Compositions containing IgE antibodies

Abstract

In one aspect, the invention relates to an anti-folate receptor alpha (FRα) immunoglobulin E (IgE) antibody for use in treating a tumor with low FRα expression in a subject.

Description

[Technical field]

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

[0002] Therapeutic antibodies currently complement conventional treatments for many malignancies, but nearly all drugs currently in development rely on only one of the nine human antibody classes, namely IgG1, the most abundant antibody class in 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, IgG1-4, IgA1, IgA2 and IgE) to perform immune surveillance and mediate the destruction of pathogens in different anatomical compartments. However, only IgG (mostly IgG1) is used in cancer immunotherapy.

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

[0004] Subsequent clinical trials with antibodies recognizing the B-cell marker CD20 supported the inference that IgG1 would be the subclass of choice 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 these studies, comparison of the anti-tumor effects of different antibody classes has been restricted to IgG and IgM in both mouse models and patients with lymphoid malignancies, although IgA has been shown to mediate ADCC in vitro and in vivo in mouse models of lymphoma (Dechant M, Valerius T (2001) IgA antibodies for cancer therapy. Crit Rev Oncol Hematol 39: 69-77).

[0005] Folate receptor alpha (FRα) is a cancer-associated antigen that is overexpressed in several types of solid cancers, including ovarian and endometrial cancers and mesotheliomas. Expression of FRα has been described in normal kidney, placenta, lung, fallopian tube, pancreas, and testis, but not in other normal tissues, including heart, liver, spleen, gastrointestinal tract, ovaries, uterus, muscle, lymphatic system, and glandular tissues (Weitman, Lark et al., Cancer Res 52(12): 3396-3401; Kelemen 2006, Int J Cancer 119(2): 243-250). In normal tissues, FRα expression is generally low or restricted to the luminal surface, making it inaccessible to circulating antibodies (Parker, Turk et al. 2005, Anal Biochem 338(2): 284-293; O'Shannessy, Yu et al. 2012, Oncotarget 3(4): 414-425). FRα expression is found in up to 40% of primary ovarian and endometrial tumors and nearly 30% of lung cancers. FRα in tumors, particularly ovarian and endometrial cancers, is known to be accessible to antibodies and can be expressed at much higher levels than in normal tissues (Antony 1996, Annu. Rev. Nutr. 16: 501-521). Because the level and location of FRα expression in normal tissues and tumors differs, FRα is considered to be an effective tumor-specific antigen (Mantovani, Miotti et al. 1994, Eur J Cancer 30A(3): 363-369; Toffoli, Cernigoi et al. 1997, Int J Cancer 74(2): 193-198).

[0006] Initial clinical trials targeting FRα with IgG antibodies have suggested good tolerability. For example, farletuzumab (MORAb-003), a humanized anti-FRα IgG antibody, was successfully administered at 12 mg / m in a phase I study in platinum-resistant epithelial ovarian cancer. 2 ~40mg / m 2(See, e.g., Konner et al., Clin Cancer Res. 2010 Nov 1;16(21):5288-95.) Several anti-FRα IgG antibodies, including antibody-drug conjugates (ADCs), are in development. For example, mirvetuximab sorafutansine is an ADC consisting of an FRα-binding antibody, a cleavable linker, and a cytotoxic payload that is being used in clinical trials for ovarian cancer.

[0007] However, phase III clinical trials of anti-FRα IgG therapy suggest that high FRα expression may be necessary for the efficacy of these antibodies. For example, a phase III trial of farletuzumab in platinum-resistant ovarian epithelial cancer failed to achieve its primary objective, i.e., improved progression-free survival (Vergote et al., Int J Gynecol Cancer. 2013;23(8 Suppl 1):11; Walters et al., Gynecol Oncol. 2013;131(2):493-498). It was suggested that this lack of efficacy may be due to the failure to include a minimum level of FRα expression as an eligibility criterion for the trial (Sato and Itamochi, OncoTargets and Therapy 2016:9 1181-1188).

[0008] Additionally, ImmunoGen's Phase 3 study of mirvetuximab soravtansine in FRα-positive platinum-resistant ovarian cancer (FORWARD I) failed to meet its primary endpoint (see Immunogen press release, ImmunoGen Announces Top-Line Results from Phase 3 FORWARD I Study of Mirvetuximab Soravtansine in Ovarian Cancer, dated March 1, 2019, available at: https: / / investor.immunogen.com / news-releases / news-release-details / immunogen-announces-top-line-results-phase-3-forward-i-study).

[0009] Eligibility criteria for the FORWARD I trial included patients with platinum-resistant ovarian cancer with intermediate or high levels of FRα who had been previously treated with up to three dosing regimens. It has been suggested that high levels of FRα expression may be necessary for efficacy (see ImmunoGen Presents Full Data from Phase 3 FORWARD I Study of Mirvetuximab Soravtansine in Ovarian Cancer at ESMO, September 29, 2019, available at: https: / / investor.immunogen.com / news-releases / news-release-details / immunogen-presents-full-data-phase-3-forward-i-study). Therefore, a further Phase III trial (SORAYA) focused only on high expression patients and implemented a more specific diagnostic test (see ClinicalTrials.gov Identifier: NCT04296890, A Study of Mirvetuximab Soravtansine in Platinum-Resistant, Advanced High-Grade Epithelial Ovarian, Primary Peritoneal, or Fallopian Tube Cancers With High Folate Receptor-Alpha Expression (SORAYA)).Other studies of mirvetuximab soravtansine in combination with additional chemotherapeutic agents also suggest that high FRα expression is required (see, e.g., Cristea et al., A phase I study of mirvetuximab soravtansine (MIRV) and gemcitabine (G) in patients (Pts) with selected FRα-positive solid tumors: Results in the ovarian cancer (EC) cohort; Journal of Clinical Oncology 2021 39:15_suppl, 5542).

[0010] Similarly, the anti-FRα antibody MOv18-IgG1 was shown to induce tumor cell killing in cancer cells with high FRα expression but not in those with low FRα expression by combining antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) functions (Cheung et al., Clin Cancer Res. 2018 October 15; 24(20): 5098-5111). The authors of Cheung et al. suggest that the lack of cell-mediated killing by MOv18-IgG1 in cells with low FRα expression may be important to avoid target / extratumoral toxic effects. Therefore, improved therapies are needed for tumors that do not express FRα at high levels, e.g., ovarian cancer.

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

[0012] A mouse / human chimeric IgE antibody specific for FRα (MOv18 IgE) has been shown to have superior antitumor efficacy compared to otherwise identical IgG in syngeneic immunocompetent animals (Gould et al., Eur J Immunol 1999; 29:3527-37; Josephs et al., Cancer Res. 2017 Mar 1; 77(5):1127-1141; Karagiannis et al., Cancer Res. 2017 Jun 1;77(11):2779-2783). TNFα / MCP-1 signaling was identified as an IgE-mediated mechanism of monocyte and macrophage activation and recruitment to tumors. These findings exemplify the potent macrophage activating function employed by IgE against parasites, but not an allergic IgE mechanism. If the antitumor activity of MOv18 IgE in these preclinical experiments can be reproduced in patients, the potential clinical application of IgE-derived drugs in clinical oncology is clear.

[0013] However, the lack of clinical trial data on the therapeutic use of IgE antibodies in humans means that there are still no suitable therapeutic methods and uses involving IgE antibodies. In particular, it is unclear which subgroups of cancer patients may benefit from therapeutic IgE antibodies. Since anti-FRα IgG antibodies are primarily indicated for high FRα expressers, there is a particular need for improved treatments for other subgroups of cancer patients, including ovarian cancer. However, it is unclear how the methods and uses developed for the administration of other therapeutic antibody isotypes (e.g., IgG) can be adapted to IgE antibodies, and whether they can be used to treat subgroups of patients with the same or different IgG and IgE antibodies. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Weiner LM, Surana R, Wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10: 317-327 [Non-Patent Document 2] Bruggemann M, Williams GT, Bindon CI, Clark MR, Walker MR, Jefferis R, Waldmann H, Neuberger MS (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J Exp Med 166: 1351-1361 [Non-Patent Document 3] Alduaij W, Illidge TM (2011) The future of anti-CD20 monoclonal antibodies: are we making progress? Blood 117: 2993-3001

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[0015] Thus, in one aspect, the invention provides an anti-folate receptor alpha (FRα) immunoglobulin E (IgE) antibody for use in treating a tumor with low FRα expression in a subject.

[0016] In one embodiment, a "low FRα expressing tumor" means that fewer than 50% of the tumor cells in a subject express FRα. Preferably, fewer than 40%, 30%, 20%, or 10% of the tumor cells in a subject express FRα.

[0017] In another embodiment, less than 50% of the tumor cells in the subject exhibit detectable FRα expression, e.g., detectable membrane (i.e., cytoplasmic membrane) FRα expression. Preferably, less than 50%, 40%, 30%, 25%, 20%, or 10% of the tumor cells in the subject exhibit detectable membrane FRα expression. FRα expression (e.g., membrane expression) is typically detected using immunohistochemistry, i.e., detectable expression refers to immunohistochemical detection of FRα.

[0018] In another embodiment, typically, using immunohistochemical detection of FRα, less than 50%, 40%, 30%, 25%, 20%, or 10% of tumor cells in a subject show moderate or high (2+) intensity staining for (e.g., membrane) FRα.

[0019] In another embodiment, the tumor cells in the subject are classified according to membrane FRα staining intensity. FRα staining intensity can be classified on a scale of 0 (undetectable FRα) to 3 (high FRα staining intensity), e.g., 1 indicates low FRα staining intensity and 2 indicates moderate FRα staining intensity. In some embodiments, the tumor cells in the subject are further classified according to the percentage of tumor cells that are FRα positive (e.g., 0-100%).

[0020] Preferably, the subject's overall tumor FRα expression score is determined as the product of membrane FRα staining intensity score and the percentage of FRα positive tumor cells. For example, in some embodiments, the subject's overall tumor FRα expression score may be less than 100, preferably less than 90, 80, 70, 60, 50, or 40, more preferably less than 30 or less than 20.

[0021] In an alternative embodiment, the tumor FRα expression in the subject can be compared with the tumor FRα expression in other cancer subjects, for example, other subjects suffering from the same type of cancer. Thus, the relative level of tumor FRα expression in the subject can be ascertained. In a preferred embodiment, the tumor (e.g., membrane) FRα expression in the subject is lower than the tumor FRα expression in at least 50% of the cancer subjects. Preferably, the (e.g., membrane) FRα expression in the tumor cells of the subject is lower than the tumor FRα expression in at least 60%, at least 70%, or at least 80% of the cancer subjects suffering from, for example, the same form of cancer, preferably ovarian cancer. More preferably, the tumor (e.g., membrane) FRα expression in the subject is lower than the tumor FRα expression in at least 50%, 60%, 70%, 80%, or at least 90% of the FRα-expressing tumors, preferably FRα-expressing ovarian tumors.

