Anti-HER2 antibodies for use in the treatment of low-HER2-expressing tumors in the target population.

Abstract

In one embodiment, the present invention relates to an anti-HER2 immunoglobulin E (IgE) antibody for use in the treatment of tumors with low HER2 expression in a subject. In another embodiment, the present invention provides an immunoglobulin that binds to HER2.

Description

[Technical Field]

[0001] This invention relates to the field of therapeutic antibodies and their use, particularly immunoglobulin E (IgE) antibodies for use in the treatment of cancer. The invention also relates to methods for treating diseases, such as cancer, using such IgE antibodies. [Background technology]

[0002] Therapeutic antibodies now complement conventional treatments for many malignant diseases, but almost all drugs currently under development rely on only one of the nine human antibody classes, namely IgG1, the most abundant antibody class in the blood (Weiner LM, Surana R, Wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10: 317-327). The human immune system naturally employs nine antibody classes and subclasses (IgM, IgD, IgG1-4, IgA1, IgA2, and IgE) to perform immune surveillance and mediate the destruction of pathogens in different anatomical compartments. However, only IgG (mostly IgG1) is used in cancer immunotherapy.

[0003] One reason for this is likely that IgG antibodies (especially IgG1) constitute the largest portion of circulating antibodies in human blood. The selection of antibody classes is also based on pioneering research from the late 1980s that compared a panel of chimeric antibodies of the same specificity, each possessing an Fc region belonging to one of nine antibody classes and subclasses (Bruggemann M, Williams GT, Bindon CI, Clark MR, Walker MR, Jefferis R, Waldmann H, Neuberger MS (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J Exp Med 166: 1351-1361). Antibodies were evaluated for their ability to bind to complement and their efficacy in mediating hemolysis and cytotoxicity of antigen-expressing target cells in the presence of complement. In combination with human peripheral blood mononuclear cells (PBMCs), IgG1 was the most effective IgG subclass in in vitro complement-dependent cell death, while IgA and IgE antibodies were completely inactive.

[0004] Subsequent clinical trials using antibodies that recognize the B-cell marker CD20 supported the inference that IgG1 would be the optimal subclass for immunotherapy in patients with B-cell malignancies, such as non-Hodgkin lymphoma (Alduaij W, Illidge TM (2011) The future of anti-CD20 monoclonal antibodies: are we making progress? Blood 117: 2993-3001). Since these studies, comparisons of antitumor effects by different antibody classes have been limited to IgG and IgM in both mouse models and patients with lymphoid malignancies, although IgA has been shown to mediate ADCC in vitro and in vivo in mouse models of lymphoma (Dechant M, Valerius T (2001) IgA antibodies for cancer therapy. Crit Rev Oncol Hematol 39: 69-77).

[0005] The HER receptor tyrosine kinase family are important mediators of cell proliferation, differentiation, and survival. This receptor family includes the epidermal growth factor receptor (EGFR, ErbB1, or HER1) and HER2 (ErbB2 or pl85). neu It includes four distinct members, including HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

[0006] EGFR, encoded by the erbB1 gene, is involved as a causative agent in human malignancies. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck, and gastric cancers, as well as glioblastoma. Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor receptor alpha (TGF-α), by the same tumor cells, leading to receptor activation via the autocrine stimulation pathway (Baselga and Mendelsohn, Pharmac. Ther. 64:127-154 (1994)). Monoclonal antibodies against EGFR or its ligands, TGF-α and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies (see, for example, Baselga and Mendelsohn, above; Masui et al. Cancer Research 44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905 (1995)).

[0007] The second member of the HER family, HER2, was initially identified as a transformant product from chemically treated rat neuroblastoma. The activated form of the neu proto-oncogene is due to a point mutation (valine to glutamate) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu has been observed in breast and ovarian cancers and is associated with poor prognosis (Slamon et al., Science, 235: 177-182 (1987); Slamon et al., Science, 244: 707-712 (1989); and U.S. Patent No. 4,968,603). HER2 overexpression (often due to gene amplification, but not always) has also been observed in other cancers, including those of the stomach, endometrium, salivary glands, lung, kidney, colon, thyroid, pancreas, and bladder (see, for example, Ross et al Cancer 79:2162-70 (1997); and Sadasivan et al J. Urol. 150:126-31 (1993)).

[0008] IgG antibodies against human HER2 protein products are described. See, for example, Drebin et al, Cell 41:695-706 (1985) and U.S. Patent No. 5,824,311. Hudziak et al, Mol. Cell. Biol. 9(3): 1165-1172 (1989) describes the construction of a panel of HER2 antibodies characterized using the human mammary tumor cell line SKBR-3. It has been further found that antibody 4D5 sensitizes HER2-overexpressing mammary tumor cells to the cytotoxic effects of TNF-α (see U.S. Patent No. 5,677,171). Human recombinant mouse IgG anti-HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab, or HERCEPTIN®) has been shown to be effective in patients with HER2-overexpressing metastatic breast cancer who have received extensive prior anticancer therapy (Baselga et al, J. Clin. Oncol. 14:737-744 (1996)). Trastuzumab received marketing authorization from the U.S. Food and Drug Administration in 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein. Other anti-HER2 IgG antibodies with various properties are described, for example, Tagliabue et al. Int. J. Cancer 47:933-937 (1991); International Publication No. 94 / 00136; U.S. Patent No. 5,783,186; and Klapper et al. Oncogene It is described in 14:2099-2109 (1997).

[0009] Homology screening has identified other HER2 receptor family members, including HER3 (see U.S. Patent Nos. 5,183,884 and 5,480,968) and HER4 (Plowman et al., Nature, 366:473-475 (1993)). All of these receptors show increased expression in at least some breast cancer cell lines. HER receptors are generally found in various combinations within cells, and heterodimerization is thought to increase the diversity of cellular responses to various HER ligands. For example, EGF stimulates EGFR and HER2 to form heterodimers, which activate EGFR, leading to transphosphorylation of HER2 in the heterodimer. Dimerization and / or transphosphorylation appear to activate HER2 tyrosine kinase. See Earp et al., above. Similarly, when HER3 is co-expressed with HER2, an active signaling complex is formed, and antibodies against HER2 can disrupt this complex (Sliwkowski et al, J. Biol. Chem., 269(20): 14661-14665 (1994)).

[0010] To target the HER signaling pathway, rhuMAb 2C4 (pertuzumab, PERJETA®) was developed as a humanized IgG antibody that inhibits the dimerization of HER2 with other HER receptors, thereby inhibiting ligand-driven phosphorylation and activation, as well as downstream activation of the RAS and AKT pathways. Pertuzumab binds to subdomain II (dimerization region) of the extracellular portion of the HER2 receptor, while trastuzumab binds to subdomain IV (near-membrane region). Pertuzumab received FDA approval in 2012 for the treatment of HER2-overexpressing metastatic breast cancer. Pertuzumab can also be administered in combination with trastuzumab and docetaxel for the same indication.

[0011] IgE class antibodies play a central role in allergic reactions and possess many properties that may be useful for cancer therapy. IgE-based active and passive immunotherapy approaches have been shown to be effective in both in vitro and in vivo cancer models, suggesting the potential use of these approaches in humans (Leoh et al., Curr Top Microbiol Immunol. 2015; 388: 109-149). Therefore, IgE therapeutic antibodies may provide enhanced immune surveillance against cancer cells and superior effector cell efficacy.

[0012] Karagiannis et al. 2009 (Cancer Immunol Immunother. 2009 June; 58(6): 915-930) described an engineered IgE that contains the same light and heavy chain variable regions as trastuzumab IgG, but has epsilon (i.e., the IgE heavy chain constant region) instead of the IgG gamma-1 heavy chain constant region of trastuzumab. Trastuzumab IgE was shown to induce antibody-dependent cell-mediated cytotoxicity (ADCC), induce mast cell degranulation in the presence of HER2-expressing tumor cells, and mediate tumor cell proliferation arrest at a level comparable to that of trastuzumab IgG.

[0013] Fully human anti-HER2 IgE has also been developed using the variable region of single-stranded Fv C6MH3-B1 (Daniels TR et al (2012) Targeting HER2 / neu with a fully human IgE to harness the allergic reaction against cancer cells. Cancer Immunol Immunother. 61: 991-1003). C6MH3-B1 induced in vitro degranulation of RBL SX-38 cells expressing human FcεRI in the presence of mouse mammary cancer cells expressing human HER2 / neu (D2F2 / E2), but not in parental D2F2 cells lacking HER2 / neu expression, or HER2 / neu (ECD HER2 The response was not induced in the presence of the shedding (soluble) extracellular domain of HER2 IgE. These results suggest that anti-HER2 IgE can induce an acute inflammatory response (type I hypersensitivity) in the tumor microenvironment where the HER2 / neu antigen is overexpressed at high levels on the surface of cancer cells (Pegram M, Ngo D. (2006) Application and potential limitations of animal models utilized in the development of trastuzumab (Herceptin): a case study. Adv Drug Deliv Rev. 58: 723-734.), facilitate FcεRI crosslinking, and induce degranulation of effector cells.

[0014] Currently, known IgG anti-HER2 antibodies used clinically (e.g., trastuzumab and pertuzumab) are only effective in treating cancers with high HER2 expression. For this reason, one of the challenges in developing anti-HER2 antibodies was identifying and selecting patient populations that might respond to the drug. Therefore, there was a need to develop accurate and reliable diagnostic assays to detect HER2 protein overexpression in tumors.

[0015] In the clinical development of trastuzumab, a clinical trial assay (CTA) developed by Genentech was initially used to select HER2-positive patients. However, during the Phase III clinical trial of trastuzumab, a newly optimized IHC assay, HercepTest®, was designed and developed by Dako. In September 1998, the U.S. Food and Drug Administration (FDA) granted simultaneous approval for trastuzumab and HercepTest®. Thus, HercepTest® became the first companion diagnostic approved by the FDA. The FDA also approved a specific immunohistochemistry (IHC) scoring technique for the assay, which requires strong, complete membrane staining (classified as HER2 IHC3+) in more than 10% of tumor cells to indicate trastuzumab treatment. This staining requirement has been used in all critical cancer clinical trials with trastuzumab, including metastatic and adjuvant indications, as well as in subsequent clinical trials involving other HER2 inhibitors, such as pertuzumab, and antibody-drug conjugates, such as ado-trastuzumab emtansine (see Jorgensen et al., (2021) Front. Oncol. 11:676939; Nicolo et al. Ther Adv Med Oncol 2023, Vol. 15: 1-16). [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] U.S. Patent No. 4,968,603 [Patent Document 2] U.S. Patent No. 5,824,311 [Patent Document 3] U.S. Patent No. 5,677,171 [Patent Document 4] International Publication No. 94 / 00136 Pamphlet [Patent Document 5] U.S. Patent No. 5,783,186 [License 6] U.S. Patent No. 5,183,884 [License 7] U.S. Patent No. 5,480,968 [Non-licensed literature]

[0017] [Non-licensed Document 1] Weiner LM, Surana R, Wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10: 317-327 [Non-licensed Document 2] Bruggemann M, Williams GT, Bindon CI, Clark MR, Walker MR, Jefferis R, Waldmann H, Neuberger MS (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J Exp Med 166: 1351-1361 [Non-licensed Document 3] Alduaij W, Illidge TM (2011) The future of anti-CD20 monoclonal antibodies: are we making progress? Blood 117: 2993-3001 [Non-licensed Document 4] Dechant M, Valerius T (2001) IgA antibodies for cancer therapy. Crit Rev Oncol Hematol 39: 69-77 [Non-licensed Document 5] Baselga and Mendelsohn, Pharmac. Ther. 64:127-154 (1994) [Non-licensed Document 6] Masui et al. Cancer Research 44:1002-1007 (1984)

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[0018] However, many cancer patients (including more than half of breast cancer patients) have tumors that express low levels of HER2, for example, with an immunohistochemical assay classification of IHC2+ or lower. Known anti-HER2-based therapies are not applicable to or effective in this subgroup. Therefore, there remains a clear and unmet need for novel therapies that can target cancers, including breast cancer, with low HER2 expression. [Means for solving the problem]

[0019] Accordingly, in one embodiment, the present invention provides an anti-HER2 immunoglobulin E (IgE) antibody for use in the treatment of low-HER2-expressing tumors in subjects.

[0020] In one embodiment, less than 10% of tumor cells in a sample from the subject show strong complete membrane staining for HER2 when immunohistochemical detection of HER2 is used. Preferably, there are no tumor cell clusters (five or more cells) that show strong complete membrane staining for HER2.

[0021] In one embodiment, immunohistochemical detection of HER2 reveals that at least 10% of tumor cells or tumor cell clusters (5 or more cells) in a sample from a subject exhibit weak to moderate complete membrane staining for HER2. In another embodiment, at least 10% of tumor cells or tumor cell clusters (5 or more cells) in a sample from a subject exhibit faint or barely recognizable partial membrane staining for HER2.

[0022] In one embodiment, immunohistochemical detection of HER2 in a sample from a subject is performed using Dako's anti-HER2 immunohistochemistry system (HercepTest®). In some embodiments, tumor samples from a subject have a HER2 immunohistochemical staining (HER2 IHC) score of 2+ or less (e.g., 2+, 1+, or 0), more preferably 2+ or 1+.

