CN117203237A - Compositions comprising IgE antibodies - Google Patents

Abstract

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

Description

Compositions comprising IgE antibodies

Technical Field

The present invention relates to the field of therapeutic antibodies and their uses, in particular to immunoglobulin E (IgE) antibodies for the treatment of cancer. The invention also relates to methods of using such IgE antibodies to treat diseases such as cancer.

Background

Therapeutic antibodies are now complementary to conventional treatments for many malignant diseases, but almost all drugs currently developed rely on only one of the nine classes of human antibodies, namely IgG1, which is the most abundant class of antibodies in the blood (Weiner LM, surana R, wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunology. Nat Rev Immunol 10:317-327). The human immune system naturally performs immune surveillance using nine antibody classes and subclasses (IgM, igD, igG1-4, igAl, igA2 and IgE) and mediates destruction of pathogens in different anatomical compartments. However, only IgG (most commonly IgG 1) is used for immunotherapy of cancer.

One reason may be that the IgG antibody (particularly IgG 1) is the largest in the human blood circulation antibody. The selection of antibody classes was also based on an open work in the late 80 s of the 20 th century that compared a set of chimeric antibodies of identical specificity, each having an Fc region belonging to one of 9 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 anti-bodies J Exp Med 166:1351-1361). Antibodies were evaluated for their ability to bind complement and their efficacy in mediating hemolysis and cytotoxicity of antigen-expressing target cells in the presence of complement. IgG1 combined with human Peripheral Blood Mononuclear Cells (PBMC) is the most effective subclass of IgG in complement-dependent cell killing in vitro, whereas IgA and IgE antibodies are completely ineffective.

Subsequent clinical trials with antibodies recognizing the B cell marker CD20 support the conclusion that IgG1 is the subset most suitable for immunotherapy in patients with B cell malignancies such as non-hodgkin's lymphoma (Alduaij W, illindge 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 was limited to IgG and IgM in both mouse models and lymphoid malignancy patients, while IgA has been demonstrated to mediate ADCC in mouse lymphoma models in vitro and in vivo (Dechant M, valerius T (2001) IgA antibodies for cancer therapy.crit Rev Oncol Hematol 39:69-77).

Folate receptor alpha (fra) is a cancer-associated antigen that is overexpressed in a variety of solid cancer types, including ovarian cancer, endometrial cancer, and mesothelioma. It has been reported that FR alpha is expressed in normal kidney, placenta, lung, fallopian tube, pancreas and testis, but not in other normal tissues, including heart, liver, spleen, gastrointestinal tract, ovary, uterus, muscle, lymph 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, the FR alpha expression levels are generally low or restricted to the luminal surface and are therefore less likely to be approached by circulating antibodies (Parker, turk et al 2005,Anal Biochem338 (2): 284-293; O' Shannessesy, yu et al 2012,Oncotarget 3 (4): 414-425.). Fra expression was found in up to 40% of primary ovarian and endometrial tumors and nearly 30% of lung cancers. It is known that FR.alpha.in tumors, particularly ovarian and endometrial cancers, are readily accessible by antibodies and that their expression levels may be much higher than in normal tissues (Antonny 1996, annu. Rev. Nutr. 16:501-521). FR alpha is considered a potent tumor-specific antigen because of the different levels and locations of FR alpha expression in normal tissues and tumors (Mantovani, miotti et al 1994,Eur JCancer 30A (3): 363-369; toffoli, cernigoi et al 1997,Int J Cancer 74 (2): 193-198).

Preliminary clinical trials using IgG antibodies to target fra showed good tolerability. For example, in phase I studies of platinum-resistant epithelial ovarian cancer, falletuzumab (Morlet-003), a humanized anti-FR alpha IgG antibody, was administered at 12mg/m ² To 40mg/m ² Has good tolerability over the dosage range (see, e.g., konner et al, clin Cancer Res.2010Nov 1;16 (21): 5288-95). Many anti-fraigg antibodies are under development, including Antibody Drug Conjugates (ADCs). For example, sorrow-rituximab (mirvetuximab soravtansine) is an ADC consisting of a fra-binding antibody, a cleavable linker, and a cytotoxic payload, which has been used in ovarian cancer assays.

However, phase III clinical trials of anti-fra IgG therapies indicate that the efficacy of these antibodies may require high fra expression. For example, phase III trials of trastuzumab on platinum-resistant epithelial ovarian cancer failed to achieve its primary goal, namely 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 the lowest level of FR alpha expression in this trial as a qualification criterion (Sato and Itamochi, oncoTargets and Therapy 2016:91181-1188).

Furthermore, the phase III test (FORWARD I) of ImmunoGen for treatment of FR alpha positive platinum resistant ovarian cancer failed to reach its primary endpoint (see news manuscript by Immunogen at 3 months 1 day 2019. ImmunoGen published the leading edge results of the phase 3 FORWARD I study of Sofos-Mituximab for treatment of ovarian cancer, available from https:// inventor. Immunogen. Com/news-release/immune n-options-top-line-ends-phase-3-FORWARD-I-stud).

Qualification criteria for the FORWARD I test include patients with platinum-resistant ovarian cancer expressing medium or high levels of FR alpha who received up to three prior regimens. This suggests that high levels of FR alpha expression may be required to achieve efficacy (see news manuscript at 29 th month 2019 by Immunogen, which shows complete data in the ovarian cancer stage 3 FORWARD I study of ESMO-rituximab, available from https:// inventor. Immunogen. Com/news-release-targets/presentation-full-data-phase-3-FORWARD-I-study). Thus, further phase III tests (SORAYA) focused only on highly expressed patients and performed more specific diagnostic tests (see ClinicalTrials. Gov Identifier: NCT04296890, A Study of Mirvetuximab Soravtansine in Platinum-resistance, advanced High-Grade Epithelial Ovarian, primary Peritoneal, or Fallopian Tube Cancers With High Folate Receptor-Alpha Expression (SORAYA)). Other studies with the use of sorrow-rituximab in combination with other chemotherapeutic agents have also indicated the need for high FR alpha expression (see, e.g., cristea et al, aphase I study of mirvetuximab soravtansine (MIRV) and gemcitabine (G) in fractions (Pts) with selected FR alpha-positive solid tumors: results in the ovarian cancer (EC) mask; journal of Clinical Oncology 2021 39:15_suppl,5542).

Similarly, the anti-FR alpha antibody MOv18-IgG1 has been shown to induce tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP) functions in tumor cells that express high FR alpha but not low FR alpha (Cheung et al Clin Cancer Res.2018October 15;24 (20): 5098-5111). The authors of Cheung et al showed that the lack of cell-mediated killing of low FR alpha expressing cells by MOvl8-IgGl may be important to avoid non-tumor targeting (on-target/off-tumor) toxic effects. Thus, there is a need for improved therapies for tumors that do not express high levels of FR alpha, such as ovarian cancer.

IgE class antibodies play a central role in allergic reactions and have many characteristics that may be beneficial for cancer therapy. Active and passive IgE-based immunotherapy approaches have been shown to be effective in both in vitro and in vivo models of cancer, suggesting potential uses for these approaches in humans (Leoh et al Curr Top Microbiol Immunol.2015; 388:109-149). Thus, igE therapeutic antibodies may provide enhanced immune surveillance and excellent efficacy of effector cells against cancer cells.

The mouse/human chimeric IgE antibody (MOv 18 IgE) has specificity for FR.alpha.and has been demonstrated to have excellent antitumor efficacy in syngeneic immunocompetent animals compared to otherwise identical IgG (Gould et al, eur J Immunol 1999;29:3527-37; josephs et al, cancer Res.2017Mar1; 77 (5): 1127-1141; karagianis et al, cancer Res.2017Jun1;77 (11): 2779-2783). tnfα/MCP-1 signaling is thought to be a mechanism by which IgE mediates monocyte and macrophage activation and recruitment to tumors. These findings correspond to the powerful macrophage activating function employed by IgE against parasites rather than the allergic IgE mechanism. If the antitumor activity of MOv18 IgE in these preclinical experiments is replicated in patients, the potential clinical use of IgE-derived drugs in clinical oncology is established.