[0022] In a preferred embodiment, the tumor expresses FRα, i.e., the tumor exhibits at least some FRα expression. Preferably, the tumor cells in the subject exhibit detectable membrane FRα expression, for example by immunohistochemistry. More preferably, at least 1% or at least 5% of the tumor cells in the subject exhibit detectable (e.g., membrane) FRα expression, for example using immunohistochemical detection of FRα. In other embodiments, at least 10%, 15%, or 20% of the tumor cells in the subject exhibit detectable membrane FRα expression.

[0023] In one embodiment, the antibody is a MOv18 IgE antibody.

[0024] In one embodiment, the IgE antibody is used to treat and / or delay the progression of cancer in a subject. For example, the antibody can be used to delay the progression of a low FRα-expressing tumor in a subject.

[0025] In one embodiment, the tumor or cancer is an ovarian tumor or ovarian cancer.

[0026] In one embodiment, the antibody lacks a cytotoxic moiety. Thus, the antibody may comprise, consist of, or consist essentially of one or more (e.g., four) polypeptide chains, such as (only) immunoglobulin (preferably IgE) heavy and / or light chains. In particular, it is preferred that the antibody is not an antibody drug conjugate (ADC). Thus, the antibody may lack an additional drug or group with a cytotoxic effect, such as a chemotherapeutic agent or drug capable of (directly) killing cancer cells. The antibody may further lack a linker or other group for conjugating a cytotoxic moiety to the polypeptide.

[0027] In one embodiment, the weekly dose of IgE antibody administered to the subject is less than 50 mg, 25 mg, 10 mg, 3 mg, or 1 mg, hi another embodiment, the weekly dose of IgE antibody is between 10 μg and 50 mg, between 70 μg and 30 mg, between 70 μg and 3 mg, between 500 μg and 1 mg, or about 700 μg.

[0028] Preferably, the IgE antibody is administered to the subject once a week or once every two weeks. In one embodiment, the IgE antibody is administered to the subject for up to 12 weeks. In another embodiment, the IgE antibody is administered to the subject (i) once a week for 6 weeks, followed by (ii) once every two weeks for 6 weeks.

[0029] In one embodiment, the IgE antibody is administered to the subject at a dose of less than 1 mg / kg, less than 0.1 mg / kg, or less than 0.03 mg / kg per dose, hi another embodiment, the IgE antibody is administered to the subject at a dose of less than 1 mg / kg / week, less than 0.1 mg / kg / week, or less than 0.03 mg / kg / week.

[0030] In a further aspect, the present invention provides a method for treating cancer and / or delaying the progression of cancer in a subject having a tumor with low FRα expression, comprising the step of administering to the subject a therapeutically effective amount of an anti-folate receptor alpha (FRα) immunoglobulin E (IgE) antibody as defined above.

[0031] In a further aspect, the present invention provides a pharmaceutical composition for use in treating a tumor with low FRα expression in a subject, comprising an anti-folate receptor alpha (FRα) immunoglobulin E (IgE) antibody as defined above and one or more pharma- ceutically acceptable excipients, carriers, or diluents.

[0032] Preferably, the composition contains less than 50 mg of IgE antibody. More preferably, the composition contains less than 30 mg, less than 25 mg, less than 10 mg, less than 5 mg, less than 3 mg, or less than 1 mg of IgE antibody. In other embodiments, the composition contains between 10 μg and 50 mg, between 70 μg and 30 mg, between 70 μg and 3 mg, between 500 μg and 1 mg, or about 700 μg of IgE antibody.

[0033] In one embodiment, the composition is in the form of a liquid. Preferably, the composition is an aqueous solution having a concentration of IgE antibodies of 0.1 mg / ml to 10 mg / ml, 0.5 mg / ml to 2 mg / ml, or about 1 mg / ml. Preferably, the pharma- ceutically acceptable excipient is selected from sodium citrate, L-arginine, sucrose, polysorbate 20, and / or sodium chloride.

[0034] In one embodiment, the composition is suitable for intravenous or subcutaneous injection. Preferably, the composition is suitable for intravenous or subcutaneous injection at a maximum total dose of up to 50 mg / week, 25 mg / week, 10 mg / week, 3 mg / week, or 1 mg / week. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 shows the amino acid sequence of MOv18 IgE light (L) chain (SEQ ID NO:1), with mouse VL in bold and human CL in plain text. [Diagram 2] FIG. 2 shows the amino acid sequence of MOv18 IgE heavy (H) chain (SEQ ID NO:2), with the mouse VH in bold and the human CH in plain text. [Diagram 3] FIG. 3 shows the amino acid sequence of MOv18 IgE light chain variable domain (VL) (SEQ ID NO:3). [Figure 4] FIG. 1 shows the amino acid sequence of MOv18 IgE heavy chain variable domain (VH) (SEQ ID NO:4). [Diagram 5] FIG. 1 shows pharmacokinetics (serum concentrations) of MOv18 IgE following intravenous administration of the antibody. [Figure 6]Figure 1 shows CT scan images and tumor measurements taken from the CT scan images showing tumor size reduction in ovarian cancer subjects treated with MOv18 IgE antibody at the 700 μg dose level. Tumors (shown in the area within the oval in each image) are shown at baseline (left panel) and after 6 weeks of treatment (right panel). Target and non-target lesion dimensions and status in subjects were determined before and after cycles of antibody treatment and after a maintenance period. [Figure 7] FIG. 1 shows a significant decrease in serum concentrations of the ovarian cancer antigen CA125 during treatment of a patient with six weekly doses of 700 μg of MOv18 IgE antibody followed by three further 700 μg doses of the antibody at two-week intervals. [Figure 8] Figure 1 shows plots of change in Response Evaluation Criteria in Solid Tumors (RECIST) scores in individual ovarian cancer subjects treated with MOv18 IgE antibody. Each line represents the percentage change in RECIST score in an individual patient from the start of treatment (i.e., 6 weeks after treatment and 12 weeks after treatment). RECIST scores that increase or decrease by less than 20% indicate stable disease. [Figure 9] Figure 1 shows a waterfall plot of the change in RECIST score in individual ovarian cancer subjects after 6 weeks of treatment with MOv18 IgE antibody. Each vertical bar represents the change in RECIST score in an individual subject at 6 weeks. A total of 20 subjects were treated. If no vertical bar is shown, it indicates that there was no change in RECIST score in that subject after 6 weeks (this occurred in 4 subjects, indicated by the gap between the vertical bars along the x-axis). [Figure 10]Figure 1 shows a waterfall plot of the change in RECIST score in individual ovarian cancer subjects after 6 or 12 weeks of treatment with MOv18 IgE antibody. The same subjects shown in Figure 9 are presented in the same order. Only a few subjects (5) continued treatment beyond 6 weeks. Each vertical bar with an asterisk (*) represents the change in RECIST score in an individual subject treated for up to 12 weeks. The remaining vertical bars without an asterisk represent the change in RECIST score in individual subjects treated for up to 6 weeks as shown in Figure 9. No vertical bar is shown to indicate a 0% change in RECIST score in a subject (this occurred in two subjects and is indicated by the gap in the vertical bars along the x-axis). [Figure 11] Figure 1 shows a waterfall plot of the change in RECIST (Response Evaluation Criteria in Solid Tumors) score in individual ovarian cancer subjects after 6 weeks of treatment with MOv18 IgE antibody compared to the FRα expression score in each subject. Each vertical bar represents the change in RECIST score in an individual subject at 6 weeks. A total of 20 subjects were treated. If no vertical bar is shown, this indicates that the subject had no change in RECIST score after 6 weeks (this occurred in 4 subjects, indicated by the gap between the vertical bars along the x-axis). PD indicates progressive disease and SD indicates stable disease. The overall FRα expression score for each subject is calculated as the product of the membrane staining intensity score (membrane score) and the percentage of tumor cells positive for FRα (%membrane+). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] It has surprisingly been found that anti-FRα IgE antibodies can provide an effective treatment for tumors with low FRα expression. In particular, anti-FRα IgE (MOv18 IgE) has been found to be effective in treating or delaying the progression of ovarian cancer in subjects with very low FRα membrane expression scores.

[0037] This finding is particularly surprising because a comparable IgG antibody (i.e., MOv18 IgG1) is known to kill tumors with high but not low FRα expression, and this selectivity was thought to be important in avoiding extratumoral toxic effects (Cheung et al., Clin Cancer Res. 2018 October 15; 24(20): 5098-5111). Additionally, anti-FRα therapeutic approaches using IgG antibodies have focused on treating high FRα expressers and / or combining the antibody with a cytotoxic moiety in another combination therapy using agents such as, for example, ADCs and / or gemcitabine (see Martin et al. Gynecol Oncol. 2017 Nov; 147(2): 402-407; Sato and Itamochi, OncoTargets and Therapy 2016:9 1181-1188 and Cristea et al., Journal of Clinical Oncology 2021 39:15_suppl, 5542).

[0038] In contrast, anti-FRα IgE antibodies can treat low FRα expressing tumors as monotherapy, i.e., without being incorporated into an ADC or combined with additional chemotherapeutic agents. Thus, the present invention represents a significant contribution to the art in addressing unmet medical needs, particularly in the subgroup of cancer patients who are low FRα expressers.

[0039] Furthermore, it has been found that methods, compositions, dosage forms, and administration regimens typically used for IgG antibodies are not necessarily transferable to IgE antibodies. In particular, it has been demonstrated herein that the minimum dose of IgE antibodies required for efficacy (e.g., anti-tumor effect in subjects with low FRα expression) can be much lower than the typical effective dose of IgG antibodies. For example, as shown in the Examples below, anti-folate receptor alpha (FRα) IgE antibodies have been found to have anti-tumor effects at unit doses as low as 700 μg (about 0.01 mg / kg), which is several orders of magnitude lower than typical IgG therapeutic antibody doses (e.g., around 150-2000 mg per dose or 2-20 mg / kg).

[0040] This result indicates that due to differences in the pharmacology and pharmacokinetics of IgG and IgE, methods, uses, and compositions (such as dosing regimens, unit dosage forms, and subgroups of cancer patients treated) developed for IgG antibodies are not necessarily applicable to IgE. The inventors have thus developed new uses and dosing regimens that are particularly applicable to therapeutic IgE administration, for example in the treatment of cancer in patients with low FRα expression.

[0041] therapeutic antibodies An antibody is a polypeptide ligand that contains at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen, such as an epitope of FRα or a fragment thereof. Antibodies are typically composed of a heavy chain and a light chain, each of which has a variable region called the heavy chain variable (VH) region and the light chain variable (VL) region. Together, the VH and VL regions are responsible for binding to the antigen recognized by the antibody.