[0023] In some embodiments, amplification of the HER2 coding gene (erbB2) is analyzed in tumor samples from subjects, for example, using fluorescence in situ hybridization (FISH), and the erbB2 copy number is optionally reported in comparison to the centromere 17 (CEN17) copy number. In some embodiments, the tumor has a HER2 / CEN17 ratio < 2.0 or does not show detectable erbB2 gene amplification when determined by FISH.

[0024] In some embodiments, tumor HER2 expression in a subject is lower than tumor HER2 expression in at least 50% of subjects with cancer. Preferably, membrane HER2 expression in the tumor cells of a subject is lower than membrane HER2 expression in at least 60%, at least 70%, or at least 80% of subjects with the same form of cancer; more preferably, tumor HER2 expression in a subject is lower than tumor HER2 expression in at least 50%, at least 70%, or at least 90% of HER2-expressing tumors (preferably HER2-expressing breast tumors).

[0025] In some embodiments, the tumor expresses HER2. Preferably, at least 1%, 5%, 10%, 15%, or 20% of the tumor cells in the subject show detectable membrane HER2 expression using immunohistochemical detection of HER2.

[0026] In one embodiment, the antibody binds to subdomain II of the extracellular domain of HER2. Preferably, the antibody at least partially competes with pertuzumab IgG for binding to HER2.

[0027] In another embodiment, the antibody binds to subdomain IV of the extracellular domain of HER2. Preferably, the antibody at least partially competes with trastuzumab IgG for binding to HER2.

[0028] In further embodiments, the antibody may comprise an amino acid sequence defined in any one of SEQ ID NOs: 1 to 10. For example, the antibody may comprise trastuzumab IgE. In further embodiments, the antibody may comprise an amino acid sequence defined in any one of SEQ ID NOs: 11 to 40. For example, the antibody may comprise one to six CDR sequences selected from the following groups: (i) SEQ ID NOs: 3, 4, 5, 8, 9, and 10; (ii) SEQ ID NOs: 13, 14, 15, 18, 19, and 20; (iii) SEQ ID NOs: 23, 24, 25, 28, 29, and 30; or (iv) SEQ ID NOs: 33, 34, 35, 38, 39, and 40.

[0029] Therefore, the antibody may include (i) a heavy chain variable domain sequence defined as any one of sequence numbers 2, 12, 22, or 32; (ii) a light chain variable domain sequence defined as any one of sequence numbers 7, 17, 27, or 37; (iii) a heavy chain sequence defined as any one of sequence numbers 1, 11, 21, or 31; and / or (iv) a light chain sequence defined as any one of sequence numbers 6, 16, 26, or 36.

[0030] In some embodiments, antibodies may be used to treat cancer in a subject and / or to slow the progression of cancer. Preferably, the tumor or cancer is a breast tumor or breast cancer, or a gastric tumor or gastric cancer. Most preferably, the tumor or cancer is a breast tumor or breast cancer.

[0031] In some embodiments, the antibody lacks a cytotoxic moiety. Preferably, the antibody is not an antibody-drug conjugate (ADC).

[0032] In a further embodiment, the present invention provides a method for treating cancer and / or delaying cancer progression in a subject having a low-HER2-expressing tumor, comprising the step of administering an anti-HER2 immunoglobulin E (IgE) antibody as defined herein to the subject in a therapeutically effective dose.

[0033] In another aspect, the present invention provides a pharmaceutical composition for use in the treatment of low-HER2-expressing tumors in a subject, comprising an anti-HER2 immunoglobulin E (IgE) antibody as defined herein and one or more pharmaceutically acceptable excipients, carriers, or diluents.

[0034] In a further embodiment, the present invention provides an immunoglobulin or functional fragment thereof comprising (i) SEQ ID NOs: 13, 14, 15, 18, 19, and 20; (ii) SEQ ID NOs: 23, 24, 25, 28, 29, and 30; or (iii) SEQ ID NOs: 33, 34, 35, 38, 39, and 40, comprising one to six CDR sequences selected from SEQ ID NOs: 33, 34, 35, 38, 39, and 40.

[0035] In one embodiment, the immunoglobulin comprises (i) a heavy chain variable domain sequence defined as any one of sequence numbers 12, 22, or 32; (ii) a light chain variable domain sequence defined as any one of sequence numbers 17, 27, or 37; (iii) a heavy chain sequence defined as any one of sequence numbers 11, 21, or 31; and / or (iv) a light chain sequence defined as any one of sequence numbers 6, 16, 26, or 36.

[0036] Preferably, the immunoglobulin is an isotype IgE immunoglobulin, for example, the antibody contains one or more Cε1, Cε2, Cε3 and / or Cε4 domains, or variants or functional fragments thereof. Preferably, the immunoglobulin contains at least Cε2, Cε3 and / or Cε4 domains.

[0037] In some embodiments, the immunoglobulin is a chimeric or humanized antibody. For example, the immunoglobulin may include (i) one or more human framework regions; and / or (ii) one or more human IgE heavy chain and / or light chain constant domains. Preferably, the immunoglobulin includes human Cε1, Cε2, Cε3 and / or Cε4 domains. [Brief explanation of the drawing]

[0038] [Figure 1]This figure shows the HER2 expression levels on the surface of JIMT1 and SKBR3 cell lines, as analyzed using flow cytometry. [Figure 2] This figure shows the antitumor efficacy of trastuzumab-IgE, a HER2-targeted IgE, against JIMT1 tumors in a humanized mouse model of PBMC. [Figure 3] This figure shows the HER2 expression levels on the surface of MTLn3 cells, as analyzed using flow cytometry. [Figure 4] This figure shows the antitumor efficacy of anti-HER2 IgE antibodies V20, V23, and V26 against MTLn3 tumors in a syngeneic Fischer 344 rat model. [Figure 5] This figure shows immune cell infiltration into MTLn3 tumors in rats treated with either control (PBS) or V26 IgE antibody. [Figure 6] This figure shows immune cell (monocytes and neutrophils) infiltration and the appearance of apoptotic cells in MTLn3 tumors in rats treated with either control (PBS) or V26 IgE antibody. [Figure 7] This figure shows the increased MTLn3 tumor cell death in rats treated with either control (PBS) or V26 IgE antibody. [Figure 8] This figure shows that anti-HER2 IgE (V26, referred to as EPS226 in Figure 8) has antitumor activity in an in vivo mouse model of triple-negative breast cancer. [Modes for carrying out the invention]

[0039] Surprisingly, anti-HER2 IgE antibodies were found to significantly inhibit the growth of low-HER2-expressing tumors in vivo. In particular, in syngeneic rat and immunodeficient humanized mouse models, anti-HER2 IgE antibodies showed high efficacy against tumor cells corresponding to HER2 IHC 1+ and HercepTest score 2+. Therefore, anti-HER2 IgE may be used for the treatment of cancer or to delay cancer progression in subjects with low HER2 membrane expression scores.

[0040] This finding is particularly surprising because anti-HER2 IgG antibodies (e.g., trastuzumab and pertuzumab) are typically applied only to the treatment of high-HER2-expressing tumors (e.g., those classified as HER2 IHC3+ using HercepTest®). Therefore, anti-HER2 therapeutic approaches using IgG antibodies focus on treating high-HER2-expressing individuals and / or combinations of antibodies with cytotoxic moieties, such as antibody-drug conjugates (ADCs).

[0041] For example, ado-trastuzumab emtansine (Kadcyla®, Roche / Genentech) is an antibody-drug conjugate consisting of trastuzumab IgG covalently bound to the cytotoxic agent DM1. However, this ADC still showed poor activity against breast cancer with low HER2 expression. More novel anti-HER2 IgG ADCs are being developed. These include trastuzumab deruxtecan (Enhertu®, trastuzumab IgG conjugated to deruxtecan, a topoisomerase I inhibitor, via a tetrapeptide-based cleavable linker), trastuzumab duocalmycin (trastuzumab IgG conjugated to a duocalmycin payload, a DNA alkylating agent, via a cleavable linker), disitamab vedotin (hertuzumab, an anti-HER2 IgG humanized antibody, conjugated to a monomethyl auristatin E (MMAE) payload, a microtubule inhibitor, via a cleavable linker), and MRG002 (modified trastuzumab IgG conjugated to an MMAE payload via a cleavable linker). Therefore, it has been suggested that methods for targeting low-HER2-expressing tumors involve using anti-HER2 IgG ADCs that have a more potent cytotoxic payload or a higher drug-to-antibody ratio (DAR) than ado-trastuzumab emtansine, or that have the ability to induce a so-called bystander effect (see Nicolo et al. Ther Adv Med Oncol 2023, Vol. 15: 1-16).

[0042] On the other hand, many ADCs exhibit excessive toxicity and unfavorable risk-benefit profiles. A substantial proportion of treated patients may require dose reduction, treatment delay, or discontinuation due to unacceptable ADC-related toxicity. This often limits ADC dosages to levels below those necessary for optimal anticancer efficacy (see, for example, Nguyen et al., Cancers (Basel). 2023 Feb; 15(3): 713).

[0043] In contrast, as shown herein, anti-HER2 IgE antibodies can inhibit the growth of low-HER2-expressing tumors in vivo as monotherapy, i.e., without being incorporated into an ADC or in combination with further chemotherapeutic agents. This can circumvent the drawbacks of ADCs and may result in reduced toxicity, improved tolerability, and an improved safety profile. Therefore, the present invention makes a significant contribution to the art in addressing unmet medical needs, particularly in subgroups of cancer patients who are low-HER2-expressing.

[0044] therapeutic antibodies Antibodies are polypeptide ligands that contain at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen, such as an epitope or fragment thereof of HER2. Antibodies are typically composed of a heavy chain and a light chain, which each has variable regions called the heavy chain variable (VH) region and the light chain variable (VL) region, respectively. Collectively, the VH and VL regions are responsible for binding to the antigen recognized by the antibody.

[0045] Antibodies include intact immunoglobulins known in the art, as well as variants and portions of antibodies, provided that such fragments retain at least one function of IgE, e.g., capable of binding to an Fcε receptor. Antibodies also include genetically modified forms, e.g., chimeric humanized antibodies (e.g., humanized antibodies containing mouse sequences in their variable regions) or human antibodies, heteroconjugate antibodies (e.g., bispecific antibodies), e.g., those described in Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0046] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). Nine major isotypes or classes, corresponding to heavy chain types α, δ, ε, γ, and μ, are IgA1-2, IgD, IgE, IgG1-4, and IgM, which determine the functional activity of antibody molecules. In other words, the heavy chain type present defines the class of antibody. The distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. Differences in the constant region of each heavy chain type are attributed to differences in the effector function of each antibody isotype, thanks to their selective binding to specific types of receptors (e.g., Fc receptors). Therefore, in embodiments of the present invention, the antibody preferably comprises an epsilon (ε) heavy chain; that is, the antibody is of isotype IgE that binds to the Fcε receptor.

[0047] The heavy and light chains each contain a constant region and a variable region (these regions are also known as "domains"). Together, the heavy and light chain variable regions bind specifically to the antigen. The light and heavy chain variable regions contain a "framework" region interspersed with three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs is defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The Kabat database is currently maintained online. The sequences of different light or heavy chain framework regions are relatively conserved within a species, e.g., humans. The antibody framework region, which is the combined framework region of the constituent light and heavy chains, plays a role in arranging and aligning the CDRs in three-dimensional space.

[0048] CDRs (Cellular Derived Layers) are primarily responsible for binding to the antigen's epitope. The CDRs on each chain are typically numbered sequentially from the N-terminus and referred to as CDR1, CDR2, and CDR3, and are typically identified by the chain on which they are located. Therefore, VH CDR3 is found in the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is a CDR1 from the variable domain of the light chain of the antibody in which it is found.

[0049] Antibodies may have specific VH and VL region sequences, and therefore specific CDR sequences. Antibodies with different specificities (i.e., different combination sites for different antigens) have different CDRs. While the CDR varies from antibody to antibody, the positions of a limited number of amino acids within the CDR are directly involved in antigen binding. These positions within the CDR are called specificity-determining residues (SDRs). "VH" refers to the variable region of the immunoglobulin heavy chain. "VL" refers to the variable region of the immunoglobulin light chain.

[0050] A "monoclonal antibody" is an antibody produced by a single clone of a B lymphocyte or by cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immunosplenic cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0051] A "chimeric antibody" contains sequences derived from two different antibodies, which typically originate from different species. For example, a chimeric antibody may contain human and mouse antibody domains, such as a human constant region and a mouse variable region (e.g., from a mouse antibody that specifically binds to a target antigen).

[0052] Chimeric antibodies are typically constructed by fusing variable and constant regions from light-chain and heavy-chain immunoglobulin genes belonging to different species, for example, through genetic engineering. For instance, a variable segment of a gene from a mouse monoclonal antibody can be linked to a human constant segment, such as kappa and epsilon. In one example, a therapeutic chimeric antibody is a hybrid protein composed of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, such as the Fc (effector) domain from a human IgE antibody. However, other mammalian species can be used, or the variable region can be generated by molecular technology. Methods for producing chimeric antibodies are publicly known in this art; see, for example, U.S. Patent No. 5,807,715.