However, the lack of clinical trial data related to the therapeutic use of IgE antibodies in humans suggests that there is still a lack of suitable therapeutic methods and uses involving IgE antibodies. In particular, it is not clear which subgroups of therapeutic IgE antibodies may be effective in cancer patients. Since anti-fra IgG antibodies are primarily used for high fra expressors, there is a clear need for improved treatment of subgroups of other cancer (including ovarian cancer) patients. However, it is not clear how methods and uses developed for administration of other therapeutic antibody isotypes (e.g., igG) can be adapted for IgE antibodies, nor is it clear whether IgG and IgE antibodies can be used to treat the same or different patient subgroups.

Disclosure of Invention

Accordingly, in one aspect, the invention provides an anti-folate receptor alpha (fra) immunoglobulin E (IgE) antibody for use in treating a low fra expressing tumor in a subject.

In one embodiment, a "low FR alpha expressing tumor" means that less than 50% of the tumor cells of the subject express FR alpha. Preferably, less than 40%, 30%, 20% or 10% of the tumor cells of the subject express fra.

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

In another embodiment, less than 50%, 40%, 30%, 25%, 20% or 10% of the tumor cells of the subject exhibit medium or high (2+) intensity staining of (e.g., membrane) FR alpha, typically when detecting FR alpha using immunohistochemistry.

In another embodiment, the tumor cells of the subject are classified according to membrane fra staining intensity. The fra staining intensity may be classified on a scale from 0 (no detectable fra) to 3 (high fra staining intensity), e.g. 1 for low fra staining intensity and 2 for medium fra staining intensity. In some embodiments, the subject's tumor cells are further classified according to the percentage of tumor cells that are FR alpha positive (e.g., from 0% to 100%).

Preferably, the overall tumor froc expression score of the subject is determined as the product (product) of the membrane froc staining intensity score and the percentage of froc positive tumor cells. For example, in some embodiments, the subject's overall tumor FR alpha 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.

In alternative embodiments, the subject's tumor FR alpha expression can be compared to tumor FR alpha expression of other cancer subjects (e.g., other subjects with the same type of cancer). The relative level of tumor fα expression in the subject can thus be determined. In preferred embodiments, the subject's tumor (e.g., membrane) fα expression is less than at least 50% of cancer subjects. Preferably, the (e.g. membrane) FR alpha expression in the tumor cells of the subject is lower than at least 60%, at least 70% or at least 80% of cancer subjects, e.g. cancer subjects suffering from the same form of cancer, preferably ovarian cancer. More preferably, the subject's tumor (e.g., membrane) expresses less than at least 50%, 60%, 70%, 80% or at least 90% of the tumor expressing FR alpha (preferably ovarian tumor expressing FR alpha).

In a preferred embodiment, the tumor expresses fra, i.e., the tumor displays at least some fra expression. Preferably, the tumor cells of the subject exhibit detectable membrane froc expression, e.g., as detected by immunohistochemistry. More preferably, at least 1% or at least 5% of the tumor cells of the subject exhibit detectable (e.g., membrane) expression of fra, e.g., detection of fra using immunohistochemistry. In other embodiments, at least 10%, 15% or 20% of the tumor cells of the subject exhibit detectable membrane froc expression.

In one embodiment, the antibody is a MOv18 IgE antibody.

In one embodiment, igE antibodies are used to treat cancer in a subject and/or delay progression of cancer in a subject. For example, antibodies can be used to delay progression of a low FR alpha expressing tumor in a subject.

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

In one embodiment, the antibody lacks a cytotoxic moiety. Thus, an antibody may comprise, consist of, or consist essentially of (only) one or more (e.g., four) polypeptide chains, such as 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 further drugs or groups (groups) with cytotoxic effects, e.g. chemotherapeutics or drugs capable of (directly) killing cancer cells. Antibodies may also lack linkers or other groups for conjugating the cytotoxic moiety to the polypeptide.

In one embodiment, the weekly dose of IgE antibodies administered to a subject is less than 50mg, 25mg, 10mg, 3mg, or 1mg. In another embodiment, the weekly dose of IgE antibodies is 10 μg to 50mg, 70 μg to 30mg, 70 μg to 3mg, 500 μg to 1mg, or about 700 μg.

Preferably, igE antibodies are administered to a subject once a week or once every two weeks. In one embodiment, igE antibodies are administered to a subject for up to 12 weeks. In another embodiment, igE antibody is administered to a subject (i) once a week for 6 weeks; followed by (ii) once every two weeks for 6 weeks.

In one embodiment, the IgE antibody is administered to the subject at a dose of less than 1mg/kg, less than 0.1mg/kg, or less than 0.03mg/kg per administration. In 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.

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

In a further aspect, the invention provides a pharmaceutical composition for use in treating a low FR alpha expressing tumor in a subject, the composition comprising an anti-folate receptor alpha (FR alpha) immunoglobulin E (IgE) antibody as defined above and one or more pharmaceutically acceptable excipients, carriers or diluents.

Preferably, the composition comprises less than 50mg IgE antibody. More preferably, the composition comprises less than 30mg, less than 25mg, less than 10mg, less than 5mg, less than 3mg, or less than 1mg of IgE antibody. In other embodiments, the composition comprises 10 μg to 50mg, 70 μg to 30mg, 70 μg to 3mg, 500 μg to 1mg, or about 700 μg of IgE antibody.

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

In one embodiment, the composition is suitable for intravenous injection or subcutaneous injection. Preferably, the composition is suitable for intravenous or subcutaneous injection, with a maximum total dose of 50 mg/week, 25 mg/week, 10 mg/week, 3 mg/week or 1 mg/week.

Brief description of the drawings

FIG. 1 shows the amino acid sequence of the light (L) chain of MOv18 IgE (SEQ ID NO: 1); mouse VL is shown in bold and human CL is shown in standard font.

FIG. 2 shows the amino acid sequence of the heavy (H) chain of MOv18 IgE (SEQ ID NO: 2); mouse VH is shown in bold and human CH is shown in standard font.

FIG. 3 shows the amino acid sequence of the MOv18 IgE light chain variable domain (VL) (SEQ ID NO: 3).

FIG. 4 shows the amino acid sequence of the MOv18 IgE heavy chain variable domain (VH) (SEQ ID NO: 4).

Figure 5 shows the pharmacokinetics (serum concentration) of MOv18 IgE following intravenous administration.

Figure 6 shows CT scan images and tumor measurements obtained from CT scan images, indicating a decrease in tumor size in ovarian cancer subjects treated with MOv18 IgE antibodies at a dose level of 700 μg. Tumors (shown in oval areas of each image) at baseline (left panel) and 6 weeks after treatment (right panel) were depicted. The target lesion size and non-target lesion size and status of the subject were determined before and after multiple rounds of treatment with the antibody and after a maintenance period.

Figure 7 shows that serum concentration of ovarian cancer antigen CA125 was significantly reduced during treatment of patients with 6 700 μg weekly doses of MOv18 IgE antibody followed by further 3 doses of 700 μg every 2 weeks.

Fig. 8 shows a graph of the variation in RECIST (solid tumor response assessment criteria) scores of individual ovarian cancer subjects treated with MOv18 IgE antibodies. Each line represents the percent change in RECIST score for individual patients from the beginning of treatment (i.e., 6 weeks after treatment and 12 weeks after treatment). An increase or decrease of less than 20% in RECIST score indicates stable disease.

Figure 9 shows a waterfall plot of changes in RECIST (solid tumor response assessment criteria) scores of individual ovarian cancer subjects after 6 weeks of treatment with MOv18 IgE antibodies. Each vertical bar represents the change in RECIST score for an individual subject at 6 weeks. A total of 20 subjects were treated. If no vertical bars are shown, this indicates that there is no change in RECIST score for the subjects after 6 weeks (this occurs in 4 subjects, indicated by the spacing between vertical bars along the x-axis).

Figure 10 shows a waterfall plot of changes in RECIST (solid tumor response assessment criteria) scores of individual ovarian cancer subjects after 6 or 12 weeks of treatment with MOv18 IgE antibodies. The same subjects as shown in fig. 9 are represented in the same order. Only some (5) subjects continued to receive treatment for more than 6 weeks. Each vertical bar marked with asterisks represents the change in RECIST score for individual subjects from treatment to 12 weeks. The remaining vertical bars without asterisks represent changes in RECIST scores for individual subjects treated for 6 weeks, as shown in fig. 9. If vertical bars are not shown, this indicates that the RECIST score of the subject varies to 0% (this occurs in two subjects, indicated by the vertical bar separation along the x-axis).