[0042] Antibodies include intact immunoglobulins known in the art, as well as variants and portions of antibodies, provided that such fragments retain at least one function of IgE, e.g., capable of binding to an Fcε receptor. Antibodies also include genetically engineered forms, such as chimeric, humanized (e.g., humanized antibodies containing mouse sequences in the variable regions) or human antibodies, heteroconjugate antibodies (e.g., bispecific antibodies), such as those described in Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0043] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are nine major isotypes or classes that determine the functional activity of the antibody molecule, namely IgA1-2, IgD, IgE, IgG1-4 and IgM, which correspond to the heavy chain types α, δ, ε, γ and μ. Thus, the heavy chain type present defines the class of the antibody. The distinct heavy chains differ in size and composition, with α and γ comprising approximately 450 amino acids, whereas μ and ε have approximately 550 amino acids. Differences in the constant regions of each heavy chain type result in different effector functions of each antibody isotype, thanks to their selective binding to specific types of receptors (e.g. Fc receptors). Thus, in an embodiment of the invention, the antibody preferably comprises an epsilon (ε) heavy chain, i.e., the antibody is of isotype IgE, which binds to Fcε receptors.

[0044] Each heavy and light chain contains a constant region and a variable region (these regions are also known as "domains"). In combination, the heavy and light chain variable regions bind specifically to an antigen. The 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 the CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, e.g., humans. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0045] 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 from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is in the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

[0046] An antibody may have particular VH and VL region sequences and therefore particular 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. Those 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.

[0047] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those of skill in the art, such as by creating hybrid antibody-forming cells from the fusion of a myeloma cell with an immune spleen cell. Monoclonal antibodies include humanized monoclonal antibodies.

[0048] A "chimeric antibody" comprises sequences derived from two different antibodies, typically from different species. For example, a chimeric antibody can contain human and mouse antibody domains, such as a human constant region and a mouse variable region (e.g., from a mouse antibody that specifically binds to a target antigen).

[0049] Chimeric antibodies are typically constructed by fusing, e.g., by genetic engineering, the variable and constant regions from light and heavy immunoglobulin genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody can be linked to human constant segments, e.g., kappa and epsilon. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, e.g., the Fc (effector) domain from a human IgE antibody, although other mammalian species can be used or the variable regions can be generated by molecular techniques. Methods for producing chimeric antibodies are known in the art, see, e.g., U.S. Pat. No. 5,807,715.

[0050] A "humanized" antibody is an antibody that comprises a human framework region and one or more CDRs from a non-human (e.g. mouse, rat or synthetic) antibody. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". In one embodiment, all of the CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region is typically substantially identical to a human immunoglobulin constant region, i.e. at least about 85-90%, such as about 95% or more identical. Thus, all parts of a humanized immunoglobulin other than the CDRs are substantially identical to the corresponding parts of a native human immunoglobulin sequence.

[0051] 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 with amino acids from the donor framework. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0052] Humanized immunoglobulins can be constructed by genetic engineering (see, for example, U.S. Patent No. 5,585,089). Typically, humanized monoclonal antibodies are produced by transferring donor antibody complementarity determining regions from the variable heavy and light chains of a mouse immunoglobulin into human variable domains, followed by substitution of human residues in the framework regions of the donor counterpart. 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, in Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.

[0053] A "human" antibody (also called a "fully human" antibody) is an antibody that contains human framework regions and all of the CDRs from a human immunoglobulin. In one example, the framework and CDRs are from the same originating human heavy and / or light chain amino acid sequence. However, a framework from one human antibody can be engineered to contain CDRs from a different human antibody.

[0054] In embodiments of the invention, the antibodies may be monoclonal or polyclonal, including chimeric, humanized or fully human antibodies.

[0055] Anti-FRα antibody In some embodiments, the antibody specifically binds to folate receptor alpha (FRα) to form an immune complex. Typically, the antibody may comprise an antigen-binding region (e.g., one or more variable regions, or one to six CDRs) derived from an antibody known to bind to FRα, preferably human FRα, such as MOv18 IgE.

[0056] FRα (also known as folate receptor 1 or FOLR1) is overexpressed in several types of solid cancers, including ovarian and endometrial cancers and mesotheliomas. The antigen is characterized as being virtually tumor-specific, and clinical trials targeting FRα with IgG and IgE antibodies have shown a favorable tolerability profile. MOv18 IgG and IgE antibodies that bind to FRα and their properties are described, for example, in Coney, LR, A. Tomassetti, et al. (1991). Cancer Res 51(22): 6125-6132; Gould, HJ, GA Mackay, et al. (1999). Eur J Immunol 29(11): 3527-3537; Karagiannis, SN, Q. Wang, et al. (2003). Eur J Immunol 33(4): 1030-1040.

[0057] In certain embodiments, the antibody comprises a variable region (e.g., a heavy chain variable domain (VH) and / or a light chain variable domain (VL)) or at least one, two, three, four, five or six CDRs (e.g., three heavy chain CDRs or three light chain CDRs) from MOv18 IgG or IgE, such as the CDRs in SEQ ID NO:4 and / or SEQ ID NO:3, where the CDR sequences may be defined according to the methods of Kabat, Chothia or IMGT (see, e.g., Dondelinger, Front Immunol. 2018; 9: 2278 and references cited therein, which are incorporated by reference herein). For example, the CDRs can be numbered according to Kabat (see Kabat EA, et al. (US) NI of H. Sequences of Immunoglobulin Chains: Tabulation Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain BP-Microglobulins, 1979), or 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 (Giudicelli V et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res. 1997 Jan 1; 25(1):206-11 or Lefranc MP, Unique database numbering system for immunogenetic analysis, Immunol Today. 1997 Nov; 18(11):509). The amino acid sequences of the VH and VL domains of MOv18 IgE are set forth in SEQ ID NO:4 and SEQ ID NO:3, respectively. In another embodiment, the antibody is a chimeric, humanized, or fully human antibody that specifically binds to an epitope bound by MOv18 IgE.Most preferably, the therapeutic antibody is MOv18 IgE, eg, the antibody comprises a light chain amino acid sequence set forth in SEQ ID NO:1 and / or a heavy chain amino acid sequence set forth in SEQ ID NO:2.

[0058] In another example, the antibody comprises a variable region (e.g., a heavy chain variable domain and / or a light chain variable domain) derived from a human B cell clone that recognizes an epitope found, for example, on FRα, preferably human FRα, or at least one, two, three, four, five or six CDRs (e.g., three heavy chain CDRs or three light chain CDRs).

[0059] In one embodiment, the antibody comprises one or more human constant regions, such as one or more human heavy chain constant domains (e.g., ε constant domains) and / or human light chain (e.g., κ or λ) constant domains. The amino acid sequence of a human light (κ) chain constant domain is shown in SEQ ID NO:1 (non-bolded text). The amino acid sequence of a human heavy chain constant domain is shown in SEQ ID NO:2 (non-bolded text). In one embodiment, the antibody comprises one or more human framework regions within the VH and / or VL domains.

[0060] In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 75%, at least about 85%, at least about 99% or at least about 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Human framework regions and the mutations that can be made in humanized antibody framework regions are known in the art (see, e.g., U.S. Pat. No. 5,585,089).

[0061] Additional antibodies against specific antigens, such as FRα, can also be produced by established methods, and at least the variable regions or CDRs from such antibodies can be used in the antibodies of the present invention (e.g., the produced antibodies can be used to provide CDR or variable region sequences for an IgE acceptor sequence). Methods for synthesizing polypeptides and immunizing host animals are known in the art. Typically, a host animal (e.g., a mouse) is inoculated intraperitoneally with an amount of immunogen (e.g., a polypeptide comprising FRα or an immunogenic fragment thereof) and (in the case of monoclonal antibody production) a hybridoma prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 25 6:495-497.

[0062] The sequence of human FRα is known (see, e.g., UniProt database accession number P15328), and thus human FRα can be, for example, purified from natural sources or expressed using recombinant techniques for use in such methods. The amino acid and nucleic acid sequences of human FRα are set forth below in SEQ ID NOs: 5 and 6, respectively: SEQ ID NO:5 - Human FRα amino acid sequence: MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKER VLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS SEQ ID NO:6 - Human FRα nucleic acid sequence: atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaaggattgcatgggccaggactgagcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagtgtcgaccctggaggaagaatgcctgctgttctaccaacaccagccaggaagcccataaggatgtttcctacctatatagattcaactggaaccactgtggagagatggcacctgcctgcaaacggcatttcatccaggacacctgcctctacgagtgctcccccaacttggggccctggatccagcaggtggatcagagctggcgcaaagagcgggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagattgtcgcacctcctacacctgcaagagcaactggcacaagggctggaactggacttcagggtttaacaagtgcgcagtgggagctgcctgccaacctttccatttctacttccccacacccactgttctgtgcaatgaaatctggactcactcctacaaggtcagcaactacagccgagggagtggccgctgcatccagatgtggttcgacccagcccagggcaaccccaatgaggaggtggcgaggttctatgctgcagccatgagtggggctgggccctgggcagcctggcctttcctgcttagcctggccctaatgctgctgtggctgctcagc

[0063] Hybridomas producing suitable antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture medium or body fluids, if desired, by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration. Undesirable activity, if present, can be removed, for example, by passing the preparation through an adsorbent made of immunogen bound to a solid phase, eluting or releasing the desired antibody from the immunogen. If desired, the desired antibody (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The antibody-encoding sequence can be maintained in a vector within a host cell, and the host cell can then be expanded and frozen for future use.

[0064] Phage display technology, such as that described in U.S. Pat. No. 5,565,332 and other published literature, can be used to select and generate 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 the relevant disorder). For example, existing antibody phage display libraries can be panned in parallel against large collections of synthetic polypeptides. According to this technique, antibody V domain genes are cloned in frame into major or minor coat protein genes of filamentous bacteriophages, such as M13 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 genes encoding antibodies exhibiting these properties. Thus, antibody sequences selected using phage display from human libraries can contain human CDR or variable region sequences that confer specific binding to a particular antigen, e.g., FRα, and can be used to provide fully human antibodies for use in the present invention.

[0065] Methods for deriving heavy and light chain sequences from human B cell and plasma cell clones are also known in the art and are typically performed using polymerase chain reaction (PCR) techniques, examples of which are described in Kuppers R, Methods Mol Biol. 2004;271:225-38; Yoshioka M et al., BMC Biotechnol. 2011 Jul 21;11:75; Scheeren FA et al., PLoS ONE 2011, 6(4): e17189. doi:10.1371 / journal.pone.0017189; Wrammert J et al., Nature 2008 453, 667-671; Kurosawa N et al., BMC Biotechnol. 2011 Apr 13;11:39; Tiller et al., J Immunol Methods. 2008 January 1; 329(1-2): 112-124. Thus, antibody sequences selected using B cell clones can contain human CDR or variable region sequences that confer specific binding to, for example, FRα, and can be used to provide fully human antibodies for use in the present invention.