[0053] A “humanized” antibody is an antibody comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody. The non-human immunoglobulin providing the CDRs is called the “donor,” and the human immunoglobulin providing the framework is called the “acceptor.” In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region is typically substantially identical to the constant region of human immunoglobulin, i.e., at least about 85–90%, for example, about 95% or more identical. Thus, all parts of the humanized immunoglobulin other than the CDRs are substantially identical to the corresponding parts of the natural human immunoglobulin sequence.

[0054] Humanized antibodies typically contain a humanized immunoglobulin light chain and a humanized immunoglobulin heavy chain. Humanized antibodies typically bind to the same antigens as the donor antibody that provides the CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions from the donor framework. Humanized or other monoclonal antibodies may have additional conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions.

[0055] Humanized immunoglobulins can be constructed through genetic engineering (see, for example, U.S. Patent No. 5,585,089). Typically, humanized monoclonal antibodies are produced by transferring the donor antibody complementarity-determining regions from the variable heavy and light chains of mouse immunoglobulin to human variable domains, and then substituting human residues in the framework region of the donor counterpart. The use of antibody components derived from humanized monoclonal antibodies prevents potential problems associated with the immunogenicity of the constant region of the donor antibody. Techniques for generating humanized monoclonal antibodies are described, for example, in Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.

[0056] A "human" antibody (also called a "fully human" antibody) is an antibody that contains all of the human framework region and CDRs from human immunoglobulins. In one example, the framework and CDRs are from the same human heavy and / or light chain amino acid sequence. However, the framework from one human antibody can be manipulated to contain CDRs from different human antibodies.

[0057] In embodiments of the present invention, the antibody may be a monoclonal or polyclonal antibody, including a chimeric, humanized, or fully human antibody.

[0058] Anti-HER2 antibody In some embodiments, the antibody or immunoglobulin specifically binds to HER2 to form an immune complex. Typically, the antibody or immunoglobulin may include an antigen-binding region (e.g., one or more variable regions, or one to six CDRs) derived from an antibody known to bind to HER2, preferably human HER2.

[0059] "HER2" refers to the human epidermal growth factor receptor 2. HER2 may also be called the receptor tyrosine-protein kinase erbB-2 or CD340 (differentiation antigen group 340). In humans, HER2 is encoded by the erbB2 (erythroblastic leukemia tumor gene B2) or neu gene. The amino acid and nucleotide sequences encoding human HER2 / erbB2 are listed in public databases and are available, for example, through database deposit number P04626-1 (UniProt), NM_001005862.3 and NP_001005862.1 (NCBI Ref Seq).

[0060] Anti-HER2 IgE antibodies, including humanized trastuzumab IgE (see Table 1 and SEQ ID NOs: 1-10) and complete human anti-HER2 IgE, are known and described, for example, Karagianis et al. (2009), Cancer Immunol Immunother. 58(6): 915-930) and Daniels TR et al. (2012), Cancer Immunol Immunother. 61: 991-1003. Further anti-HER2 IgE antibodies are described herein (see Table 1 and SEQ ID NOs: 11-40).

[0061] In a particular embodiment, the antibody comprises a variable region (e.g., a heavy chain variable domain (VH) and / or a light chain variable domain (VL)) or at least one, two, three, four, five, or six CDRs (e.g., three heavy chain CDRs or three light chain CDRs) derived from any known anti-HER2 IgG or IgE antibody. The CDR sequences may be defined according to the methods of Kabat, Chothia, or IMGT (see, for example, Dondelinger, Front Immunol. 2018; 9: 2278 and the references cited herein as incorporated herein by reference). For example, CDR follows Kabat (see Kabat EA, et al. (US) NI of H. Sequences of Immunoglobulin Chains: Tabulation Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain BP-Microglobulins, 1979), or Chothia (see Chothia C, et al, Canonical structures for the hypervariable regions of immunoglobulins, J Mol Biol. 1987 Aug 20; 196(4):901-1), or IMGT (see Giudicelli V et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res. 1997 Jan 1; 25(1):206-11 or Lefranc MP, Unique database numbering system for immunogenetic analysis, Immunol Today. 1997 Nov; (See 18(11):509) It may be prescribed.

[0062] In one embodiment, the antibody comprises at least 1, 2, 3, 4, 5, or 6 CDRs (e.g., 3 heavy-chain CDRs or 3 light-chain CDRs) derived from trastuzumab or pertuzumab. In another embodiment, the antibody or immunoglobulin comprises at least 1, 2, 3, 4, 5, or 6 CDRs (e.g., 3 heavy-chain CDRs or 3 light-chain CDRs) derived from trastuzumab IgE, V20 IgE, V23 IgE, or V26 IgE as described herein, preferably V20 IgE, V23 IgE, or V26 IgE, more preferably V23 IgE or V26 IgE, and most preferably V26 IgE. For example, an antibody or immunoglobulin may comprise at least one, two, three, four, five, or six CDRs (e.g., three heavy chain CDRs or three light chain CDRs) selected from one of the following groups: (i) SEQ ID NOs: 3, 4, 5, 8, 9, and 10; (ii) SEQ ID NOs: 13, 14, 15, 18, 19, and 20; (iii) SEQ ID NOs: 23, 24, 25, 28, 29, and 30; or (iv) SEQ ID NOs: 33, 34, 35, 38, 39, and 40. In this embodiment, a CDR may be defined according to the method of Chothia (above).

[0063] In another embodiment, the antibody or immunoglobulin comprises at least one, two, three, four, five, or six CDRs (e.g., three heavy chain CDRs or three light chain CDRs) present in any one of SEQ ID NOs: 1, 2, 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31, 32, 36, or 37, where, for example, CDRs are defined according to Chothia's method. In another embodiment, the antibody or immunoglobulin comprises (i) a heavy chain variable domain sequence defined in any one of SEQ ID NOs: 2, 12, 22, or 32; (ii) a light chain variable domain sequence defined in any one of SEQ ID NOs: 7, 17, 27, or 37; (iii) a heavy chain sequence defined in any one of SEQ ID NOs: 1, 11, 21, or 31; and / or (iv) a light chain sequence defined in any one of SEQ ID NOs: 6, 16, 26, or 36.

[0064] For example, antibodies or immunoglobulins are (a) at least one, two, three, four, five, or six CDRs selected from sequence numbers 3, 4, 5, 8, 9, and 10; heavy chain and / or light chain variable domain sequences as defined in sequence number 2 and / or 7; and / or heavy chain and / or light chain sequences as defined in sequence number 1 or 6; (b) at least one, two, three, four, five, or six CDRs selected from sequence numbers 13, 14, 15, 18, 19, and 20; heavy chain and / or light chain variable domain sequences as defined in sequence number 12 and / or 17; and / or heavy chain and / or light chain sequences as defined in sequence number 11 or 16: (c) at least one, two, three, four, five, or six CDRs selected from SEQ ID NOs: 23, 24, 25, 28, 29, and 30; heavy chain and / or light chain variable domain sequences as defined in SEQ ID NOs: 22 and / or 27; and / or heavy chain and / or light chain sequences as defined in SEQ ID NOs: 21 or 26; (d) at least one, two, three, four, five, or six CDRs selected from sequence numbers 33, 34, 35, 38, 39, and 40; heavy chain and / or light chain variable domain sequences defined in sequence number 32 and / or 37; and / or heavy chain and / or light chain sequences defined in sequence number 31 or 36 It may include.

[0065] In another embodiment, the antibody or immunoglobulin is a chimeric, humanized, or fully human antibody that specifically binds to the epitope to which the anti-HER2 antibody binds, such as trastuzumab, pertuzumab, V20 IgE, V23 IgE, or V26 IgE (as defined in Table 1). For example, the antibody or immunoglobulin may compete (at least partially) with trastuzumab or pertuzumab for binding to human HER2. The (IgE) antibody or immunoglobulin may bind to the same epitope as a known anti-HER2 antibody, or to an epitope that (for example, at least partially) overlaps with the epitope of a known anti-HER2 antibody, or to an epitope proximal to the epitope of a known anti-HER2 antibody (e.g., trastuzumab or pertuzumab). Therefore, in competitive assays, (IgE) antibodies or immunoglobulins may show partial or complete competition with anti-HER2 antibodies (e.g., trastuzumab or pertuzumab) for binding to HER2.

[0066] Assays for determining the competitive binding of an antibody to a target antigen, and methods for quantifying the binding of an antibody to HER2, are known in the art and are described, for example, in Shu et al., Nature Scientific Reports volume 10, Article number: 2986(2020); Fu et al., MAbs. 2014 Jul 1; 6(4): 978-990; and Rudkouskaya et al., Molecules. 2020 Dec; 25(24): 5976. The antibody or immunoglobulin may bind to, for example, subdomain II or subdomain IV of the extracellular domain of human HER2. In some embodiments, the antibody or immunoglobulin cross-reacts with both human and rodent HER2, for example, rat and / or mouse HER2.

[0067] In this specification, it has been shown that in syngeneic rat tumor models, anti-HER2 IgE antibodies exhibited varying degrees of growth inhibition against HER2 IHC 1+ tumor cells (see, for example, Figure 4). V26 IgE showed the highest antitumor effect, followed by V23 IgE and V20 IgE. V26 IgE binds to subdomain II of the extracellular domain of HER2 and competes with pertuzumab IgG for binding to HER2. V23 IgE binds to subdomain IV of the extracellular domain of HER2 and competes with trastuzumab IgG for binding to HER2. V20 IgE does not compete with pertuzumab or trastuzumab for binding to HER2.

[0068] Therefore, in a preferred embodiment, the antibody or immunoglobulin binds to subdomain II of the extracellular domain of HER2 and / or competes (at least partially) with pertuzumab IgG for binding to HER2, for example, the antibody or immunoglobulin comprises at least one, two, three, four, five, or six CDRs selected from SEQ ID NOs: 33, 34, 35, 38, 39, and 40; heavy chain and / or light chain variable domain sequences defined in SEQ ID NOs: 32 and / or 37; and / or heavy chain and / or light chain sequences defined in SEQ ID NOs: 31 or 36. Most preferably, the antibody or immunoglobulin comprises a chimeric, humanized, or whole human antibody that binds to subdomain II of the extracellular domain of HER2 and / or competes (at least partially) with pertuzumab IgG for binding to HER2. For example, the antibody or immunoglobulin may be a humanized antibody comprising at least one, two, three, four, five, or six CDRs selected from SEQ ID NOs: 33, 34, 35, 38, 39, and 40, as well as one or more human framework regions, and / or one or more human IgE heavy chain and / or light chain constant domains. Alternatively, the antibody or immunoglobulin may be a chimeric antibody comprising heavy chain and / or light chain variable domain sequences defined in SEQ ID NOs: 32 and / or 37, and one or more human IgE heavy chain and / or light chain constant domains.

[0069] In alternative embodiments, the antibody or immunoglobulin binds to subdomain IV of the extracellular domain of HER2 and / or competes (at least partially) with trastuzumab IgG for binding to HER2, for example, the antibody or immunoglobulin comprises at least one, two, three, four, five, or six CDRs selected from SEQ ID NOs: 23, 24, 25, 28, 29, and 20; heavy chain and / or light chain variable domain sequences as defined in SEQ ID NOs: 22 and / or 27; and / or heavy chain and / or light chain sequences as defined in SEQ ID NOs: 21 or 26. More preferably, the antibody or immunoglobulin comprises a chimeric, humanized, or whole human antibody that binds to subdomain IV of the extracellular domain of HER2 and / or competes (at least partially) with trastuzumab IgG for binding to HER2. For example, the antibody or immunoglobulin may be a humanized antibody comprising at least one, two, three, four, five, or six CDRs selected from SEQ ID NOs. 23, 24, 25, 28, 29, and 30, as well as one or more human framework regions, and / or one or more human IgE heavy chain and / or light chain constant domains. Alternatively, the antibody or immunoglobulin may be a chimeric antibody comprising heavy chain and / or light chain variable domain sequences defined in SEQ ID NOs. 22 and / or 27, and one or more human IgE heavy chain and / or light chain constant domains.

[0070] In one embodiment, the antibody includes one or more human constant regions, for example, one or more human heavy chain constant domains (e.g., ε-constant domains) and / or human light chain (e.g., κ or λ) constant domains. The amino acid sequence of the human heavy chain constant domain is shown in SEQ ID NO: 41 (the text that is not bold and underlined, which is present in SEQ ID NO: 1). The amino acid sequence of the human light (κ) chain constant domain is shown in SEQ ID NO: 42 (the text that is not bold and underlined, which is present in SEQ ID NO: 6). In one embodiment, the antibody includes one or more human framework regions within the VH and / or VL domains.

[0071] In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 75%, at least about 85%, at least about 99%, or at least about 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Mutations that can be created in the human framework region and the humanized antibody framework region are known in the art (see, for example, U.S. Patent No. 5,585,089).