Fig. 11 shows a waterfall plot of changes in RECIST (solid tumor response assessment criteria) scores of individual ovarian cancer subjects after 6 weeks of treatment with MOv18 IgE antibodies, as compared to the fra expression score of each subject. Each vertical bar represents the change in RECIST score for an individual subject at 6 weeks. A total of 20 subjects received treatment. If vertical bars are not shown, this indicates that there is no change in RECIST score for the subjects after 6 weeks (this occurs in four subjects, indicated by the vertical bar separation along the x-axis). PD represents progressive disease and SD represents stable disease. The overall FR expression score for each subject was calculated by the computer as the product of the membrane staining intensity score (membrane score) and the percentage of FR positive tumor cells (% membrane+).

Detailed Description

It has surprisingly been found that anti-FR alpha IgE antibodies can provide effective treatment for low FR alpha expressing tumors. In particular, anti-fraige (MOv 18 IgE) was found to be effective in treating or delaying progression of ovarian cancer in subjects with very low fra membrane expression scores.

This finding is particularly surprising because the corresponding IgG antibody (i.e., MOv18 IgG 1) is known to kill high FR alpha expressing tumors but not low FR alpha expressing tumors, and this selectivity is believed to be important in avoiding off-tumor toxic effects (Cheung et al Clin Cancer Res.2018, 10, 15; 24 (20): 5098-5111). Furthermore, anti-FR alpha treatment methods using IgG antibodies have focused on the treatment of high FR alpha expressitors and/or combinations of antibodies with cytotoxic moieties, e.g., in ADC and/or in separate combination therapies with drugs such as gemcitabine (see Martin et al Gynecol Oncol.2017Nov;147 (2): 402-407; sato and Itamochi, oncoTargets and Therapy 2016:9 1181-1188; and Cristea et al Journal of Clinical Oncology 2021:15_suppl, 5542).

In contrast, anti-FR alpha IgE antibodies are capable of treating low FR alpha expressing tumors as monotherapy, i.e. without the need to be incorporated into an ADC or in combination with other chemotherapeutic drugs. The present invention therefore represents a significant contribution to the art in addressing unmet medical needs, particularly in a subset of cancer patients who are under-expressed in FR alpha.

Furthermore, it has been found that methods, compositions, dosage forms and protocols generally used for IgG antibodies are not necessarily applicable 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 on subjects with low FR alpha expression) can be much lower than the generally effective dose of IgG antibodies. For example, as shown in the examples below, anti-folate receptor alpha (FR alpha) IgE antibodies were 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., about 150-2000mg or 2-20mg/kg per dose).

The results indicate that methods, uses and compositions developed for IgG antibodies (such as dosage regimens, unit dosage forms and subgroups of cancer patients to be treated) are not necessarily transferable to IgE due to differences in pharmacology and pharmacokinetics of IgG and IgE. The inventors have thus developed new uses and dosage regimens particularly suitable for therapeutic IgE administration, for example in the treatment of cancer in patients with low FR alpha expression.

Therapeutic antibodies

Antibodies are polypeptide ligands comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and specifically binds to an epitope of an antigen (e.g., fα) or a fragment thereof. Antibodies are typically composed of heavy and light chains, each chain having a variable region, known as the heavy chain Variable (VH) region and the light chain Variable (VL) region. The VH and VL regions are collectively responsible for binding to the antigen recognized by the antibody.

Antibodies, including intact immunoglobulins as well as variants and portions of antibodies, are well known in the art, provided that such fragments retain at least one function of IgE, e.g., are capable of binding to fcs receptors. Antibodies also include genetically engineered forms, such as chimeric, humanized (e.g., humanized antibodies containing murine sequences in the variable regions) or human antibodies, heteroconjugate antibodies (e.g., bispecific antibodies), as described in Kuby, j., immunology, 3 rd edition, w.h.freeman & co., new York, 1997.

Typically, naturally occurring immunoglobulins have a heavy (H) chain and a light (L) chain interconnected by disulfide bonds. There are two types of light chains, lambda and kappa. There are nine major isoforms or classes that can determine the functional activity of an antibody molecule: igA1-2, igD, igE, igG1-4 and IgM correspond to the heavy chain types α, δ, ε, γ and μ. Thus, the type of heavy chain present defines the class of antibodies. Different heavy chains differ in size and composition; alpha and gamma contain about 450 amino acids, while mu and epsilon contain about 550 amino acids. The difference in constant regions for each heavy chain type results in a different effector function for each antibody isotype because they selectively bind to a particular type of receptor (e.g., fc receptor). Thus, in embodiments of the invention, the antibody preferably comprises an epsilon heavy chain, i.e., the antibody is an isotype IgE that binds to the fcs receptor.

Each heavy and light chain comprises a constant region and a variable region (these regions are also referred to as "domains"). The heavy and light chain variable regions combine to specifically bind antigen. The light and heavy chain variable regions comprise a "framework" region, which is interrupted by three hypervariable regions, also known as "complementarity determining regions" or "CDRs. The framework regions and CDR ranges have been established (see, kabat et al, sequences of Proteins of Immunological Interest, U.S. device of Health and Human Services, 1991). The Kabat database is now maintained online. The framework region sequences of different light or heavy chains are relatively conserved within a species (e.g., human). The framework regions of antibodies, i.e., the combined framework regions of the constitutive light and heavy chains, are used to position and align the CDRs in three-dimensional space.

CDRs are mainly responsible for binding to epitopes of antigens. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, VH CDR3 is located in the variable domain of the antibody heavy chain in which it is located, while VL CDR1 is CDR1 of the variable domain of the antibody light chain in which it is located.

Antibodies may have specific VH and VL region sequences, and thus specific CDR sequences. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. These positions in 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.

A "monoclonal antibody" is an antibody produced by a single B lymphocyte clone 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 skilled in the art, for example, by fusing myeloma cells with immune spleen cells to produce hybrid antibody-forming cells. Monoclonal antibodies include humanized monoclonal antibodies.

"chimeric antibodies" include sequences derived from two different antibodies, which are typically derived from different species. For example, chimeric antibodies can include human and mouse antibody domains, such as human constant regions and mouse variable regions (e.g., from a murine antibody that specifically binds a target antigen).

Chimeric antibodies are typically constructed by fusing (e.g., by genetic engineering) the variable and constant regions of light and heavy chain immunoglobulin genes belonging to different species. For example, variable fragments of genes from mouse monoclonal antibodies can be ligated to human constant fragments, such as kappa and epsilon. In one embodiment, the therapeutic chimeric antibody is thus a hybrid protein consisting of a variable domain or antigen binding domain from a mouse antibody and a constant domain or effector domain from a human antibody (e.g., an Fc (effector) domain from a human IgE antibody), although other mammalian species may be used, or the variable region may be produced by molecular techniques. Methods of preparing chimeric antibodies are well known in the art, see, for example, U.S. patent No. 5,807,715.

A "humanized" antibody is one that includes 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 referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, all CDRs in the humanized immunoglobulin are from a donor immunoglobulin. The constant region is generally substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of the humanized immunoglobulin, except the CDRs, are substantially identical to the corresponding parts of the native human immunoglobulin sequence.

Humanized antibodies typically comprise a humanized immunoglobulin light chain and a humanized immunoglobulin heavy chain. Humanized antibodies typically bind the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acid substitutions taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function.

Humanized immunoglobulins may be constructed by genetic engineering (see, e.g., U.S. Pat. No. 5,585,089). Typically, humanized monoclonal antibodies are prepared by transferring the donor antibody complementarity determining regions from the heavy and light variable chains of a mouse immunoglobulin into a human variable domain and then replacing the human residues in the framework regions of the donor counterpart. The use of antibody components derived from humanized monoclonal antibodies avoids potential problems associated with the immunogenicity of the donor antibody constant regions. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al, nature 321:522,1986; riechmann et al Nature 332:323,1988; verhoeyen et al Science 239:1534,1988; carter et al, proc.Nat' l Acad.Sci.U.S. A.89:4285,1992; sandhu, crit. Rev. Biotech.12:437,1992; and Singer et al J.Immunol.150:2844,1993.