[0066] IgE antibodies The therapeutic antibodies administered to the subject are IgE antibodies, i.e., antibodies of the isotype IgE. There are several fundamental structural differences between IgE and IgG that have functional effects. IgE shares the same basic molecular structure with other classes of antibodies, but the heavy chain of IgE has one more domain than the heavy chain of IgG. The most distinct feature of IgE is the Cε2 domain, since the Cε3 and Cε4 domains of IgE are homologous in sequence and similar in structure to the Cγ2 and Cγ3 domains of IgG. The Cε2 domain is found to fold back against the heavy chain IgE and make extensive contacts with the Cε3 domain. This curved structure of the IgE heavy chain allows it to adopt either an open or closed conformation. Unbound IgE dimers have one chain in an open conformation and one chain in a closed conformation. Binding of FcεRI to IgE is believed to be biphasic, with initial binding to an open Cε chain followed by extensive structural rearrangements to allow binding to closed Cε chains. Binding between IgE dimers and FcεRI occurs with a 1:1 stoichiometry, despite the presence of two identical Cε chains. This rearrangement results in a very tight interaction between IgE and FcεRI and a much greater affinity of IgE for the Fc receptor than is found with IgG and FcγR (McDonnell, JM, R. Calvert, et al. (2001) Nat Struct Biol 8(5): 437-441).

[0067] The antibodies used in the present invention are typically capable of binding to Fcε receptors, such as FcεRI and / or FcεRII receptors. Preferably, the antibodies are capable of binding at least to FcεRI (i.e. high affinity Fcε receptor) or at least to FcεRII (CD23, low affinity Fcε receptor). Typically, the antibodies are also capable of activating Fcε receptors expressed, for example, on cells of the immune system, to initiate IgE-mediated effector functions.

[0068] The epsilon (ε) heavy chain is crucial for IgE antibodies and contains an N-terminal variable domain, VH, and four constant domains, Cε1 to Cε4. As with other antibody isotypes, the variable domain confers antigen specificity and the constant domains recruit isotype-specific effector functions.

[0069] IgE differs from the more abundant IgG isotype in that it cannot fix complement and does not bind to the Fc receptors FcγRI, RII and RIII expressed on the surface of monocytes, NK cells and neutrophils. However, IgE binds to the "high affinity" IgE receptor (FcεRI, Ka.10) on various immune cells, such as mast cells, basophils, monocytes / macrophages and eosinophils. 11 M -1 ), and the "low affinity" receptor FcεRII (Ka.10), also known as CD23, which is expressed on inflammatory and antigen-presenting cells (e.g., monocytes / macrophages, platelets, dendritic cells, T and B lymphocytes). 7 M -1 ) and can have a highly specific interaction.

[0070] The sites of IgE responsible for these receptor interactions have been mapped to peptide sequences on the Cε chain and are distinct. The FcεRI site is in the cleft created by residues between Gln301 and Arg376 and includes the junction between the Cε2 and Cε3 domains (Helm, B. et al. (1988) Nature 331, 180183). The FcεRII binding site is located within residue Val370 surrounding Cε3 (Vercelli, D. et al. (1989) Nature 338, 649-651). The main difference that distinguishes the two receptors is that FcεRI binds monomeric Cε, whereas FcεRII binds only dimeric Cε, i.e. two Cε chains must be associated. IgE is glycosylated in vivo but this is not necessary for binding to FcεRI and FcεRRII. In the absence of glycosylation, binding is actually slightly stronger (Vercelli, D. et al. (1989) et. cit.).

[0071] Thus, binding to the Fcε receptor and the associated effector functions are typically mediated by the heavy chain constant domains of the antibody, in particular by the domains which together form the Fc region of the antibody. The antibodies described herein typically comprise at least a portion of an IgE antibody, such as one or more constant domains derived from IgE, preferably human IgE. In a particular embodiment, the antibody comprises one or more domains (derived from IgE) selected from Cε1, Cε2, Cε3 and Cε4. In one embodiment, the antibody comprises at least Cε2 and Cε3, more preferably at least Cε2, Cε3 and Cε4, preferably the domains are derived from human IgE. In one embodiment, the antibody comprises an epsilon (ε) heavy chain, preferably a human ε heavy chain.

[0072] Amino acid sequences of constant domains from human IgE are shown, for example, in Figures 1 and 2 (SEQ ID NOs: 1 and 2, non-bold text). Nucleotide sequences encoding constant domains from human IgE, in particular the Cε1, Cε2, Cε3 and Cε4 domains, are also disclosed, for example, in WO 2013 / 050725. Amino acid sequences of other human and mammalian IgEs and their domains, including the human Cε1, Cε2, Cε3 and Cε4 domains and human ε heavy chain sequences, are known in the art and available from publicly accessible databases. For example, a database of human immunoglobulin sequences can be accessed from the International ImMunoGeneTics Information System (IMGT®) website at http: / / www.imgt.org. As an example, the sequences of various human IgE heavy (ε) chain alleles and their respective constant domains (Cε1-4) can be accessed at http: / / www.imgt.org / IMGT_GENE-DB / GENElect?query=2+IGHE&species=Homo+sapiens.

[0073] Preferred anti-FRα IgE antibodies In one embodiment, the anti-FRα antibody comprises a VH domain comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:4, e.g., at least 20, 30, 50 or 100 amino acids of SEQ ID NO:4, or the entire length of SEQ ID NO:4, or one, two or three CDRs present in SEQ ID NO:4 (e.g., as defined according to Kabat, Chothia or IMGT).

[0074] In one embodiment, the anti-FRα antibody comprises a VL domain comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:3, e.g., at least 20, 30, 50 or 100 amino acids of SEQ ID NO:3, or the entire length of SEQ ID NO:3, or one, two or three CDRs present in SEQ ID NO:3 (e.g., as defined according to Kabat, Chothia or IMGT).

[0075] Generally, functional fragments of the sequences defined above can be used in the present invention. Functional fragments can 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 FRα and / or Fcε receptors).

[0076] Variants of the above amino acid and nucleotide sequences may also be used in the present invention, provided that the resulting antibody binds to the Fcε receptor. Typically, such variants have a high degree of sequence identity with one of the sequences identified above.

[0077] 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 higher the similarity between the two sequences. Homologs or variants of amino acid or nucleotide sequences have a relatively high degree of sequence identity when aligned using standard methods.

[0078] Methods for aligning sequences for comparison are 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. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988 and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed discussion of sequence alignment methods and homology calculations.

[0079] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.), and on the Internet, for use in conjunction 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 Internet at the NCBI website.

[0080] Homologs and variants of antibodies (e.g., anti-FRα antibodies or domains thereof, such as VL, VH, CL or CH domains) typically have at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the original sequence (e.g., the sequence defined above), counted over the full length alignment with the amino acid sequence of the antibody or domain thereof, e.g., using NCBI Blast2.0, gapped blastp set to default parameters. For comparison of amino acid sequences of more than about 30 amino acids, the Blast2 sequence function is used with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and gap cost per residue of 1). When aligning short peptides (less than about 30 amino acids), the alignment should be performed using the Blast2 sequence function with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). Proteins with even greater similarity to the reference sequence, when assessed by this method, will show increasing percentage identities, e.g., 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 compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10-20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on the similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the Internet at the NCBI website. Those skilled in the art will recognize that these sequence identity ranges are provided for guidance only. It is entirely possible that very significant homologs will be obtained that fall outside the ranges provided.

[0081] Typically, a variant may contain one or more conservative amino acid substitutions compared to the original amino acid or nucleic acid sequence. A conservative substitution is a substitution that does not substantially affect or reduce the affinity of the antibody with the target antigen (e.g., FRα) and / or Fcε receptor. For example, a human antibody that specifically binds to FRα may contain up to 1, 2, 5, 10, or 15 conservative substitutions compared to the original sequence (e.g., as defined above) and retain specific binding to the FRα polypeptide. The term conservative variation also includes the use of substituted amino acids in place of unsubstituted parent amino acids, provided that the antibody specifically binds to the target antigen (e.g., FRα). A non-conservative substitution is one that reduces the activity or binding to the target antigen (e.g., FRα) and / or Fcε receptor.

[0082] Functionally similar amino acids that can be replaced by conservative substitutions are known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other: 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).

[0083] In some embodiments, the IgE antibody may be conjugated to a cytotoxic moiety, e.g., a chemotherapeutic agent capable of (directly) killing cancer cells, to produce an antibody-drug conjugate (ADC). The antibody may be directly conjugated to the cytotoxic moiety or through a linker, as known in the art. For example, one example of such a cytotoxic agent is the maytansinoid DM4, a potent tubulin-targeting agent present in the IgG ADC mirvetuximab sora ftansine. In other embodiments, the IgE antibody may be administered to a subject in combination with another (e.g., simultaneous or sequential) administration of a chemotherapeutic agent, e.g., gemcitabine (2',2'-difluoro-2'-deoxycytidine).

[0084] However, in a preferred embodiment, the IgE antibody lacks a cytotoxic moiety and / or is administered as monotherapy. It has surprisingly been found that anti-FRα IgE antibodies can treat tumors with low FRα expression without the need for administration of cytotoxic drugs (either in the form of ADCs or in combination with chemotherapy).

[0085] Thus, an antibody may comprise, consist or consist essentially of a (optionally glycosylated) polypeptide chain. For example, an antibody may comprise, consist or consist essentially of one or more (preferably four) polypeptide chains, e.g., two heavy immunoglobulin chains and optionally two light immunoglobulin chains. Preferably, the heavy and / or light chains comprise one or more domains of an IgE antibody.

[0086] In particular, it is preferred that the antibody is not an antibody drug conjugate (ADC). Thus, the antibody may lack an additional drug or group that has a cytotoxic effect, such as a chemotherapy drug that can (directly) kill cancer cells. The antibody may further lack a linker or other group for conjugating a cytotoxic moiety to the polypeptide.

[0087] More IgE antibodies As noted above, in a preferred embodiment, the IgE antibody binds to FRα. Preferably, the IgE antibody is capable of inducing cytotoxicity (e.g., ADCC) and / or phagocytosis (ADCP), particularly against cancer cells expressing such antigens.

[0088] In some embodiments, one or more of the variable domains and / or one or more of the CDRs, preferably at least three CDRs, or more preferably all six CDRs, may be derived from one or more of the following antibodies: MOv19 (Coney et al., Cancer Res. 1991 Nov 15;51(22):6125-32; Coney et al., Cancer Res. 1994 May 1;54(9):2448-55), mirvetuximab soravtansine (IMGN853, as described in Ab et al., Molecular Cancer Therapeutics 14(7):1605-13, July 2015), or farletuzumab (MORAb-003, Ebel et al., Cancer Immun. 2007;7:6; Sato and Itamochi, OncoTargets and Therapy 2016:9 Mirvetuximab sorafutansine (IMGN853) refers to an immunoconjugate comprising the humanized MOv19 (M9346A) antibody, a sulfoSPDB (N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoic acid) linker, and DM4 maytansinoid (N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)maytansine).