[0072] Further antibodies against specific antigens, such as HER2, can be produced by established methods, and at least the variable region or CDR from such antibodies can be used in the antibodies of the present invention (for example, the produced antibody can be used to provide a CDR or variable region sequence to the IgE acceptor sequence). Methods for synthesizing polypeptides and immunizing host animals are known in the art. Typically, a host animal (e.g., a mouse) is intraperitoneally inoculated with a certain amount of immunogen (e.g., a polypeptide containing HER2 or its immunogenic fragment) and (in the case of monoclonal antibody production) a hybridoma prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 25 6:495-497.

[0073] The sequence of human HER2 is publicly known (see, for example, UniProt database accession number P04626-1), and therefore human HER2 can be purified from natural sources, for example, or expressed using recombinant technology for use in such a manner. The amino acid and nucleic acid sequences of human HER2 are shown in SEQ ID NOs. 43 and 44 below, respectively: Sequence ID No. 43 - Human HER2 amino acid sequence: 1 MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ 61 VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK 121 GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE 181 DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA 241 LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR 301 CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA 361 PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI 421 SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA 481 CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ 541 NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC 601 THSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL 661 LQETELVEPL TPSGAMPNQA QMRILKETEL RKVKVLGSGA FGTVYKGIWI PDGENVKIPV 721 AIKVLRENTS PKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVQLVTQL MPYGCLLDHV 781 RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR LVHRDLAARN VLVKSPNHVK ITDFGLARLL 841 DIDETEYHAD GGKVPIKWMA LESILRRRFT HQSDVWSYGV TVWELMTFGA KPYDGIPARE 901 IPDLLEKGER LPQPPICTID VYMIMVKCWM IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ 961 NEDLGPASPL DSTFYRSLLE DDDMGDLVDA EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS 1021 STRSGGGDLT LGLEPSEEEA PRSPLAPSEG AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ 1081 RYSEDPTVPL PSETDGYVAP LTCSPQPEYV NQPDVRPQPP SPREGPLPAA RPAGATLERP 1141 KTLSPGKNGV VKDVFAFGGA VENPEYLTPQ GGAAPQPHPP PAFSPAFDNL YYWDQDPPER 1201 GAPPSTFKGT PTAENPEYLG LDVPV Sequence ID 44 - Human HER2 nucleic acid sequence: 1 gttctttatt ctactctccg ctgaagtcca cacagtttaa attaaagttc ccggattttt 61 gtgggcgcct gccccgcccc tcgtccccct gctgtgtcca tatatcgagg cgatagggtt 121 aagggaaggc ggacgcctga tgggttaatg agcaaactga agtgttttcc atgatctttt 181 ttgagtcgca attgaagtac cacctcccga gggtgattgc ttccccatgc ggggtagaac 241 ctttgctgtc ctgttcacca ctctacctcc agcacagaat ttggcttatg cctactcaat 301 gtgaagatga tgaggatgaa aacctttgtg atgatccact tccacttaat gaatggtggc 361 aaagcaaagc tatattcaag accacatgca aagctactcc ctgagcaaag agtcacagat 421 aaaacggggg caccagtaga atggccagga caaacgcagt gcagcacaga gactcagacc 481 ctggcagcca tgcctgcgca ggcagtgatg agagtgacat gtactgttgt ggacatgcac 541 aaaagtgagt gtgcaccggc acagacatga agctgcggct ccctgccagt cccgagaccc 601 acctggacat gctccgccac ctctaccagg gctgccaggt ggtgcaggga aacctggaac 661 tcacctacct gcccaccaat gccagcctgt ccttcctgca ggatatccag gaggtgcagg 721 gctacgtgct catcgctcac aaccaagtga ggcaggtccc actgcagagg ctgcggattg 781 tgcgaggcac ccagctcttt gaggacaact atgccctggc cgtgctagac aatggagacc 841 cgctgaacaa taccacccct gtcacagggg cctccccagg aggcctgcgg gagctgcagc 901 ttcgaagcct cacagagatc ttgaaaggag gggtcttgat ccagcggaac ccccagctct 961 gctaccagga cacgattttg tggaaggaca tcttccacaa gaacaaccag ctggctctca 1021 cactgataga caccaaccgc tctcgggcct gccacccctg ttctccgatg tgtaagggct 1081 cccgctgctg gggagagagt tctgaggatt gtcagagcct gacgcgcact gtctgtgccg 1141 gtggctgtgc ccgctgcaag gggccactgc ccactgactg ctgccatgag cagtgtgctg 1201 ccggctgcac gggcccccaag cactctgact gcctggcctg cctccacttc aaccacagtg 1261 gcatctgtga gctgcactgc ccagccctgg tcacctacaa cacagacacg tttgagtcca 1321 tgcccaatcc cgagggccgg tatacattcg gcgccagctg tgtgactgcc tgtccctaca 1381 actacctttc tacggacgtg ggatcctgca ccctcgtctg ccccctgcac aaccaagagg 1441 tgacagcaga ggatggaaca cagcggtgtg agaagtgcag caagccctgt gccccgagtgt 1501 gctatggtct gggcatggag cacttgcgag aggtgagggc agttaccagt gccaatatcc 1561 aggagtttgc tggctgcaag aagatctttg ggagcctggc atttctgccg gagagctttg 1621 atgggaccc agcctccaac actgccccgc tccagccaga gcagctccaa gtgtttgaga 1681 ctctggaaga gatcacaggt tacctataca tctcagcatg gccggacagc ctgcctgacc 1741 tcagcgtctt ccagaacctg caagtaatcc ggggacgaat tctgcacaat ggcgcctact 1801 cgctgaccct gcaagggctg ggcatcagct ggctggggct gcgctcactg agggaactgg 1861 gcagtggact ggccctcatc caccataaca cccacctctg cttcgtgcac acggtgccct 1921 gggaccagct ctttcggaac ccgcaccaag ctctgctcca cactgccaac cggccagagg 1981 acgagtgtgt gggcgagggc ctggcctgcc accagctgtg cgcccgaggg cactgctggg 2041 gtccagggcc cacccagtgt gtcaactgca gccagttcct tcggggccag gagtgcgtgg 2101 aggaatgccg agtactgcag gggctcccca gggagtatgt gaatgccagg cactgtttgc 2161 cgtgccaccc tgagtgtcag ccccagaatg gctcagtgac ctgttttgga ccggaggctg 2221 accagtgtgt ggcctgtgcc cactataagg accctccctt ctgcgtggcc cgctgcccca 2281 gcggtgtgaa acctgacctc tcctacatgc ccatctggaa gtttccagat gaggagggcg 2341 catgccagcc ttgccccatc aactgcaccc actcctgtgt ggacctggat gacaagggct 2401 gccccgccga gcagagagcc agccctctga cgtccatcat ctctgcggtg gttggcattc 2461 tgctggtcgt ggtcttgggg gtggtctttg ggatcctcat caagcgacgg cagcagaaga 2521 tccggaagta cacgatgcgg agactgctgc aggaaacgga gctggtggag ccgctgacac 2581 ctagcggagc gatgcccaac caggcgcaga tgcggatcct gaaagagacg gagctgagga 2641 aggtgaaggt gcttggatct ggcgcttttg gcacagtcta caagggcatc tggatccctg 2701 atggggagaa tgtgaaaatt ccagtggcca tcaaagtgtt gagggaaaac acatccccca 2761 aagccaacaa agaaatctta gacgaagcat acgtgatggc tggtgtgggc tccccatatg 2821 tctcccgcct tctgggcatc tgcctgacat ccacggtgca gctggtgaca cagcttatgc 2881 cctatggctg cctcttagac catgtccggg aaaaccgcgg acgcctgggc tcccaggacc 2941 tgctgaactg gtgtatgcag attgccaagg ggatgagcta cctggaggat gtgcggctcg 3001 tacacaggga cttggccgct cggaacgtgc tggtcaagag tcccaaccat gtcaaaatta 3061 cagacttcgg gctggctcgg ctgctggaca ttgacgagac agagtaccat gcagatgggg 3121 gcaaggtgcc catcaagtgg atggcgctgg agtccattct ccgccggcgg ttcacccacc 3181 agagtgatgt gtggagttat ggtgtgactg tgtgggagct gatgactttt ggggccaaac 3241 cttacgatgg gatcccagcc cgggagatcc ctgacctgct ggaaaagggg gagcggctgc 3301 cccagccccc catctgcacc attgatgtct acatgatcat ggtcaaatgt tggatgattg 3361 actctgaatg tcggccaaga ttccgggagt tggtgtctga attctcccgc atggccaggg 3421 acccccagcg ctttgtggtc atccagaatg aggacttggg cccagccagt cccttggaca 3481 gcaccttcta ccgctcactg ctggaggacg atgacatggg ggacctggtg gatgctgagg 3541 agtatctggt accccagcag ggcttcttct gtccagaccc tgccccgggc gctgggggca 3601 tggtccacca caggcaccgc agctcatcta ccaggagtgg cggtggggac ctgacactag 3661 ggctggagcc ctctgaagag gaggccccca ggtctccact ggcaccctcc gaaggggctg 3721 gctccgatgt atttgatggt gacctgggaa tgggggcagc caaggggctg caaagcctcc 3781 ccacacatga ccccagccct ctacagcggt acagtgagga ccccacagta cccctgccct 3841 ctgagactga tggctacgtt gcccccctga cctgcagccc ccagcctgaa tatgtgaacc 3901 agccagatgt tcggccccag cccccttcgc cccgagaggg ccctctgcct gctgcccgac 3961 ctgctggtgc cactctggaa aggcccaaga ctctctcccc agggaagaat ggggtcgtca 4021 aagacgtttt tgcctttggg ggtgccgtgg agaaccccga gtacttgaca ccccagggag 4081 gagctgcccc tcagccccac cctcctcctg ccttcagccc agccttcgac aacctctatt 4141 actgggacca ggacccacca gagcgggggg ctccacccag caccttcaaa gggacaccta 4201 cggcagagaa cccagagtac ctgggtctgg acgtgccagt gtgaaccaga aggccaagtc 4261 cgcagaagcc ctgatgtgtc ctcagggagc agggaaggcc tgacttctgc tggcatcaag 4321 aggtgggagg gccctccgac cacttccagg ggaacctgcc atgccaggaa cctgtcctaa 4381 ggaaccttcc ttcctgcttg agttcccaga tggctggaag gggtccagcc tcgttggaag 4441 aggaacagca ctggggagtc tttgtggatt ctgaggccct gcccaatgag actctagggt 4501 ccagtggatg ccacagccca gcttggccct ttccttccag atcctgggta ctgaaagcct 4561 tagggaagct ggcctgagag gggaagcggc cctaagggag tgtctaagaa caaaagcgac 4621 ccattcagag actgtccctg aaacctagta ctgccccccca tgaggaagga acagcaatgg 4681 tgtcagtatc caggctttgt acagagtgct tttctgttta gtttttactt tttttgtttt 4741 gtttttttaa agatgaaata aagacccagg gggagaatgg gtgttgtatg gggaggcaag 4801 tgtggggggt ccttctccac acccactttg tccatttgca aatatatttt ggaaaaca

[0074] Hybridomas that produce suitable antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture medium or body fluids, if desired, by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by passing the preparation through an adsorbent made of an immunogen bound to a solid phase, thereby eluting or releasing the desired antibody from the immunogen. If desired, the desired antibody (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or proliferation. The sequence encoding the antibody can be maintained in a vector within host cells, and the host cells can then be expanded and frozen for future use.

[0075] Phage display techniques, such as those described in U.S. Patent No. 5,565,332 and other published literature, can be used to in vitro select and generate human antibodies and antibody fragments from an immunoglobulin variable (V) domain gene repertoire from non-immunized donors (e.g., from human subjects including patients with the relevant disorders). For example, an existing antibody phage display library can be sorted in parallel against a large collection of synthetic polypeptides. According to this technique, antibody V domain genes are cloned in frame onto major or minor coat protein genes of filamentous bacteriophages, e.g., M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies exhibiting those properties. Thus, antibody sequences selected from a human library using phage display may include human CDR or variable region sequences that give specific binding to a particular antigen, e.g., HER2, which can be used to provide fully human antibodies for use in the present invention.

[0076] Methods for deriving heavy and light chain sequences from human B cells and plasma cell clones are also known in this technology, and are typically performed using polymerase chain reaction (PCR) technology. Examples of this method include: Kuppers R, Methods Mol Biol. 2004;271:225-38; Yoshioka M et al., BMC Biotechnol. 2011 Jul 21;11:75; Scheeren FA et al., PLoS ONE 2011, 6(4): e17189. doi:10.1371 / journal.pone.0017189; Wrammert J et al., Nature 2008 453, 667-671; Kurosawa N et al., BMC Biotechnol. 2011 Apr 13;11:39; Tiller et al., J Immunol Methods. 2008 January 1; This is described in 329(1-2): 112-124. Therefore, the antibody sequence selected using B cell clones may include, for example, a human CDR or variable region sequence that gives specific binding to HER2, which can be used to provide a fully human antibody for use in the present invention.

