composition

Abstract

In one aspect, the present invention relates to immunoglobulin E (IgE) antibody for use in treating and / or delaying progression of cancer in a subject, wherein the antibody has a dissociation constant (Kd) for binding to a target antigen of 10 nM or less. In another aspect, immunoglobulins that bind to HER2 are provided.

Description

[0001] COMPOSITION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of therapeutic antibodies and uses thereof and in particular to immunoglobulin E (IgE) antibodies for use in treating cancer. The present invention also relates to methods of treating diseases such as cancer using such IgE antibodies.

[0004] BACKGROUND

[0005] Therapeutic antibodies now complement conventional treatments for a number of malignant diseases, but almost all agents currently developed 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 deploys nine antibody classes and subclasses (IgM, IgD, IgGl-4, IgAl, IgA2 and IgE) to perform immune surveillance and to mediate destruction of pathogens in different anatomical compartments. Yet only IgG (most often IgGl) has been applied in immunotherapy of cancers.

[0006] One reason may be that IgG antibodies (particularly IgGl), constitute the largest fraction of circulating antibodies in human blood. The choice of antibody class is also based on pioneering work in the late 1980s, comparing a panel of chimaeric antibodies of the same specificity, each with Fc regions belonging to one of the 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. JExp Med 166: 1351-1361). Antibodies were evaluated for their ability to bind complement and their potency to mediate haemolysis and cytotoxicity of antigen-expressing target cells in the presence of complement. IgGl in combination with human peripheral blood mononuclear cells (PBMC) was the most effective IgG subclass in complement-dependent cell killing in vitro, while the IgA and IgE antibodies were completely inert.

[0007] Subsequent clinical trials with antibodies recognising the B cell marker CD20 supported the inference that IgGl would be the subclass best suited for immunotherapy of patients with B cell malignancies such as non-Hodgkin’s lymphoma (Alduaij W, Illidge TM (2011) The future of anti-CD20 monoclonal antibodies: are we making progress? Blood 117: 2993-3001). Since those studies, comparisons of anti-tumour effects by different antibody classes have been confined to IgG and IgM in both murine models and patients with lymphoid malignancies, while 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).

[0008] The HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbBl, or HER1), HER2 (ErbB2 or pl85neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

[0009] EGFR, encoded by the erb gene, has been causally implicated in human malignancy. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas. Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF-a), by the same tumor cells resulting in receptor activation by an autocrine stimulatory pathway (Baselga and Mendelsohn, Pharmac. Ther. 64:127-154 (1994)). Monoclonal antibodies directed against the EGFR or its ligands, TGF-a and EGF have been evaluated as therapeutic agents in the treatment of such malignancies (see e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research 44:1002-1007 (1984); and Wu et al. J. Clin. Invest.

[0010] 95:1897-1905 (1995)).

[0011] The second member of the HER family, i.e. HER2, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu is observed in breast and ovarian cancers and correlates with a poor prognosis (Slamon et al., Science, 235: 177-182 (1987); Slamon et al., Science, 244:707-712 (1989); and US Pat No.

[0012] 4,968,603). Overexpression of HER2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder (see e.g. Ross et al Cancer 79:2162-70 (1997); and Sadasivan et al J. Urol. 150:126-31 (1993)).

[0013] IgG antibodies directed against human HER2 protein products have been described. See, for example, Drebin et al, Cell 41:695-706 (1985) and U. S. Patent 5,824,311. Hudziak et al, Mol. Cell. Biol. 9(3): 1165-1172 (1989) describe the generation of a panel of HER2 antibodies which were characterized using the human breast tumor cell line SKBR-3. The antibody 4D5 was further found to sensitize HER2- overexpressing breast tumor cell lines to the cytotoxic effects of TNF-a (see U. S. Patent 5,677,171). A recombinant humanized version of the murine IgG anti-HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, Trastuzumab or HERCEPTIN® was shown to be effective in patients with HER2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al, J. Clin. Oncol. 14:737-744 (1996)). Trastuzumab received marketing approval from the US 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 have been described in e.g. Tagliabue et al. Int. J. Cancer 47:933-937 (1991); W094 / 00136; U. S. Patent 5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

[0014] Homology screening has resulted in the identification of other HER receptor family members, including HER3 (see US Patents 5,183,884 and 5,480,968) and HER4 (Plowman et al, Nature, 366:473-475 (1993)). Both of these receptors display increased expression on at least some breast cancer cell lines. The HER receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of HER ligands. For instance, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and results in transphosphorylation of HER2 in the heterodimer. Dimerization and / or transphosphorylation appears to activate the HER2 tyrosine kinase. See Earp et al., supra. Likewise, when HER3 is co-expressed with HER2, an active signaling complex is formed and antibodies directed against HER2 are capable of disrupting this complex (Sliwkowski et al, J. Biol. Chem., 269(20): 14661-14665 (1994)).

[0015] 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, and downstream activation of the RAS and AKT pathways. Pertuzumab binds to subdomain II (the dimerization region) of the extracellular part of the HER2 receptor, in contrast to trastuzumab, which binds to subdomain IV (the juxtamembrane region). Pertuzumab received US FDA approval for the treatment of HER2-overexpressing metastatic breast cancer in 2012. Pertuzumab may also be administered in combination with trastuzumab and docetaxel for the same indication.

[0016] Antibodies of the IgE class play a central role in allergic reactions and have many properties that may be advantageous for cancer therapy. IgE-based active and passive immunotherapeutic approaches have been shown to be effective in both in vitro and in vivo models of cancer, suggesting the potential use of these approaches in humans (Leoh et al., Curr Top Microbiol Immunol. 2015; 388: 109-149). Thus IgE therapeutic antibodies may offer enhanced immune surveillance and superior effector cell potency against cancer cells.

[0017] Karagiannis et al. 2009 (Cancer Immunol Immunother. 2009 June; 58(6): 915-930) described an engineered IgE comprising the same light- and heavy-chain variable-regions as trastuzumab IgG, but with an epsilon (i.e. IgE heavy chain constant region) in place of the IgG gamma-1 heavy chain constant region of trastuzumab. Trastuzumab IgE was shown to trigger antibodydependent cell-mediated cytotoxicity (ADCC) and trigger mast cell degranulation in the presence of HER2-expressing tumour cells, and to mediate comparable levels of tumour cell growth arrest to trastuzumab IgG.

[0018] A fully human anti-HER2 IgE has also been developed using the variable regions of the singlechain 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 FcsRI in the presence of murine mammary carcinoma cells expressing human HER2 / wew (D2F2ZE2) but not in the presence of the parental D2F2 cells that lack HER2 / wew expression or shed (soluble) extracellular domain of HER2 / wew(ECDHER2). These results suggest that anti-HER2 IgE could trigger an acute inflammatory response (type I hypersensitivity) within the tumour microenvironment, where the HER2 / wew 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.), facilitating FcsRI cross-linking and triggering effector cell degranulation.

[0019] However the relevance of antibody affinity for a target antigen in the function of IgE anti cancer antibodies has not previously been defined. In the context of allergy, it is known that IgE antibodies that bind allergens with low affinity (e.g. around 100 nM) are capable of activating mast cells and driving allergic reactions (see e.g. Chang et al., Allergy. 2021;76:2565-2574). The reason for this is thought to be that multiple IgE molecules on the surface of a single mast cell may bind individual allergens bivalently, leading to cross-linking via multiple epitopes on the allergen and high-avidity interactions. Antibody avidity represents the overall strength of the antibody-antigen interactions and is influenced by several factors, in particular multivalent binding. Thus in contrast to IgG antibodies, in the case of IgE it has been suggested that avidity rather than affinity is the key determinant of antibody potency in promoting allergic responses (see e.g. Bachmann et al., Allergy. 2024;00:1-10).

[0020] These and other properties of IgE could potentially be relevant to anti-cancer therapy using IgE antibodies. For instance, it is known that the affinity of IgE for FcsRI is typically 100- to 10,000-fold higher than that of the clinically used IgG subclasses for their Fey receptors. Additionally, the avidity of IgE for trimeric CD23 is comparable to that measured with IgG-FcyRI complexes. These properties mean that IgE can persist on immune cells in the absence of antigen complex formation. Moreover, the half-lives of IgE and IgG antibodies vastly differ in the circulation and tissues: 1.5 days for IgE and 2-3 weeks for IgG in the serum, partly due to the lack of FcRn binding by IgE. The opposite is true in tissues such as the skin, where the half-life of IgE is approximately two weeks compared with 2-3 days for IgG. Consequently compared to IgG, IgE antibodies are more likely to be transported into a tumour microenvironment and presented to cancer antigens on the surface of immune cells. If IgE antibodies are directed against cancer antigens, these features could be highly beneficial in ensuring potent effector functions, long persistence and immune surveillance at tumour sites (see Sutton et al., Antibodies 2019, 8(1), 19).

[0021] In summary, the ability of IgE antibodies to generate immune responses in the context of either allergy or cancer was thought to be linked to avidity and the high-affinity binding of IgE to Fes receptors, rather than affinity of binding to a target antigen. The role of affinity of IgE antibodies for cancer antigens in promoting anti-tumor responses had not been studied and thus was unknown. However, based on the studies described above increased affinity of IgE antibodies was not expected to lead to an increased potency in functional effects against tumor antigens.

[0022] There thus remains a need for improved treatments for cancer, having enhanced properties compared to existing IgG- and / or IgE-based treatments.

[0023] SUMMARY OF THE INVENTION

[0024] Accordingly, in one aspect the present invention provides an immunoglobulin E (IgE) antibody having a high affinity for a target antigen, e.g. a dissociation constant (Kd) for binding to a target epitope or antigen of 10 nM or less. The IgE antibody may be used for treating and / or delaying progression of cancer in a subject. In one embodiment, the antibody is an affinity-matured antibody. For example, the antibody may have an increased affinity for binding to the target epitope or antigen compared to a parent antibody from which the antibody is derived. In one embodiment, the parent antibody has a Kd for binding to the target antigen of 20 nM or higher.

[0025] In some embodiments, the affinity of the affinity matured antibody for the target antigen is increased by at least 2-fold, preferably at least 3 -fold, at least 10-fold, at least 100-fold or at least 1000-fold, compared to the parent antibody. For instance, the affinity matured antibody may have a Kd for the target antigen that is less than 50%, less than 30%, less than 10%, less than 1% or less than 0.1% of the corresponding Kd of the parent antibody for the target antigen. In some embodiments, the antibody has a Kd for binding to the target antigen of 1 pM to 10 nM, 100 pM to 5 nM or 0.5 to 3 nM.

[0026] In some embodiments, the antibody has an increased anticancer potency compared to a parent or reference antibody. In particular, the increased affinity of the antibody for binding to the target antigen may lead to an increased anticancer potency of the antibody compared to the parent antibody.

[0027] In one embodiment, the antibody binds specifically to HER2. Preferably the antibody binds to subdomain II of the extracellular domain of the HER2, more preferably the antibody at least partially competes with pertuzumab IgG for binding to HER2.

[0028] In one embodiment the antibody comprises an amino acid sequence as defined in any one of SEQ ID NO:s 1 to 27. In another embodiment, the antibody comprises one or more CDR sequences selected from SEQ ID NOs: 13 and / or 14. The antibody may further comprise CDR sequences selected from SEQ ID NO:s 3, 4, 5 and / or 8. For instance, the antibody may comprise (i) a light chain variable domain sequence as defined in SEQ ID NO: 12; and / or (ii) a light chain sequence as defined in SEQ ID NO: 11. In some embodiments, the antibody may further comprise (iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 2; and / or (iv) a heavy chain sequence as defined in SEQ ID NO: 1. Preferably the CDRs are defined according to the method of IMGT.

[0029] In another embodiment, the antibody comprises a CDR sequence as defined in SEQ ID NO: 13. The antibody may further comprises CDR sequences selected from SEQ ID NO:s 3, 4, 5, 8 and / or 10. For instance, the antibody may comprise (i) a light chain variable domain sequence as defined in SEQ ID NO: 20; and / or (ii) a light chain sequence as defined in SEQ ID NO: 19. In some embodiments, the antibody may further comprise (iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 2; and / or (iv) a heavy chain sequence as defined in SEQ ID NO: 1. Preferably the CDRs are defined according to the method of IMGT.

[0030] In another embodiment, the antibody comprises one or more CDR sequences selected from SEQ ID NOs: 23, 24, 13 and / or 27. The antibody may further comprise CDR sequences selected from SEQ ID NO:s 3 and / or 8. For instance, the antibody may comprise (i) a light chain variable domain sequence as defined in SEQ ID NO: 26; and / or (ii) a light chain sequence as defined in SEQ ID NO: 25. In some embodiments, the antibody may further comprise (iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 22; and / or (iv) a heavy chain sequence as defined in SEQ ID NO: 21. Preferably the CDRs are defined according to the method of IMGT.

[0031] In one embodiment, the parent antibody comprises an amino acid sequence as defined in any one of SEQ ID NO:s 1 to 10. In another embodiment, the parent antibody comprises one to six CDR sequences selected from SEQ ID NOs: 3, 4, 5, 8, 9 and 10, e.g. wherein the CDRs are defined according to the method of IMGT. In another embodiment, the parent antibody comprises (i) a heavy chain variable domain sequence as defined in SEQ ID NO: 2; (ii) a light chain variable domain sequence as defined in SEQ ID NO: 7; (iii) a heavy chain sequence as defined in SEQ ID NOs: 1; and / or (iv) a light chain sequence as defined in SEQ ID NOs: 6.

