the combination of trastuzumab and pertuzumab significantly reduced proliferation. The mean H-scores were 129.6 (control), 7.4 (trastuzumab plus pertuzumab), and 97.6 (trastuzumab plus mAb104) (Figure 16D). These findings are similar to previous reports (Brockhoff G, (2007) Cell proliferation. 40(4):488-507).

[0802] When compared with the control antibody, 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.

[0803] Stomach cancer

[0804] Example 13: Antiproliferative effect of mAb104 in vitro

[0805] In vitro, trastuzumab significantly (p<0.0001) inhibited the growth of NCI-N87 cells and affected the proliferation of OE-19 gastric cancer cells. Figure 17 This is consistent with previous reports (Gravalos C et al., (2008) Annals of oncology. 19(9): 1523-9). Compared with the control antibody, pertuzumab as a monotherapy did not have any significant antiproliferative activity in the evaluated cell lines (NCI-N87 p = 0.02; and OE19 p = 0.96). These findings are consistent with other studies, although the incubation time and dosage differ (Brockhoff G et al., (2007) Cell proliferation. 40(4): 488-507; Tokuda Y et al., (1996) British journal of cancer. 73(11): 1362; Yamashita-Kashima Y et al., (2011) Clinical Cancer Research. 17(15): 5060-70; Nahta R et al., (2004) Cancerresearch. 64(7): 2343-6; Gong SJ et al., (2004) Cancer letters. 214(2): 215-24; Ko BK et al., (2015) Molecular oncology. 9(2): 398-408; Tomioka H et al., (2012) International journal of oncology. 41(2): 551-8).

[0806] Compared with the isotype control antibody, the monoclonal antibody mAb104 did not show significant growth inhibition in vitro (NCI-N87 p = 0.34; and OE19 p = 0.12). Figure 17This 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).

[0807] The combination of trastuzumab and pertuzumab significantly inhibited proliferation in the evaluated gastric / GEJ cell lines. Figure 18 However, there was no statistically significant difference compared to trastuzumab monotherapy. The effect of mAb104 in combination with trastuzumab or pertuzumab on proliferation was evaluated; the addition of mAb104 did not increase antiproliferative activity compared to the individual antibodies. Significantly, the combination of mAb104 and trastuzumab was not statistically different from the combination of trastuzumab and pertuzumab (NCI-N87 p = 0.29; and OE19 p = 0.14).

[0808] Example 14: Effects of mAb104 on ErbB receptors and downstream signal transduction pathways

[0809] 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, consistent with previous reports (KoB-K et al., (2015) Molecular oncology.9(2):398-408; Tomioka H et al., (2012) International journal of oncology.41(2):551-8) (Figures 19A and B).

[0810] Combination therapy with trastuzumab and pertuzumab or mAb104 in the NCI-N87 gastric cancer cell line resulted in downregulation of phosphorylated akt and phosphorylated p44 / p42 MAPK, while total protein levels remained unchanged (Figures 19C and D).

[0811] Under ligand-stimulated conditions (Figs. 20A and B, and 20C and D), mAb104 did not affect the MAPK or Akt signaling pathways in the evaluated cell lines. This lack of effect was also observed when mAb104 was used in combination with trastuzumab and pertuzumab (Figs. 20C and D).

[0812] The changes in signal transduction described in this cell line treated with a combination of trastuzumab and pertuzumab 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 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 signal transduction. Conversely, no changes in total protein or activated ErbB family proteins or MAPK and Akt signaling cascades were observed in OE19 when treated with a combination of trastuzumab with pertuzumab or mAb104. In both gastric cancer cell lines, combination therapy with pertuzumab and mAb104 had no effect on signaling cascades or total or phosphorylated ErbB protein levels.

[0813] Example 15: Effect of mAb104 on apoptosis of gastric cancer cells

[0814] Flow cytometry was used to determine the apoptosis-inducing activity of mAb104 in the NCI-N87 (Fig. 21A and B) and OE-19 cell lines (Fig. 21C), and early and late apoptotic cell fractions were quantified by quadrant analysis (Fig. 21). Compared to the control antibody, cells treated with trastuzumab did not induce apoptosis; in contrast, treatment with pertuzumab produced more cells in the late stage of apoptosis. These findings are consistent with other published studies. mAb104 increased the late apoptotic cell population compared to trastuzumab or pertuzumab monotherapy. In OE-19, no antibody, as a monotherapy or combination, induced any apoptosis compared to the control antibody (Fig. 21C).