[0077] IgE antibody The therapeutic antibody administered to the target may be an IgE antibody, specifically an isotype IgE antibody. There are several fundamental structural differences between IgE and IgG, which have functional effects. While IgE shares the same basic molecular structure as other classes of antibodies, the IgE heavy chain has one more domain than the IgG heavy chain. The Cε3 and Cε4 domains of IgE are sequence homologous and structurally similar to the Cγ2 and Cγ3 domains of IgG; therefore, the most clearly distinguishable feature of IgE is the Cε2 domain. The Cε2 domain has been found to fold back relative to the heavy chain IgE and be in broad contact with the Cε3 domain. This bent structure of the IgE heavy chain allows it to take on either an open or closed conformation. An unbound IgE dimer has one chain in an open conformation and one chain in a closed conformation. The binding of FcεRI to IgE is thought to be biphasic, involving a wide range of structural rearrangements after the initial binding to an open Cε chain, enabling binding to a closed Cε chain. Binding between the IgE dimer and FcεRI occurs 1:1 stoichiometrically, despite the presence of two identical Cε chains. This rearrangement results in a very close interaction between IgE and FcεRI, and a considerably greater affinity of IgE for the Fc receptor than is found with IgG and FcγR (McDonnell, JM, R. Calvert, et al. (2001) Nat Struct Biol 8(5): 437-441).

[0078] The antibodies used in this invention can typically bind to Fcε receptors, such as FcεRI and / or FcεRII receptors. Preferably, the antibody can bind to at least FcεRI (i.e., a high-affinity Fcε receptor) or to at least FcεRII (CD23, a low-affinity Fcε receptor). Typically, the antibody can also activate Fcε receptors expressed on cells of the immune system, for example, to initiate IgE-mediated effector functions.

[0079] The epsilon (ε) heavy chain is crucial for IgE antibodies and contains an N-terminal variable domain VH and four constant domains Cε1-Cε4. As with other antibody isotypes, the variable domain confers antigen specificity, while the constant domains mobilize isotype-specific effector functions.

[0080] IgE differs from the more abundant IgG isotypes in that it cannot fix complement and does not bind to Fc receptors FcγRI, RII, and RIII, which are expressed on the surface of mononuclear cells, NK cells, and neutrophils. However, IgE does not bind to "high affinity" IgE receptors (FcεRI, Ka.10) on various immune cells, such as mast cells, basophils, monocytes / macrophages, and eosinophils. 11 M -1 ), as well as the “low affinity” receptor FcεRII (Ka.10), also known as CD23, which is expressed on inflammatory and antigen-presenting cells (e.g., monocytes / macrophages, platelets, dendritic cells, T and B lymphocytes). 7 M -1 It can have a very specific interaction with ).

[0081] The IgE sites responsible for these receptor interactions are mapped to different peptide sequences on the Cε chain. The FcεRI site is located in the cleavage created by the residues between Gln301 and Arg376, and includes the junction between the Cε2 and Cε3 domains (Helm, B. et al. (1988) Nature 331, 180183). The FcεRII binding site is located within the Cε3 periphery residue Val370 (Vercelli, D. et al. (1989) Nature 338, 649-651). The main difference that distinguishes the two receptors is that FcεRI binds to monomeric Cε, while FcεRII binds only to dimeric Cε, meaning the two Cε chains must associate. IgE is glycosylated in vivo, but this is not necessary for binding to FcεRI and FcεRII. Without glycosylation, the bond is actually slightly stronger (Vercelli, D. et al. (1989) et al. previously published).

[0082] Therefore, binding to the Fcε receptor and related effector functions are typically mediated by the constant domains of the antibody's heavy chain, particularly by domains that co-form the antibody's Fc region. The antibodies described herein typically include at least a portion of an IgE antibody, for example, one or more constant domains derived from IgE, preferably human IgE. In certain embodiments, the antibody includes one or more domains (derived from IgE) selected from Cε1, Cε2, Cε3, and Cε4. In one embodiment, the antibody includes at least Cε2 and Cε3, more preferably at least Cε2, Cε3, and Cε4, and preferably the domains are derived from human IgE. In one embodiment, the antibody includes an epsilon (ε) heavy chain, preferably a human ε heavy chain.

[0083] The amino acid sequences of the constant domains derived from human IgE are shown, for example, in Table 1 (non-bold text for SEQ ID NOs. 41 and 42, and SEQ ID NOs. 1 and 6). The nucleotide sequences encoding the constant domains derived from human IgE, particularly the Cε1, Cε2, Cε3, and Cε4 domains, are also disclosed, for example, in International Publication No. 2013 / 050725. The amino acid sequences of other human and mammalian IgE, as well as those of the human Cε1, Cε2, Cε3, and Cε4 domains and their respective domains, including the human ε-heavy chain sequence, are known in this art and are available from publicly accessible databases. For example, the database of human immunoglobulin sequences is accessible from the International ImmunoGeneTics Information System (IMGT®) website at http: / / www.imgt.org. As an example, sequences of various human IgE heavy (ε) chain alleles and their individual constant domains (Cε1-4) can be accessed at http: / / www.imgt.org / IMGT_GENE-DB / GENElect?query=2+IGHE&species=Homo+sapiens.

[0084] Preferred anti-HER2 IgE antibodies and their variants / fragments In one embodiment, the anti-HER2 antibody includes at least a portion of the amino acid sequence defined in any one of SEQ ID NOs: 2, 12, 22, or 32, for example, at least 20, 30, 50, or 100 amino acids of any one of SEQ ID NOs: 2, 12, 22, or 32, or the full length of any one of SEQ ID NOs: 2, 12, 22, or 32, or a VH domain containing one, two, or three CDRs (for example, defined according to Kabat, Chothia, or IMGT) present in any one of SEQ ID NOs: 2, 12, 22, or 32.

[0085] In one embodiment, the anti-HER2 antibody includes at least a portion of the amino acid sequence defined in any one of SEQ ID NOs: 7, 17, 27, or 37, for example, at least 20, 30, 50, or 100 amino acids of any one of SEQ ID NOs: 7, 17, 27, or 37, or the full length of any one of SEQ ID NOs: 7, 17, 27, or 37, or a VL domain containing one, two, or three CDRs (defined according to, for example, Kabat, Chothia, or IMGT) present in any one of SEQ ID NOs: 7, 17, 27, or 37.

[0086] In general, functional fragments of the sequences defined above can be used in the present invention. The functional fragments may be of any length specified above (e.g., at least 50, 100, 300 or 500 nucleotides, or at least 50, 100, 200 or 300 amino acids), provided that they retain the activity required when present in an antibody (e.g., specific binding to HER2 and / or Fcε receptors).

[0087] The above-mentioned amino acid and nucleotide sequence variants can also be used in the present invention, provided that the resulting antibody binds to the Fcε receptor. Typically, such variants have high sequence identity with one of the sequences specified above.

[0088] The similarity between amino acid or nucleotide sequences is expressed in terms of similarity between sequences, otherwise it is called sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology). A higher percentage indicates greater similarity between two sequences. Homologs or variants of amino acid or nucleotide sequences exhibit a relatively high degree of sequence identity when aligned using standard methods.

[0089] Methods for aligning sequences for comparison are publicly known in this art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed discussion of sequence alignment methods and homology calculations.

[0090] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.), and via the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to determine sequence identity using this program are available on the NCBI website.

[0091] Antibody homologs and variants (e.g., anti-HER2 antibodies or their domains, e.g., VL, VH, CL, or CH domains) are typically found to have at least approximately 75% sequence identity with the original sequence (e.g., the sequences specified above), e.g., at least approximately 80%, 90%, 95%, 96%, 97%, 98%, or 99%, when counted across the full-length alignment of the antibody or its domain with the amino acid sequence using, for example, NCBI Blast2.0, with gapped blastp set to default parameters. For comparison of amino acid sequences longer than approximately 30 amino acids, the Blast2 sequence function is used with the default BLOSUM62 matrix set to default parameters (gap cost 11 and gap cost per residue 1). When aligning short peptides (less than approximately 30 amino acids), the alignment should be performed using the Blast2 sequence function with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). Proteins with greater similarity to the reference sequence will exhibit increased percentage identity when evaluated by this method, showing, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When comparing less than the entire sequence for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10–20 amino acids, and depending on their similarity to the reference sequence, they may have at least 85%, or at least 90% or 95% sequence identity. Methods for determining sequence identity over such short windows are available on the NCBI website. Those skilled in the art will recognize that these ranges of sequence identity are presented for guidance purposes only. It is entirely possible that very significant homologs may be obtained that fall outside the presented range.

[0092] Typically, a variant may contain one or more conserved amino acid substitutions compared to the original amino acid or nucleic acid sequence. Conserved substitutions are those that do not substantially affect or reduce the antibody's affinity for the target antigen (e.g., HER2) and / or Fcε receptor. For example, a human antibody that specifically binds to HER2 may contain up to 1, 2, 5, 10, or 15 conserved substitutions compared to the original sequence (e.g., as defined above) and retain specific binding to the HER2 polypeptide. The term "conservative variation" also includes the use of substituted amino acids at the location of the unsubstituted parent amino acids, provided that the antibody specifically binds to the target antigen (e.g., HER2). Non-conservative substitutions are those that reduce the activity or binding to the target antigen (e.g., HER2) and / or Fcε receptor.

[0093] Functionally analogous amino acids that can be substituted by conservative substitution are known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conserved substitutions with one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0094] In preferred embodiments, the IgE antibody lacks a cytotoxic moiety and / or is administered as monotherapy. It has been surprisingly found that anti-HER2 IgE antibodies can target low-HER2-expressing tumors without the need for the administration of cytotoxic drugs (either in the form of ADCs or in combination with chemotherapy). This can circumvent the drawbacks of ADCs, potentially resulting in reduced toxicity, improved tolerability, and an improved safety profile.

[0095] Therefore, an antibody may consist of, or essentially consist of, a polypeptide chain (which may be glycosylated). For example, an antibody may consist of, or essentially consist of, one or more (preferably four) polypeptide chains, such as two immunoglobulin heavy chains and optionally two immunoglobulin light chains. Preferably, the heavy chain and / or light chains contain one or more domains of an IgE antibody.

[0096] In particular, it is preferable that the antibody is not an antibody-drug conjugate (ADC). Therefore, the antibody may lack further drugs or groups that have cytotoxic effects, such as chemotherapeutic drugs that can (directly) kill cancer cells. The antibody may further lack linkers or other groups for conjugating the cytotoxic portion to a polypeptide.

[0097] Further IgE antibodies As described above, in preferred embodiments, the IgE antibody binds to HER2. Preferably, the IgE antibody can induce cytotoxicity (e.g., ADCC) and / or phagocytosis (ADCP) particularly against cancer cells expressing such antigens.

[0098] In some embodiments, one or more variable domains and / or one or more CDRs, preferably at least three CDRs, or more preferably all six CDRs, may be derived from one or more of the following antibodies: trastuzumab, pertuzumab, or margetuximab (see, for example, Ling et al., Front Immunol. 2018; 9: 469; Rugo et al., JAMA Oncology. 2021;7(4):573-584).

[0099] In another embodiment, the antibody is a chimeric, humanized, or fully human antibody that specifically binds to the epitope to which one of the antibodies described above binds. The IgE antibody may further contain one or more IgE constant domains, such as Cε1-Cε4 domains, as described above.

[0100] Antibody and nucleic acid production Nucleic acid molecules (also known as polynucleotides) encoding polypeptides (including, but not limited to, antibodies and their functional fragments) as described herein can be readily generated by those skilled in the art using the amino acid sequences, sequences available in the art, and genetic codes described herein. Furthermore, those skilled in the art can readily construct various clones containing functionally equivalent nucleic acids, for example, nucleic acids encoding the same effector molecule or antibody sequence but with different sequences. Thus, nucleic acids encoding antibodies are provided in the present invention.

[0101] Nucleic acid sequences encoding antibodies or functional fragments thereof that specifically bind to a target antigen (e.g., HER2) can be synthesized, for example, by cloning a suitable sequence, or by using an automated synthesizer such as the one described in Narang et al., Meth. Enzymol. 68:90-99, 1979, the phosphotriester method of Brown et al., Meth. Enzymol. 68:109-151, 1979, the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981, or by using an automated synthesizer such as the one described in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984. It can be prepared by any suitable method, including direct chemical synthesis by the solid-phase phosphoramidite triester method described in 1981 and the solid-phase supported method described in U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization using DNA polymerase with the single strand as a template. Those skilled in the art recognize that the chemical synthesis of DNA is generally limited to sequences of about 100 bases, and longer sequences can be obtained by ligation of shorter sequences.

[0102] Exemplary nucleic acids encoding antibodies or their functional fragments can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques, as well as sufficient instructions for those skilled in the art to perform many clones, can be found, for example, in *Molecular Cloning: A Laboratory Manual*, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and *Current Protocols in Molecular Biology* (Ausubel et al., eds 1995 supplement). Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to those skilled in the art.

[0103] The antibodies described herein can be formed by modifying nucleic acids encoding natural antibodies. Modification by site-directed mutagenesis is known in the art. Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and self-sustained sequence replication system (3SR). A wide range of cloning methods, host cell and in vitro amplification methods are known to those skilled in the art.