[0032] In one embodiment, the tumor or cancer is a breast tumor or breast cancer.

[0033] In a further aspect, the present invention provides a method for treating and / or delaying progression of cancer in a subject, the method comprising a step of administering an immunoglobulin E (IgE) antibody as defined above (e.g. having a dissociation constant (Kd) for binding to a target antigen of 10 nM or less) to the subject in a therapeutically-effective amount.

[0034] In a further aspect, the present invention provides a pharmaceutical composition for use in treating and / or delaying progression of cancer in a subject, comprising an immunoglobulin E (IgE) antibody as defined above (e.g. having a dissociation constant (Kd) for binding to a target antigen of 10 nM or less) and one or more pharmaceutically acceptable excipients, carriers or diluents.

[0035] In a further aspect, the present invention provides an immunoglobulin, or a functional fragment thereof, comprising one or more CDR sequences selected from SEQ ID NOs: 13 and / or 14. In one embodiment, the immunoglobulin further comprises one or more CDR sequences selected from SEQ ID NO:s 3, 4, 5 and / or 8. In another embodiment, the immunoglobulin comprises (i) a light chain variable domain sequence as defined in SEQ ID NO: 12; and / or (ii) a light chain sequence as defined in SEQ ID NO: 11. The immunoglobulin may further comprise (iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 2; and / or (iv) a heavy chain sequence as defined in SEQ ID NO: 1. Preferably the immunoglobulin is of isotype IgE.

[0036] In a further aspect, the present invention provides an immunoglobulin, or a functional fragment thereof, comprising a CDR sequence as defined in SEQ ID NO: 13. In one embodiment, the immunoglobulin further comprises one or more CDR sequences selected from SEQ ID NO:s 3, 4, 5, 8 and / or 10. In another embodiment, the immunoglobulin comprises (i) a light chain variable domain sequence as defined in SEQ ID NO: 20; and / or (ii) a light chain sequence as defined in SEQ ID NO: 19. The immunoglobulin may further comprise (iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 2; and / or (iv) a heavy chain sequence as defined in SEQ ID NO: 1. Preferably the immunoglobulin is of isotype IgE.

[0037] In a further aspect, the present invention provides an immunoglobulin, or a functional fragment thereof, comprising one or more CDR sequences selected from SEQ ID NOs: 23, 24, 13 and / or 27. In one embodiment, the immunoglobulin further comprises one or more CDR sequences selected from SEQ ID NO:s 3 and / or 8. In another embodiment, the immunoglobulin comprises (i) a light chain variable domain sequence as defined in SEQ ID NO: 26; and / or (ii) a light chain sequence as defined in SEQ ID NO: 25. The immunoglobulin may further comprise (iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 22; and / or (iv) a heavy chain sequence as defined in SEQ ID NO: 21. Preferably the immunoglobulin is of isotype IgE.

[0038] In further embodiments, the immunoglobulin is a chimaeric or humanized antibody, preferably comprising one or more human framework regions and / or one or more human IgE heavy and / or light chain constant domains.

[0039] In a further aspect, the present invention provides a method for increasing anti-cancer potency of an IgE antibody. The method may comprise a step of increasing affinity of the antibody for a target antigen. In one embodiment, the method comprises a step of in vitro affinity maturation of the antibody. The method may comprise increasing the affinity of the antibody for the target antigen by at least 2-fold, preferably by at least 3 -fold, at least 10-fold, at least 100-fold or at least 1000-fold.

[0040] In one embodiment, the method reduces a dissociation constant (Kd) of the antibody for binding to a target antigen, for instance to 10 nM or less, or to a value within the range of 1 pM to 10 nM, 100 pM to 5 nM or 0.5 to 3 nM. In some embodiments, the antibody has a Kd for binding to the target antigen of 20 nM or higher before performing the method. In further embodiments, the antibody obtained by the method is an antibody as described herein, e.g. in the embodiments described above or in the Examples. In some embodiments, the method increases anti -cancer potency of the IgE antibody by at least 5%, 10%, 50%, 75% or 95%.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the effects of varying concentrations of affinity-matured IgE antibody EPS 226.6, compared to a parent EPS 226 (wild type, WT) IgE antibody and anti-NIP IgE isotype control, on mast cell activation in a Hoxb8 degranulation assay. NIP refers to 4-hydroxy-3-i odo-5 -nitrophenyl acetic acid, i.e. a small molecule substrate not found in humans useful as an isotype control. EPS 226.6 IgE has an improved potency and results in an overall increase in mast cell activation compared to EPS 226 IgE.

[0043] Figure 2 shows the effects of affinity-matured IgE antibody PAP322 (EPS 232) at doses of 2 or 10 mg / kg, compared to a parent EPS 226 IgE antibody and NIP IgE isotype control, on tumour growth in female NXG mice implanted subcutaneously with SKBR3 cells. Data are expressed as mean tumour growth inhibition at day 28 + / - sem of n=10-12 mice per group (3 PBMC donors). One-way ANOVA performed on raw data and statistical comparison to PBS control performed. **** p<0.0001, ** p<0.01, no star is not statistically significant. EPS 232 at both 2mg / kg and lOmg / kg delivered a significantly greater tumour growth inhibition compared to EPS 226.

[0044] Figure 3 shows the effects of affinity-matured IgE antibody PAP322 (EPS 232) at doses of 2 or 10 mg / kg, compared to a parent EPS 226 IgE antibody and NIP IgE isotype control, on tumour growth in female Fischer rats implanted subcutaneously with MTLn3 cells.

[0045] DETAILED DESCRIPTION OF THE INVENTION It has surprisingly been found herein that IgE antibodies with enhanced affinity for target antigen are capable of producing improved antitumor responses. In particular, affinity-matured IgE antibodies showed improved potency and greater activity against tumor cells both in vitro and in vivo compared to a reference (parent) antibody from which they were derived. This represents the first demonstration that affinity maturation can be employed to enhance the therapeutic potential of IgE antibodies.

[0046] This effect was particularly unexpected because in the case of IgE, it was previously thought that avidity for a target antigen and the high-affinity binding of IgE to Fes receptors were the main determinants of potency. Monovalent affinity for a target antigen was not thought to be relevant to anti-cancer effects of IgE in vivo, because low-affinity IgE antibodies were known to be capable of potently stimulating effector functions via high-avidity interactions with antigen. Moreover, the high-affinity binding of IgE to Fes receptors, even in the absence of antigen, suggested that IgE is transported to and persists in the tumor microenvironment on the surface of immune cells, leading to increased presentation to cancer antigens compared to IgG. Accordingly, it was thought that high affinity for antigen was not necessary for IgE anticancer activity, and might even be detrimental due to increased binding of antibody to non-tumour targets.

[0047] Antibody

[0048] Antibodies are polypeptide ligands comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and specifically binds an epitope of an antigen, such as HER2, or a fragment thereof. Antibodies are typically composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.

[0049] Antibodies include intact immunoglobulins and the variants and portions of antibodies well known in the art, provided that such fragments retain at least one function of IgE, e.g. are capable of binding an Fee receptor. Antibodies also include genetically engineered forms such as chimaeric, humanized (for example, humanized antibodies with murine sequences contained in the variable regions) or human antibodies, heteroconjugate antibodies (such as, bispecific antibodies), e.g. as described in Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (k). There are nine main isotypes or classes which determine the functional activity of an antibody molecule: IgAl-2, IgD, IgE, IgGl-4 and IgM, corresponding to the heavy chain types a, 5, e, y, and p. Thus, the type of heavy chain present defines the class of antibody. Distinct heavy chains differ in size and composition; a and y contain approximately 450 amino acids, while p and e have approximately 550 amino acids. The differences in the constant regions of each heavy chain type account for the different effector functions of each antibody isotype, by virtue of their selective binding to particular types of receptor (e.g. Fc receptors). Accordingly, in embodiments of the present invention the antibody preferably comprises an epsilon (e) heavy chain, i.e. the antibody is of the isotype IgE which binds to Fee receptors.

[0050] Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The extent of the framework region and CDRs has been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U. S. Department of Health and Human Services, 1991). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0051] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

[0052] Antibodies may have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). References to “VH” refer to the variable region of an immunoglobulin heavy chain. References to “VL” refer to the variable region of an immunoglobulin light chain.

[0053] A “monoclonal antibody” is an antibody produced by a single clone of B -lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0054] A “chimaeric antibody” comprises sequences derived from two different antibodies, which are typically derived from different species. For example, chimaeric antibodies may include human and murine antibody domains, e.g. human constant regions and murine variable regions (e.g. from a murine antibody that specifically binds to a target antigen).

[0055] Chimaeric antibodies are typically constructed by fusing variable and constant regions, e.g. by genetic engineering, from light and heavy chain immunoglobulin genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and epsilon. In one example, a therapeutic chimaeric antibody is thus a hybrid protein composed of the variable or antigenbinding domain from a mouse antibody and the constant or effector domain from a human antibody, e.g. an Fc (effector) domain from a human IgE antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimaeric antibodies are well known in the art, e.g., see U. S. Pat. No. 5,807,715.

[0056] A “humanized” antibody is an antibody including human framework regions and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) antibody. The non-human immunoglobulin providing the CDRs is termed a “donor”, and the human immunoglobulin providing the framework is teamed an “acceptor”. In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. The constant regions are typically substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody typically comprises a humanized immunoglobulin light chain and a humanized immunoglobulin heavy chain. A humanized antibody typically binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

[0057] Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U. S. Pat. No. 5,585,089). Typically humanized monoclonal antibodies are produced by transferring donor antibody complementarity determining regions from heavy and light variable chains of a mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the donor counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. Techniques for producing humanized monoclonal antibodies are described, for example, by 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. U. S. A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.

[0058] A “human” antibody (also called a “fully human” antibody) is an antibody that includes human framework regions and all of the CDRs from a human immunoglobulin. In one example, the framework and the CDRs are from the same originating human heavy and / or light chain amino acid sequence. However, frameworks from one human antibody can be engineered to include CDRs from a different human antibody.

[0059] In embodiments of the present invention, the antibodies may be monoclonal or polyclonal antibodies, including chimaeric, humanized or fully human antibodies.

[0060] Target antigen

[0061] The antibodies described herein may bind specifically (i.e. via their variable domains or the complementarity determining regions (CDRs) thereof) to one or more target antigens useful in treating cancer. For instance, the antibodies may bind specifically to one or more cancer antigens (i.e. antigens expressed selectively or overexpressed on cancer cells). The antibodies may enhance cytotoxicity, phagocytosis (e.g. ADCC and / or ADCP) and other cancer cell- killing function of immune system cells (e.g. monocytes / macrophages and natural killer cells). For example, the antibodies may bind specifically e.g. to EGF-R (epidermal growth factor receptor), VEGF (vascular endothelial growth factor) or erbB2 receptor (Her2 / neu). One example of an antibody comprising variable domains that bind selectively to Her2 / neu is trastuzumab (Herceptin).

[0062] Anti-HER2 antibodies

[0063] In preferred embodiments, the antibody or immunoglobulin binds specifically to HER2 to form an immune complex. Typically the antibody or immunoglobulin may comprise an antigenbinding region (e.g. one or more variable regions, or one to 6 CDRs) derived from an antibody which is known to bind HER2, preferably human HER2.

[0064] “HER2” refers to human epidermal growth factor receptor 2. HER2 may also be referred to as receptor tyrosine-protein kinase erbB-2 or CD340 (cluster of differentiation 340). HER2 is encoded in humans by the erbB2 (erythroblastic oncogene B2) or neu gene. Amino acid and nucleotide sequences encoding human HER2 / erZ> B2 are described in public databases and available e.g. under database accession numbers P04626-1 (UniProt), NM_001005862.3 and NP_001005862.1 (NCBI Ref Seq).

[0065] In one specific embodiment, the antibody comprises a part of a variable region (e.g. a heavy chain variable domain (VH) and / or a light chain variable domain (VL)) or one, two, three, four or five CDRs (e.g. up to 3 heavy chain CDRs or up to 3 light chain CDRs) derived from any known anti-HER2 IgG or IgE antibody. Preferably the antibody comprises at least one CDR (e.g. one light chain CDR or one heavy chain CDR) as described herein, e.g. a modified CDR sequence obtained by affinity maturation of a known IgE antibody. CDR sequences may be defined according to the method of Kabat, Chothia or IMGT (see e.g. Dondelinger, Front Immunol. 2018; 9: 2278 and references cited therein, which are incorporated herein by reference). For instance, CDRs may be defined according to Kabat: see Kabat EA, et al. (U. S.) NI of H. Sequences of Immunoglobulin Chains: Tabulation Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain BP-Mi croglobulins, 1979; or according to 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 according to IMGT: see Giudicelli V et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res.

[0066] 1997 Jan 1; 25(l):206-ll or Lefranc MP, Unique database numbering system for immunogenetic analysis, Immunol Today. 1997 Nov; 18(11):509. In one embodiment, the antibody or immunoglobulin comprises at least one, two, three, four or five CDRs (e.g. up to 3 heavy chain CDRs or up to 3 light chain CDRs) derived from EPS 226 IgE as described herein. For instance, the antibody or immunoglobulin may comprise at least one, two, three, four or five CDRs (e.g. up to 3 heavy chain CDRs or up to 3 light chain CDRs) selected from SEQ ID NOs: 3, 4, 5, 8, 9 and 10. In this embodiment, the CDRs may be defined according to the method of IMGT (supra).