[0815] Compared to the combination of trastuzumab and mAb104, the combination of trastuzumab and pertuzumab resulted in significantly more apoptotic cells. After treatment with trastuzumab and pertuzumab, 61.4% of cells survived, compared to 90.5% after treatment with mAb104 (Figure 21A). The combination of trastuzumab and mAb104 did not induce apoptosis compared to the control antibody or other monotherapy.

[0816] A significant finding was the observation 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 increased twofold after mAb014 treatment compared to the other treatment groups. Interestingly, treatment with mAb104 in combination with trastuzumab did not result in an increase in the number of cells undergoing necrosis.

[0817] Example 16: Effect of mAb104 on migration (wound healing) test

[0818] 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 typically initially attach to islands and then proliferate into dense plaques, making accurate estimation of confluence difficult, and therefore were not used. At 72 h post-treatment, the addition of antibody did not delay the migration of OE-19 cells compared to a 100 μg / mL dose of the control antibody. Figure 22 ).

[0819] The breast cancer cell line BT-474 typically forms tightly packed, multilayered colonies that rarely fuse, making it unsuitable for this assay.

[0820] Example 17: Efficacy of mAb104 monotherapy in HER2-overexpressing / amplified gastric cancer xenografts

[0821] The inventors tested the antitumor activity of mAb104 in mice with xenografts of the HER2-overexpressing gastric cancer cell line NCI-N87. Mice were treated with mAb104, trastuzumab, pertuzumab, and control IgG, injected three times a week for three weeks. In all experiments, when the average tumor volume was 100-120 mmHg... 3 Treatment began at that time. Preliminary experiments involving the NCI-N87 xenograft model used different treatment doses to confirm the efficacy of mAb104 in the tumor model.

[0822] Using an initial treatment dose of 1 mg, the results were as follows: Figure 23 As shown. At the end of treatment (day 28), all tumors in the treatment group were significantly smaller than those in the control group (ANOVA p < 0.0001). Post-hoc tests were performed using the Bonferroni method. Tumors in all treatment groups were significantly smaller (p < 0.0001); the mean tumor volume was 333.9 mm. 3 (Control) 56.1mm 3 (Trastuzumab), 77.9mm 3 (Pertuzumab) and 66.1mm 3 (mAb104), no statistically significant difference in growth inhibition was observed between different treatment groups (p>0.05).

[0823] Based on the efficacy observed at a 1 mg dose, the experiment was repeated using a lower dose of 0.5 mg / treatment. Tumor growth curves are shown below. Figure 24 As shown. At the end of treatment (day 32), all tumors in the treatment group were significantly smaller than those in the control group (ANOVA p<0.0001). Post-hoc tests using the Bonferroni method showed that all tumors in the treatment group were significantly smaller than those in the control group (p<0.0001). The mean tumor volume was 280.8 mm. 3 (Control) 104.0mm 3 (Trastuzumab), 152.2mm 3 (pertuzumab) and 105.7mm 3 (mAb104). No statistically significant difference in growth inhibition was observed between treatment groups. Tumor growth cessation was evident from the start of treatment until day 50 in all antibody groups, and 22 days after treatment cessation.

[0824] Mice treated with the anti-HER2 antibody had significantly longer survival than the control group (ANOVA p < 0.0002; post-hoc tests showed that all treatment groups had significantly longer survival than control mice, p < 0.0001). At the end of day 100, mAb104 demonstrated a significant survival advantage, with 60% of animals surviving, compared to 20% with trastuzumab, and none in the other groups. The median survival was 59 days (control), 91 days (trastuzumab), and 83 days (pertuzumab), and was not reached in mice treated with mAb104 at the end of the experiment (day 100). This observation is likely due to the expected longer half-life of mouse mAb104 in mouse circulation compared to the humanized construct.

[0825] When the experiment was repeated in established NCI-N87 gastric cancer xenografts using a dose of 0.1 mg / treatment, similar efficacy in vivo was observed. Figure 25 At the end of treatment (day 28), all tumors in the treatment group were significantly smaller than those in the control group (ANOVA p < 0.0001. The mean tumor volume was 340.4 mm). 3 (Control) 136.3mm 3 (Trastuzumab), 147.5mm 3 (pertuzumab) and 104.4mm 3 (mAb104). No statistically significant difference in growth inhibition was observed between the treatment groups. Figure 25 A).

[0826] Log-rank analysis showed that mice treated with 0.1 mg / dose of anti-HER2 antibody had significantly longer survival than the control group (p<0.0001; post-hoc tests showed that all treatment groups had significantly longer survival than control mice (p<0.0002)). Figure 25 B). For mice treated with mAb104, the median survival was 62 days (control), 83 days (trastuzumab), 79 days (pertuzumab), and 83 days.