A "human" antibody (also referred to as a "fully human" antibody) is an antibody that includes all CDRs from the human framework regions and from the human immunoglobulin. In one example, the framework and CDRs are from identical human heavy and/or light chain amino acid sequences. However, the framework from one human antibody may be engineered to contain CDRs from a different human antibody.

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

anti-FR alpha antibodies

In some embodiments, the antibody specifically binds to a folate receptor (fα) to form an immune complex. Typically, the antibody may comprise an antigen binding region (e.g., one or more variable regions or 1 to 6 CDRs) derived from an antibody (e.g., MOv18 IgE) known to bind to fra (preferably human fra).

Fα (also known as folate receptor 1 or FOLR 1) is overexpressed in several solid cancer types, including ovarian cancer, endometrial cancer, and mesothelioma. The antigen has been characterized as having potent tumor specificity, and clinical trials targeting fα using IgG and IgE antibodies demonstrated favorable tolerability profiles. MOv18 IgG and IgE antibodies that bind to fαand their properties are described, for example, in Coney, L.R., A.Tomassetti et al (1991) Cancer Res 51 (22): 6125-6132; gould, H.J., G.A.Mackay et al (1999) Eur J Immunol 29 (11): 3527-3537; karagiannis, S.N., Q.Wang et al (2003) Eur J Immunol 33 (4): 1030-1040.

In a specific embodiment, the antibody comprises a variable region (e.g., heavy chain variable domain (VH) and/or light chain variable structure (VL)) or at least one, two, three, four, five or six CDRs (e.g., 3 heavy chain CDRs or 3 light chain CDRs) from a MOv18 IgG or IgE, e.g., CDRs present in SEQ ID No. 4 and/or SEQ ID No. 3, wherein CDR sequences can be defined according to methods of Kabat, chothia or IMGT (see, e.g., domdinger, front immunol.2018;9:2278 and references cited therein). For example, CDRs can be defined according to Kabat: see Kabat EA et al (U.S.) NI of H.sequences of Immunoglobulin Chains: tabulation Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain BP-Microglobulins,1979; or according to Chothia definition: see Chothia C et al Canonical structures for the hypervariable regions of immunoglobulins, J Mol biol 1987Aug 20;196 (4) 901-1; or according to IMGT definition: see Giudielli V et al, IMGT, the international ImMunoGeneTics database, nucleic Acids Res.1997Jan 1;25 (1) 206-11 or Lefranc MP, unique database numbering system for immunogenetic analysis, immunol today.1997Nov;18 (11):509. The amino acid sequences of the VH and VL domains of MOv18 IgE are shown 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 to which MOv18 IgE binds. Most preferably, the therapeutic antibody is MOv18 IgE, e.g. the antibody comprises the light chain amino acid sequence defined in SEQ ID No. 1 and/or the heavy chain amino acid sequence defined in SEQ ID No. 2.

In another embodiment, the antibody comprises a variable region (e.g., a heavy chain variable domain and/or a light chain variable domain) or at least one, two, three, four, five, or six CDRs (e.g., 3 heavy chain CDRs or 3 light chain CDRs) derived from a human B cell clone that recognizes an epitope found, for example, on fra (preferably human fra).

In one embodiment, the antibody comprises one or more human constant regions, such as one or more human heavy chain constant domains (e.g., epsilon constant domains) and/or human light chain (e.g., kappa or lambda) constant domains. The amino acid sequence of the human light chain (k) constant domain is shown in SEQ ID NO. 1 (not bolded). The amino acid sequence of the human heavy chain constant domain is shown in SEQ ID NO. 2 (not bold). In one embodiment, the antibody comprises one or more human framework regions within the VH and/or VL domains.

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

Further antibodies against specific antigens (e.g., fα) may also be produced by mature methods, and at least the variable regions or CDRs from such antibodies may be used in the antibodies of the invention (e.g., the produced antibodies may be used to contribute CDRs or variable region sequences to IgE receptor sequences). Methods for synthesizing polypeptides and immunizing host animals are well known in the art. Typically, a host animal (e.g., a mouse) is intraperitoneally inoculated with an amount of an immunogen (e.g., fra or a polypeptide comprising an immunogenic fragment thereof) and (in the case of monoclonal antibody production) hybridomas made from its lymphocytes and immortalized myeloma cells using the general somatic hybridization techniques of Kohler, b. And Milstein, c. (1975) Nature 25:495-497.

The sequence of human FR alpha is well known (see, e.g., uniProt database accession No. P15328), so human FR alpha can be purified, e.g., from natural sources or expressed using recombinant techniques for such methods. The amino acid and nucleic acid sequences of human FR alpha are shown below in SEQ ID NOs: 5 and 6, respectively:

SEQ ID NO: 5-human FR alpha amino acid sequence:

MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS

SEQ ID NO. 6-human FR alpha nucleic acid sequence:

atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaaggattgcatgggccaggactgagcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagtgtcgaccctggaggaagaatgcctgctgttctaccaacaccagccaggaagcccataaggatgtttcctacctatatagattcaactggaaccactgtggagagatggcacctgcctgcaaacggcatttcatccaggacacctgcctctacgagtgctcccccaacttggggccctggatccagcaggtggatcagagctggcgcaaagagcgggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagattgtcgcacctcctacacctgcaagagcaactggcacaagggctggaactggacttcagggtttaacaagtgcgcagtgggagctgcctgccaacctttccatttctacttccccacacccactgttctgtgcaatgaaatctggactcactcctacaaggtcagcaactacagccgagggagtggccgctgcatccagatgtggttcgacccagcccagggcaaccccaatgaggaggtggcgaggttctatgctgcagccatgagtggggctgggccctgggcagcctggcctttcctgcttagcctggccctaatgctgctgtggctgctcagc

hybridomas producing suitable antibodies can be cultured in vitro or in vivo using known methods. If desired, monoclonal antibodies can be isolated from the culture medium or body fluids by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration. The undesired activity, if present, can be removed, for example, by passing the preparation through an adsorbate made of an immunogen attached to a solid phase, and eluting or releasing the desired antibody from the immunogen. If desired, the antibody of interest (monoclonal or polyclonal) may be sequenced and the polynucleotide sequence may then be cloned into an expression or amplification vector. The sequences encoding the antibodies may be maintained in vectors in host cells, which may then be amplified and frozen for future use.

Phage display techniques, such as described in U.S. Pat. No. 5,565,332 and other publications, can be used to select and prepare human antibodies and antibody fragments in vitro from libraries of immunoglobulin variable (V) domain genes from non-immunized donors (e.g., human subjects, including patients with related diseases). For example, existing antibody phage display libraries can be screened in parallel against a large number of synthetic polypeptides. According to this technique, the antibody V domain gene is cloned in-frame into a major or minor capsid protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as a functional antibody fragment on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody will also result in selection of genes encoding antibodies with these properties. Thus, antibody sequences selected from a human repertoire using phage display may include human CDR or variable region sequences that confer specific binding to a particular antigen (e.g., fra), which may be used to provide fully human antibodies for use in the invention.

Methods for deriving heavy and light chain sequences from human B-cell and plasma cell clones are well 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.201110ul 21;11:75; scheeren FA et al, PLoS ONE 2011,6 (4): e17189.Doi: 10.1371/journ. Fine. 0017189; wrammert J et al, nature 2008 453,667-671; kurosawa N et al, BMC biotechnol.2011apr13; 11:39; tiller et al, J Immunol methods.20088 January 1;329 (1-2):112-124. Thus, antibody sequences selected using B cell cloning may include human CDR or variable region sequences that confer specific binding to, for example, fra, which may be used to provide fully human antibodies for use in the invention.