[0089] For example, an IgE antibody may comprise the following variable domain sequence, or one to six CDRs derived therefrom (e.g., as defined according to Kabat, Chothia, or IMGT): Farletuzumab VH domain: EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSS (SEQ ID NO: 7) Farletuzumab VL(V κ )domain: DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK (SEQ ID NO: 8)

[0090] In other embodiments, an IgE antibody may comprise one or more variable domain sequences or CDRs of an anti-FRα (i.e., anti-FOLR1) antibody or antigen-binding fragment thereof, e.g., as defined in U.S. 2012 / 0009181 or WO 2018 / 213260, the contents of which are incorporated herein by reference. For example, an IgE antibody may comprise the following variable domain sequence, or one to six CDRs derived therefrom (e.g., as defined according to Kabat, Chothia, or IMGT): huMOv19 VH Domain (SEQ ID NO:9): QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSS huMOV19 VL Domain (SEQ ID NO:10): DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR

[0091] For example, an IgE antibody may comprise one or more (e.g., all six) of the following CDR sequences (as defined according to Kabat): huMOv19 VH-CDR1 (SEQ ID NO: 11): GYFMN huMOv19 VH-CDR2 (SEQ ID NO: 12): RIHPYDGDTFYNQKFQG huMOv19 VH-CDR3 (SEQ ID NO: 13): YDGSRAMDY huMOv19 VL-CDR1 (SEQ ID NO: 14): KASQSVSFAGTSLMH huMOv19 VL-CDR2 (SEQ ID NO: 15): RASNLEA huMOv19 VL-CDR3 (SEQ ID NO: 16): QQSREYPYT

[0092] In another embodiment, the antibody is a chimeric, humanized, or fully human antibody that specifically binds to the epitope bound by mirvetuximab or farletuzumab. The IgE antibody may further comprise one or more IgE constant domains, e.g., Cε1-Cε4 domains, as described above.

[0093] Antibody and Nucleic Acid Production Nucleic acid molecules (also known as polynucleotides) encoding the polypeptides provided herein (including, but not limited to, antibodies and functional fragments thereof) can be readily generated by one of skill in the art using the amino acid sequences provided herein, sequences available in the art, and the genetic code. Moreover, one of skill in the art can readily construct various clones containing functionally equivalent nucleic acids, e.g., nucleic acids that differ in sequence but encode the same effector molecule or antibody sequence. Thus, nucleic acids encoding antibodies are provided in the present invention.

[0094] Nucleic acid sequences encoding antibodies or functional fragments thereof that specifically bind to a target antigen (e.g., FRα) can be prepared, for example, by cloning appropriate sequences or by synthesis using, for example, 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 diethyl phosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981, or an automated synthesizer as described, for example, in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984, by the method of Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, It can be prepared by any suitable method, including direct chemical synthesis by the solid-phase phosphoramidite triester method described by Roberts, et al., 1981, and the solid-phase support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides. This can be converted into double-stranded DNA by hybridization with complementary sequences, or by polymerization using DNA polymerase with the single strand as a template. Those skilled in the art recognize that chemical synthesis of DNA is generally limited to sequences of about 100 bases, and longer sequences can be obtained by ligation of shorter sequences.

[0095] Exemplary nucleic acids encoding antibodies or functional fragments thereof can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques, as well as sufficient instructions to enable one of skill in the art to perform many cloning procedures, can be found, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and Current Protocols in Molecular Biology (Ausubel et al., eds 1995 supplement)). Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to those of skill in the art.

[0096] Nucleic acids encoding native antibodies can be modified to form the antibodies described herein. Modification by site-directed mutagenesis is known in the art. Nucleic acids can also be prepared by amplification techniques. Amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methods are known to those of skill in the art.

[0097] In one embodiment, the antibody is prepared by inserting a cDNA encoding one or more antibody domains (e.g., a murine IgG1 heavy chain variable region that binds to human FRα) into a vector containing a cDNA encoding one or more additional antibody domains (e.g., the human heavy chain ε constant region) such that the antibody domains are read in frame as one contiguous polypeptide comprising a functional antibody region.

[0098] In one embodiment, the cDNA encoding the heavy chain constant region is ligated to the heavy chain variable region such that the constant region is located at the carboxyl terminus of the antibody. The heavy chain variable and / or constant region can then be ligated to the light chain variable and / or constant region of the antibody using disulfide bonds.

[0099] Once the nucleic acid encoding the antibody or functional fragment thereof has been isolated and cloned, the desired protein can be expressed in recombinantly engineered cells, such as bacteria, plant, yeast, insect and mammalian cells. Those of skill in the art will be aware of the numerous expression systems available for protein expression, including E. coli, other bacterial hosts, yeast and various higher eukaryotic cells, such as COS, CHO, HeLa and myeloma cell lines.

[0100] One or more DNA sequences encoding an antibody or fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny are not identical to the parent cell due to possible 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 desired antibodies are also included in the present disclosure.

[0101] 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 can be either constitutive or inducible, and then incorporating it into an expression cassette. The cassette is suitable for replication and integration in either prokaryotes or eukaryotes. A typical expression cassette contains specific sequences useful for regulating the expression of the DNA encoding the protein. For example, the expression cassette can contain an appropriate promoter, enhancer, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of the protein coding sequence, splicing signals for introns, maintenance of the correct reading frame of the gene to allow for correct translation of the mRNA, and a stop codon.

[0102] To obtain high expression levels of the cloned genes, it is desirable to construct an expression cassette that contains, at a minimum, a strong promoter to drive transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator. For E. coli, this includes a promoter, such as the T7, trp, lac, or lambda promoter, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include promoters and / or enhancers, such as those derived from immunoglobulin genes, SV40, or cytomegalovirus, and polyadenylation sequences, and may further include splice donor and acceptor sequences. The cassette can be transferred into the selected host cell by known methods, such as transformation or electroporation for E. coli, and calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed with the cassette can be selected by antibiotic resistance conferred by genes contained in the cassette, such as the amp, gpt, neo, and hyg genes.

[0103] If the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical procedures, e.g., microinjection, electroporation, insertion of a plasmid coated in a liposome, or viral vectors can be used. Eukaryotic cells can be co-transformed with polynucleotide sequences encoding an antibody, a labeled antibody or a functional fragment thereof, and a second foreign DNA molecule encoding a selective trait, e.g., the herpes simplex thymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors, e.g., simian virus 40 (SV40) or bovine papilloma virus, to express the protein (see, e.g., Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Those skilled in the art can readily use expression systems, e.g., plasmids and vectors, useful for producing proteins in higher eukaryotic cells, e.g., cells including COS, CHO, HeLa, and myeloma cell lines.

[0104] Modifications can be made to the nucleic acid encoding the polypeptides described herein (e.g., human FRα-specific IgE antibodies) without diminishing their biological activity. Some modifications can be made to facilitate cloning, expression, or incorporation of targeting molecules into fusion proteins. Such modifications are known to those of skill in the art and include, for example, stop codons, methionine added to the amino terminus to provide an initiation site, amino acids added to either end to create restriction sites at convenient locations, or amino acids added to aid in purification steps (e.g., polyHis). In addition to recombinant methods, the antibodies of the present disclosure can also be constructed in whole or in part using standard peptide synthesis known in the art.

[0105] Once expressed, recombinant antibodies can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, NY, 1982). The antibodies, immunoconjugates and effector molecules need not be 100% pure. Once purified, partially or to homogeneity as desired, the polypeptides should be substantially free of endotoxin if used therapeutically.

[0106] Frequently, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization with strong denaturants followed by refolding. During the solubilization step, a reducing agent must be present to break disulfide bonds, as known in the art. An exemplary buffer containing a reducing agent is 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M dithioerythritol (DTE). Reoxidation of disulfide bonds can occur with 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 in particular in Buchner et al., supra.

[0107] Renaturation is typically accomplished by diluting the denatured and reduced protein (e.g., 100-fold) in a 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.

[0108] As a modification of the two-chain antibody purification protocol, the heavy and light chain regions are solubilized and reduced separately and then combined in the refolding solution. The illustrated yields are obtained when the two proteins are mixed in a molar ratio that does not exceed a 5-fold molar excess of one protein over the other. Excess oxidized glutathione or other oxidizing low molecular weight compounds can be added to the refolding solution after redox shuffling is complete.

[0109] In addition to recombinant methods, the antibodies, labeled antibodies and functional fragments thereof disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of polypeptides 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. The technique of solid phase synthesis is 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. Longer proteins can be synthesized by condensation of the amino and carboxyl termini of shorter fragments.

[0110] Methods for forming peptide bonds by activation of the carboxyl-terminal end (eg, by using the coupling reagent N,N'-dicyclohexylcarbodiimide) are known in the art.

[0111] In one embodiment, the antibody, nucleic acid, expression vector, host cell, or other biological product is isolated. "Isolated" means that the product has been separated or purified substantially away from other biological components in the environment (e.g., a cell) in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles. "Isolated" nucleic acids and antibodies include nucleic acids and antibodies purified by standard purification methods. The term also encompasses nucleic acids and antibodies prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0112] Compositions and Treatment Methods Compositions comprising a carrier and one or more therapeutic IgE antibodies or functional fragments thereof are provided herein. The compositions can be prepared in unit dosage form for administration to a subject. The antibodies can be formulated for systemic or local (e.g., intratumoral) administration. In one example, the therapeutic IgE antibodies are formulated for parenteral administration, such as intravenous or subcutaneous administration.

[0113] Compositions for administration may comprise a solution of the antibody (or functional fragment thereof) dissolved in a pharma- ceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers may be used, e.g., buffered saline, and the like. These solutions are sterile and substantially free of undesirable matter. These compositions may be sterilized by conventional and well-known sterilization techniques. The compositions may contain pharma- cetically acceptable auxiliary substances, as necessary, to approximate physiological conditions, e.g., pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentrations of antibody and excipients in these formulations may vary and are selected according to the particular mode of administration selected and the needs of the subject, based primarily on fluid volumes, viscosities, body weight, and the like. Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).

[0114] In a preferred embodiment, the composition is provided as a unit dosage form containing a defined amount of IgE antibody suitable for administration to a subject, e.g., in a single dose. The unit dosage form may be packaged individually, e.g., in a single container, vial, pre-filled syringe, etc. The unit dosage form may be suitable for immediate administration to a subject (e.g., may contain a physiologically acceptable concentration of salt), or the unit dosage form may be provided in a concentrated or lyophilized form (e.g., for dilution with sterile saline prior to use).