[0067] In another embodiment, the antibody or immunoglobulin comprises at least one, two, three, four or five CDRs (e.g. up to 3 heavy chain CDRs or up to 3 light chain CDRs) present in any one or more of SEQ ID NOs: 1, 2, 6 or 7, e.g. wherein the CDRs are defined according to the method of IMGT. Preferably the antibody or immunoglobulin comprises at least one CDR sequence (e.g. two or three CDR sequences) present in SEQ ID NO: 11 or SEQ ID NO: 12.

[0068] In each of the above embodiments, the antibody preferably comprises at least one of the CDR sequences of SEQ ID NOs: 13, 14, 23, 24 and / or 27, e.g. wherein the CDRs are defined according to the method of IMGT. In one embodiment, the antibody comprises at least one of the CDR sequences of SEQ ID NOs: 13 and / or 14. In one embodiment, the antibody comprises the CDR sequences of SEQ ID NOs: 13 and / or 14 in combination with SEQ ID NO:s 3, 4, 5 and / or 8, e.g. wherein the CDRs are defined according to the method of IMGT.

[0069] In one embodiment, the antibody comprises a CDR sequence as defined in SEQ ID NO: 13. In one embodiment, the antibody comprises the CDR sequence of SEQ ID NO: 13 in combination with SEQ ID NO:s 3, 4, 5, 8 and / or 10 e.g. wherein the CDRs are defined according to the method of IMGT.

[0070] In another embodiment, the antibody or immunoglobulin comprises (i) a heavy chain variable domain sequence as defined in SEQ ID NO: 2; and / or (ii) a heavy chain sequence as defined in SEQ ID NO: 1. In these embodiments, the antibody preferably comprises (iii) a light chain variable domain sequence as defined in SEQ ID NO: 12 or 20; and / or (iv) a light chain sequence as defined in SEQ ID NO: 11 or 19.

[0071] For instance, the antibody or immunoglobulin may comprise six CDRs as defined in SEQ ID NOs: 3, 4, 5, 8, 13 and 14, e.g. wherein the CDRs are defined according to the method of IMGT; a heavy chain variable domain as defined in SEQ ID NO:2 and a light chain variable domain sequence as defined in SEQ ID NO: 12; and / or a heavy chain sequence as defined in SEQ ID NO: 1 and a light chain sequence as defined in SEQ ID NO: 11. In another embodiment, the antibody or immunoglobulin may comprise six CDRs as defined in SEQ ID NOs: 3, 4, 5, 8, 13 and 10, e.g. wherein the CDRs are defined according to the method of IMGT; a heavy chain variable domain as defined in SEQ ID NO:2 and a light chain variable domain sequence as defined in SEQ ID NO: 20; and / or a heavy chain sequence as defined in SEQ ID NO: 1 and a light chain sequence as defined in SEQ ID NO: 19.

[0072] In another embodiment, the antibody or immunoglobulin comprises (i) a heavy chain variable domain sequence as defined in SEQ ID NO: 22; and / or (ii) a heavy chain sequence as defined in SEQ ID NO: 21. In these embodiments, the antibody preferably comprises (iii) a light chain variable domain sequence as defined in SEQ ID NO: 26; and / or (iv) a light chain sequence as defined in SEQ ID NO: 25.

[0073] For instance, the antibody or immunoglobulin may comprise six CDRs as defined in SEQ ID NOs: 3, 23, 24, 8, 13 and 27, e.g. wherein the CDRs are defined according to the method of IMGT; a heavy chain variable domain as defined in SEQ ID NO:22 and a light chain variable domain sequence as defined in SEQ ID NO: 26; and / or a heavy chain sequence as defined in SEQ ID NO: 21 and a light chain sequence as defined in SEQ ID NO: 25.

[0074] In another embodiment, the antibody or immunoglobulin is a chimaeric, humanized or fully human antibody that specifically binds an epitope bound by a known antibody, e.g. EPS 226 IgE. EPS 226 IgE binds to subdomain II of the extracellular domain of the HER2 and competes with pertuzumab IgG for binding to HER2. Thus the antibody or immunoglobulin may (at least partially) compete with EPS 226 IgE 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 (e.g. at least partially) overlapping with that of a known anti-HER2 antibody, or to an epitope in the vicinity of that of a known anti-HER2 antibody (e.g. pertuzumab or EPS 226 IgE). Thus in competition assays, the (IgE) antibody or immunoglobulin may show partial or complete competition with a known antibody (e.g. pertuzumab or EPS 226 IgE) for binding to a target antigen (e.g. HER2).

[0075] Assays for determining competitive binding of antibodies to target antigens, as well as other methods for quantifying binding of antibodies to HER2 are well 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; Rudkouskaya et al., Molecules. 2020 Dec; 25(24): 5976. The antibody or immunoglobulin may bind e.g. to 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, e.g. rat and / or mouse HER2.

[0076] Accordingly, in a preferred embodiment the antibody or immunoglobulin binds to subdomain II of the extracellular domain of the HER2 and / or (at least partially) competes with pertuzumab IgG for binding to HER2, e.g. the antibody or immunoglobulin comprises at least one, two, three, four or five CDRs selected from SEQ ID NOs: 3, 4, 5, 8, 9 and 10 and at least one CDR selected from SEQ ID NOs 13, 14, 23, 24 and / or 27; a heavy chain variable domain as defined in SEQ ID NO:2 or 22 and a light chain variable domain sequence as defined in SEQ ID NO: 12, 20 or 26; and / or a heavy chain sequence as defined in SEQ ID NO: 1 or 21 and a light chain sequence as defined in SEQ ID NO: 11, 19 or 25.

[0077] Most preferably, the antibody or immunoglobulin comprises a chimaeric, humanized or fully human antibody that binds to subdomain II of the extracellular domain of the HER2 and / or (at least partially) competes with pertuzumab IgG for binding to HER2. For instance, the antibody or immunoglobulin may be a humanized antibody comprising at least one, two, three, four or five or CDRs selected from SEQ ID NOs: 3, 4, 5, 8, 9 and 10, at least one CDR selected from SEQ ID NOs 13, 14, 23, 24 and / or 27, and one or more human framework regions and / or one or more human IgE heavy and / or light chain constant domains. Alternatively the antibody or immunoglobulin may be a chimaeric antibody comprising a heavy chain variable domain as defined in SEQ ID NO:2 or 22 and a light chain variable domain sequence as defined in SEQ ID NO: 12, 20 or 26 and one or more human IgE heavy and / or light chain constant domains.

[0078] In one embodiment, the antibody comprises one or more human constant regions, e.g. one or more human heavy chain constant domains (e.g. e constant domains) and / or a human light chain (e.g. K or X constant domain. An amino acid sequence of a human heavy chain constant domain is shown in SEQ ID NO: 15 (non-bold and non-underlined text present in SEQ ID NO:1). An amino acid sequence of a human light (K) chain constant domain is shown in SEQ ID NO: 16 (non-bold and non-underlined text present in SEQ ID NO:6). In one embodiment the antibody comprises one or more human framework regions within the VH and / or VL domains.

[0079] In one embodiment, the sequence of a humanized immunoglobulin heavy chain variable region framework can 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 can 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. Human framework regions, and mutations that can be made in a humanized antibody framework regions, are known in the art (see, for example, U. S. Pat. No. 5,585,089).

[0080] Further antibodies against a specific antigen, e.g. HER2, may also be generated by well-established methods, and at least the variable regions or CDRs from such antibodies may be used in the antibodies of the present invention (e.g. the generated antibodies may be used to donate CDR or variable region sequences into IgE acceptor sequences). Methods for synthesizing polypeptides and immunizing a host animal are well known in the art. Typically, the host animal (e.g. a mouse) is inoculated intraperitoneally with an amount of immunogen (e.g. HER2 or a polypeptide comprising an immunogenic fragment thereof), and (in the case of monoclonal antibody production) hybridomas prepared from its lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497.

[0081] The sequence of human HER2 is well known (see e.g. UniProt database accession no. P04626-1) and thus human HER2 may, for example, be purified from a natural source or expressed using recombinant techniques for use in such methods. The amino acid and nucleic acid sequences of human HER2 are shown below in SEQ ID NO:s 17 and 18 respectively:

[0082] SEQ ID NO: 17 - Human HER2 amino acid sequence:

[0083] 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 SEQ ID NO: 18 - Human HER2 nucleic acid sequence:

[0084] 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 gggccccaag 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 gcccgagtgt 1501 gctatggtct gggcatggag cacttgcgag aggtgagggc agttaccagt gccaatatcc 1561 aggagtttgc tggctgcaag aagatctttg ggagcctggc atttctgccg gagagctttg 1621 atggggaccc 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 ctgcccccca tgaggaagga acagcaatgg 4681 tgtcagtatc caggctttgt acagagtgct tttctgttta gtttttactt tttttgtttt 4741 gtttttttaa agatgaaata aagacccagg gggagaatgg gtgttgtatg gggaggcaag 4801 tgtggggggt ccttctccac acccactttg tccatttgca aatatatttt ggaaaaca

[0085] Hybridomas that produce suitable antibodies may be grown in vitro or in vivo using known procedures. Monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen. If desired, the antibody (monoclonal or polyclonal) of interest may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use.

[0086] Phage display technology, for instance as described in US 5,565,332 and other published documents, may be used to select and produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors (e.g. from human subjects, including patients suffering from a relevant disorder). For example, existing antibody phage display libraries may be panned in parallel against a large collection of synthetic polypeptides. According to this technique, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus antibody sequences selected using phage display from human libraries may include human CDR or variable region sequences conferring specific binding to a specific antigen such as HER2, which may be used to provide fully human antibodies for use in the present invention.

[0087] Methods for deriving heavy and light chain sequences from human B cell and plasma cell clones are also well known in the art and typically performed using polymerase chain reaction (PCR) techniques, examples of the methods are described in: Kuppers R, Methods Mol Biol.

[0088] 2004;271:225-38; Yoshioka M et al., BMC Biotechnol. 2011 Jul 21; 11:75; Scheeren FA et al., PLoS ONE 2011, 6(4): el7189. 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; 329(1-2): 112-124. Thus antibody sequences selected using B cell clones may include human CDR or variable region sequences conferring specific binding to e.g. HER2, which may be used to provide fully human antibodies for use in the present invention.

[0089] IgE antibodies

[0090] The therapeutic antibody to be administered to the subject may be an IgE antibody, i.e. an antibody of the isotype IgE. There are some fundamental structural differences between IgEs and IgGs, and these have functional effects. While IgE shares the same basic molecular architecture as antibodies of other classes, the heavy chain of IgE contains one more domain than the heavy chain of IgG. The Cs3 and Cs4 domains of IgE are homologous in sequence, and similar in structure, to the Cy2 and Cy3 domains of IgG, so that it is the Cs2 domains that are the most obvious distinguishing feature of IgE. The Cs2 domain has been found to be folded back against the heavy chain IgE and to make extensive contact with the Cs3 domain. This bent structure of the IgE heavy chain allows it to adopt an open or closed conformation. The unbound IgE dimer has one chain in the open and one chain in the closed conformation. Binding of FcsRI to IgE is biphasic and is thought to involve initial binding to the open Cs chain followed by extensive structural rearrangement to allow binding to the closed Cs chain. The binding between the IgE dimer and the FcsRI occurs with 1:1 stoichiometry despite the presence of two identical Cs-chains. This rearrangement results in a very tight interaction between IgE and FcsRI, and a much greater affinity of IgE for its Fc receptor than found with IgG and FcyRs (McDonnell, J. M., R. Calvert, et al. (2001) Nat Struct Biol 8(5): 437-441). The antibodies used in the present invention are typically capable of binding to Fee receptors, e.g. to the FceRI and / or the FceRII receptors. Preferably the antibody is at least capable of binding to FceRI (i.e. the high affinity Fee receptor) or is at least capable of binding to FceRII (CD23, the low affinity Fee receptor). Typically the antibodies are also capable of activating Fee receptors, e.g. expressed on cells of the immune system, in order to initiate effector functions mediated by IgE.

[0091] The epsilon (e) heavy chain is definitive for IgE antibodies, and comprises an N-terminal variable domain VH, and four constant domains Cel -Ce4. As with other antibody isotypes, the variable domains confer antigen specificity and the constant domains recruit the isotype-specific effector functions.

[0092] IgE differs from the more abundant IgG isotypes, in that it is unable to fix complement and does not bind to the Fc receptors FcyRI, RII and RIII expressed on the surfaces of mononuclear cells, NK cells and neutrophils. However, IgE is capable of very specific interactions with the “high affinity” IgE receptor on a variety of immune cells such as mast cells, basophils, monocytes / macrophages, eosinophils (FceRI, Ka. 1011M-1), and with the “low affinity” receptor, Fee RII (Ka. 107M-1), also known as CD23, expressed on inflammatory and antigen presenting cells (e.g. monocytes / macrophages, platelets, dendritic cells, T and B lymphocytes.