IgE antibodies

The therapeutic antibody to be administered to the subject is an IgE antibody, i.e., an antibody to isotype IgE. There are some fundamental structural differences between IgE and IgG, which have a functional impact. Although IgE has the same basic molecular structure as antibodies of other classes, the heavy chain of IgE comprises one more domain than the heavy chain of IgG. The C3 and C4 domains of IgE are homologous in sequence and similar in structure to the C2 and C3 domains of IgG, whereby the C2 domain is the most distinct distinguishing feature of IgE. The C epsilon 2 domain has been found to fold back relative to the IgE heavy chain and to make extensive contact with the C epsilon 3 domain. This bent structure of the IgE heavy chain allows it to adopt either an open or a closed conformation. Unbound IgE dimers have one strand in an open conformation and one strand in a closed conformation. The binding of fceri to IgE is biphasic and is believed to involve initial binding to the open C epsilon chain followed by extensive structural rearrangement to allow binding to the closed C epsilon chain. Although two identical C epsilon chains were present, the binding between IgE dimer and fceri was performed at a 1:1 stoichiometric ratio. This rearrangement results in a very tight interaction between IgE and FcεRI, and a much greater affinity between IgE and its Fc receptor than between IgG for FcγR (McDonnell, J.M., R.Calvert et al, (2001) Nat Struct Biol 8 (5): 437-441).

Antibodies for use in the present invention are generally capable of binding to fceri receptors, e.g., binding to fceri and/or fceri receptors. Preferably, the antibody is capable of binding at least fceri (i.e., high affinity fceri receptor) or at least fceri (CD 23, low affinity fceri receptor). Typically, the antibodies are also capable of activating fcs receptors, such as fcs receptors expressed on cells of the immune system, to initiate IgE-mediated effector functions.

Epsilon heavy chains are well defined for IgE antibodies and include an N-terminal variable domain VH and four constant domains C epsilon 1-C epsilon 4. Like other antibody isotypes, the variable domains confer antigen specificity and the constant domains confer isotype-specific effector functions.

IgE differs from the more abundant IgG isotype in that it is unable to fix complement and is unable to bind to Fc receptors fcyri, RII and RIII expressed on the surface of monocytes, NK cells and neutrophils. However, igE is capable of very specific interactions with "high affinity" IgE receptors on a variety of immune cells (e.g. mast cells, basophils, monocytes/macrophages, eosinophils) (fceri, ka.10) ¹¹ M ^-1 ) And "low affinity" receptors (fceri, also known as CD23, ka.10) expressed on inflammatory and antigen presenting cells (e.g., monocytes/macrophages, platelets, dendritic cells, T and B lymphocytes) ⁷ M ^- 1)。

The sites on IgE responsible for these receptor interactions have been indicated on the peptide sequence on the C epsilon chain and are different. The fceri site is located in the cleft formed by residues between Gln 301 and Arg 376 and includes the linkage between the C2 and C3 domains (Helm, b. Et al, (1988) nature331, 180183). The Fc epsilon RII binding site is located around residue Val 370 within C epsilon 3 (Vercelli, D. Et al, (1989) Nature 338, 649-651). One major difference distinguishing between these two receptors is that fceri binds to the C monomer, whereas fceri binds only to dimerized C, i.e. both C chains have to be bound. Although IgE is glycosylated in vivo, this is not necessary for its binding to fceri and fcrrii. In fact, the binding force was slightly stronger in the absence of glycosylation (Vercelli, D.et al, (1989) et. supra).

Thus, binding to the fcs receptor and related effector functions are typically mediated by the heavy chain constant domain of the antibody, particularly by the domains that together form the Fc region of the antibody. Antibodies described herein generally comprise at least a portion of an IgE antibody, e.g., one or more constant domains derived from IgE (preferably human IgE). In specific embodiments, the antibodies comprise one or more domains (derived from IgE) selected from the group consisting of C epsilon l, C epsilon 2, C epsilon 3, and C epsilon 4. In one embodiment, the antibody comprises at least C epsilon 2 and C epsilon 3, more preferably at least C epsilon 2, C epsilon 3 and C epsilon 4, preferably wherein the domain is derived from human IgE. In one embodiment, the antibody comprises an (epsilon) heavy chain, preferably a human epsilon heavy chain.

The amino acid sequence of a constant domain derived from human IgE is shown, for example, in FIGS. 1 and 2 (SEQ ID NOS: 1 and 2, not bolded). Nucleotide sequences encoding constant domains derived from human IgE, in particular the C epsilon 1, C epsilon 2, C epsilon 3 and C epsilon 4 domains, are also disclosed in, for example, WO 2013/050725. The amino acid sequences of other human and mammalian IgE and domains thereof (including human C epsilon l, C epsilon 2, C epsilon 3 and C epsilon 4 domains and human epsilon heavy chain sequences) are known in the art and are available from publicly accessible databases. For example, a database of human immunoglobulin sequences can be obtained from the International ImmunoGeneTics information SystemThe website is accessed, and the website address is http:// www.imgt.org. As an example, the sequences of the various human IgE heavy (ε) chain alleles and their respective constant domains (Cε 1-4) can be found in http:// www.imgt.org/IMGT_GENE-DB/GENElectquery=2+IGHE&species=homo+sapiens access.

Preferred anti-FR alpha IgE antibodies

In one embodiment, the anti-FR alpha antibody comprises a VH domain comprising at least a portion of the amino acid sequence defined in SEQ ID NO. 4, e.g. comprising at least 20, 30, 50 or 100 amino acids of SEQ ID NO. 4, or the full 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).

In one embodiment, the anti-FR alpha antibody comprises a VL domain comprising at least a portion of the amino acid sequence defined in SEQ ID NO. 3, e.g., comprising at least 20, 30, 50 or 100 amino acids of SEQ ID NO. 3, or the full 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).

In general, functional fragments of the sequences defined above are useful in the present invention. The functional fragment may be any length as described 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 activity desired in the presence of the antibody (e.g., specific binding to the FR alpha and/or fcs receptor).

Variants of the above amino acid and nucleotide sequences may also be used in the present invention provided that the resulting antibodies bind to the fcs receptor. Typically, such variants have a high level of sequence identity to one of the sequences described above.

Similarity between amino acid or nucleotide sequences is expressed as similarity between sequences, also known as sequence identity. Sequence identity is typically measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences. Homologs or variants of the amino acid or nucleotide sequences will have a relatively high level of sequence identity when aligned using standard methods.

Sequence alignment methods for comparison are well known in the art. Various procedures and alignment algorithms are described in Smith and Waterman, adv. Appl. Math.2:482,1981; needleman and Wunsch, J.mol.biol.48:443,1970; pearson and Lipman, proc.Natl. Acad.Sci.U.S.A.85:2444,1988; higgins and Sharp, gene 73:237,1988; higgins and Sharp, CABIOS 5:151,1989; corpet et al, nucleic Acids Research 16:10881,1988; and Pearson and Lipman, proc.Natl. Acad.Sci.U.S.A.85:2444,1988.Altschul et al Nature Genet.6:119,1994 gives detailed information on sequence alignment and homology calculations.

NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J.mol. Biol.215:403,1990) is available from a variety of sources, including the national center for Biotechnology information (NCBI, bethesda, md.) and the Internet, along with sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Instructions on how to use this procedure to determine sequence identity are available at the NCBI website on the internet.

Homologs and variants of antibodies (e.g., anti-FR alpha antibodies or domains thereof, such as VL, VH, CL or CH domains) typically have at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the original sequence (e.g., the sequence defined above), e.g., calculated over a full length alignment with the amino acid sequence of the antibody or domain thereof using NCBI Blast 2.0 (notch blastp set as default parameters). For comparisons of amino acid sequences greater than about 30 amino acids, blast 2 sequence functions were used, using a default BLOSUM62 matrix (gap penalty of 11, gap per residue of 1) set as default parameters. When aligning short peptides (less than about 30 amino acids), the Blast 2 sequence functions should be used for alignment, using PAM30 matrix (open gap penalty 9, extended gap penalty 1) set as default parameters. Proteins having even greater similarity to the reference sequence will exhibit an increased percentage of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, when assessed by this method. When comparing sequence identity to less than complete sequences, homologues and variants generally 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 their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on NCBI websites on the internet. Those skilled in the art will appreciate that these ranges of sequence identity are provided for guidance only; it is entirely possible to obtain very significant homologs that fall outside the ranges provided.