[0115] The anti-FRα IgE antibody may be administered in any suitable dose. However, in preferred embodiments described herein, a typical unit dose of the pharmaceutical composition (e.g., for intravenous administration) contains less than 50 mg of IgE antibody. For example, the composition (i.e., in unit dosage form) may contain less than 40 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, or 1 mg of IgE antibody. The composition may contain at least 10 μg, 100 μg, 200 μg, 300 μg, 500 μg, 700 μg, 1 mg, 3 mg, 5 mg, or 10 mg of IgE antibody. In preferred embodiments, the composition comprises 10 μg-50 mg, 70 μg-30 mg, 300 μg-50 mg, 300 μg-30 mg, 300 μg-3 mg, 500 μg-50 mg, 500 μg-30 mg, 500 μg-10 mg, 500 μg-3 mg, 700 μg-50 mg, 700 μg-30 mg, 700 μg-10 mg, 700 μg-3 mg, 500 μg-5 mg, 500 μg-1 mg, or about 700 μg of IgE antibody. In some embodiments, the composition may comprise one or more amounts of IgE antibody within the above ranges, except for one or more of the following amounts: 1 μg, 5 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, or 15 mg. For example, the composition may contain from 2 μg to 9 μg, from 11 μg to 99 μg, from 101 μg to 499 μg, from 501 to 999 μg or from 2 mg to 9 mg.

[0116] 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, for example, less than 1 mg / kg. Preferably, the IgE antibody may be administered to the subject at a dose of, for example, 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 (per administration). 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 can 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 can be within one or more of the ranges set forth above, except for one or more of the following doses: 1 μg / kg, 10 μg / kg, 100 μg / kg, or 0.5 mg / kg. For example, the dose of the IgE antibody can be 2-9 μg / kg, 11-99 μg / kg, 101-499 μg / kg, or 0.51-0.7 mg / kg.

[0117] In an embodiment of the present invention, the unit dosage form of the IgE antibody is administered at most once a week, for example, the maximum weekly dose of the IgE antibody is 50 mg, 40 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg or 1 mg. For example, the weekly dose of the IgE antibody can be 10 μg to 50 mg, 70 μg to 30 mg, 300 μg to 50 mg, 300 μg to 30 mg, 300 μg to 3 mg, 500 μg to 50 mg, 500 μg to 30 mg, 500 μg to 10 mg, 500 μg to 3 mg, 700 μg to 50 mg, 700 μg to 30 mg, 700 μg to 10 mg, 700 μg to 3 mg, 500 μg to 5 mg, 500 μg to 1 mg, or about 700 μg. The weekly dose of the IgE antibody can also be determined according to the body weight of the subject, for example, the IgE antibody can be administered to the subject at a dose of, for example, 0.7 mg / kg / week, 0.5 mg / kg / week, 0.3 mg / kg / week, 0.1 mg / kg / week, 0.07 mg / kg / week, 0.05 mg / kg / week, 0.03 mg / kg / week, or less than 0.01 mg / kg / week. In a preferred embodiment, the dose of the IgE antibody administered to the subject can 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 an IgE antibody administered to a subject can be within one or more of the ranges set forth above, excluding one or more of the following doses: 1 μg / kg / day (7 μg / kg / week), 10 μg / kg / day (70 μg / kg / week), or 100 μg / kg / day (0.7 mg / kg / week). For example, the dose of an IgE antibody can be 2-6 μg / kg / week, 8-69 μg / kg / week, or 71-699 μg / kg / week.

[0118] In one embodiment, the pharmaceutical composition is a liquid comprising one or more excipients selected from sodium citrate, L-arginine, sucrose, polysorbate 20 and / or sodium chloride. Preferably, the composition has a pH of 6.0 to 8.0, for example about 6.5. Preferred concentrations of the excipients include 0.05 to 0.5 M (for example about 0.1 M) sodium citrate, 10 to 50 g / L (for example about 30 g / L) L-arginine, 10 to 100 g / L (for example about 50 g / L) sucrose, 0.01 to 0.05% w / w (for example 0.02% w / w) polysorbate 20. In one embodiment, the IgE antibodies are present in such formulations in a concentration of about 0.1 mg / ml to 10 mg / ml or 0.5 mg / ml to 2 mg / ml, for example about 1 mg / ml. In some embodiments, such compositions can be formulated as unit dosage forms, e.g., in about a 1 ml volume of solution containing about 1 mg of IgE antibody, e.g., in a 2 ml type I glass vial. The compositions can be diluted in sterile saline (0.9% w / v) prior to administration to a subject, e.g., in a volume of 1 ml of composition in 250 ml of saline.

[0119] Antibodies can be provided in lyophilized form and rehydrated with sterile water prior to administration, but can also be provided in a sterile solution of known concentration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and administered to the subject. There is considerable experience in the art with the administration of antibody drugs, which have been commercially available in the United States since the approval of RITUXAN® in 1997. Antibodies can be administered by intravenous infusion or slow infusion rather than bolus administration. In one example, a larger loading dose is administered, followed by a lower level maintenance dose. For example, an initial loading dose can be infused over a period of 90 minutes or so, followed by weekly maintenance doses for 4-8 weeks, if the previous doses were well tolerated, over a period of 30 minutes.

[0120] The antibody (or functional fragment thereof) can be administered to slow or inhibit the growth of cells, e.g., cancer cells. In these applications, a therapeutically effective amount of the antibody is administered to a subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells, or to inhibit a sign or symptom of cancer. In some embodiments, the antibody is administered to a subject to inhibit or prevent the development of metastasis, or to reduce the size or number of metastases, e.g., micrometastases, e.g., micrometastases to regional lymph nodes (Goto et al., Clin. Cancer Res. 14(11):3401-3407, 2008).

[0121] Thus, in some embodiments, the IgE antibodies are used to treat cancer and / or delay or prevent the progression of cancer. "Delaying or preventing the progression" of cancer means, for example, that the cancer is at least stable for a period of time after administration of the antibody, for example at least 6 weeks, at least 12 weeks, at least 6 months, or at least 12 months. "Stable" disease can be defined, for example, as a change in RECIST score of less than 20%.

[0122] RECIST (Response Evaluation Criteria in Solid Tumors) assessment is a simple method to determine whether a patient's disease has improved, remained the same, or worsened after treatment with a cancer drug, and is commonly used in clinical trials of anticancer drugs. RECIST criteria are specified, for example, 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 the diameters of target lesions, with reference to the smallest sum examined. Stable disease is defined as neither sufficient regression to be a partial response (at least a 30% reduction in the sum of the diameters of target lesions) nor sufficient increase to be a PD, i.e., an increase of <20% is defined as stable disease.

[0123] It is recognized that in this context, "at least stable" includes an increase or decrease in RECIST score of less than 20%. Thus, the antibody may delay or prevent disease progression (e.g., delay or prevent the appearance of one or more signs or symptoms of cancer and / or inhibit the proliferation of cancer cells and / or prevent or reduce metastasis) or ameliorate the disease or promote disease remission (e.g., reduce or inhibit one or more signs or symptoms of cancer and / or kill cancer cells).

[0124] subject Suitable subjects may include subjects diagnosed with cancer, such as cancers that express FRα, including, but not limited to, skin cancer (e.g., melanoma), lung cancer, prostate cancer, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), breast cancer (including, but not limited to, basal breast cancer, ductal carcinoma, and lobular breast cancer), leukemia (e.g., acute myeloid leukemia and 11g23-positive acute leukemia), lymphoma, neural crest tumor (e.g., astrocytoma, glioma, or neuroblastoma), ovarian cancer, colon cancer, gastric cancer, pancreatic cancer, bone cancer (e.g., chordoma), glioma, or sarcoma (e.g., chondrosarcoma). Preferably, the antibody is administered to treat a solid tumor. Preferably, the subject is a human.

[0125] The therapeutically effective amount of the antibody will depend on the severity of the disease and the general state of the patient's health. A therapeutically effective amount of the antibody is one that produces either a subjective alleviation of the disease or an objectively identifiable improvement that is noted by a physician or other qualified observer. These compositions can be administered simultaneously or sequentially with other chemotherapeutic agents.

[0126] Anti-FRα antibodies are used to treat subjects suffering from tumors with low FRα expression. Such subjects may be referred to as "low FRα expressers" in contrast to moderate or high FRα expressers. The terms "tumors with low FRα expression" and "low FRα expressers" are well understood in the field of cancer therapy and are frequently used to describe certain patient populations (see, e.g., Cristea et al., Journal of Clinical Oncology 2021 39:15_suppl, 5542; Martin et al. Gynecol Oncol. 2017 Nov; 147(2): 402-407). Thus, the subgroup of patients with low tumor FRα expression is clearly distinct from high FRα expressers (see, e.g., SORAYA trial, ClinicalTrials.gov Identifier: NCT04296890).

[0127] Detection of low FRα expression Low tumor FRα expression can be determined using known standard techniques.Typically, FRα expression is determined in a biopsy sample from tumor.The techniques for obtaining biopsy samples from tumor tissue are known in the art, as are the histopathological techniques for processing such samples.For example, biopsy tissue samples can be fixed in formalin and embedded in paraffin, or freshly processed before sectioning, and placed on microscope slides for light microscopic analysis and imaging.

[0128] Hematoxylin and eosin (H&E) staining of paraffin-embedded sections is the default technique for visualizing tissues on glass slides for pathology analysis. Immunohistochemistry (IHC) staining is a known approach for identifying protein expression on cells in pathology tissue slides. Upon staining, tissues in which the target protein is overexpressed compared to normal typically have a brown appearance. For example, the expression level can be detected by using an antibody against FRα.

[0129] Suitable techniques for the detection of FRα expression in tumor samples are described, for example, in Zhao et al., “Development and Application of An Immunohistochemistry-based Clinical Assay for Evaluating Folate Receptor Alpha (FRα) Expression in the Clinical Setting”, Abstract 3400A, AACR annual meeting, April 18-22, 2015; Ab et al. (2015), Molecular Cancer Therapeutics 14(7):1605-13; Altwerger et al., Mol Cancer Ther. 2018 May; 17(5): 1003-1011 and Martin et al. “Gynecol Oncol. 2017 Nov; 147(2): 402-407. For example, FRα expression can be determined using the Ventana FOLR1 (FOLR-2.1) CDx assay, i.e. cells are cultured in Discovery 1000 cells from Ventana Medical Systems. Using an Ultra instrument, staining was performed with antibody FOLR1-2.1 (see above citation and ClinicalTrials.gov Identifier: NCT04296890).