[0093] The sites on IgE responsible for these receptor interactions have been mapped to peptide sequences on the Ce chain, and are distinct. The FceRI site lies in a cleft created by residues between Gin 301 and Arg 376, and includes the junction between the Ce2 and Ce3 domains (Helm, B. et al. (1988) Nature 331, 180183). The FceRII binding site is located within Ce3 around residue Vai 370 (Vercelli, D. et al. (1989) Nature 338, 649-651). A major difference distinguishing the two receptors is that FceRI binds monomeric Ce, whereas FceRII will only bind dimerised Ce, i.e. the two Ce chains must be associated. Although IgE is glycosylated in vivo, this is not necessary for its binding to FceRI and FceRRII. Binding is in fact marginally stronger in the absence of glycosylation (Vercelli, D. et al. (1989) et. Supra).

[0094] Thus binding to Fee receptors and related effector functions are typically mediated by the heavy chain constant domains of the antibody, in particular by domains which together form the Fc region of the antibody. The antibodies described herein typically comprise at least a portion of an IgE antibody e.g. one or more constant domains derived from an IgE, preferably a human IgE. In particular embodiments, the antibodies comprise one or more domains (derived from IgE) selected from Cel, Ce2, Ce3 and Ce4. In one embodiment, the antibody comprises at least Ce2 and Ce3, more preferably at least Ce2, Ce3 and Ce4, preferably wherein the domains are derived from a human IgE. In one embodiment, the antibody comprises an epsilon (e) heavy chain, preferably a human e heavy chain.

[0095] The amino acid sequences of constant domains derived from human IgE are shown in e.g. Table 1 (SEQ IDNO:s 15 and 16, non-bold text in SEQ ID Nos: 1 and 6). Nucleotide sequences encoding constant domains derived from human IgE, in particular Cel, Ce2, Ce3 and Ce4 domains, are also disclosed in e.g. WO 2013 / 050725. The amino acid sequences of other human and mammalian IgEs and domains thereof, including human Cel, Ce2, Ce3 and Ce4 domains and human e heavy chain sequences, are known in the art and are available from public-accessible databases. For instance, databases of human immunoglobulin sequences are accessible from the International ImMunoGeneTics Information System (IMGT®) website at http: / / www.imgt.org. As one example, the sequences of various human IgE heavy (e) chain alleles and their individual constant domains (Cel -4) are accessible at http: / / www.imgt.org / IMGT_GENE-DB / GENElect? query=2+IGHE&species=Homo+sapiens.

[0096] Preferred anti-HER2 IgE antibodies and variants / fragments thereof

[0097] In one embodiment, the anti-HER2 antibody comprises a VH domain comprising at least a portion of the amino acid sequence as defined in SEQ ID NO: 2 or 22, e.g. comprising at least 20, 30, 50 or 100 amino acids of SEQ ID NO: 2 or 22, or the full length of SEQ ID NO: 2 or 22, or one, two or three CDRs present in SEQ ID NO: 2 or 22 (e.g. defined according to Kabat, Chothia or IMGT).

[0098] In one embodiment, the anti-HER2 antibody comprises a VL domain comprising at least a portion of the amino acid sequence as defined in SEQ ID NO: 12, 20 or 26 e.g. comprising at least 20, 30, 50 or 100 amino acids of SEQ ID NO: 12, 20 or 26 or the full length of SEQ ID NO: 12, 20 or 26 or one, two or three CDRs present in SEQ ID NO: 12, 20 or 26 (e.g. defined according to Kabat, Chothia or IMGT).

[0099] In general, functional fragments of the sequences defined above may be used in the present invention. Functional fragments may be of any length as specified above (e.g. at least 50, 100, 300 or 500 nucleotides, or at least 50, 100, 200 or 300 amino acids), provided that the fragment retains the required activity when present in the antibody (e.g. specific binding to a target antigen such as HER2 and / or a Fee receptor). Variants of the above amino acid and nucleotide sequences may also be used in the present invention, provided that the resulting antibody binds an Fee receptor. Typically such variants have a high degree of sequence identity with one of the sequences specified above.

[0100] The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of the amino acid or nucleotide sequence will possess a relatively high degree of sequence identity when aligned using standard methods.

[0101] Methods of alignment of sequences for comparison are well known in the 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. U. S. A. 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. U. S. A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0102] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0103] Homologs and variants of the antibody (e.g. anti-HER2 antibody or a domain thereof, e.g. a VL, VH, CL or CH domain) typically have at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the original sequence (e.g. a sequence defined above), for example counted over the full length alignment with the amino acid sequence of the antibody or domain thereof using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0104] Typically variants may contain one or more conservative amino acid substitutions compared to the original amino acid or nucleic acid sequence. Conservative substitutions are those substitutions that do not substantially affect or decrease the affinity of an antibody to the target antigen (e.g. HER2) and / or Fee receptors. For example, a human antibody that specifically binds HER2 may include up to 1, up to 2, up to 5, up to 10, or up to 15 conservative 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 a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody specifically binds the target antigen (e.g. HER2). Non-conservative substitutions are those that reduce an activity or binding to the target antigen (e.g. HER2) and / or Fee receptors.

[0105] Functionally similar amino acids which may be exchanged by way of conservative substitution are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for 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).

[0106] Thus the antibody may comprise, consist of or consist essentially of (optionally glycosylated) polypeptide chains. For instance the antibody may comprise, consist of or consist essentially of one or more (preferably four) polypeptide chains, e.g. two immunoglobulin heavy chains and optionally two immunoglobulin light chains. Preferably the heavy and / or light chains comprises one or more domains from an IgE antibody. Further IgE antibodies

[0107] As described above, in preferred embodiments the IgE antibody binds to HER2. Preferably the IgE antibodies are capable of inducing cytotoxicity (e.g. ADCC) and / or phagocytosis (ADCP), particularly against cancer cells expressing such an antigen.

[0108] In another embodiment, the antibody is a chimaeric, humanized or fully human antibody that specifically binds the epitope bound by one of the above antibodies. The IgE antibody may further comprise one or more IgE constant domains, e.g. Cel-Ce4 domains, as described above.

[0109] In alternative embodiments, the IgE antibodies of the present application can exhibit binding affinity and specificity towards one or more other antigens associated with cancer cells. For example, in one or more embodiments, the antibodies can exhibit affinity and specificity towards cancer-related antigens including but not limited to HER1 (anti-HERl antibodies), HER3 (anti-HER3 antibodies), HER4 (anti-HER4 antibodies), EGFR (anti-EGFR antibodies), VEGFR (anti-VEGFR antibodies), CD47 (anti- CD47 antibodies), FGFR (anti-FGFR antibodies), carcinoembryonic antigen (CEA) (anti-CEA antibodies), Bladder Tumor Antigen (BTA) (anti-BTA antibodies), CA125 (anti-CA125 antibodies), PDGFR (anti-PDGFR antibodies), IGFR (anti-IGFR antibodies), CAI 5- 3 / CA27.29 (anti-CA15-3 / CA27.29 antibodies), CAI 9-9 (anti-CA19-9 antibodies), CA27.29 (anti-CA27.29-antibodies), programmed death ligand 1 (PD-L1) (anti-PD-Ll antibodies), PD- L2 (anti-PD-L2 antibodies) CTL4 (anti-CTL4 antibodies), CD3 (anti-CD3 antibodies), CD 19 (anti-CD19 antibodies), CD20 (anti-CD20 antibodies), CD22 (anti-CD22 antibodies), CD25 (anti-CD25 antibodies), CD27 (anti-CD27 antibodies), CD30 (anti-CD30 antibodies), CD33 (anti-CD33 antibodies), CD37 (anti-CD37 antibodies), CD38 (anti-CD38 antibodies), CD40 (anti-CD40 antibodies), CD48 (anti-CD48 antibodies), CD52 (anti-CD52 antibodies), B7-H3 (anti-B7-H3 antibodies), TIM-3 (anti-TIM-3 antibodies), LAG-3 (anti-LAG-3 antibodies), V- domain Ig suppressor of T cell activation (VISTA) (anti- VISTA antibodies), HVEM (anti- HVEM antibodies), ICOS (anti-ICOS antibodies), 4- IBB (anti-4-lBB antibodies), 0X40 (anti-OX40 antibodies), RANKE (anti-RANKL antibodies) and GITR (anti-GITR antibodies), epithelial and mesenchymal markers of circulating tumor cells, Prostatic Acid Phosphatase (PAP) (anti -PAP antibodies), prostate-specific antigen (PSA) (anti-PSA antibodies), soluble mesothelin-related peptides (SMRP) (anti-SMRP antibodies), somatostatin receptor (SR) (anti- SR antibodies), Urokinase plasminogen activator (uPA) (anti-uPA antibodies), plasminogen activator inhibitor (PAI-1) (anti -P Al- 1 antibodies), TCR (e.g., MHC class I or class II molecules) (anti-MHC I / II antibodies), A2a Receptor (anti-A2aR antibodies), MICA family (anti-MICA / B antibodies), RAET1 / ULBP family (anti-RAETl / ULBP antibodies), or HLA-E (anti-HLA-E antibodies).

[0110] Production of antibodies and nucleic acids

[0111] Nucleic acid molecules (also referred to as polynucleotides) encoding the polypeptides provided herein (including, but not limited to antibodies and functional fragments thereof) can readily be produced by one of skill in the art, using the amino acid sequences provided herein, sequences available in the art, and the genetic code. In addition, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same effector molecule or antibody sequence. Thus, nucleic acids encoding antibodies are provided herein.

[0112] Nucleic acid sequences encoding the antibodies that specifically bind the target antigen (e.g. HER2), or functional fragments thereof, can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts.

[0113] 22(20): 1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham -VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U. S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

[0114] Exemplary nucleic acids encoding antibodies, or functional fragments thereof, can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found see, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989); and Current Protocols in Molecular Biology (Ausubel et al., eds 1995 supplement)). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R& D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N. J.), 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 one of skill.

[0115] Nucleic acids encoding native antibodies can be modified to form the antibodies described herein. Modification by site-directed mutagenesis is well known in the art. Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3 SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

[0116] In one embodiment, antibodies are prepared by inserting a cDNA which encodes one or more antibody domains (e.g. a mouse IgGl heavy chain variable region which binds human HER2) into a vector which comprises a cDNA encoding one or more further antibody domains (e.g. a human heavy chain e constant region). The insertion is made so that the antibody domains are read in frame that is in one continuous polypeptide which contains a functional antibody region.

[0117] In one embodiment, cDNA encoding a heavy chain constant region is ligated to a 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 regions can subsequently be ligated to a light chain variable and / or constant region of the antibody using disulfide bonds.

[0118] Once the nucleic acids encoding the antibody or functional fragment thereof have been isolated and cloned, the desired protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of proteins including E. coh. other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

[0119] One or more DNA sequences encoding the antibody or fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.

[0120] The expression of nucleic acids encoding the isolated antibodies and antibody fragments described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

[0121] To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator. For E. coh. this includes a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and / or an enhancer derived from, for example, an immunoglobulin gene, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and acceptor sequences. The cassettes can be transferred into the chosen host cell by well-known methods such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

[0122] When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding the antibody, labelled antibody, or functional fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One of skill in the art can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

[0123] Modifications can be made to a nucleic acid encoding a polypeptide described herein (e.g., a human HER2-specific IgE antibody) without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps. In addition to recombinant methods, the antibodies of the present disclosure can also be constructed in whole or in part using standard peptide synthesis well known in the art.

[0124] Once expressed, the recombinant antibodies can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N. Y., 1982). The antibodies, immunoconjugates and effector molecules need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

[0125] Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, as is well known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, and especially as described by Buchner et al., supra. Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0126] As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. Excess oxidized glutathione or other oxidizing low molecular weight compounds can be added to the refolding solution after the redox-shuffling is completed.

[0127] In addition to recombinant methods, the antibodies, labelled antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by 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. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments.

[0128] Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N’ -di cylohexylcarbodimide) are well known in the art.

[0129] In one embodiment, the antibodies, nucleic acids, expression vectors, host cells or other biological products are isolated. By “isolated” it is meant that the product has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and antibodies that have been “isolated” include nucleic acids and antibodies purified by standard purification methods. The term also embraces nucleic acids and antibodies prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0130] High affinity antibodies In embodiments of the present invention, the IgE antibody binds to a target antigen with high affinity. Typically by this it is meant that the antibody binds to the target antigen (e.g. HER2) with a dissociation constant (Kd) of 10 nM or less. For instance, the Kd of the antibody / antigen binding interaction may be less than 8nM, less than 5 nM, less than 3 nM, less than 1 nM, less than 0.5 nM, less than 300 pM, less than 100 pM or less than 10 pM. The antibody may bind to the target antigen with a Kd of at least 1 pM, e.g. at least 10 pM, at least 100 pM, at least 500 pM or at least 1 nM. Thus in preferred embodiments, the Kd of the antibody / antigen interaction may be in the range of e.g. 1 pM to 10 nM, 10 pM to 8 nM, 100 pM to 5 nM, 500 pM to 3 nM, or 1 nM to 3 nM.

[0131] As is known in the art, “affinity” is a measure of the tightness with a particular ligand (e.g., an antibody) binds to its partner (e.g., an epitope on an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koff and kon, respectively). The Kd of an antibody for its target epitope is inversely related to its affinity constant (Ka). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Antibody affinity may be measured using standard techniques such as by immunoassay, whole cell panning, surface plasmon resonance (SPR), e.g. using standard instrumentation such as a BIAcore instrument (GE Healthcare) and biolayer interferometry (BLI). The immunoassay can be a radioimmunoassay, enzyme linked immunosorbent assay (ELISA) or electrochemiluminescence assay. Antibody affinity may be measured under standard conditions (e.g. as described in the BLI study in the Examples below).