[0104] In one embodiment, the antibody is prepared by inserting cDNA encoding one or more antibody domains (e.g., the mouse IgG1 heavy chain variable region that binds to human HER2) into a vector containing cDNA encoding one or more further antibody domains (e.g., the human heavy chain ε constant region). The insertion is performed so that the antibody domains are read in frame as a single continuous polypeptide containing the functional antibody region.

[0105] In one embodiment, a cDNA encoding the heavy chain constant region is ligated to the heavy chain variable region so that the constant region is located at the carboxyl terminus of the antibody. The heavy chain variable and / or constant region can then be ligated to the light chain variable and / or constant region of the antibody using disulfide bonds.

[0106] Once an antibody or nucleic acid encoding a functional fragment thereof is isolated and cloned, the desired protein can be expressed in recombinant cells, such as bacteria, plants, yeast, insects, and mammalian cells. Those skilled in the art will likely know of numerous expression systems available for protein expression, including Escherichia coli (E. coli), other bacterial hosts, yeast, and various higher eukaryotic cells, such as COS, CHO, HeLa, and myeloma cell lines.

[0107] One or more DNA sequences encoding an antibody or a fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. This term also includes any offspring of the target host cell. It is understood that not all offspring will be identical to the parent cells due to mutations that may occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in this technique. Hybridomas expressing the desired antibody are also included in this disclosure.

[0108] The expression of nucleic acids encoding isolated antibodies and antibody fragments described herein can be achieved by operably ligating DNA or cDNA to a promoter (which can be constitutive or inductive) and then incorporating it into an expression cassette. The cassette may be suitable for replication and incorporation in either prokaryotes or eukaryotes. A typical expression cassette contains specific sequences useful for regulating the expression of protein-coding DNA. For example, an expression cassette may include a suitable promoter, enhancer, transcription and translation terminators, a start sequence, a start codon (i.e., ATG) preceding the protein-coding sequence, a splicing signal for introns, maintenance of the correct reading frame of the gene to enable correct translation of mRNA, and a stop codon.

[0109] To obtain high expression levels of cloned genes, it is desirable to construct an expression cassette containing a strong promoter to drive transcription, a ribosome binding site for translation initiation, and a minimal transcription / translation terminator. For E. coli, this includes a promoter, e.g., T7, trp, lac, or lambda promoter, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the regulatory sequence may include, for example, a promoter and / or enhancer derived from an immunoglobulin gene, SV40, or cytomegalovirus, and a polyadenylation sequence, and may further include splice donor and acceptor sequences. The cassette can be transferred to host cells selected by known methods, e.g., transformation or electroporation for E. coli, and calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed with the cassette can be selected for antibiotic resistance provided by genes contained in the cassette, e.g., amp, gpt, neo, and hyg genes.

[0110] When the host is a eukaryote, calcium phosphate coprecipitation, conventional mechanical procedures such as microinjection, electroporation, DNA transfection methods such as insertion of liposome-coated plasmids, or viral vectors may be used. Eukaryotic cells can be co-transformed with an antibody, a labeled antibody or a functional fragment thereof, and a polynucleotide sequence encoding a second foreign DNA molecule encoding a selective trait, such as the herpesthymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors, such as simian virus 40 (SV40) or bovine papillomavirus, to express proteins (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Those skilled in the art can readily use expression systems, such as plasmids and vectors, that are useful for generating proteins in higher eukaryotic cells, including cells such as COS, CHO, HeLa, and myeloma cell lines.

[0111] Modifications can be made to nucleic acids encoding polypeptides described herein (e.g., human HER2-specific IgE antibodies) without attenuating their biological activity. Some modifications can be made to facilitate cloning, expression, or incorporation of targeted molecules into fusion proteins. Such modifications are known to those skilled in the art and include, for example, stop codons, methionine added to the amino terminus to provide an initiation site, amino acids added to either terminus to create a restriction site at a convenient location, or amino acids (e.g., polyHis) added to assist in purification steps. In addition to recombinant methods, the antibodies of this disclosure can also be constructed whole or partially using standard peptide synthesis known in the art.

[0112] Once expressed, recombinant antibodies can be purified according to standard procedures in the technique, including ammonium sulfate precipitation, affinity columns, and column chromatography (see R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, NY, 1982 for general information). Antibodies, immunoconjugates, and effector molecules do not need to be 100% pure. Once partially or homogeneously purified as desired, polypeptides should not substantially contain endotoxins if used therapeutically.

[0113] Frequently, functional heterologous proteins from E. coli or other bacteria require isolation from inclusion bodies, solubilization using strong denaturants, and subsequent refolding. During the solubilization step, a reducing agent must be present to separate the disulfide bonds, as is known in this technique. An exemplary buffer containing a reducing agent is 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, and 0.3 M dithioerythritol (DTE). Re-oxidation of disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized forms, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, and particularly in Buchner et al., previously mentioned.

[0114] Regeneration is typically achieved by diluting the denatured and reduced protein in a refolding buffer (e.g., 100-fold). An example buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0115] As a modification of the double-chain antibody purification protocol, the heavy and light chain regions are solubilized and reduced separately, and then combined in a refolding solution. The exemplary yield is obtained when the two proteins are mixed in a molar ratio in which one protein does not exceed a 5-fold molar excess of the other. Excess oxidized glutathione or other oxidative low molecular weight compounds can be added to the refolding solution after the redox shuffling is complete.

[0116] In addition to recombinant methods, the antibodies, labeled antibodies, and their functional fragments disclosed herein can also be constructed whole or partially using standard peptide synthesis. Solid-phase synthesis of polypeptides less than approximately 50 amino acids in length can be achieved by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence. The techniques of solid-phase synthesis are described in Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963 and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Longer proteins can be synthesized by condensation of the amino and carboxyl terms of shorter fragments.

[0117] Methods for forming peptide bonds by activating the carboxyl terminus (for example, by using the coupling reagent N,N'-dicyclohexylcarbodiimide) are known in this art.

[0118] In one embodiment, antibodies, nucleic acids, expression vectors, host cells, or other biological products are isolated. “Isolated” means that the product is separated or purified substantially from other biological components in the environment in which the component naturally exists (e.g., cells), namely other chromosomes and extrachromosomal DNA and RNA, proteins, and organelles. “Isolated” nucleic acids and antibodies include nucleic acids and antibodies purified by standard purification methods. This term also includes nucleic acids and antibodies prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0119] Compositions and Therapeutic Methods The present invention provides compositions comprising a carrier and one or more therapeutic IgE antibodies or functional fragments thereof. The compositions can be prepared in unit dosage forms for administration to a subject. The antibodies may be formulated for systemic or topical (e.g., intratumor) administration. In one example, the therapeutic IgE antibody may be formulated for parenteral administration, such as intravenous or subcutaneous administration.

[0120] The compositions for administration may comprise a solution of an antibody (or a functional fragment thereof) dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as buffered saline, can be used. These solutions are sterile and substantially free of undesirable substances. These compositions can be sterilized by conventional and known sterilization techniques. The compositions may, if necessary, contain pharmaceutically acceptable adjuvants, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., to approximate physiological conditions. The concentrations of the antibody and excipients in these formulations can vary and are selected according to the specific dosage form and the needs of the target, primarily based on the volume, viscosity, body weight, etc. of the fluid. Practical methods for preparing administerable compositions are known or evident to those skilled in the art and are described in detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).

[0121] In a preferred embodiment, the composition is provided as a unit dosage form containing a specified amount of IgE antibody suitable for administration to a subject, for example, in a single dose. The unit dosage form may be individually packaged, for example, in a single container, vial, or pre-filled syringe. The unit dosage form may be suitable for immediate administration to a subject (for example, containing a physiologically acceptable concentration of salt), or the unit dosage form may be provided in a concentrated or lyophilized form (for example, for dilution with sterile saline before use).

[0122] Anti-HER2 IgE antibodies can be administered in any appropriate dose. In the preferred embodiments described herein, a typical unit dose of a pharmaceutical composition (e.g., for intravenous administration) contains less than 50 mg of IgE antibody. For example, a composition (i.e., a unit dosage form) may contain 40 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, or less than 1 mg of IgE antibody. A composition may contain at least 10 μg, 100 μg, 200 μg, 300 μg, 500 μg, 700 μg, 1 mg, 3 mg, 5 mg, or 10 mg of IgE antibody. In preferred embodiments, the composition contains 10 μg to 50 mg, 70 μg to 30 mg, 300 μg to 50 mg, 300 μg to 30 mg, 300 μg to 3 mg, 500 μg to 50 mg, 500 μg to 30 mg, 500 μg to 10 mg, 500 μg to 3 mg, 700 μg to 50 mg, 700 μg to 30 mg, 700 μg to 10 mg, 700 μg to 3 mg, 500 μg to 5 mg, 500 μg to 1 mg, or about 700 μg of IgE antibody. In some embodiments, the composition may contain one or more amounts of IgE antibody within the above ranges, except for one or more of the following amounts: 1 μg, 5 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, or 15 mg. For example, the composition may contain 2 μg to 9 μg, 11 μg to 99 μg, 101 μg to 499 μg, 501 to 999 μg, or 2 mg to 9 mg.

[0123] The dose of IgE antibody administered to a subject may be based on the subject's body weight. Therefore, the dose of IgE antibody administered to a subject may be, for example, less than 1 mg / kg. Preferably, the IgE antibody can be administered to a subject in doses (per administration) of less than, for example, 0.7 mg / kg, 0.5 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.07 mg / kg, 0.05 mg / kg, 0.03 mg / kg, or 0.01 mg / kg. The dose of IgE antibody administered to a subject may be at least 0.001 mg / kg, 0.003 mg / kg, 0.005 mg / kg, 0.007 mg / kg, 0.01 mg / kg, 0.05 mg / kg, or 0.1 mg / kg. In preferred embodiments, the dose of IgE antibody administered to the subject may be 0.001-1 mg / kg, 0.003-0.7 mg / kg, 0.005-0.5 mg / kg, 0.005-0.1 mg / kg, 0.005-0.05 mg / kg, 0.007-0.03 mg / kg, or 0.007-0.15 mg / kg. In some embodiments, the dose of IgE antibody administered to the subject may be within one or two or more of the above ranges, excluding one or two or more of the following doses: 1 μg / kg, 10 μg / kg, 100 μg / kg, or 0.5 mg / kg. For example, the dose of IgE antibody may be 2-9 μg / kg, 11-99 μg / kg, 101-499 μg / kg, or 0.51-0.7 mg / kg.

[0124] In embodiments of the present invention, the above-mentioned IgE antibody unit dosage form is administered at most once a week, and for example, the maximum weekly dose of IgE antibody is 50 mg, 40 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, or 1 mg. For example, the weekly dose of IgE antibody may be 10 μg to 50 mg, 70 μg to 30 mg, 300 μg to 50 mg, 300 μg to 30 mg, 300 μg to 3 mg, 500 μg to 50 mg, 500 μg to 30 mg, 500 μg to 10 mg, 500 μg to 3 mg, 700 μg to 50 mg, 700 μg to 30 mg, 700 μg to 10 mg, 700 μg to 3 mg, 500 μg to 5 mg, 500 μg to 1 mg, or about 700 μg. The weekly dose of IgE antibody can also be determined according to the subject's body weight. For example, IgE antibody can be administered to the subject at doses of, for example, 0.7 mg / kg / week, 0.5 mg / kg / week, 0.3 mg / kg / week, 0.1 mg / kg / week, 0.07 mg / kg / week, 0.05 mg / kg / week, 0.03 mg / kg / week, or less than 0.01 mg / kg / week. In a preferred embodiment, the dose of IgE antibody administered to the subject may be 0.001 to 1 mg / kg / week, 0.003 to 0.7 mg / kg / week, 0.005 to 0.5 mg / kg / week, 0.005 to 0.1 mg / kg / week, 0.005 to 0.05 mg / kg / week, 0.007 to 0.03 mg / kg / week, or 0.007 to 0.15 mg / kg / week. In some embodiments, the dose of IgE antibody administered to the subject may be within the range of 1 or 2 or more specified above, excluding the following doses: 1 μg / kg / day (7 μg / kg / week), 10 μg / kg / day (70 μg / kg / week), or 100 μg / kg / day (0.7 mg / kg / week). For example, the dose of IgE antibody may be 2-6 μg / kg / week, 8-69 μg / kg / week, or 71-699 μg / kg / week.

[0125] In one embodiment, the pharmaceutical composition is a liquid comprising one or more excipients selected from sodium citrate, L-arginine, sucrose, polysorbate 20, and / or sodium chloride. Preferably, the composition has a pH of 6.0 to 8.0, for example, about 6.5. Preferred concentrations of the excipients include 0.05 to 0.5 M (e.g., about 0.1 M) of sodium citrate, 10 to 50 g / L (e.g., about 30 g / L) of L-arginine, 10 to 100 g / L (e.g., about 50 g / L) of sucrose, and 0.01 to 0.05% w / w (e.g., 0.02% w / w) of polysorbate 20. In one embodiment, IgE antibodies are present in such a formulation at a concentration of about 0.1 mg / ml to 10 mg / ml or 0.5 mg / ml to 2 mg / ml, for example, about 1 mg / ml. In some embodiments, such compositions can be formulated as a unit dosage form in a 2 ml type I glass vial, for example, in a 1 ml volume solution containing about 1 mg of IgE antibody. The composition can be diluted in sterile saline (0.9% w / v) before administration to the subject, for example, by 1 ml of the composition in 250 ml of saline.