[0130] Any suitable anti-FRα antibody can be used in such methods, for example, an anti-FRα IgG antibody (polyclonal or monoclonal). A variety of anti-FRα antibodies suitable for use in immunohistochemistry are available from commercial sources, such as Thermofisher / Invitrogen (catalog number PA5-42004), Leica Biosystems (e.g., BN3.2), Enzo Life Sciences (e.g., clone 548908, catalog number ENZ-ABS378-0100), Abcam (e.g., ab67422), and Immunogen (353.2.1, FOLR1-2.1). Preferably, the anti-FRα antibody used to detect FRα is BN3.2, as described, for example, in Smith et al., "A novel monoclonal antibody for detection of folate receptor alpha in paraffin-embedded tissues", Hybridoma (Larchmt). 2007 Oct;26(5):281-8, doi: 10.1089 / hyb.2007.0512.

[0131] A "tumor with low FRα expression" typically means that less than 50% of the tumor cells in a subject express FRα. Preferably, less than 40%, 30%, 25%, 20%, or 10% of the tumor cells in a subject express FRα. These expression levels typically refer to membrane FRα expression detectable by immunohistochemistry, for example, using the methods described above.

[0132] As described in the publications cited above, FRα expression can be classified according to both the percentage of cells exhibiting membrane FRα expression in a tissue sample and the intensity of FRα staining. In some cases, FRα staining intensity can be classified on a scale of 0 (no detectable FRα above isotype control) to 3 (high / strong FRα staining intensity). Typically, 1 indicates low / weak FRα staining intensity and 2 indicates moderate FRα staining intensity. Classification by FRα staining intensity can also be combined with the determination of the percentage of tumor cells that are FRα positive (e.g., 0-100%).

[0133] For example, in some cases, less than 50%, 40%, 30%, 25%, 20%, or 10% of tumor cells in a subject exhibit medium or high (2+) intensity staining of membrane FRα. For example, in one embodiment, less than 25% of tumor cells exhibit medium or high (i.e., 2+) intensity staining of membrane FRα. Thus, in one embodiment, FRα expression levels are measured using a FRα marker, as described, for example, in Martin et al. Gynecol Oncol. 2017 Nov; 147(2): 402-407; Cristea et al., Journal of Clinical Oncology 2021 39:15_suppl, 5542; ClinicalTrials.gov Identifier: NCT02996825; and ImmunoGen Presents Full Data from Phase 3 FORWARD I Study of Mirvetuximab Soravtansine in Ovarian Cancer at 2019, available at: https: / / investor.immunogen.com / news-releases / news-release-details / immunogen-presents-full-data-phase-3-forward-i-study. It may be classified using the "PS2+" scoring method as described in ESMO or WO 2018 / 213260.

[0134] Preferably, the subject's overall tumor FRα expression score is determined. The overall tumor FRα expression score may be defined as the product of the subject's membrane FRα staining intensity score (i.e., 0-3) and the percentage of tumor cells that are FRα positive (e.g., at least 1+) (0-100). This results in a maximum overall score of 300. An overall score of 201-300 may be defined as high expression, 101-200 as moderate expression, and 100 or less as low expression. Thus, in some embodiments, the subject's overall tumor FRα expression score may be 100 or less, preferably less than 90, 80, 70, 60, 50, or 40 or less, more preferably 30 or less or 20 or less.

[0135] In other embodiments, the "H score" is calculated as described, for example, in Altwerger et al., Mol Cancer Ther. 2018 May; 17(5): 1003-1011. The H score is calculated by determining the level of FRα expression (i.e., the intensity level of membrane staining for each cell (0-3, 0=negative, 1=weak, 2=moderate, and 3=strong) and the percentage of cells in a representative area at each staining intensity (0-100%) [1×(% cells 1+)+2×(% cells 2+)+3×(% cells 3+)]) as follows. This results in a final score ranging from 0 to 300. An H score of 201-300 may be defined as high expression, 101-200 as moderate expression, and 100 or less as low expression. Thus, in some embodiments, a subject's H-score may be 100 or less, preferably less than 90, 80, 70, 60, 50, or 40 or less, and more preferably 30 or less or 20 or less.

[0136] In an alternative embodiment, tumor FRα expression in a subject can be compared with tumor FRα expression in a group of cancer subjects.Typically, a group of cancer subjects refers to other subjects suffering from the same type of cancer.Therefore, the relative level of tumor FRα expression in a subject can be confirmed by comparing with other subjects with the same type of cancer.In this method, the absolute quantification of FRα expression level is not required, but only the relative determination compared with other cancer subjects is required, thus avoiding any systematic bias due to lack of standardization of expression detection.

[0137] In a preferred embodiment, the tumor (e.g., membrane) FRα expression in the subject is at least 50% lower than that of cancer subjects.Preferably, the membrane FRα expression in the tumor cells of the subject is at least 60%, at least 70%, or at least 80% lower than that of cancer subjects, such as subjects suffering from the same form of cancer (e.g., ovarian cancer).More preferably, the tumor membrane FRα expression in the subject is at least 50%, 60%, 70%, 80%, or at least 90% lower than that of FRα-expressing tumors (e.g., in FRα-expressing ovarian tumors).

[0138] It is preferred that the tumor expresses FRα, i.e., the tumor exhibits at least some FRα expression. Typically, this means that the tumor cells in the subject exhibit detectable membrane FRα expression, for example, by immunohistochemistry. More preferably, at least 1% or at least 5% of the tumor cells in the subject exhibit detectable (e.g., membrane) FRα expression, for example, using immunohistochemical detection of FRα. In other embodiments, at least 10%, 15%, or 20% of the tumor cells in the subject exhibit detectable membrane FRα expression.

[0139] Preferably, the determination of FRα expression is performed on a recent tumor biopsy, i.e., a biopsy obtained from the subject immediately prior to initiation of anti-FRα IgE therapy (rather than an older or archived sample). For example, the biopsy can be taken, and / or FRα expression determined, less than 6 months, 3 months, 1 month, 2 weeks, 1 week, 3 days, 48 ​​hours, or 24 hours prior to initiation of anti-FRα IgE therapy.

[0140] The invention will now be further described, by way of example only, with reference to the following non-limiting embodiments. [Example]

[0141] Chimeric MOv18 IgE (MOv18 IgE) Chimeric MOv18 IgE (MOv18 IgE) is an anti-folate receptor alpha (FRα) monoclonal antibody (mAb) of the IgE class. The antibody has been shown to mediate potent antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) against FRα-expressing tumor cell lines in vitro and FRα-expressing tumor xenografts in vivo. The target antigen FRα is overexpressed in several solid cancer types, including ovarian and endometrial cancers and mesotheliomas. The antigen has been characterized as effectively tumor-specific, and clinical trials targeting FRα with IgG antibodies, including testing of chimeric MOv18 IgG1 (MOv18 IgG1), have shown a favorable tolerability profile. MOv18 IgE is believed to have a mechanism of action that involves binding to blood effector cells, including monocytes, basophils, and eosinophils. These IgE-bearing cells invade tissues and, upon encountering FRα-expressing cells, mount an IgE-mediated immune response against the tumor.The MOv18 IgG and IgE antibodies and their properties are described, for example, in Coney, LR, A. Tomassetti, et al. (1991). "Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein." Cancer Res 51(22): 6125-6132; Gould, HJ, GA Mackay, et al. (1999). "Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma." Eur J Immunol 29(11): 3527-3537; Karagiannis, SN, Q. Wang, et al. (2003). "Activity of human monocytes in IgE antibody dependent surveillance and killing of ovarian tumor cells." Eur J Immunol 33(4): It is described in 1030-1040.

[0142] Drug Prescription and Administration MOv18 IgE is a 168 kDa protein with light and heavy chain amino acid sequences as shown in Figures 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2). The MOv18 IgE drug product is supplied as a sterile, pyrogen-free, particle-free solution containing 1 mg / mL MOv18 IgE at pH 6.5, filled in 1.0 mL 2 mL vials for dilution prior to injection. Each vial of MOv18 IgE also contains excipients, namely 0.1 M sodium citrate, 30 g / L L-arginine, 50 g / L sucrose, 0.02% polysorbate 20 in water for injection.

[0143] MOv18 IgE was administered by intravenous (IV) infusion after dilution in 0.9% (w / v) saline. Additionally, subjects were assessed for risk of anaphylaxis by receiving an intradermal [ID] dose of MOv18 IgE (plus histamine and saline controls) prior to each IV dose. Alternatively, skin prick testing was used to assess anaphylaxis risk. Patients proceeded to IV administration of MOv18 IgE only if they had no skin reaction to the antibodies.

[0144] Prior to administration of the therapeutic antibody, blood samples were obtained from the subjects and subjected to a basophil activation test in the presence of MOv18 IgE (Flow CAST®, Buhlmann Laboratories AG, Schonenbuch, Switzerland).

[0145] research design MOv18 IgE was tested in an open-label, phase I, multiple-dose escalation study in FRα-positive solid tumors. Twenty-four patients with advanced solid tumors expressing FRα were enrolled in the study. Eligible subjects had adequate organ function, no history of severe allergies, and no concomitant medications or comorbidities that may increase risk in the event of anaphylaxis.

[0146] FRα expression for each patient was determined in tumor biopsies using anti-FRα BN3.2 primary antibody (available from Leica Biosystems) and the methodology described by Lawson and Scorer (Lawson, N. and P. Scorer (2010). "Evaluation of Antibody to Folate Receptor-alpha (FR-α)." 16(21): 5288-5295, available at http: / / www.leicabiosystems.com / pathologyleaders / evaluation-of-antibody-to-folate-receptoralpha-fr-%CE%B1 / ). Methods for determining FRα expression are also described, for example, in Ab et al., “IMGN853, a Folate Receptor-a (FRα)-Targeting Antibody-Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors”, Molecular Cancer Therapeutics 14(7):1605-13, July 2015. Immunohistochemistry (IHC) staining for FRα was performed on formalin-fixed paraffin-embedded tissue from tumor biopsy samples on microscope slides. Slides were baked, dewaxed, and treated with hydrogen peroxide to inactivate endogenous peroxidase. Slides were incubated with either anti-FRα mouse monoclonal antibody BN3.2 or an isotype control, visualized with specific stains, and counterstained with hematoxylin. All tissue slides were evaluated and scored by a certified pathologist. FRα staining intensity was scored on a scale of 0 to 3 (0 = no specific staining, similar to staining of the isotype control, 1 = weak staining, 2 = moderate staining, and 3 = strong staining). The percentage of tumor cells showing FRα expression (i.e., staining intensity of at least 1+) was also determined. Subjects in this study showed expression of FRα (i.e., 1+, 2+, or 3+ membrane staining) in at least 5% of tumor cells by immunohistochemistry.