[0132] Methods for increasing affinity and potency

[0133] In one aspect, the present invention provides a method for increasing anti-cancer potency of an IgE antibody, by increasing affinity of the antibody for a target antigen. The method is typically performed in vitro. In general, any method for increasing antibody affinity may be employed, e.g. in vitro affinity maturation as described below. Such methods typically lead to an improved potency of the IgE, e.g. in assays of anticancer effects either in vitro and / or in vivo, for example in animal or human cells and / or animal or human subjects.

[0134] By “improved potency” it is typically meant that the antibody provides an anticancer effect at a lower concentration compared to a reference or parent antibody. In some embodiments, the antibody may produce an increased anticancer effect compared to a reference or parent antibody when tested at the same concentration.

[0135] Potency of an antibody may be quantified by determining standard parameters such as EC50, i.e. the half maximal effective concentration (a concentration of antibody that produces an effect 50% of a maximum, or halfway between a baseline and maximum). In certain cases, potency may be indicated by IC50, i.e. the half maximal inhibitory concentration (a concentration of antibody that produces 50% inhibition of an undesirable or cancer-promoting effect).

[0136] Thus in embodiments of the present invention, the method reduces an EC50 of the antibody compared to a reference antibody, e.g. in an assay of an anticancer effect, and / or the method reduces an IC50 of the antibody compared to a reference antibody, e.g. in an assay of an undesirable or cancer-promoting effect. Preferably potency is increased by at least 5%, 10%, 50%, 75%, 90%, 95% or 99% compared to the reference antibody, e.g. the EC50 or IC50% of the antibody is reduced by at least 5%, 10%, 75%, 90%, 95% or 99% compared to the reference antibody.

[0137] Some suitable assays are described in the Examples below, involving e.g. activation of immune cell responses, such as mast cell degranulation, antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or inhibition of tumor cell survival or tumor cell killing.

[0138] Affinity maturation

[0139] In preferred embodiments, the antibody is an affinity matured antibody. As used herein, the term “affinity matured” in the context of antibodies refers to an antibody that is derived from a reference (parent) antibody, e.g., by mutation, and binds to the same antigen, preferably binds to the same epitope, as the reference antibody; and has a higher affinity for the antigen than that of the reference (parent) antibody. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antibody, e.g. using recombinant DNA and other molecular biological techniques. Thus in this context, “affinity matured” refers to in vitro affinity maturation. Typically, the affinity matured antibody binds to the same epitope as the initial reference antibody.

[0140] Affinity matured antibody variants may be conveniently generated, e.g., using phage displaybased affinity maturation techniques (see e.g. Li et al, International Journal of Biological Macromolecules 247(2023): 125733). Briefly, one or more CDR residues are mutated and the variant antigen binding molecules displayed on phage and screened for a particular biological activity (e.g. binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antigen binding molecule to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antigen binding molecule complex to identify contact points between the antibody and antigen. Such contact residues and neighbouring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0141] Thus in some embodiments, alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity, including in CDR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular may be targeted specifically. In some embodiments, the affinity of the affinity matured antibody for the target epitope or antigen is increased by at least 2-fold, preferably at least 3-fold, at least 10-fold, at least 100-fold or at least 1000-fold, compared to the reference (parent) antibody. For instance, the affinity matured antibody may have a Kd for the target epitope or antigen that is less than 50%, less than 30%, less than 10%, less than 1% or less than 0.1% of the corresponding Kd of the reference (parent) antibody.

[0142] Compositions and therapeutic methods

[0143] Compositions are provided herein that include 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 antibody can be formulated for systemic or local (such as intra-tumour) administration. In one example, the therapeutic IgE antibody is formulated for parenteral administration, such as intravenous administration or subcutaneous administration.

[0144] The compositions for administration can include a solution of the antibody (or a functional fragment thereof) dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the antibody and excipients in these formulations can vary, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington’s Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).

[0145] In preferred embodiments the compositions are provided as unit dosage forms, e.g. comprising a defined amount of the IgE antibody suitable for administration to a subject in a single dose. The unit dosage forms may be packaged individually, e.g. in single containers, vials, pre-filled syringes or the like. The unit dosage forms may be suitable for immediate administration to the subject (e.g. may comprise a physiologically acceptable concentration of salts) or the unit dosage forms may be provided in concentrated or lyophilized form (e.g. for dilution with sterile saline solution before use).

[0146] The IgE antibody may be administered at any suitable dose. In preferred embodiments described herein, a typical unit dose of the pharmaceutical composition (e.g. for intravenous administration) comprises less than 50 mg of the IgE antibody. For instance, the composition (i.e. in unit dosage form) may comprise less than 40mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, or 1 mg of the IgE antibody. The composition may comprise at least 10 pg, 100 pg, 200 pg, 300 pg, 500 pg, 700 pg, 1 mg, 3 mg, 5 mg or 10 mg of the IgE antibody. In preferred embodiments, the composition comprises comprising 10 pg to 50 mg, 70 pg to 30 mg, 300 pg to 50 mg, 300 pg to 30 mg, 300 pg to 3 mg, 500 pg to 50 mg, 500 pg to 30 mg, 500 pg to 10 mg, 500 pg to 3 mg, 700 pg to 50 mg, 700 pg to 30 mg, 700 pg to 10 mg, 700 pg to 3 mg, 500 pg to 5 mg, 500 pg to 1 mg, or about 700 pg of the IgE antibody. In some embodiments, the composition may comprise an amount of the IgE antibody within one or more of the above ranges, but excluding one or more of the following amounts: 1 pg, 5pg, lOpg, 50pg, lOOpg, 500pg, Img, 2mg, 4mg, 5mg, lOmgor 15mg. For instance, the composition may comprise 2 pg to 9 pg, 11 pg to 99 pg, 101 pg to 499 pg, 501 to 999 pg or 2 mg to 9 mg.

[0147] The dosage of the IgE antibody administered to the subject may be based on the subject’s body weight. Thus the dose of the IgE antibody administered to the subject may be e.g. less than 1 mg / kg. Preferably the IgE antibody may be administered to the subject in a dose (per administration) of e.g. less than 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 the IgE antibody administered to the 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 the 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 the IgE antibody administered to the subject may be within one or more of the above defined ranges, but excluding one or more of the following dosages: Ipg / kg, lOpg / kg, lOOpg / kg or 0.5 mg / kg. For instance, the dose of the IgE antibody may be 2 to 9 pg / kg, 11 to 99 pg / kg, 101 to 499 pg / kg or 0.51 to 0.7 mg / kg.

[0148] In embodiments of the present invention, the unit dosages of the IgE antibody described above are at administered at most once a week, e.g. the maximum weekly dose of the IgE antibody is 50 mg, 40mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, or 1 mg. For instance the weekly dose of the IgE antibody may be 10 pg to 50 mg, 70 pg to 30 mg, 300 pg to 50 mg, 300 pg to 30 mg, 300 pg to 3 mg, 500 pg to 50 mg, 500 pg to 30 mg, 500 pg to 10 mg, 500 pg to 3 mg, 700 pg to 50 mg, 700 pg to 30 mg, 700 pg to 10 mg, 700 pg to 3 mg, 500 pg to 5 mg, 500 pg to 1 mg, or about 700 pg. The weekly dose of the IgE antibody may also be determined according to the subj ect’ s body weight, e.g. the IgE antibody may be administered to the subj ect in a dose of e.g. less than 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 0.01 mg / kg / week. In preferred embodiments, the dose of the IgE antibody administered to the subject may be 0.001-1 mg / kg / week, 0.003-0.7 mg / kg / week, 0.005-0.5 mg / kg / week, 0.005-0.1 mg / kg / week, 0.005-0.05 mg / kg / week, 0.007-0.03 mg / kg / week or 0.007-0.15 mg / kg / week. In some embodiments, the dose of the IgE antibody administered to the subject may be within one or more of the above defined ranges, but excluding one or more of the following dosages: Ipg / kg / day (7 pg / kg / week), lOpg / kg / day (70 pg / kg / week) or lOOpg / kg / day (0.7 mg / kg / week). For instance, the dose of the IgE antibody may be 2 to 6 pg / kg / week, 8 to 69 pg / kg / week, or 71 to 699 pg / kg / week.

[0149] 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, e.g. about 6.5. Preferred concentrations of the excipients include: 0.05 to 0.5 M (e.g. about 0.1 M) sodium citrate; 10 to 50 g / L (e.g. about 30 g / L) L-arginine; 10 to 100 g / L (e.g. about 50 g / L) sucrose; 0.01 to 0.05% w / w (e.g. 0.02% w / w) polysorbate 20. In one embodiment, the IgE antibody is 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, e.g. about 1 mg / ml. In some embodiments, such a composition may be formulated as a unit dosage form e.g. in a volume of about 1 ml of solution comprising about 1 mg of the IgE antibody, for instance in a 2 ml type I glass vial. The composition may be diluted with sterile saline (0.9% w / v) before administration to the subject, e.g. in an amount of 1 ml of the composition in 250 ml of saline.

[0150] Antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and administered to the subject. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U. S. since the approval of RITUXAN (Registered trademark) in 1997. Antibodies can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks infused over a 30 minute period if the previous dose was well tolerated.

[0151] The antibody (or functional fragment thereof) can be administered to slow or inhibit the growth of cells, such as cancer cells. In these applications, a therapeutically effective amount of an antibody is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer. In some embodiments, the antibodies are administered to a subject to inhibit or prevent the development of metastasis, or to decrease the size or number of metasases, such as micrometastases, for example micrometastases to the regional lymph nodes (Goto et al., Clin. Cancer Res.

[0152] 14(11):3401-3407, 2008).

[0153] Thus in some embodiments, the IgE antibody is used to treat cancer and / or to delay or prevent the progression of cancer. By “delay or prevent the progression” of cancer it is meant that, for example, the cancer is at least stable for a period of time after administration of the antibody, e.g. for at least 6 weeks, at least 12 weeks, at least 6 months or at least 12 months. “Stable” disease may be defined e.g. as a change in the RECIST score of less than 20%.

[0154] RECIST (Response Evaluation Criteria in Solid Tumours) evaluation is a simple method for determining whether a patient’s disease has improved, stayed about the same, or worsened following treatment with a cancer therapeutic, and is commonly used in clinical trials of anticancer agents. The RECIST criteria are specified e.g. in Eisenhauer et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1), European Journal Of Cancer 45 (2009) 228 - 247. RECIST defines Progressive Disease (PD) as at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study. Stable disease is defined as neither sufficient shrinkage to qualify for Partial Response (at least a 30% decrease in the sum of diameters of target lesions), nor sufficient increase to qualify for PD, i.e. an increase of <20% is defined as stable disease.

[0155] It will be appreciated that in this context “at least stable” includes an increase or decrease in the RECIST score of less than 20%. Thus the antibody may delay or prevent progression of the disease (e.g. delay or prevent appearance of one or more signs or symptoms or cancer, and / or inhibit the growth of cancer cells and / or prevent or reduce metastases), or ameliorate or promote remission of the disease (e.g. reduce or inhibit one or more sign or symptom of cancer, and / or kill cancer cells).

[0156] Subjects

[0157] Suitable subjects may include those diagnosed with cancer, e.g. a cancer that expresses HER2, such as, but not limited to, skin cancer (e.g. melanoma), lung cancer, prostate cancer, squamous cell carcinoma (such as head and neck squamous cell carcinoma), breast cancer (including, but not limited to basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), leukemia (such as acute myelogenous leukemia and 11g23-positive acute leukemia), lymphoma, a neural crest tumour (such as an astrocytoma, glioma or neuroblastoma), ovarian cancer, colon cancer, stomach cancer (e.g. gastric or gastroesophageal junction (GEJ) adenocarcinoma), pancreatic cancer, bone cancer (such as a chordoma), glioma or a sarcoma (such as chondrosarcoma).

[0158] Preferably the antibody is administered to treat a solid tumour. In some embodiments the cancer is breast cancer or gastric cancer, preferably breast cancer. Preferably the subject is human.

[0159] In some embodiments, the subject is suffering from a metastatic cancer. For instance, 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.

[0160] A therapeutically effective amount of antibody will depend upon the severity of the disease and the general state of the patient’s health. A therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. These compositions can be administered in conjunction with another chemotherapeutic agent, either simultaneously or sequentially.

[0161] Low HER2 expression

[0162] In some embodiments, an anti-HER2 antibody as described herein may be used to treat a subject suffering from a low HER2-expressing tumor. Such subjects may be referred to as “low HER2 expressors”, in contrast to high HER2 expressors. The terms “low HER2-expressing tumor” and “low HER2 expressor” are well understood in the field of cancer therapy and are frequently used to describe specific patient groups (see e.g. Nicolo et al. Ther Adv Med Oncol 2023, Vol. 15: 1 -16; Tarantino et al., J Clin Oncol 2020; 38: 1951-1962). Thus the subgroup of patients having low tumor HER2 expression is clearly distinguished from high HER2 expressors.