[0126] Antibodies are supplied in lyophilized form and can be rehydrated with sterile water before administration, but they can also be supplied in sterile solutions of known concentrations. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and administered to the subject. There is considerable experience in the technology of administering antibody drugs, and these have been commercially available in the United States since the approval of RITUXAN® in 1997. Antibodies can be administered by slow infusion rather than intravenous infusion or rapid infusion. In one example, a higher loading dose is administered, followed by a lower level of maintenance dose. For example, an initial loading dose can be infused over a period of about 90 minutes, and then weekly maintenance doses can be infused over a period of 30 minutes for 4 to 8 weeks, if the previous dose is well tolerated.

[0127] Antibodies (or functional fragments thereof) can be administered to delay or inhibit the proliferation of cells, such as cancer cells. In these applications, a therapeutically effective dose of the antibody is administered to the subject in an amount sufficient to inhibit the proliferation, replication, or metastasis of cancer cells, or to inhibit the signs or symptoms of cancer. In some embodiments, the antibody is administered to the subject to inhibit or prevent the development of metastasis, or to reduce the size or number of metastases, such as micrometastases, such as micrometastases to regional lymph nodes (Goto et al., Clin. Cancer Res. 14(11):3401-3407, 2008).

[0128] Therefore, in some embodiments, IgE antibodies are used to treat cancer and / or slow or prevent the progression of cancer. "Slowing or preventing the progression of cancer" means, for example, that the cancer is stable for a period of time after antibody administration, e.g., at least 6 weeks, at least 12 weeks, at least 6 months, or at least 12 months. A "stable" disease can be defined, for example, as a change in the RECIST score of less than 20%.

[0129] RECIST (Guidelines for Evaluating the Response of Solid Tumors) evaluation is a simple method for determining whether a patient's disease has improved, remained largely the same, or worsened after treatment with cancer drugs, and is commonly used in clinical trials of anticancer drugs. The RECIST criteria are specified, for example, in Eisenhauer et al., New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1), European Journal of Cancer 45 (2009) 228-247. RECIST defines progressive disease (PD) as an increase of at least 20% in the total diameter of target lesions, with the smallest sum examined as reference. Stable disease is defined as neither regression sufficient to constitute a partial response (a reduction of at least 30% in the total diameter of target lesions) nor increase sufficient to constitute PD; i.e., an increase of <20% is defined as stable disease.

[0130] In this context, "at least stable" is recognized to include an increase or decrease in the RECIST score of less than 20%. Therefore, antibodies may delay or prevent disease progression (e.g., delay or prevent the appearance of one or more signs or symptoms of cancer, and / or inhibit the proliferation of cancer cells, and / or prevent or reduce metastasis), or improve the disease or promote remission of the disease (e.g., reduce or prevent one or more signs or symptoms of cancer, and / or kill cancer cells).

[0131] subject Appropriate subjects may include those diagnosed with cancer, such as HER2-expressing cancers, including, but not limited to, skin cancer (e.g., melanoma), lung cancer, prostate cancer, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), breast cancer (including, but not limited to, basal breast cancer, ductal carcinoma, and lobular breast carcinoma), leukemia (e.g., acute myeloid leukemia and 11g23-positive acute leukemia), lymphoma, crest tumors (e.g., astrocytoma, glioma, or neuroblastoma), ovarian cancer, colorectal cancer, gastric cancer (e.g., gastric or gastroesophageal junction (GEJ) adenocarcinoma), pancreatic cancer, bone cancer (e.g., chordoma), glioma, or sarcoma (e.g., chondrosarcoma).

[0132] Preferably, the antibody is administered to treat a solid tumor. In some embodiments, the cancer is breast cancer or gastric cancer, preferably breast cancer. Preferably, the subject is human.

[0133] In some embodiments, the subject has metastatic cancer. For example, in some embodiments, the cancer is metastatic breast cancer or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma. Most preferably, the cancer is metastatic breast cancer.

[0134] The therapeutically effective dose of antibodies depends on the severity of the disease and the patient's overall health condition. A therapeutically effective dose of antibodies results in either a subjective reduction of the disease or an objectively identifiable improvement noticed by a physician or other qualified observer. These compositions can be administered concurrently or sequentially with other chemotherapeutic agents.

[0135] Low HER2 expression Anti-HER2 antibodies are used to treat patients with low-HER2-expressing tumors. These patients are sometimes referred to as "low-HER2-expressing" patients, in contrast to high-HER2-expressing patients. The terms "low-HER2-expressing tumors" and "low-HER2-expressing patients" are well understood in the field of cancer therapy and are frequently used to describe specific patient groups (see, for example, Nicolo et al. Ther Adv Med Oncol 2023, Vol. 15: 1-16; Tarantino et al. J Clin Oncol 2020; 38: 1951-1962). Therefore, the subgroup of patients with low tumor HER2 expression is clearly distinguished from high-HER2-expressing patients.

[0136] Low tumor HER2 expression can be determined using known standard techniques. Typically, HER2 expression is determined in biopsy or surgical specimens from tumors. Techniques for obtaining surgical biopsy specimens from tumor tissue are known in the art, as are histopathological techniques for processing such specimens. For example, biopsy tissue specimens may be fixed in formalin and embedded in paraffin, or freshly processed before sectioning and placed on microscope slides for light microscopy analysis and imaging.

[0137] HER2 expression levels can be detected using immunohistochemistry techniques. Hematoxylin and eosin (H&E) staining of paraffin-embedded sections is a default technique for visualizing tissue on glass slides for pathological analysis. Immunohistochemistry (IHC) staining is a known approach for identifying protein expression on cells within pathological tissue slides. Staining typically results in a brown appearance in tissue where the target protein is overexpressed compared to normal. For example, its expression level can be detected by using an antibody against HER2.

[0138] Low HER2 expression can be defined as a HER2 IHC score less than 3+, for example, 0, 1+, or 2+. Preferably, the tumor has a HER2 IHC score of 1+ or 2+, more preferably 1+. In an alternative embodiment, the tumor has a HER2 IHC score of 0. Immunohistochemical detection of HER2 in a sample from a subject can be performed using Dako's anti-HER2 immunohistochemistry system (i.e., HercepTest®). The HercepTest® method is described, for example, in FDA premarket authorization application P980018 approved on September 25, 1998, and P980018 / S010 approved on October 20, 2010, as well as in Dako's HercepTest®, Code K5204. Instructions for Use, (PD04086US_02 / K520421-5). The HercepTest™ method and alternative methods for detecting HER2 expression in tumors are also described, for example, in Jorgensen et al., (2021) Front. Oncol. 11:676939.

[0139] Various studies describe methods for detecting HER2 in tumor samples and standardization of the results. In particular, see the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer (2018 edition and 2023 update; see Wolff et al. Arch Pathol Lab Med (2023), Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer, available at https: / / doi.org / 10.5858 / arpa.2023-0950-SA and www.asco.org / breast-cancer-guidelines). Also, for example, Fitzgibbons et al., Arch Pathol Lab Med. 2014;138:595-601; Canda et al., Eur J Breast Health. 2018 Jul; 14(3): 160-165; Dowsett et al., Modern Pathology. 2007;20:584-591; Zhang et al. Curr Oncol Rep See also 2020 Apr 29;22(5):51.

[0140] The FDA-approved classifications for HER2 IHC staining scores are listed below: [Table 1]

[0141] Any suitable anti-HER2 antibody, such as an anti-HER2 IgG antibody (polyclonal or monoclonal), can be used in this manner. A variety of anti-HER2 antibodies suitable for use in immunohistochemistry are available from commercial sources.

[0142] Therefore, "low HER2-expressing tumors" typically mean that less than 10% of tumor cells in a sample from a subject show strong, complete membrane staining for HER2 (i.e., corresponding to a HER2 IHC score of less than 3+). Preferably, the tumor sample lacks tumor cell clusters (≥5 cells) with strong, complete membrane staining for HER2. These expression levels typically refer to membrane HER2 expression detectable by immunohistochemistry, for example, using the method described above.

[0143] For example, in some embodiments, at least 10% of tumor cells or tumor cell clusters (five or more cells) in a sample from a subject show weak to moderate complete membrane staining for HER2, i.e., membrane staining corresponding to a HER2 IHC score of 2+, when immunohistochemical detection for HER2 is used. In some embodiments, tumor cell clusters (five or more cells) in a sample from a subject may show weak to moderate complete membrane staining for HER2.

[0144] In other embodiments, at least 10% of tumor cells in a sample from the subject show faint or barely recognizable partial (incomplete) membrane staining for HER2, i.e., membrane staining corresponding to a HER2 IHC score of 1+, when immunohistochemical detection of HER2 is used. In some embodiments, tumor cell clusters (five or more cells) from the subject may show faint or barely recognizable partial (incomplete) membrane staining for HER2. In these embodiments, the tumor does not meet the criterion for a HER2 IHC score of 2+ or higher, i.e., less than 10% of tumor cells in a sample from the subject show weak to moderate complete membrane staining for HER2 (and there are no tumor cell clusters (five or more cells) showing weak to moderate complete membrane staining for HER2).

[0145] In an alternative embodiment, tumor HER2 expression in a subject can be compared to tumor HER2 expression in a population of cancer subjects. Typically, a population of cancer subjects refers to other subjects with the same type of cancer. Thus, the relative level of tumor HER2 expression in a subject can be determined by comparing it to other subjects with the same type of cancer. This method does not require absolute quantification of HER2 expression levels, but only a relative determination in comparison to other cancer subjects, thus avoiding any systematic bias due to the lack of standardization of expression detection.

[0146] In a preferred embodiment, tumor (e.g., membrane) HER2 expression in the subject is lower than tumor HER2 expression in at least 50% of cancer subjects. Preferably, membrane HER2 expression in the tumor cells of the subject is lower than membrane HER2 expression in at least 60%, at least 70%, or at least 80% of cancer subjects, e.g., those with the same form of cancer (e.g., ovarian cancer). More preferably, tumor membrane HER2 expression in the subject is lower than tumor membrane HER2 expression in at least 50%, 60%, 70%, 80%, or at least 90% of HER2-expressing tumors (e.g., in HER2-expressing breast tumors).

[0147] In some embodiments, it is preferable that the tumor expresses HER2, i.e., exhibits at least some degree of HER2 expression. Typically, this means that the tumor cells in the subject exhibit membrane HER2 expression detectable, for example, by immunohistochemistry. More preferably, at least 1% or at least 5% of the tumor cells in the subject exhibit detectable (e.g., membrane) HER2 expression, for example, using immunohistochemical detection of HER2. In other embodiments, at least 10%, 15%, or 20% of the tumor cells in the subject exhibit detectable membrane HER2 expression. Thus, the tumor may have a HER2 IHC score of 1+ or 2+ (rather than 0).

[0148] Preferably, HER2 expression is determined using a recent tumor biopsy sample, i.e., a biopsy sample obtained from the subject immediately before the initiation of anti-HER2 IgE therapy (not an older or stored sample). For example, the biopsy sample may be collected and / or determined within 6 months, 3 months, 1 month, 2 weeks, 1 week, 3 days, 48 ​​hours, or less than 24 hours prior to the initiation of anti-HER2 IgE therapy.

[0149] In some embodiments, amplification of the erbB2(neu) gene may also be monitored. For example, in situ hybridization (including ISH, e.g., fluorescent ISH or FISH) can be performed on a sample from the subject to detect HER2 coding gene amplification. In FISH, the HER2 gene copy number in the relevant tumor cells may be determined and reported by comparing it with the copy number of a centromere 17 (CEN17) reference probe determined in the same cells (e.g., using the PathVysion® HER2 DNA probe kit (Abbott Diagnostics) or HER2 FISH pharmDx (Dako)). A HER2 / CEN17 ratio of ≥2.0 may be used as a cutoff value to indicate HER2 amplification by FISH (see, for example, D'Alfonso et al., J Clin Pathol (2013) 66:409-14; and Jorgensen and Winther, The Development of the HercepTest - From Bench to Bedside. In: Jorgensen JT, Winther H, editors. Molecular Diagnostics - The Key Driver of Personalized Cancer Medicine. Singapore: Pan Stanford Publishing (2010)).

[0150] Therefore, in some embodiments, the tumor lacks erbB2 gene amplification. For example, tumor samples from a subject may not show detectable amplification of the HER2 coding gene (erbB2) when determined, for example, by FISH, or may have a HER2 / CEN17 ratio < 2.0.