[0147] Patients received a total of six doses of MOv18 IgE, starting at a fixed dose of 70 μg, once a week as an IV infusion. Dose escalation occurred through defined dose levels (including 70 μg, 250 μg, 500 μg, 700 μg, 1.5 mg, and 3 mg) to a maximum dose of 50 mg. During this phase, three weeks of treatment were considered one cycle. Patients in either cohort who appeared to benefit from MOv18 IgE continued to receive up to three additional doses of MOv18 IgE at two-week intervals at the same dose level (unless dose-limiting toxicity occurred or the patient developed progressive disease). This additional period was considered the maintenance phase.

[0148] Initially, patients were enrolled in single-patient cohorts (cohorts 1–4; 70–700 μg doses of MOv18 IgE) because the planned doses were very low and unlikely to result in significant biological responses. For cohorts 5–10 (1.5–50 mg doses of MOv18 IgE), three patients were enrolled per cohort, with an additional three patients added to the cohort if necessary due to toxicity. Cohorts were expanded to six patients to further explore the safety and efficacy of MOv18 IgE. No further dose escalation was performed after cohort 10 (i.e., 50 mg was the highest dose evaluated).

[0149] result Ten patients received MOv18 IgE early in the study, of which two patients were treated at the 500 μg dose level. Anti-drug antibodies were detected in three of the 21 patients evaluated at 6 weeks and / or off-study follow-up (>8 weeks) (ADA was detected in two patients, plus one patient had suspected ADA). One patient treated at the 500 μg dose level experienced a grade 3 anaphylactic episode shortly after receiving MOv18 IgE. This patient responded to standard anaphylactic treatment according to protocol and fully recovered.

[0150] The pharmacokinetics (serum concentrations) of MOv18 IgE following intravenous administration for the specific dose cohorts of interest are shown in Figure 5. Cohort 1: 70 μg; Cohort 2: 250 μg; Cohort 3: 500 μg; Cohort 4: 700 μg; Cohort 5: 1.5 mg.

[0151] Figures 6 and 7 show that administration of MOv18 IgE antibody at a unit dose of 700 μg resulted in an anti-tumor effect. Figure 6 shows the results of tumor measurements taken from CT scan images showing a reduction in tumor size in an ovarian cancer subject treated with MOv18 IgE antibody at a dose level of 700 μg. Figure 7 shows a significant reduction in serum concentration of ovarian cancer antigen CA125 during treatment in a patient who received six weekly doses of 700 μg of MOv18 IgE antibody, followed by three additional doses of the antibody at two-week intervals. Reduction of CA125 during ovarian cancer treatment has been shown to be associated with positive treatment outcomes (see, e.g., Yang, Z., Zhao, B. & Li, L. The significance of the change pattern of serum CA125 level for judging prognosis and diagnosing recurrences of epithelial ovarian cancer. J Ovarian Res 9, 57 (2016)). The decrease in CA125 levels shown in FIG. 7 exceeds the threshold that defines response to chemotherapy in ovarian cancer according to the Gynecologic Oncology Group (GOG) criteria (see, e.g., Rustin et al. Defining response of ovarian carcinoma to initial chemotherapy according to serum CA 125, Journal of Clinical Oncology 1996 14:5, 1545-1551).

[0152] Figures 8-10 show that the majority of patients treated with low doses of MOv18 IgE antibody experienced stable disease. Figure 8 is a plot of the change in RECIST score in individual ovarian cancer subjects treated with MOv18 IgE antibody from the beginning to the end of treatment (weeks 0-12). Figures 9 and 10 show the change in RECIST score in individual subjects at 6 and 12 weeks of treatment, respectively. A change in RECIST score of less than 20% indicates stable disease. After 6 weeks of treatment, 70% (14 / 20) of treated patients had stable disease. 60% (3 / 5) of patients treated up to 12 weeks still had stable disease. Note that the dosing frequency was reduced from once a week to once every 2 weeks during the period between 6 and 12 weeks.

[0153] Stable disease (i.e., less than 20% change in RECIST score) is a key driver of the primary efficacy endpoint, Progression Free Survival (PFS). The study shows disease control rates of 70% at 6 weeks and 60% at 12 weeks. Thus, these results demonstrate that IgE antibodies can be used in humans at very low doses to treat and / or delay the progression of cancer in subjects.

[0154] Figure 11 shows that MOv18 IgE is effective in treating subjects with low FRα antigen levels. Inclusion criteria for this study required that at least 5% of tumor cells be FRα positive by immunohistochemistry. However, of the subjects who showed stable disease at 6 weeks, 6 / 14 were low FRα expressers, as indicated by an overall FRα expression score of 100 or less. In each of these subjects, 50% or less of the tumor cells were FRα positive.

[0155] Of the six subjects who showed no change or a decrease in RECIST score at 6 weeks, four were low FRα expressors (an overall FRα expression score of 100 or less and 50% or less FRα-positive tumor cells). Furthermore, the two best responders in this study (both of whom showed a decrease in RECIST score) were both low FRα expressors. The best responder (as shown in Figures 6 and 7) had a very low overall FRα expression score of 20 and only 10% FRα-positive tumor cells.

[0156] The results indicate that MOv18 IgE is well suited for anti-cancer therapy and administration is well tolerated by most patients. Most strikingly, this antibody shows anti-tumor activity even at very low doses (e.g., 700 μg) in subjects with low FRα expression. These results are the first to support the safety and efficacy of IgE as a treatment for low FRα-expressing tumors, including unit doses of less than 50 mg (less than 1 mg / kg / week).

[0157] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems 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 certain preferred embodiments, it will 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 apparent to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. A composition comprising an anti-folate receptor alpha (FRα) immunoglobulin E (IgE) antibody for use in treating a tumor with low FRα expression in a subject.

2. A composition comprising an anti-folate receptor alpha (FRα) immunoglobulin E (IgE) antibody for treating cancer and / or delaying the progression of cancer in a subject having a tumor with low FRα expression.

3. The composition of claim 1 or 2, wherein less than 50% of tumor cells in the subject express FRα, preferably less than 40%, 30%, 20%, or 10% of tumor cells in the subject express FRα.

4. 3. The composition of claim 1 or 2, wherein less than 50%, 30%, or 20% of the tumor cells in the subject exhibit detectable membrane FRα expression using immunohistochemical detection of FRα.

5. The composition of claim 1 or 2, wherein, using immunohistochemical detection of FRα, less than 50%, 30%, or 20% of tumor cells in the subject show moderate or high (2+) intensity staining for membrane FRα.

6. 3. The composition of claim 1 or 2, wherein tumor cells in a subject are classified according to (i) membrane FRα staining intensity on a scale of 0 (FRα undetectable) to 3 (high FRα), and (ii) the percentage of tumor cells that are FRα positive (0-100%), and the subject's overall tumor FRα expression score is determined as the product of (i) and (ii), preferably wherein the subject's overall tumor FRα expression score is less than 100, more preferably less than 50, and even more preferably less than 30.

7. The composition of claim 1 or 2, wherein tumor FRα expression in the subject is lower than tumor FRα expression in at least 50% of cancer subjects, preferably wherein membrane FRα expression in the subject's tumor cells is lower than tumor FRα expression in at least 60%, at least 70%, or at least 80% of subjects suffering from the same form of cancer, more preferably wherein tumor FRα expression in the subject is lower than tumor FRα expression in at least 50%, at least 70%, or at least 90% of FRα-expressing tumors (preferably FRα-expressing ovarian tumors).

8. The composition of claim 1 or 2, wherein the tumor expresses FRα, and preferably the tumor cells in the subject exhibit membrane FRα expression detectable by immunohistochemistry.

9. The composition of claim 1 or 2, wherein at least 1% of tumor cells in the subject show detectable membrane FRα expression using immunohistochemical detection of FRα, and preferably at least 5%, 10%, 15%, or 20% of tumor cells in the subject show detectable membrane FRα expression.

10. The composition described in claim 1 or 2, wherein the anti-FRα IgE antibody is a MOv18 IgE antibody.

11. 10. The composition of claim 1 for use in treating cancer and / or delaying the progression of cancer in a subject.

12. The composition of claim 1 or 2, wherein the tumor or cancer is an ovarian tumor or ovarian cancer.

13. The composition of claim 1 or 2, wherein the anti-FRα IgE antibody lacks a cytotoxic moiety and is preferably not an antibody-drug conjugate (ADC).

14. 3. The composition of claim 1 or 2, wherein the maximum weekly dose of anti-FRα IgE antibody administered to the subject is 50 mg, 25 mg, 10 mg, 3 mg, or 1 mg.

15. The composition of claim 1 or 2, wherein the weekly dose of anti-FRα IgE antibody is 10 μg to 50 mg, 70 μg to 30 mg, 70 μg to 3 mg, 500 μg to 1 mg, or about 700 μg.

16. The composition described in claim 1 or 2, wherein the anti-FRα IgE antibody is administered to the subject once a week or once every two weeks.

17. The composition of claim 1 or 2, wherein the anti-FRα IgE antibody is administered to the subject once a week or once every two weeks and for up to 12 weeks.

18. The composition of claim 1 or 2, wherein the anti-FRα IgE antibody is administered to the subject (i) once weekly for six weeks, and then (ii) once every two weeks for six weeks.

19. The composition described in claim 1 or 2, wherein the anti-FRα IgE antibody is administered to the subject at a dose of less than 1 mg / kg, less than 0.1 mg / kg, or less than 0.03 mg / kg per administration.

20. The composition described in claim 1 or 2, wherein the anti-FRα IgE antibody is administered to the subject at a dose of less than 1 mg / kg / week, less than 0.1 mg / kg / week, or less than 0.03 mg / kg / week.

21. The composition of claim 1 or 2, which is a pharmaceutical composition and further comprises one or more pharma- ceutically acceptable excipients, carriers, or diluents.

22. 22. The composition of claim 21, comprising less than 50 mg of anti-FRα IgE antibody.

23. 22. The composition of claim 21, comprising less than 30 mg, less than 25 mg, less than 10 mg, less than 5 mg, less than 3 mg, or less than 1 mg of anti-FRα IgE antibody.

24. 22. The composition of claim 21, comprising between 10 μg and 50 mg, between 70 μg and 30 mg, between 70 μg and 3 mg, between 500 μg and 1 mg, or about 700 μg of anti-FRα IgE antibody.

25. 22. The composition of claim 21 in the form of a liquid.

26. 22. The composition of claim 21, comprising an aqueous solution having a concentration of anti-FRα IgE antibody of 0.1 mg / ml to 10 mg / ml, 0.5 mg / ml to 2 mg / ml, or about 1 mg / ml.

27. The composition of claim 21, wherein the pharma- ceutically acceptable excipient is selected from sodium citrate, L-arginine, sucrose, polysorbate 20, and / or sodium chloride.

28. 22. The composition of claim 21, suitable for intravenous injection, preferably for intravenous injection up to a maximum total dose of 50 mg / week, 25 mg / week, 10 mg / week, 3 mg / week, or 1 mg / week.

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