[0163] Low tumor HER2 expression may be determined using well known and standard techniques. Typically HER2 expression is determined in a biopsy or surgical specimen from the tumor. Techniques for obtaining surgical biopsy samples from tumor tissue are known in the art, as are histopathological techniques for processing such samples. For instance, biopsy tissue samples may be fixed with formalin and embedded in paraffin or processed fresh before sectioning and placement on microscope slides for light microscopy analysis and imaging.

[0164] HER2 expression levels may be detected using immunohistochemical techniques. Haemotoxylin and eosin (H& E) staining of paraffin-embedded sections is the default technology to visualize tissue on a glass slide for pathology analysis. Immunohistochemistry (IHC) staining is a well-known approach to identify expression of proteins on cells in pathology tissue slides. The staining results in a typical brown appearance of tissue where the targeted protein is overexpressed as compared to normal. For example, by using an antibody against HER2, its expression levels can be detected.

[0165] Low HER2 expression may be defined as e.g. a HER2 IHC score of less than 3+, e.g. 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 the subject may be performed using a Dako anti-HER2 immunohistochemistry system (i.e. HercepTest™). The HercepTest™ method is described e.g. in FDA Premarket approval applications P980018, approved 25 September 1998 and P980018 / S010, approved 20 October 2010, and in Dako HercepTest™, Code K5204. Instruction for Use, (PD04086US 02 / K520421-5). The HercepTest™ method and alternative methods for detecting HER2 expression in tumors are also described in e.g. Jorgensen et al., (2021) Front. Oncol. 11:676939.

[0166] Various studies describe methods for detecting HER2 in tumor samples and the standardization of the results thereof. See in particular 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 version 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). See also e.g. 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.

[0167] 2007;20:584-591; Zhang et al. Curr Oncol Rep 2020 Apr 29;22(5):51.

[0168] The FDA-approved classification of HER2 IHC staining scores is described below:

[0169] Score Surgical Specimen - Biopsy Specimen -Staining HER2

[0170] Staining Pattern Pattern Overexpression Assessment

[0171] 0 No reactivity observed or No reactivity or no Negative

[0172] weak membranous membranous reactivity in any

[0173] reactivity observed in < (or < 5 clustered) tumor cell

[0174] 10% of tumor cells

[0175] 1+ Faint / barely perceptible membranous Tumor cell Negative membranous reactivity in > cluster (> 5 cells) with a

[0176] 10% of tumor cells; cells are faint / barely perceptible

[0177] reactive only in part of their reactivity irrespective of

[0178] membrane percentage of tumor cells

[0179] stained

[0180] 2+ Weak to moderate complete, Tumor cell cluster (> 5 cells) Equivocal basolateral or lateral with a weak to moderate

[0181] membranous reactivity in complete, basolateral or lateral

[0182] >10% of tumor cells membranous reactivity

[0183] irrespective of percentage of

[0184] tumor cells stained

[0185] 3+ Strong complete, basolateral Tumor cell cluster (> 5 cells) Positive

[0186] or lateral membranous with a strong complete,

[0187] reactivity in > 10% of tumor basolateral or lateral

[0188] cells membranous reactivity

[0189] irrespective of percentage of

[0190] tumor cells stained

[0191] Any suitable anti-HER2 antibody may be used in such methods, e.g. an anti-HER2 IgG antibody (polyclonal or monoclonal). Various anti-HER2 antibodies suitable for use in immunohistochemistry are available from commercial sources.

[0192] Thus by “low HER2-expressing tumor” it is typically meant that less than 10% of tumor cells in a sample from the 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 a tumor cell cluster (> 5 cells) with strong complete membrane staining for HER2. These levels of expression typically refer to membrane HER2 expression that is detectable by immunohistochemistry, e.g. using the methods described above. For instance in some embodiments, at least 10% of tumor cells or a tumor cell cluster (5 or more cells) in a sample from the subject show weak to moderate complete membrane staining for HER2, i.e. corresponding to a HER2 IHC score of 2+ using immunohistochemical detection of HER2. In some embodiments, a tumor cell cluster (5 or more cells) in a sample from the subject may show weak to moderate complete membrane staining for HER2.

[0193] In other embodiments, at least 10% of tumor cells in a sample from the subject show faint or barely perceptible partial (incomplete) membrane staining for HER2, i.e. corresponding to a HER2 IHC score of 1+ using immunohistochemical detection of HER2. In some embodiments, a tumor cell cluster (5 or more cells) from the subject may show faint or barely perceptible partial (incomplete) membrane staining for HER2. In these embodiments, the tumor does not meet the criteria for a HER2 IHC score of 2+ or above, i.e. less than 10% of tumor cells in a sample from the subject show weak to moderate complete membrane staining for HER2 (and no tumor cell cluster (5 or more cells) showing weak to moderate complete membrane staining for HER2 is present).

[0194] In alternative embodiments, tumor HER2 expression in the subject may be compared to that in a population of cancer subjects. Typically the population of cancer subjects refers to other subjects suffering from the same type of cancer. Thus the relative level of tumor HER2 expression in the subject, compared to other subjects with the same type of cancer, may be ascertained. Since this method does not require an absolute quantitation of HER2 expression levels, but only a relative determination compared to other cancer subjects, any systematic bias due to lack of standardization of expression detection is avoided.

[0195] In preferred embodiments, tumor (e.g. membrane) HER2 expression in the subject is lower than in at least 50% of cancer subjects. Preferably membrane HER2 expression in tumor cells of the subject is lower than in at least 60%, at least 70% or at least 80% of cancer subjects, e.g. in subjects suffering from the same form of cancer (e.g. ovarian cancer). More preferably tumor membrane HER2 expression in the subject is lower than in at least 50%, 60%, 70%, 80% or at least 90% of HER2-expressing tumors (e.g. in HER2-expressing breast tumors).

[0196] In some embodiments the tumor expresses HER2, i.e. the tumor shows at least some HER2 expression. Typically this means that tumor cells in the subject show detectable membrane HER2 expression, e.g. by immunohistochemistry. More preferably at least 1% or at least 5% of tumor cells in the subject show detectable (e.g. membrane) HER2 expression, e.g. using immunohistochemical detection of HER2. In other embodiments at least 10%, 15% or 20% of tumor cells in the subject show detectable membrane HER2 expression. Thus the tumor may have a HER 2 IHC score of 1+ or 2+ (in preference to 0).

[0197] Preferably the determination of HER2 expression is made on a recent tumor biopsy sample, i.e. a biopsy sample obtained from the subject shortly before the start of anti-HER2 IgE treatment (rather than an old or archived sample). For instance, the biopsy sample may be obtained and / or HER2 expression determined less than 6 months, 3 months, 1 month, 2 weeks, 1 week, 3 days, 48 hours or 24 hours before the start of anti- HER2 IgE treatment.

[0198] In some embodiments, amplification of the erbB2 (neu) gene may also be monitored. For instance, in situ hybridization (ISH, including e.g. fluorescence ISH or FISH) can be performed on a tumor sample from the subject to detect HER2-encoding gene amplification. In FISH, the HER2 gene copy number in relevant tumor cells may be determined and reported relative to 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 > 2.0 may be used as cut-off value indicative of HER2 amplification by FISH (see e.g. 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).

[0199] Thus in some embodiments, the tumor lacks erbB2 gene amplification. For example, a tumor sample from the subject may show no detectable amplification of a HER2-encoding gene (erbB2 e.g. as determined by FISH, or may show a HER2 / CEN17 ratio < 2.0.

[0200] Additional diagnostic methods for analysing HER2 expression in combination with anti-HER2 treatment have been described. The table below shows FDA approved companion diagnostics assays for HER2 targeted drugs. This includes IHC and (fluorescent) in situ hybridization assays, as well as newer next generation sequencing (NGS) assays.

[0201] Assay Manufacturer Breast cancer Gastric cancer IHC Assays

[0202] Bond Oracle HER2 IHC System Leica Biosystems Trastuzumab

[0203] HercepTest™ Agilent Technologies / Dako Trastuzumab Trastuzumab Denmark Pertuzumab

[0204] Ado-trastuzumab emtansme

[0205] InSite™ Her-2 / neu KIT Biogenex Laboratories

[0206]

[0207] Trastuzumab PATHWAY anti-Her2 / neu Ventana Medical Systems Trastuzumab

[0208] Ado-trastuzumab emtansine

[0209] ISH Assays

[0210] HER2 FISH pharmDx Kit Agilent Technologies / Dako trastuzumab 1’rastuzumab Denmark

[0211] Pertuzumab

[0212] Ado-trastuzumab emtansine

[0213] HER2 CISH Agilent Technologies / Dako Trastuzumab

[0214] pharmDx Kit Denmark Ventana Medical Trastuzumab

[0215] INFORM™ HER- Systems Trastuzumab

[0216] 2 / neu Ado-trastuzumab emtansine INFORM™ HER2 Dual ISH DNA Ventana Medical Systems

[0217] Probe Cocktail

[0218] PathVysion® HER-2 DNA Probe Abbott Molecular Trastuzumab

[0219] Kit

[0220] SPOT-LIGHT® HER2 CISH Kit Life Technologies Corporation Trastuzumab

[0221] VENTANA HER2 Dual ISH DNA Ventana Medical Systems Trastuzumab

[0222] Probe Cocktail

[0223] NGS Assays

[0224] FoundationOne® CDx Foundation Medicine trastuzumab

[0225] Pertuzumab

[0226]

[0227] Ado-trastuzumab emtansine

[0228] The invention will now be further described by way of example only, with reference to the following non-limiting embodiments.

[0229] EXAMPLES

[0230] Anti-HER2 IgE antibodies

[0231] The following anti-HER2 IgE antibodies were used in the examples.

[0232] Table 1

[0233] Heavy chain (HC) CDR loops HC Light chains (LC) CDR loops LC - variable domain (VH) is (IMGT) - variable domain (IMGT)

[0234] bold and underlined (VL) is bold and

[0235] - constant (CH) domain is underlined

[0236] standard text - constant (CL)

[0237] domain is standard

[0238]

[0239] text EPS QVQLVQSGAEVKKPGA CDRH1 QSVLTQPASVSG CDRL1

[0240] 226 SVKVSCKASGYTFTSYA SYAMH SPGQSITISCTGT TGTSSDVGSYN IgE MHWVRQAPGQRLEWI (SEQ ID NOG) SSDVGSYNLVS LVS GWINAGNGNTKYSQKF WYQQHPGKAP (SEQ ID NOG) QGRVTITRDTSASTAY CDRH2 KLMIYEVSNRPS MELSSLRSEDTAVYYC WINAGNGNTKYS GVSNRFSGSKSG CDRL2 ARDFSSQVATAAVDYW QKFQG NTASLTISGLQA EVSNRPS GQGTLVTVSS VS VKAP S (SEQ ID NO:4) EDEAD YYCSSY (SEQ ID NO: 9) LYPLKPCSSENTASVTLG TSSSTLVFGGGT CLVKDYFPDPVTVTWYS CDRH3 KLTVLGQPKSTP CDRL3 DSLNTSTMNFPSVGSDL DFSSQVATAAVDY TLTVFPPSTEELQ SSYTSSSTL KTTTSQMTSWGKSAKNF (SEQ ID NOG) GNKATLVCLISD (SEQ ID NO: 10) TCHVTHAPSTFVSDLTIR FYPSDVEVAWK ARPVNITKPTVDLLHSSC ANGAPISQGVDT DPNAFHSTIQLYCFVYGH ANPTKQGNKYIA IQNDVSIHWLMDDRKIY SSFLRLTAEQWR ETHAQNVLIKEEGKLAST SRNSFTCQVTHE YSRLNITQQQWMSESTF GNTVEKSLSPAE TCKVTSQGENYWAHTR CV RCSDDEPRGVITYLIPPSP LDLYENGTPKLTCLVLD LC -SEQ ID NOG LESEENITVTWVRERKKS VL -SEQ ID NO:7 IGSASQRSTKHHNATTSI TSILPVDAKDWIEGEGYQ CRVDHPHFPKPIVRSITK APGKRSAPEVYVFLPPEE EEKDKRTLTCLIQNFFPE DISVQWLQDSKLIPKSQH STTTPLKYNGSNQRFFIFS RLEVTKALWTQTKQFTC RVIHEALREPRKLERTIS KSLGNTSLRPSQASM HC - SEQ ID NO:1