In general, a variant may comprise one or more conservative amino acid substitutions compared to the original amino acid or nucleic acid sequence. Conservative substitutions are those substitutions that do not substantially affect or reduce the affinity of the antibody for the target antigen (e.g., fα) and/or fcs receptor. For example, a human antibody that specifically binds to a FR alpha may include up to 1, up to 2, up to 5, up to 10, or up to 15 conservative substitutions as compared to the original sequence (as defined above), and retains specific binding to the FR alpha polypeptide. The term conservative variation also includes the use of a substituted amino acid instead of the unsubstituted parent amino acid, provided that the antibody specifically binds to the target antigen (e.g., fα). Non-conservative substitutions are those that reduce activity or binding to the target antigen (e.g., fα) and/or fcs receptor.

Functionally similar amino acids that can be exchanged by conservative substitutions are well known to those of ordinary skill in the art. The following six groups are considered examples of amino acids that are conservative substitutions for one another: 1) Alanine (a), serine (S), threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

In some embodiments, igE antibodies may be conjugated to a cytotoxic moiety, such as a chemotherapeutic agent that (directly) kills cancer cells, in order to produce an Antibody Drug Conjugate (ADC). Antibodies may be conjugated to a cytotoxic moiety directly or via a linker, as known in the art. For example, one example of such a cytotoxic agent is maytansinoid DM4 (maytansinoid DM 4), a potent tubulin targeting agent, found in IgG ADC sorrow-rituximab. In other embodiments, igE antibodies may be administered to a subject in combination with the separate (e.g., simultaneous or sequential) administration of a chemotherapeutic agent, such as gemcitabine (2 ',2' -difluoro 2' deoxycytidine).

However, in preferred embodiments, igE antibodies lack a cytotoxic moiety and/or are administered as monotherapy. It has surprisingly been found that anti-FR alpha IgE antibodies are capable of treating low FR alpha expressing tumors without the need to administer cytotoxic drugs (in ADC form or in a chemotherapeutic combination).

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

In particular, it is preferred that the antibody is not an antibody-drug conjugate (ADC). Thus, the antibody may lack other drugs or groups that have cytotoxic effects, e.g., chemotherapeutic agents or drugs that are capable of (directly) killing cancer cells. Antibodies may also lack linkers or other groups for conjugating the cytotoxic moiety to the polypeptide.

Other IgE antibodies

As described above, in a preferred embodiment, igE antibodies bind to FR alpha. Preferably, igE antibodies are capable of inducing cytotoxicity (e.g., ADCC) and/or phagocytosis (ADCP), particularly against cancer cells expressing such antigens.

In some embodiments, one or more variable domains and/or one or more CDRs, preferably at least three CDRs, or more preferably all six CDRs, can be derived from one or more of the following antibodies: MOv19 (Coney et al, cancer Res.1991Nov 15;51 (22): 6125-32; coney et al, cancer Res.1994May 1;54 (9): 2448-55), sofossa-Mituximab (IMGN 853, as described in Ab et al, molecular Cancer Therapeutics (7): 1605-13, july 2015) or Fatuzumab (MORAB-003, as described in Ebel et al, cancer Immun.2007;7:6; sato and Itamochi, oncoTargets and Therapy 2016:9 1-1188). Sofoster-mitoximab (IMGN 853) refers to immunoconjugates comprising a humanized MOv19 (M9346A) antibody, a sulfoSPDB (N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutyrate) linker, and a DM4 maytansinoid (N2 '-deacetylated-N2' - (4-mercapto-4-methyl-l-oxopentyl) maytansine).

For example, an IgE antibody may comprise the following variable domain sequences or 1 to 6 CDRs derived therefrom (e.g., as defined according to Rabat, chothia or IMGT):

the trastuzumab VH domain:

EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLE WVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYF CARHGDDPAWFAYWGQGTPVTVSS(SEQ ID NO:7)

fatuzumab VL (V) _κ ) Domain:

DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKP WIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPY MYTFGQGTKVEIK(SEQ ID NO:8)

in other embodiments, igE antibodies may comprise one or more variable domain sequences or CDRs from an anti-froc (i.e., anti-FOLR 1) antibody or antigen-binding fragment thereof, as defined, for example, in US 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 sequences or 1 to 6 CDRs derived therefrom (e.g., as defined according to Rabat, chothia or IMGT):

huMOv19 VH domain (SEQ ID NO: 9):

QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSS

huMOV19 VL domain (SEQ ID NO: 10):

DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR

for example, an IgE antibody may comprise one or more (e.g., all six) of the following CDR sequences (defined according to Rabat):

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

in another embodiment, the antibody is a chimeric, humanized or fully human antibody that specifically binds to an epitope to which rituximab or pertuzumab binds. IgE antibodies may also comprise one or more IgE constant domains as described above, e.g. the C epsilon 1-C epsilon 4 domain.

Production of antibodies and nucleic acids

Nucleic acid molecules (also referred to as polynucleotides) encoding polypeptides provided herein, including but not limited to antibodies and functional fragments thereof, can be readily produced by one of skill in the art using the amino acid sequences, sequences available in the art, and genetic codes provided herein. Furthermore, one skilled in the art can readily construct various clones comprising functionally equivalent nucleic acids (e.g., nucleic acids that differ in sequence but encode the same effector molecule or antibody sequence). Thus, provided herein are nucleic acids encoding antibodies.

The nucleic acid sequence encoding an antibody or functional fragment thereof that specifically binds to a target antigen (e.g., FR alpha) can be prepared by any suitable method, including, for example, cloning of the appropriate sequence or by direct chemical synthesis by methods such as the phosphotriester method of Narag et al, meth. Zymol.68:90-99,1979; the phosphodiester method of Brown et al, meth. Enzymol.68:109-151, 1979; beaucage et al, tetra. Lett.22:1859-1862,1981, diethyl phosphoramidite method; beaucage & Caruthers, tetra. Letts.22 (20): 1859-1862,1981, using, for example, an automated synthesizer as described in, for example, needleham-Van Devanter et al, nucl. Acids Res.12:6159-6168, 1984; and U.S. Pat. No. 4,458,066. Chemical synthesis produces single stranded oligonucleotides. This can be converted to double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using a single strand as a template. Those skilled in the art will recognize that while chemical synthesis of DNA is typically limited to sequences of about 100 bases, longer sequences can be obtained by ligating shorter sequences.

Exemplary nucleic acids encoding antibodies or functional fragments thereof may be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques and instructions sufficient to guide the skilled artisan through many cloning exercises are described, for example, in Molecular Cloning: A Laboratory Manual, 2 nd edition, volumes 1-3, editions by Sambrook et al Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 1989); and Current Protocols in Molecular Biology (Ausubel et al, edited 1995 journal). Product information from manufacturers of biological reagents and experimental equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, mo.), R & DSystems (Minneapolis, minn.), pharmacia Amersham (Piscataway, n.j.), CLONTECH Laboratories, inc (Palo Alto, calif.), chem Genes corp, aldrich Chemical Company (Milwaukee, wis.), glen Research, inc, GIBCO BRL Life Technologies, inc (Gaithersburg, md.), fluka Chemica-Biochemika Analytika (Fluka Chemie AG, buchs, switzerland), invitrogen (Carlsbad, calif.) and Applied Biosystems (Foster City, calif.), as well as many other commercial sources known to those skilled in the art.

Nucleic acids encoding natural antibodies can be modified to form antibodies described herein. Modification by site-directed mutagenesis is well known in the art. Nucleic acids can also be prepared by amplification methods. Amplification methods include Polymerase Chain Reaction (PCR), ligase Chain Reaction (LCR), transcription-based amplification systems (TAS), and autonomous sequence replication systems (3 SR). The skilled artisan is familiar with a variety of cloning methods, host cells and in vitro amplification methods.

In one embodiment, the antibody is prepared by inserting a cDNA encoding one or more antibody domains (e.g., a mouse IgG1 heavy chain variable region that binds human FR alpha) into a vector comprising a cDNA encoding one or more other antibody domains (e.g., a human heavy chain epsilon constant region). Insertion is performed such that the antibody domains are in-frame (i.e., in one continuous polypeptide comprising the functional antibody region).

In one embodiment, the cDNA encoding the heavy chain constant region is linked to the heavy chain variable region such that the constant region is at the carboxy terminus of the antibody. Disulfide bonds may then be used to link the heavy chain variable and/or constant regions to the light chain variable and/or constant regions of the antibody.