[0827] Example 18: The efficacy of mAb104 monotherapy in xenografts for HER2-overexpressing / amplified gastroesophageal cancer effect

[0828] The inventors tested the in vivo antitumor activity of mAb104 in mice carrying OE-19 xenografts. Mice were treated with 1 mg / antibody dose of mAb104, trastuzumab, pertuzumab, and control IgG, three times a week for three weeks. The tumor activity was assessed when the mean tumor volume was 100-120 mm. 3 Treatment began at that time. In this rapidly growing tumor model, the tumor volume exceeded the ethically approved 1000 mm. 3 Mice treated with the isotype control antibody were eliminated before the treatment plan was completed (day 19; antibody dose received -5).

[0829] The results are as follows Figure 26 As shown. On day 19, all tumors in the treatment groups were significantly smaller than those in the control group (ANOVA p<0.0001). Post-hoc tests were performed using the Bonferroni method. Tumors in each treatment group were significantly smaller than those in the control group (p<0.0001). At the end of the study, there was no statistically significant difference in tumor growth inhibition among trastuzumab, pertuzumab, or mAb104 (p=0.16).

[0830] The inventors repeated the experiment using a lower dose of 0.5 mg / treatment, and the tumor growth curve is shown below. Figure 27 In the mean tumor volume of 100-120 mm. 3 Treatment began at that time. At the end of the study (day 25), the mean tumor volume in the treatment group was 974.8 mm. 3 (Comparison), 782.0mm 3 (mAb104), 474.5mm 3 (Trastuzumab), 832.0mm 3(Pertuzumab). Tumors treated with trastuzumab were significantly smaller than those in the control group (p<0.001). Although tumors were numerically smaller in both the mAb104 and pertuzumab groups, the difference in growth inhibition did not reach statistical significance after the Bonferroni method. No statistically significant difference in growth inhibition was observed between pertuzumab and mAb104 (p=0.39, both comparisons were two-tailed). Trastuzumab was more effective than both pertuzumab groups (p=0.024; two-tailed) and mAb104 (p=0.004; two-tailed). Figure 27 A).

[0831] Log-rank analysis showed that mice treated with anti-HER2 antibody had significantly longer survival than the control group (p<0.0005, with post-hoc tests showing significantly longer survival in all treatment groups compared to the control group (p<0.006)). The median survival was 25 days (control), 30 days (pertuzumab), 30 days (mAb104), and 35 days (trastuzumab). Figure 27 B).

[0832] Example 19: Efficacy of mAb104 combined with trastuzumab in NCI-N87 gastric cancer xenografts

[0833] The inventors evaluated the efficacy of the combination of mAb104 and trastuzumab in the NCI-N87 xenograft model. Mice were treated with a total dose of 0.5 mg of mAb104 and trastuzumab, trastuzumab and pertuzumab, or control IgG alone. Each mouse received 0.25 mg of trastuzumab and 0.25 mg of mAb104 or pertuzumab to achieve a total treatment dose of 0.5 mg. The efficacy was evaluated when the mean tumor volume was 100-120 mm. 3 Treatment began on day 9, and the tumor growth curve is shown below. Figure 28 middle.

[0834] Immediate antitumor efficacy was observed with the start of treatment in all antibody therapies, and continued tumor growth elimination was observed after treatment cessation on day 24. A more prolonged antitumor efficacy was observed in the combination therapy group. On day 57, the mean tumor volume in the control group reached the ethically approved 1000 mmHg. 3 At this time point, mice in the control group were removed....

Claims

1. A HER2 / ErbB2 binding protein comprising an antigen binding domain, wherein: The antigen binding domain specifically binds to an epitope within domain II of HER2, including residues 293 to 309 of the mature normal or wild-type human HER2 sequence shown in SEQ ID NO: 27, and the epitope is exposed in response to HER2 amplification or activation, and wherein the epitope is expressed in tumorigenic, hyperproliferative or abnormal cells but not in normal or wild-type cells, wherein the HER2 binding protein comprises: (i) a heavy chain variable region sequence (VH) comprising CDR1 consisting of the sequence shown in SEQ ID NO: 14, CDR2 consisting of the sequence shown in SEQ ID NO: 16, and A CDR3 consisting of the sequence shown in SEQ ID NO: 18; and A light chain variable region sequence (VL) comprising CDR1 consisting of the sequence shown in SEQ ID NO:20, CDR2 consisting of the sequence shown in SEQ ID NO:22, and A CDR3 consisting of the sequence shown in SEQ ID NO:24; or (ii) a heavy chain variable region sequence (VH) comprising CDR1 consisting of the sequence shown in SEQ ID NO:15, CDR2 consisting of the sequence shown in SEQ ID NO: 17, and CDR3 consisting of the sequence shown in SEQ ID NO: 19; and A light chain variable region sequence (VL) comprising CDR1 consisting of the sequence shown in SEQ ID NO:21, CDR2 consisting of the sequence shown in SEQ ID NO:23, and CDR3 consisting of the sequence shown in SEQ ID NO:

25.