[0241] VH - SEQ ID NO:2

[0242] EPS QVQLVQSGAEVKKPGA CDRH1 QSVLTQPASVSG CDRL1

[0243] 232 SVKVSCKASGYTFTSYA SYAMH SPGQSITISCTGT TGTSSDVGSYN IgE MHWVRQAPGQRLEWI (SEQ ID NOG) SSDVGSYNLVS LVS GWINAGNGNTKYSQKF WYQQHPGKAP (SEQ ID NOG) QGRVTITRDTSASTAY CDRH2 KLMIYTVSNRPS MELSSLRSEDTAVYYC WINAGNGNTKYS GVSNRFSGSKSG CDRL2 ARDFSSQVATAAVDYW QKFQG NTASLTISGLQA TVSNRPS GQGTLVTVSSVSVKAP S (SEQ ID NO:4) EDEAD YYCSSY (SEQ ID NO: 13) LYPLKPCSSENTASVTLG TSSSSLVFGGGT CLVKDYFPDPVTVTWYS CDRH3 KLTVLGQPKSTP CDRL3 DSLNTSTMNFPSVGSDL DFSSQVATAAVDY TLTVFPPSTEELQ SSYTSSSSL KTTTSQMTSWGKSAKNF (SEQ ID NOG) GNKATLVCLISD (SEQ ID NO: 14) TCHVTHAPSTFVSDLTIR FYPSDVEVAWK ARPVNITKPTVDLLHSSC ANGAPISQGVDT (mutations in DPNAFHSTIQLYCFVYGH ANPTKQGNKYIA CDRL2 and IQNDVSIHWLMDDRKIY SSFLRLTAEQWR CDRL3 of EPS ETHAQNVLIKEEGKLAST SRNSFTCQVTHE 232 compared to YSRLNITQQQWMSESTF GNTVEKSLSPAE EPS 226 are TCKVTSQGENYWAHTR CV shown in bold and RCSDDEPRGVITYLIPPSP underlined) LDLYENGTPKLTCLVLD LC -SEQ ID NO: 11 LESEENITVTWVRERKKS VL -SEQ ID NO: 12 IGSASQRSTKHHNATTSI TSILPVDAKDWIEGEGYQ

[0244]

[0245] CRVDHPHFPKPIVRSITK APGKRSAPEVYVFLPPEE

[0246] EEKDKRTLTCLIQNFFPE DISVQWLQDSKLIPKSQH STTTPLKYNGSNQRFFIFS RLEVTKALWTQTKQFTC RVIHEALREPRKLERTIS KSLGNTSLRPSQASM HC - SEQ ID NO:1

[0247] VH - SEQ ID NO:2

[0248] EPS QVQLVQSGAEVKKPGA CDRH1 QSVLTQPASVSG CDRL1 226.6 SVKVSCKASGYTFTSYA SYAMH SPGQSITISCTGT TGTSSDVGSYN IgE MHWVRQAPGQRLEWI (SEQ ID NOG) SSDVGSYNLVS LVS GWINAGNGNTKYSQKF WYQQHPGKAP (SEQ ID NO: 8) QGRVTITRDTSASTAY CDRH2 KLMIYTVSNRPS MELSSLRSEDTAVYYC WINAGNGNTKYS GVSNRFSGSKSG CDRL2 ARDFSSQVATAAVDYW QKFQG NTASLTISGLQA TVSNRPS GQGTLVTVSS VS VKAP S (SEQ ID NO:4) EDEAD YYCSSY (SEQ ID NO: 13) LYPLKPCSSENTASVTLG TSSSTLVFGGGT CLVKDYFPDPVTVTWYS CDRH3 KLTVLGQPKSTP CDRL3 DSLNTSTMNFPSVGSDL DFSSQVATAAVDY TLTVFPPSTEELQ SSYTSSSTL KTTTSQMTSWGKSAKNF (SEQ ID NOG) GNKATLVCLISD (SEQ ID NO: 10) TCHVTHAPSTFVSDLTIR FYPSDVEVAWK ARPVNITKPTVDLLHSSC ANGAPISQGVDT (mutation in DPNAFHSTIQLYCFVYGH ANPTKQGNKYIA CDRL2 of EPS IQNDVSIHWLMDDRKIY SSFLRLTAEQWR 226.6 compared to ETHAQNVLIKEEGKLAST SRNSFTCQVTHE EPS 226 is shown YSRLNITQQQWMSESTF GNTVEKSLSPAE in bold and TCKVTSQGENYWAHTR CV underlined) RCSDDEPRGVITYLIPPSP LDLYENGTPKLTCLVLD LC -SEQ ID NO: 19 LESEENITVTWVRERKKS VL -SEQ ID NO:20 IGSASQRSTKHHNATTSI TSILPVDAKDWIEGEGYQ CRVDHPHFPKPIVRSITK APGKRSAPEVYVFLPPEE EEKDKRTLTCLIQNFFPE DISVQWLQDSKLIPKSQH STTTPLKYNGSNQRFFIFS RLEVTKALWTQTKQFTC RVIHEALREPRKLERTIS KSLGNTSLRPSQASM HC - SEQ ID NO:1

[0249]

[0250] VH - SEQ ID NO:2 PAP QVQLVQSGAEVKKPGA CDRH1 QSVLTQPASVSG CDRL1

[0251] 317 SVKVSCKASGYTFTSYA SYAMH SPGQSITISCTGT TGTSSDVGSYN IgE MHWVRQAPGQRLEWI (SEQ ID NOG) SSDVGSYNLVS LVS GWINAGNGNTKYSTKF WYQQHPGKAP (SEQ ID NO: 8) QGRVTITRDTSASTAY CDRH2 KLMIYTVSNRPS MELSSLRSEDTAVYYC WINAGNGNTKYS GVSNRFSGSKSG CDRL2 ARDFSLQVATAAVDYW TKFQG NTASLTISGLQA TVSNRPS GQGTLVTVSS VS VKAP S (SEQ ID NO:23) EDEAD YYCSSY (SEQ ID NO: 13) LYPLKPCSSENTASVTLG TSSTLVFGGGT CLVKDYFPDPVTVTWYS CDRH3 KLTVLGQPKSTP CDRL3 SSYT- DSLNTSTMNFPSVGSDL DFSLQVATAAVD TLTVFPPSTEELQ SSTL KTTTSQMTSWGKSAKNF Y GNKATLVCLISD (SEQ ID NO:27) TCHVTHAPSTFVSDLTIR (SEQ ID NO:24) FYPSDVEVAWK ARPVNITKPTVDLLHSSC ANGAPISQGVDT (mutation in DPNAFHSTIQLYCFVYGH (mutations in CDRH2 ANPTKQGNKYIA CDRL2 of PAP IQNDVSIHWLMDDRKIY and CDRH3 of PAP SSFLRLTAEQWR 317 compared to ETHAQNVLIKEEGKLAST 317 compared to EPS SRNSFTCQVTHE EPS 226 is shown YSRLNITQQQWMSESTF 226 are shown in GNTVEKSLSPAE in bold and TCKVTSQGENYWAHTR bold and CV underlined;

[0252] RCSDDEPRGVITYLIPPSP underlined) deletion of S in LDLYENGTPKLTCLVLD LC -SEQ ID NO:25 CDRL3 of PAP LESEENITVTWVRERKKS VL -SEQ ID NO:26 317 compared to IGSASQRSTKHHNATTSI EPS 226 is shown TSILPVDAKDWIEGEGYQ as hyphen -) CRVDHPHFPKPIVRSITK APGKRSAPEVYVFLPPEE EEKDKRTLTCLIQNFFPE DISVQWLQDSKLIPKSQH STTTPLKYNGSNQRFFIFS RLEVTKALWTQTKQFTC RVIHEALREPRKLERTIS KSLGNTSLRPSQASM HC - SEQ ID NO:21

[0253]

[0254] VH - SEQ ID NO:22

[0255] A panel of 100 antibodies from phage display was triaged on the basis of binding affinity to recombinant HER2 and HER2 on cell surfaces, retained binding to FcsRl, epitope specificity, rat / human HER2 cross-reactivity, ability to degranulate human FcsRl expressing basophils, ability to kill tumour cells by ADCC and ADCP and assessments of biophysical properties (solubility and thermal stability). EPS 226 IgE was identified using such methods and selected for further study. EPS 226 competes with pertuzumab for binding to HER2, and thus binds to subdomain II (the dimerisation region) of the extracellular domain of the HER2. As described in PCT / EP2024 / 068202, EPS 226 IgE inhibits growth of low HER2-expressing tumours in vivo in animal models.

[0256] Affinity Maturation library construction and phage biopanning EPS 226 IgE as described above was selected for affinity maturation. To avoid the insertion of potential immunogenic sites in the framework regions, only the CDRs have been subjected to mutagenesis.

[0257] EPS226 CDRs have been affinity matured using a Site Saturation Mutagenesis approach. The wild type amino acids coding sequences have been systematically replaced with sequences encoding all the 19 non-wild type amino acids, single point deletions have also been included. This allowed to insertion of one mutation at the time in each heavy and light CDR, resulting in clones that bared maximum six point mutations.

[0258] A phage display library composed of 2xl09clones has been constructed and two rounds of biopanning have been performed.

[0259] The library has been panned on decreasing concentrations of the purified HER2 antigen (starting from 15 nM down to 1 nM), single colonies from Pan 2 output have been picked and binding has been assessed in phage ELISA. To further characterise the binders an off-rate screening has been performed following the protocol as described in F. Ylera et al., “Off-rate screening for selection of high- affinity anti-drug antibodies ”, Anal Biochem (2013) 441(2):208-13.

[0260] The sequences of the clones that performed the best in the off-rate ranking have been retrieved, and HEK293 cells have been transfected to produce the IgE version of the matured molecules. After the purification of the IgE from the supernatant, monovalent Kd of each IgE for HER2 has been determined using biolayer interferometry (BLI). BLI experiments were performed on the Octet R8 (Sartorius) to determine the monovalent affinity of EPS226 and affinity matured IgE for human HER2. Streptavidin SA biosensors (Sartorius) were loaded with CaptureSelect™ Biotin Anti-IgE Conjugate (CSIgE, ThermoScientific) (0.1 pg / ml), followed by capture of IgE (25 nM) and binding to tag-free human HER2 (HE2-H5212, AcroBiosystems) at varying concentrations. The following experimental setup was used: wash (60 seconds), load CSIgE (600 seconds), wash (60 seconds), baseline (30 seconds), load IgE (300 seconds), wash (60 seconds), baseline (30 seconds), association HER2 (600 seconds), dissociation (600 seconds). The same concentration of IgE was loaded on every sensor, but association was performed on HER2 ranging from 100 nM to 0.137 nM in 1:3 dilutions and a buffer blank. Phosphate buffered saline pH 7.4 with 0.05 % (v / v) Tween-20 (PBST) was used for sample preparation and dilution, and for all wash, baseline, and dissociation steps. Runs were set up using 96-well plates shaking at 1000 rpm at 30 °C. Data (n=3) were buffer blank subtracted and then fit using the Octet kinetics analysis software using a global fit (1:1). Concentration ranges covering approximately 90 % to 10 % Req / Rmax were used to determine KD and the best fit of the data.

[0261] Hoxb8 mast cell degranulation assay

[0262] Affinity matured IgE antibodies were prepared based on each clone and their potency was assessed in a mast cell degranulation assay. Hoxb8 mast cells (2.5 x 104) were seeded in a 96 well round bottom plate. The cells were sensitised to a test article (EPS 226, EPS 226.6, PAP317 or EPS 232) in a 12-point dose response (0.0001 - lOOnM). The cells were washed and exposed to JIMT cells (4:1 ratio of JIMT1: Hoxb8 cells) for 30 minutes. Degranulation was assessed by measuring CD 107a levels by flow cytometry.

[0263] The affinity and potency of each IgE antibody obtained using the above methods is shown in Table 1 below:

[0264] Table 1

[0265] KD (M) KD (M) KD (M) KD (M) Potency Molecule Alias

[0266] n=l n=2 n=3 (average) (EC50, M) Human V26 IgE EPS226 2.573E-08 2.596E-08 2.712E-08 2.627E-08 2.09E-10

[0267] Human V26 P102.3 E5 IgE EPS226.6 7.182E-09 5.047E-09 5.071E-09 5.767E-09 6.40E-11

[0268] Human V26 WT-150 / 139 IgE EPS232

[0269] 2.451E-09 3.078E-09 3.091E-09 2.873E-09 3.60E-11 (PAP322)

[0270] Human V26 WT-150 / 141 IgE 2.421E-09 2.444E-09 3.123E-09 2.663E-09 8.17E-11

[0271] Human V26 139-150 / 139 IgE 7.376E-10 7.775E-10 9.408E-10 8.186E-10 6.99E-11

[0272] Human V26 139-150 / 141 IgE PAP317 1.017E-09 9.618E-10 9.996E-10 9.928E-10 5.20E-11

[0273] Human V26 139-149 / 139 IgE 8.045E-10 1.093E-09 1.097E-09 9.982E-10 7.86E-11

[0274] Human V26 140-150 / 139 IgE 7.565E-10 7.648E-10 8.375E-10 7.863E-10 9.82E-11

[0275] Human V26 140-150 / 141 IgE 8.953E-10 1.038E-09 1.057E-09 9.968E-10 9.62E-11

[0276] Human V26 140-149 / 139 IgE 9.419E-10 7.756E-10 1.005E-09 9.075E-10 1.38E-10

[0277]

[0278] As shown in the above table, EPS 226 has a Kd for HER 2 of approximately 26 nM (2.6 E-08), whereas each of the affinity matured IgE antibodies has a Kd for HER 2 of less than 10 nM (i.e. 1.0E-08). Figure 1 shows that EPS 226.6 IgE has an improved potency and results in an overall increase in mast cell activation compared to EPS 226 IgE.

[0279] Three affinity matured IgE candidates, i.e. EPS 226.6, EPS 232 and PAP 317 were selected from the above antibodies for further study and compared with EPS 226 IgE in the assays described below.

[0280] Jurkat-FcsRI activation assay

[0281] Jurkat cells transduced to express the human FcsRI (ay?) receptor were seeded (5 x 104) in a 96-well round bottom plate and cultured with either SKBR3 or JIMT-1 target cells (1:1 ratio of Jurkat-FcsRI: target cells) in the presence of anti-HER2 IgE antibodies (6-point dose response ranging from 5 - 0.0016 nM) for 18 hours. Jurkat-FcsRI (ay?) activation was assessed by measuring CD69 expression by flow cytometry. EC50 values were calculated from the percentage of CD69-positive Jurkat-FcsRI (ay?) cells.