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 bacterial, plant, yeast, insect and mammalian cells. Those skilled in the art are expected to be familiar with a variety of expression systems useful for expressing proteins, including E.coli (E.coli), other bacterial hosts, yeast, and various higher eukaryotic cells, such as COS, CHO, heLa and myeloma cell lines.

One or more DNA sequences encoding an antibody or fragment thereof may be expressed in vitro by transferring the DNA into a suitable host cell. The cells may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It will be appreciated that all offspring may differ from the parent cell by mutations that may occur during replication. Methods of stable transfer (i.e., sustained maintenance of exogenous DNA in a host) are known in the art. Hybridomas expressing the antibody of interest are also encompassed in the present disclosure.

Expression of nucleic acids encoding the isolated antibodies and antibody fragments described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is constitutive or inducible) and then incorporating into the expression cassette. These expression cassettes may be suitable for replication and integration in prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences for regulating the expression of the DNA encoding the protein. For example, the expression cassette may comprise appropriate promoters, enhancers, transcriptional and translational terminators, initiation sequences, initiation codons (i.e., ATG) prior to the gene encoding the protein, splicing signals of introns, maintenance of the correct reading frame of the gene to allow for correct translation of the mRNA, and stop codons.

In order to obtain high levels of expression of the cloned gene, it is necessary to construct an expression cassette comprising, at a minimum, a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription/translation terminator. For E.coli (E.coli), this includes promoters such as the T7, trp, lac or lambda promoters, ribosome binding sites, and preferably transcription termination signals. For eukaryotic cells, the control sequences may include promoters and/or enhancers (derived from, for example, immunoglobulin genes, SV40, or cytomegalovirus) and polyadenylation sequences, and may further include splice donor and acceptor sequences. The expression cassette may be transferred into the selected host cell by well known methods, for example using transformation or electroporation for E.coli (E.coli), calcium phosphate treatment for mammalian cells, electroporation or lipofection. Cells transformed by the expression cassette may be selected for antibiotic resistance conferred by the genes contained in the cassette (e.g., amp, gpt, neo and hyg genes).

When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, the addition of plasmids entrapped in liposomes, or viral vectors may be used. Eukaryotic cells may also be co-transformed with a polynucleotide sequence encoding an antibody, a labeled antibody, or a functional fragment thereof, and a second exogenous DNA molecule encoding a selectable phenotype (e.g., a herpes simplex thymidine kinase gene). Another approach is to transiently infect or transform eukaryotic cells and express proteins using eukaryotic viral vectors, such as monkey virus 40 (SV 40) or bovine papilloma virus (see, e.g., eukaryotic Viral Vectors, cold Spring Harbor Laboratory, gluzman edit, 1982). Expression systems such as plasmids and vectors can be readily used by those skilled in the art for the production of proteins in cells, including higher eukaryotic cells such as COS, CHO, heLa and myeloma cell lines.

Nucleic acids encoding polypeptides described herein (e.g., human froc-specific IgE antibodies) can be modified without decreasing their biological activity. Some modifications may be made to facilitate cloning, expression or incorporation of the targeting molecule into the fusion protein. Such modifications are well known to those skilled in the art and include, for example, stop codons, methionine added at the amino terminus to provide an initiation site, other amino acids placed at either end to create restriction sites that facilitate localization, or other amino acids (e.g., polyHis) that aid in the purification step. In addition to recombinant methods, antibodies of the invention may be constructed in whole or in part using standard peptide synthesis methods well known in the art.

After expression, the recombinant antibodies may 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, n.y., 1982). Antibodies, immunoconjugates and effector molecules need not be 100% pure. After purification (partial purification or achieving the desired homogeneity), the polypeptide should be substantially free of endotoxin if used therapeutically.

Typically, functional heterologous proteins from E.coli (E.coli) or other bacteria are isolated from inclusion bodies and require solubilization with a strong denaturing agent and then refolding. During the dissolution step, a reducing agent must be present to separate the disulfide bonds, as is well known in the art. Exemplary buffers with reducing agents are: 0.1M Tris (pH 8), 6M guanidine, 2mM EDTA, 0.3M DTE (dithioerythritol). Reoxidation of disulfide bonds may occur in the presence of reduced and oxidized forms of low molecular weight thiol reagents as described by Saxena et al, biochemistry 9:5015-5021,1970, and in particular as described by Buchner et al (supra).

Renaturation is typically accomplished by dilution (e.g., 100-fold) of the denatured reduced protein into refolding buffer. Exemplary buffers are 0.1M Tris (pH 8.0), 0.5M L-arginine, 8mM glutathione oxide (GSSG), and 2mM EDTA.

As a variation on the diabody purification protocol, the heavy and light chains were dissolved and reduced separately and then combined in refolding solution. Exemplary yields are obtained when the two proteins are mixed in a molar ratio such that the molar excess of one protein relative to the other is no more than 5-fold. After the redox switch is completed, excess oxidized glutathione or other oxidative low molecular weight compounds may be added to the refolding solution.

In addition to recombinant methods, the antibodies, labeled antibodies, and functional fragments thereof disclosed herein may also be constructed in whole or in part using standard peptide synthesis methods. Solid phase synthesis of polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. The solid phase Synthesis technique consists of Barany & Merrifield, the Peptides: analysis, synthesis, biology, volume 2, special Methods in Peptide Synthesis, part A, pages 3-284; merrifield et al, J.Am.chem.Soc.85:2149-2156,1963, and Stewart et al, solid Phase Peptide Synthesis, 2 nd edition, pierce chem.Co., rockford, ill., 1984. Proteins of greater length can be synthesized by condensation of the amino and carboxyl termini of shorter fragments.

Methods for forming peptide bonds by activating the carboxyl terminus (e.g., by using the coupling reagent N, N' -dicyclohexyl carbodiamide) are well known in the art.

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

Compositions and methods of treatment

Provided herein are compositions comprising a carrier and one or more therapeutic IgE antibodies or functional fragments thereof. The composition may be prepared in unit dosage form for administration to a subject. Antibodies may be formulated for systemic or local (e.g., intratumoral) administration. In one example, the therapeutic IgE antibody is formulated for parenteral administration, such as intravenous administration or subcutaneous administration.

The composition for administration may comprise a solution of the antibody (or functional fragment thereof) dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional and well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of antibodies and excipients in these formulations can vary and will be selected based on liquid volume, viscosity, body weight, etc., primarily according to the particular mode of administration selected and the needs of the subject. Practical methods of preparing the administrable compositions are known or apparent to those skilled in the art and are described in more detail in such disclosures as Remington' sPharmaceutical Science, 19 th edition, mack Publishing Company, easton, pa. (1995).

In a preferred embodiment, the composition is provided in unit dosage form, e.g., comprising a defined amount of IgE antibodies suitable for administration to a subject in a single dose. The unit dosage forms may be packaged individually, for example in individual containers, vials, prefilled syringes, and the like. The unit dosage form may be suitable for immediate administration to a subject (e.g., may include a physiologically acceptable concentration of salt), or the unit dosage form may be provided in concentrated or lyophilized form (e.g., for dilution with a sterile saline solution prior to use).

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

The dosage of IgE antibody administered to a subject may be based on the body weight of the subject. Thus, the dosage of IgE antibodies administered to a subject may be, for example, less than 1mg/kg. Preferably, igE antibodies may be administered to a subject at a dose of, for example, less than 0.7mg/kg, 0.5mg/kg, 0.3mg/kg, 0.1mg/kg, 0.07mg/kg, 0.05mg/kg, 0.03mg/kg, or 0.01mg/kg (per administration). The dose of IgE antibody administered to a subject may be at least 0.001mg/kg, 0.003mg/kg, 0.005mg/kg, 0.007mg/kg, 0.01mg/kg, 0.05mg/kg, or 0.1mg/kg. In preferred embodiments, the dosage of IgE antibody administered to a subject may be 0.001-1mg/kg, 0.003-0.7mg/kg, 0.005-0.5mg/kg, 0.005-0.1mg/kg, 0.005-0.05mg/kg, 0.007-0.03mg/kg, or 0.007-0.15mg/kg. In some embodiments, the dosage of IgE antibodies administered to a subject may be within one or more of the ranges defined above, but excluding one or more of the following dosages: 1. Mu.g/kg, 10. Mu.g/kg, 100. Mu.g/kg or 0.5mg/kg. For example, the IgE antibody may be dosed at 2 to 9 μg/kg, 11 to 99 μg/kg, 101 to 499 μg/kg, or 0.51 to 0.7mg/kg.