[0151] Additional diagnostic methods for analyzing HER2 expression in conjunction with anti-HER2 therapy are described. The table below shows FDA-approved companion diagnostic assays for HER2-targeted drugs. These include IHC and (fluorescence) in situ hybridization assays, as well as more novel next-generation sequencing (NGS) assays. [Table 2]

[0152] The present invention will be further described below for illustrative purposes only, with reference to the following non-limiting embodiments. [Examples]

[0153] Anti-HER2 IgE antibody The following anti-HER2 IgE antibodies were used in the examples. [Table 3] JPEG2026522926000004.jpg238170 JPEG2026522926000005.jpg252170 JPEG2026522926000006.jpg252170 JPEG2026522926000007.jpg41170

[0154] Trastuzumab IgE is described in Karagiannis et al. 2009, Cancer Immunol Immunother. 2009 58(6): 915-930. V20, V23, and V26 IgE were generated using phage display. A panel of 100 antibodies from the phage display was classified based on evaluation of recombinant HER2 and its binding affinity to HER2 on the cell surface, retention of binding to FcεR1, epitope specificity, rat / human HER2 cross-reactivity, ability to degranulate human FcεR1-expressing basophils, ability to kill tumor cells by ADCC and ADCP, and biophysical properties (solubility and thermal stability).

[0155] V23 competes with trastuzumab for binding to HER2 and therefore binds to subdomain IV (near-membrane region) of the extracellular domain of HER2. V26 IgE competes with pertuzumab for binding to HER2 and therefore binds to subdomain II (dimerization region) of the extracellular domain of HER2. V20 does not compete with either trastuzumab or pertuzumab for binding to HER2.

[0156] In the following studies, the above-mentioned IgE antibodies targeting HER2 were profiled in vivo in humanized mouse and rat tumor models with low HER2 expression.

[0157] Anti-HER2 IgE treatment of HER2 IHC 2+ tumors in humanized NXG mice A tumor model using NXG mice, which were humanized with peripheral blood mononuclear cells (PBMCs), was established using JIMT1 cells. (Prkdc) scid Il2rg Tm1 / Rj(NXG) mice have loss-of-function mutations in the Prkdc and Il2rg genes on a NOD (non-obese diabetic) background and are severely immunodeficient (see, for example, Gillgrass et al., Front Immunol. 2020;11:617516; Radaelli et al., PLoS 1. 2015;10(5):e0124974). Immunodeficient mice transplanted with human PBMC can be used to analyze the human immune response (see, for example, Yaguchi et al., Cell Mol Immunol. 2018 Nov; 15(11): 953-962).

[0158] JIMT1 cells are established cancer cell lines derived from the pleural metastasis of a 62-year-old breast cancer patient who was clinically resistant to trastuzumab. JIMT1 cells have been reported to have moderate to weak HER2 expression levels (HercepTest IHC 2+ by clinically appropriate quantification methods) and were found to be resistant to both trastuzumab and pertuzumab in vitro and in xenograft tumors (Tanner et al., Mol Cancer Ther. 2004 Dec;3(12):1585-92). Analysis of HER2 expression on the cell surface using flow cytometry showed that HER2 levels were lower in JIMT1 cell lines compared to the HercepTest IHC 3+ cell line SKBR3 (see Figure 1).

[0159] Female NXG mice were subcutaneously implanted with JIMT1 cells (1x10 7 cells / mouse; 1:1 matrigel). Tumor volume was monitored by caliper three times a week throughout the study. When the tumors reached a volume of 30-50 mm 3 the mice were randomized into treatment groups of 12 mice. At this point, peripheral blood mononuclear cells (PBMC; 5 × 10 6Cells / mouse (3 donors / group) and Flt3L (10 mg) were administered intravenously. Mice were administered 20 mg / kg trastuzumab-IgE (see Table 1 for sequence) or vehicle control (PBS) intravenously simultaneously with PBMCs, and then administered twice a week for 31 days.

[0160] In these trials, trastuzumab-IgE (20 mg / kg) resulted in a statistically significant inhibition of tumor growth (60%) compared to PBS vehicle controls (Figure 2).

[0161] Anti-HER2 IgE treatment of HER2 IHC 1+ tumors in rats Three anti-HER2 IgE antibodies (V20, V23, and V26) were tested in a syngeneic Fischer rat / MTLn3 model (see, e.g., Neri et al., J Natl Cancer Inst 68:507-517). MTLn3 is a highly invasive tumor cell line that rapidly develops large tumors in rats. In this syngeneic model, the MTLn3 tumor cell line (derived from rat mammary cancer) has the same genetic background as the host rat, in contrast to the humanized NXG mouse test described above (in which humanized cell lines are transplanted into immunodeficient mice humanized by PBMCs). Therefore, the host rat has a fully functioning, genuine immune system.

[0162] HER2 levels in MTLn3 cells were evaluated by flow cytometry and were found to be lower in both JIMT1 and SKBR3 cell lines (see Figure 3). Therefore, MTLn3 cells are thought to have very low HER2 expression levels (equivalent to HercepTest IHC 1+ by clinically relevant quantitative methods).

[0163] Female Fisher 344 rats (4-5 weeks old) were given MTLn3 cells (0.7 × 10⁶ in PBS). 6 Cells (from rats) were subcutaneously transplanted proximal to the mammary fat pad. Tumor volume was monitored three times a week using calipers throughout the study. When the tumor was 50-100 mm 3Upon reaching a certain volume, the rats were randomized into treatment groups of 12-13 rats each. The rats were administered intravenously twice weekly for 2.5 weeks at a dose of 20 mg / kg of HER2-targeted IgE antibody V20, V23, or V26 (see Table 1 for sequences) or a vehicle control (PBS).

[0164] These trials showed varying degrees of tumor growth inhibition against HER2-targeted variants (ranging from 25.7% to 52.0%), with V26 > V23 > V20 (see Figure 4 and Table 2 below). [Table 4]

[0165] V26 caused increased infiltration of immune cells, including neutrophils and monocytes, into the tumor (see Figures 5 and 6). V26 also caused increased tumor cell death, as evidenced by an increase in the number of apoptotic cells (see Figures 6 and 7).

[0166] Anti-HER2 IgE treatment of HER2 IHC-0 tumors in mice MDA-MB-231, a triple-negative breast cancer cell line, exhibits extremely low levels of HER2 expression (defined as HercepTest 0). MDA-MB-231 cells (1 × 10⁻⁶) 7 Cells were subcutaneously transplanted into NXG mice in a 1:1 ratio with Matrigel. The tumors were approximately 35-50 mm in size. 3 When it reaches this stage, the mouse is given human PBMC (5×10 6 Cells were intravenously transplanted with an additional dose of FLT3L (10 μg for 3 consecutive days) to enhance bone marrow components. Mice were administered PBMCs, followed by two weekly doses. Tumor volume was assessed by caliper. Mice were administered 10 mg / kg of HER2-targeted IgE antibody V26 (see Table 1 for sequence), isotype control antibody (NIP IgE), or vehicle control (PBS) along with PBMCs, followed by two weekly doses. Tumor volume was assessed by caliper.

[0167] As shown in Figure 8 (where "EPS 226" refers to the V26 IgE antibody), V26 statistically significantly reduces tumor growth compared to PBS and isotype controls in this model. This indicates that HER2-targeted IgE can be used to treat triple-negative breast cancer.

[0168] These data demonstrate the ability of HER2-targeted IgE to significantly influence tumor growth in low HER2 expression conditions.

[0169] All publications referenced in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to certain preferred embodiments, it will be understood that the claimed invention is not unduly limited to such specific embodiments. In fact, various modifications of the described forms for carrying out the invention which will be apparent to those skilled in the art are intended within the following claims.

Claims

1. An anti-HER2 immunoglobulin E (IgE) antibody for use in the treatment of low-HER2-expressing tumors in the target population.

2. An IgE antibody for use according to claim 1, wherein less than 10% of tumor cells in a sample from a subject show strong complete membrane staining for HER2 when immunohistochemical detection of HER2 is used.

3. An IgE antibody for use according to claim 1 or 2, wherein, using immunohistochemical detection of HER2, at least 10% of tumor cells or tumor cell clusters (five or more cells) in a sample from a subject exhibit weak to moderate complete membrane staining for HER2.

4. An IgE antibody for use according to claim 1 or 2, wherein, using immunohistochemical detection of HER2, at least 10% of tumor cells or tumor cell clusters (five or more cells) in a sample from a subject show faint or barely recognizable partial membrane staining for HER2.

5. The IgE antibody for use according to any one of claims 1 to 4, wherein the immunohistochemical detection of HER2 in a sample from a subject is performed using Dako's anti-HER2 immunohistochemistry system (HercepTest®); preferably, the tumor sample from the subject has a HER2 immunohistochemical staining (HER2 IHC) score of 2+ or less, more preferably 2+ or 1+.

6. An IgE antibody for use according to any one of claims 1 to 5, wherein, as determined by fluorescence in situ hybridization, the tumor has a HER2 / CEN17 ratio < 2.0 or does not show detectable erbB2 gene amplification.

7. An IgE antibody for use according to any one of claims 1 to 6, wherein tumor HER2 expression in the subject is lower than tumor HER2 expression in at least 50% of cancer subjects, preferably membrane HER2 expression in tumor cells of the subject is lower than membrane HER2 expression in at least 60%, at least 70%, or at least 80% of subjects with the same form of cancer; more preferably tumor HER2 expression in the subject is lower than tumor HER2 expression in at least 50%, at least 70%, or at least 90% of HER2-expressing tumors (preferably HER2-expressing breast tumors).

8. An IgE antibody for use according to any one of claims 1 to 7, wherein the tumor expresses HER2; preferably, at least 1%, 5%, 10%, 15%, or 20% of the tumor cells in the subject exhibit detectable membrane HER2 expression by immunohistochemical detection of HER2.

9. An IgE antibody for use according to any one of claims 1 to 8, which binds to subdomain II of the extracellular domain of HER2 and preferably at least partially competes with pertuzumab IgG for binding to HER2.

10. An IgE antibody for use according to any one of claims 1 to 9, which binds to subdomain IV of the extracellular domain of HER2 and preferably at least partially competes with trastuzumab IgG for binding to HER2.

11. An IgE antibody for use according to any one of claims 1 to 10, wherein the antibody is trastuzumab IgE.

12. An IgE antibody for use according to any one of claims 1 to 11, comprising an amino acid sequence defined in any one of sequence numbers 1 to 40.

13. (i) Sequence IDs 3, 4, 5, 8, 9 and 10; (ii) Sequence IDs 13, 14, 15, 18, 19 and 20; (iii) Sequence IDs 23, 24, 25, 28, 29 and 30; or (iv) Sequence IDs 33, 34, 35, 38, 39 and 40 An IgE antibody for use according to claim 12, comprising one to six CDR sequences selected from the above.

14. (i) Heavy chain variable domain sequences defined in any one of sequence numbers 2, 12, 22, or 32; (ii) Light chain variable domain sequences defined as any one of sequence numbers 7, 17, 27, or 37; (iii) Heavy chain sequences defined in any one of sequence numbers 1, 11, 21, or 31; and / or (iv) Light chain sequence defined in any one of sequence numbers 6, 16, 26, or 36 An IgE antibody for use according to claim 12 or 13, comprising:

15. An IgE antibody for use according to any one of claims 1 to 14, for use in the treatment of cancer and / or delaying the progression of cancer in a subject.

16. An IgE antibody for use according to any one of claims 1 to 15, wherein the tumor or cancer is a mammary gland tumor or breast cancer.

17. An IgE antibody for use according to any one of claims 1 to 16, which lacks a cytotoxic moiety and is preferably not an antibody-drug conjugate (ADC).

18. A method for treating cancer and / or delaying the progression of cancer in a subject having a low-HER2-expressing tumor, comprising the step of administering to the subject a therapeutically effective dose of an anti-HER2 immunoglobulin E (IgE) antibody according to any one of claims 1 to 17.

19. A pharmaceutical composition for use in the treatment of low HER2-expressing tumors in a subject, comprising an anti-HER2 immunoglobulin E (IgE) antibody according to any one of claims 1 to 17, and one or more pharmaceutically acceptable excipients, carriers, or diluents.

20. (i) Sequence IDs 13, 14, 15, 18, 19 and 20; (ii) Sequence IDs 23, 24, 25, 28, 29 and 30; or (iii) Sequence IDs 33, 34, 35, 38, 39 and 40 An immunoglobulin or functional fragment thereof containing one to six CDR sequences selected from the following.

21. Immunoglobulins, (i) Heavy chain variable domain sequences defined in any one of sequence numbers 12, 22, or 32; (ii) Light chain variable domain sequences defined in any one of sequence numbers 17, 27, or 37; (iii) Heavy chain sequences defined in any one of sequence numbers 11, 21, or 31; and / or (iv) Light chain sequence defined in any one of sequence numbers 6, 16, 26, or 36 The immunoglobulin or functional fragment thereof according to claim 20, comprising:

22. The immunoglobulin or functional fragment thereof according to claim 20 or 21, wherein the immunoglobulin is an isotype IgE immunoglobulin.

23. The immunoglobulin or functional fragment thereof according to any one of claims 20 to 22, wherein the immunoglobulin is a chimeric or humanized antibody, preferably a chimeric or humanized antibody comprising one or more human framework regions and / or one or more human IgE heavy chain and / or light chain constant domains.

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