[0282] RBL-SX38 degranulation assay

[0283] RBL-SX38 cells (1 x 104) were seeded in a 96 well flat bottom plates for 16 hours. Cells were then sensitised with IgE antibodies (anti -NIP IgE, EPS 226, EPS 226.6, PAP317 or EPS 232) for one hour using an 8-point titration curve (5 - 0.0023 nM). NIP refers to 4-hydroxy-3-iodo-5-nitrophenylacetic acid, i.e. a small molecule substrate not found in humans useful as an isotype control (see e.g. Bax et al. Allergy. 2020;00:1-5). Cells were washed in buffer (HBSS 1% BSA) prior to stimulating with SKBR3 cells (3 x 104) and incubated for 30 minutes. Post incubation the supernatant was harvested and mixed with substrate 4-methyllumbelliferyl N-acetyl-b-D-glucosaminide (1:1 ratio) in black 96-well plates, before incubating at room temperature protected from light for 16 hours. The reaction was quenched with 0.5 M Tris and samples acquired using a Fluorescent Microplate Reader (350-nm excitation, 450-nm emission). Degranulation was expressed as a percentage of Triton X-100 release (100%).

[0284] RBL-SX38 ADCC assay

[0285] RBL-SX38 cells (8 x 104) and JIMT-1 cells transduced to express the Incucyte® Nuclight Rapid near infrared (NIR) Dye (1 x 104) were seeded in a flat-bottom 96-well plate and cultured with 10 nM IgE antibodies. JIMT-1 cell growth was assessed using the Incucyte® SX5 instrument (Sartorius). Four images per well were captured and the average NIR cell count across the images recorded. Images were captured after 30 minutes of incubation and every two hours subsequently for a total of 64 hours. Area under curve analysis of NIR cell count was calculated and subsequently the percentage of JIMT-1 growth inhibition relative to isotype control was calculated.

[0286] IgE-mediated inhibition of tumour cell growth

[0287] SKBR3 cells (1 x 104) were seeded in 96 well flat bottom plates in the presence of antibodies (NIP-IgE, EPS 226, EPS 226.6, PAP317 or EPS 232) at 100 nM and incubated for five days. Cell growth inhibition was measured as percentage confluency relative to isotype control by Incucyte® SX5 instrument (Sartorius) using images were captured every 2 hours.

[0288] The results are shown in Table 2 below: Table 2

[0289] IgE Yield Tm Signalling Signalling Degran Degran ADCC Direct Kd from IgE FceRI FceRI Hoxb8 RBLSX38 RBLSX38: effects (nM) 3 L (°C) Jurkat: Jurkat: JIMT1 SKBR3 JIMT1 (% SKBR3 prep SKBR3 JIMT1 (EC50 (EC50 growth (%

[0290] (EC50,

[0291] (mg) (EC50, nM) nM) Inhibition inhibition @ nM)

[0292] nM) @ 10nM) 100nM) EPS226 26.2 278 58 0.024 0.018 0.209 0.076 6.9 -1

[0293] EPS226.6 5.78 155 58 0.021 0.010 0.064 0.048 9.4 9

[0294] PAP317 0.99 210 58.5 0.020 0.013 0.052 0.067 8.3 34

[0295] EPS232 2.87 120 58 0.016 0.009 0.036 0.059 9.1 19

[0296]

[0297] The results in Table 2 show that the affinity matured IgE antibodies have surprising advantages compared to the parent antibody EPS 226 from which they were generated. For instance, EPS 226.6, PAP 317 and EPS 232 each show direct inhibition of SKBR3 tumour cell growth, whereas this was not observed for EPS 226 under the conditions tested.

[0298] In vivo anti-tumour activity

[0299] SKBR3 mouse model

[0300] Female NXG mice were implanted subcutaneously with SKBR3 cells (1x107cells per mouse; 1:1 matrigel). Tumour volume was monitored via calliper three times weekly throughout the study. Once tumours reached a volume of 30-50mm3mice were randomised into treatment groups of 12 mice. At this point peripheral blood mononuclear cells (PBMC; 5x106cells per mouse; 3 donors per group) were administered with Flt3L (10 mg) intravenously. Flt3L (10 mg) was also administered on the following two days. Mice were dosed intravenously with test article (EPS 226 or EPS 232) at 2 or 10 mg / kg, isotype control (NIP IgE; 10 mg / kg) or vehicle control (PBS) at the same time as PBMCs and twice weekly thereafter for a duration of 29 days.

[0301] The results are shown in Table 3 and Figure 2. Data are expressed as mean tumour growth inhibition at day 28 + / - sem of n=10-12 mice per group (3 PBMC donors). One-way ANOVA performed on raw data and statistical comparison to PBS control performed. **** p<0.0001, ** p<0.01, no star is not statistically significant.

[0302] Table 3 - Comparison of Tumour Growth Inhibition (TGI) vs PBS at day 28 Treatment % TGI at 2mg / kg % TGI at lOmg / kg

[0303] EPS 226 17.2 ± 5.0 43.7 ± 4.0

[0304] EPS 232 29.0 ± 4.8 51.2 ± 2.9

[0305] NIP IgE N / A 10.3 ± 6.3

[0306]

[0307] MTLn3 rat model Female Fischer rats were implanted subcutaneously in close proximity to the mammary fat pad with MTLn3 cells (7x105cells per rat). Tumour volume was monitored via calliper three times weekly throughout the study. Once tumours reached a volume of 30-50mm3rats were randomised into treatment groups of 10 rats. Rats were dosed intravenously with test article (EPS 226 or EPS 232) at 2 or 10 mg / kg, isotype control (NIP IgE; 10 mg / kg) or vehicle control (PBS) twice weekly. The results are shown in Table 4 and Figure 3.

[0308] Table 4 - Comparison of Tumour Growth Inhibition (TGI) vs PBS

[0309] Treatment % TGI at 2mg / kg % TGI at lOmg / kg

[0310] EPS 226 32.0 ± 12.5 39.9 ± 10.8

[0311] EPS 232 43.3 ± 13.9 52.2 ± 13.3

[0312] NIP IgE N / A -19.1 ± 11.2

[0313]

[0314] The data in Figures 2 and 3 and Tables 3 and 4 show that EPS 232 at both 2mg / kg and lOmg / kg delivered a significantly greater tumour growth inhibition compared to EPS 226.

[0315] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

CLAIMS1. An immunoglobulin E (IgE) antibody for use in treating and / or delaying progression of cancer in a subject, wherein the antibody has a dissociation constant (Kd) for binding to a target antigen of 10 nM or less.

2. An IgE antibody for use according to claim 1, wherein the antibody is an affinity-matured antibody.

3. An IgE antibody for use according to claim 1 or claim 2, wherein the antibody has an increased affinity for binding to the target antigen compared to a parent antibody from which the antibody is derived, preferably wherein the parent antibody has a Kd for binding to the target antigen of 20 nM or higher.

4. An IgE antibody for use according to claim 3, wherein the affinity of the affinity matured antibody for the target antigen is increased by at least 2-fold, preferably at least 3-fold, at least 10-fold, at least 100-fold or at least 1000-fold, compared to the parent antibody.

5. An IgE antibody for use according to claim 3 or claim 4, wherein the affinity matured antibody has a Kd for the target antigen that is less than 50%, less than 30%, less than 10%, less than 1% or less than 0.1% of the corresponding Kd of the parent antibody for the target antigen.

6. An IgE antibody for use according to any preceding claim, wherein the antibody has a Kd for binding to the target antigen of 1 pM to 10 nM, 100 pM to 5 nM or 0.5 to 3 nM.

7. An IgE antibody for use according to any of claims 2 to 6, wherein the increased affinity of the antibody for binding to the target antigen is associated with an increased anticancer potency of the antibody compared to the parent antibody.

8. An IgE antibody for use according to any preceding claim, wherein the antibody binds to HER2.

9. An IgE antibody for use according to any preceding claim, wherein the antibody binds to subdomain II of the extracellular domain of the HER2, preferably wherein the antibody at least partially competes with pertuzumab IgG for binding to HER2.

10. An IgE antibody for use according to any preceding claim, wherein the antibody comprises an amino acid sequence as defined in any one of SEQ ID NO:s 1 to 27.

11. An IgE antibody for use according to claim 10, wherein the antibody comprises one or more CDR sequences selected from:(i) SEQ ID NOs: 13 and / or 14, optionally in combination with one or more of SEQ ID NO:s 3, 4, 5 and / or 8;(ii) SEQ ID NO: 13, optionally in combination with one or more of SEQ ID NO:s 3, 4, 5, 8 and / or 10; or(iii) SEQ ID NOs: 23, 24, 13 and / or 27, optionally in combination with one or more of SEQ ID NO:s 3 and / or 8.

12. An IgE antibody for use according to claim 10 or claim 11, wherein the antibody comprises:(i) a light chain variable domain sequence as defined in any one of SEQ ID NOs: 12, 20 or 26; and / or(ii) a light chain sequence as defined in any one of SEQ ID NOs: 11, 19 or 25;optionally in combination with:(iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 2 or 22; and / or(iv) a heavy chain sequence as defined in SEQ ID NO: 1 or 21.

13. An IgE antibody for use according to any preceding claim, wherein the parent antibody comprises an amino acid sequence as defined in any one of SEQ ID NO:s 1 to 10.

14. An IgE antibody for use according to claim 13, wherein the parent antibody comprises one to six CDR sequences selected from SEQ ID NOs: 3, 4, 5, 8, 9 and 10.

15. An IgE antibody for use according to claim 13 or claim 14, wherein the parent antibody comprises:(i) a heavy chain variable domain sequence as defined in SEQ ID NO: 2;(ii) a light chain variable domain sequence as defined in SEQ ID NO: 7;(iii) a heavy chain sequence as defined in SEQ ID NOs: 1; and / or(iv) a light chain sequence as defined in SEQ ID NOs: 6.

16. An IgE antibody for use according to any preceding claim, wherein the tumor or cancer is a breast tumor or breast cancer.

17. A method for treating and / or delaying progression of cancer in a subject, the method comprising a step of administering an immunoglobulin E (IgE) antibody as defined in any preceding claim to the subject in a therapeutically-effective amount.

18. A pharmaceutical composition for use in treating and / or delaying progression of cancer in a subject, comprising an immunoglobulin E (IgE) antibody as defined in any of claims 1 to 15 and one or more pharmaceutically acceptable excipients, carriers or diluents.

19. An immunoglobulin, or a functional fragment thereof, comprising one to six CDR sequences selected from:(i) SEQ ID NOs: 13 and / or 14, optionally in combination with one or more of SEQ ID NO:s 3, 4, 5 and / or 8;(ii) SEQ ID NO: 13, optionally in combination with one or more of SEQ ID NO:s 3, 4, 5, 8 and / or 10; or(iii) SEQ ID NOs: 23, 24, 13 and / or 27, optionally in combination with one or more of SEQ ID NO:s 3 and / or 8.

20. An immunoglobulin or functional fragment thereof according to claim 19, wherein the immunoglobulin comprises:(i) a light chain variable domain sequence as defined in any one of SEQ ID NOs: 12, 20 or 26; and / or(ii) a light chain sequence as defined in any one of SEQ ID NOs: 11, 19 or 25;optionally in combination with:(iii) a heavy chain variable domain sequence as defined in SEQ ID NO: 2 or 22; and / or(iv) a heavy chain sequence as defined in SEQ ID NO: 1 or 21.

21. An immunoglobulin or functional fragment thereof according to claim 19 or claim 20, wherein the immunoglobulin is of isotype IgE.

22. An immunoglobulin or functional fragment thereof according to any of claims 19 to 21, wherein the immunoglobulin is a chimaeric or humanized antibody, preferably comprising one or more human framework regions and / or one or more human IgE heavy and / or light chain constant domains.

23. A method for increasing anti-cancer potency of an IgE antibody, comprising increasing affinity of the antibody for a target antigen.

24. A method according to claim 23, wherein the method comprises a step of in vitro affinity maturation of the antibody.

25. A method according to claim 23 or claim 24, wherein the method comprises increasing the affinity of the antibody for the target antigen by at least 2-fold.

26. A method according to any of claims 23 to 25, wherein the method comprises increasing the affinity of the antibody for the target antigen by at least 3 -fold, at least 10-fold, at least 100-fold or at least 1000-fold.

27. A method according to any of claims 23 to 26, wherein the method reduces a dissociation constant (Kd) of the antibody for binding to a target antigen to 10 nM or less.

28. A method according to any of claims 23 to 27, wherein the antibody has a Kd for binding to the target antigen of 1 pM to 10 nM, 100 pM to 5 nM or 0.5 to 3 nM after performing the method.

29. A method according to any of claims 23 to 28, wherein the antibody has a Kd for binding to the target antigen of 20 nM or higher before performing the method.

30. A method according to any of claims 23 to 29, wherein the antibody obtained by the method is as defined in any of claims 1 to 15.

31. A method according to any of claims 23 to 30, wherein the method increases anti-cancer potency of the IgE antibody by at least 5%, 10%, 50%, 75% or 95%.

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