2. The HER2 binding protein according to claim 1, wherein The epitope includes the amino acid sequence CPLHNQEVTAEDGTQRC (SEQ ID NO: 1).

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

25.

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

24.

5. The HER2 binding protein according to claim 4, wherein The HER2 binding protein is an antibody or an antigen-binding fragment thereof, which comprises: (i) a heavy chain variable region sequence (VH) that is at least 55% identical to the heavy chain variable region sequence of mAb104 (SEQ ID NO: 2); and / or (ii) a light chain variable region sequence (VL) that is at least 50% identical to the light chain variable region sequence of mAb104 (SEQ ID NO: 3).

6. The HER2 binding protein according to claim 5, wherein The HER2 binding protein is humanized, chimeric or deimmunized.

7. The HER2 binding protein according to claim 3, wherein The HER2 binding protein is an antibody or an antigen-binding fragment thereof, which comprises: (i) a VH that is at least 55% identical to the heavy chain variable region sequence of mAb106 (SEQ ID NO: 4); and (ii) a VL that is at least 50% identical to the light chain variable region sequence of mAb106 (SEQ ID NO: 5).

8. The HER2 binding protein of claim 7, wherein the HER2 binding protein is humanized, chimeric or deimmunized.

9. The HER2 binding protein according to claim 1, wherein The HER2 binding protein comprises: (i) a VH comprising SEQ ID NO: 2 and a VL comprising SEQ ID NO: 3; or (ii) VH comprising SEQ ID NO:4 and VL comprising SEQ ID NO:

5.

10. The HER2 binding protein according to claim 5 or 6, wherein The antigen-binding fragment 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 a heavy chain constant domain CH2 and / or CH3; or (iv) at least one of (i) and / or (ii) linked to a protein that enhances the half-life of the antibody.

11. The HER2 binding protein according to claim 7 or 8, wherein The antigen-binding fragment 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 a heavy chain constant domain CH2 and / or CH3; or (iv) at least one of (i) and / or (ii) linked to a protein that enhances the half-life of the antibody.

12. The HER2 binding protein according to claim 5 or 6, wherein The antigen-binding fragment is: (i) Diabodies; (ii) three-chain antibodies; (iii) tetrabodies; (iv) Fab; (v) F(ab′)2; (vi) Fv; or (vii) at least one of (i) to (vi) linked to a heavy chain constant region or Fc or a heavy chain constant domain CH2 and / or CH3; or (viii) at least one of (i) to (vi) linked to a protein that enhances the half-life of the antibody.

13. The HER2 binding protein according to claim 7 or 8, wherein The antigen-binding fragment is: (i) Diabodies; (ii) three-chain antibodies; (iii) tetrabodies; (iv) Fab; (v) F(ab′)2; (vi) Fv; or (vii) at least one of (i) to (vi) linked to a heavy chain constant region or Fc or a heavy chain constant domain CH2 and / or CH3; or (viii) at least one of (i) to (vi) linked to a protein that enhances the half-life of the antibody.

14. The HER2 binding protein of claim 1, which is conjugated to a detectable or functional moiety.

15. The HER2 binding protein according to claim 1, which is conjugated to a drug.

16. A composition comprising (i) a HER2 binding protein according to any one of claims 1 to 15 and (ii) an anti-HER2 antibody, a chemotherapeutic agent, or an immunotherapeutic agent or a combination thereof.

17. The composition of claim 16, wherein the immunotherapeutic agent is a radioimmunotherapeutic agent.

18. A composition comprising the HER2 binding protein according to any one of claims 1 to 15 and a suitable carrier.

19. Use of the HER2 binding protein of any one of claims 1 to 15, the composition of claim 16 or 17, or the composition of claim 18 in the preparation of a medicament for treating a cancer expressing HER2, wherein the cancer expressing HER2 is selected from biliary tract cancer, colon cancer, lung cancer, bladder cancer, endometrial cancer, ovarian cancer, breast cancer, gastric cancer, and gastroesophageal cancer.

20. Use of the HER2 binding protein of any one of claims 1 to 15 in the preparation of a composition or system for diagnosing or detecting breast cancer in a biological sample obtained from a subject, wherein the HER2 binding protein binds to HER2.

21. A complex comprising the HER2 binding protein of any one of claims 1 to 15 bound to HER2.

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