In embodiments of the invention, the unit dose of IgE antibody described above is administered up to once a week, e.g., the maximum weekly dose of IgE antibody is 50mg, 40mg, 30mg, 25mg, 20mg, 15mg, 10mg, 5mg, 3mg, or 1mg. For example, a weekly dose of IgE antibodies may be 10 μg to 50mg, 70 μg to 30mg, 300 μg to 50mg, 300 μg to 30mg, 300 μg to 3mg, 500 μg to 50mg, 500 μg to 30mg, 500 μg to 10mg, 500 μg to 3mg, 700 μg to 50mg, 700 μg to 30mg, 700 μg to 10mg, 700 μg to 3mg, 500 μg to 5mg, 500 μg to 1mg, or about 700 μg. The weekly dosage of IgE antibodies may also be determined based on the weight of the subject, e.g., igE antibodies may be administered to the subject at a dose of, e.g., less than 0.7 mg/kg/week, 0.5 mg/kg/week, 0.3 mg/kg/week, 0.1 mg/kg/week, 0.07 mg/kg/week, 0.05 mg/kg/week, 0.03 mg/kg/week, or 0.01 mg/kg/week. In preferred embodiments, the dosage of IgE antibody administered to a subject may be 0.001-1 mg/kg/week, 0.003-0.7 mg/kg/week, 0.005-0.5 mg/kg/week, 0.005-0.1 mg/kg/week, 0.005-0.05 mg/kg/week, 0.007-0.03 mg/kg/week, or 0.007-0.15 mg/kg/week. In some embodiments, the dosage of IgE antibodies administered to a subject may be within one or more of the ranges defined above, but excluding one or more of the following dosages: 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 dosage of IgE antibody may be 2 to 6. Mu.g/kg/week, 8 to 69. Mu.g/kg/week, or 71 to 699. Mu.g/kg/week.

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 pH of the composition is from 6.0 to 8.0, for example about 6.5. Preferred concentrations of excipients include: 0.05 to 0.5M (e.g., about 0.1M) sodium citrate; from 10 to 50g/L (e.g., about 30 g/L) L-arginine; 10 to 100g/L (e.g., about 50 g/L) sucrose; 0.01 to 0.05% w/w (e.g., 0.02% w/w) polysorbate 20. In one embodiment, igE antibodies are present in such formulations at a concentration of about 0.1mg/ml to 10mg/ml or 0.5mg/ml to 2mg/ml, for example about 1 mg/ml. In some embodiments, such compositions may be formulated in unit dosage form, e.g., in a volume of about 1ml of a solution comprising about 1mg IgE antibody, e.g., in a 2ml type I glass bottle. The composition may be diluted with sterile saline (0.9% w/v) prior to administration to a subject, for example, in 250ml of saline in an amount of 1ml of the composition.

Antibodies can be provided in lyophilized form and re-hydrated with sterile water prior to administration, although they are also provided in sterile solutions of known concentrations. The antibody solution was then added to an infusion bag containing 0.9% sodium chloride (USP) and administered to the subject. Since rituximab (RITUXAN, registered trademark) was approved, there has been considerable experience in the field of administration of antibody drugs that have been marketed in the united states. Antibodies may be administered by slow infusion, rather than by intravenous bolus injection or bolus injection (bolus). In one example, a higher loading dose is administered followed by a lower level of maintenance dose. For example, an initial loading dose may be infused over about 90 minutes, followed by a maintenance dose infused weekly over a 30 minute period for 4-8 weeks if the previous dose was well tolerated.

The antibody (or functional fragment thereof) may 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 the subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells, or to inhibit signs or symptoms of cancer. In some embodiments, the antibodies are administered to a subject to inhibit or prevent the progression of metastasis, or to reduce the size or number of metastases, e.g., micrometastases of regional lymph nodes (Goto et al, clin. Cancer Res.14 (11): 3401-3407, 2008).

Thus, in some embodiments, igE antibodies are used to treat cancer and/or delay or prevent progression of cancer. By "delay or prevention of cancer progression" is meant, for example, that the cancer is stable for at least a period of time, e.g., at least 6 weeks, at least 12 weeks, at least 6 months, or at least 12 months, after administration of the antibody. For example, a "stable" disease may be defined as a RECIST score that varies by less than 20%.

RECIST (solid tumor response evaluation criteria) evaluation is a simple method for determining whether a patient's disease has improved, remained unchanged or worsened after treatment with cancer therapy, 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 an increase in the sum of target lesion diameters by at least 20% (referenced to the smallest sum in the study). Disease stabilization is defined as a disease where there is not enough shrinkage to meet partial response (at least 30% reduction in the sum of target lesion diameters) nor enough increase to meet PD, i.e., an increase of less than 20% is defined as stable.

In this context, "at least stable" is understood to include an increase or decrease in RECIST score of less than 20%. Thus, an antibody may delay or prevent progression of a disease (e.g., delay or prevent appearance of one or more signs or symptoms of cancer, and/or inhibit growth of cancer cells, and/or prevent or reduce metastasis), or improve or promote remission of a disease (e.g., reduce or inhibit one or more signs or symptoms of cancer, and/or kill cancer cells).

A subject

Suitable subjects may include those diagnosed with cancer, such as cancers that express FR alpha, such as, 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 myelogenous leukemia and 11g 23-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.

The therapeutically effective amount of antibody will depend on the severity of the disease and the overall health of the patient. A therapeutically effective amount of an antibody is one that produces subjective relief of the symptom(s) or an objectively identifiable improvement as observed by a clinician or other qualified observer. These compositions may be administered simultaneously or sequentially with another chemotherapeutic agent.

anti-FR alpha antibodies are used to treat subjects with low FR alpha expressing tumors. Such subjects may be referred to as "low-FR alpha expressiors" as compared to moderate or high-FR alpha expressiors. The terms "low FR alpha expressing tumor" and "low FR alpha expressing person" are well understood in the field of cancer treatment and are often used to describe a specific patient group (see e.g. Cristea et al Journal of Clinical Oncology 202139:15_suppl,5542; martin et al, gynecol Oncol.2017Nov;147 (2): 402-407). Thus, a subset of patients with low tumor FR alpha expression can be distinguished significantly from those with high FR alpha expression (see, e.g., SORAYA assay, clinicalTrials gov identifier: NCT 04296890).

Detection of low FR alpha expression

Low tumor FR alpha expression can be determined using well known standard techniques. Typically, FR alpha expression is determined in a biopsy sample from a tumor. Techniques for obtaining biopsy samples from tumor tissue are known in the art, as are histopathological techniques for processing such samples. For example, biopsy tissue samples may be formalin fixed and embedded in paraffin or freshly processed prior to sectioning and placement on a microscope slide for optical microscopy and imaging.

Hematoxylin and eosin (H & E) staining of paraffin-embedded sections is the default technique for observing tissues on slides for pathological analysis. Immunohistochemical (IHC) staining is a well known method for recognizing protein expression on cells in pathological tissue sections. Staining results in tissues that over-express the targeted protein compared to normal exhibiting a typical brown appearance. For example, by using an antibody against fra, the expression level thereof can be detected.

Suitable techniques for detecting expression of FR alpha in a tumor sample are described, for example, in Zhao et al, "Development and Application of An Immunohistochemistry-based Clinical Assay for Evaluating Folate Receptor Alpha (FR alpha) Expression in the Clinical Setting", abstract 3400A, AACR annual meeting, 2015, 4, 18-22; ab et al (2015), molecular Cancer Therapeutics 14 (7): 1605-13; altwerger et al Mol Cancer Ther.2018May;17 (5) 1003-1011, "Gynecol Oncol.2017Nov;147 (2):402-407. For example, the Ventana FOLR1 (FOLR-2.1) CDx assay can be used to determine FR alpha expression, i.e., cells were stained with antibody FOLR1-2.1 using a Ventana Medical System Discovery Ultra instrument (see the above citation and ClinicalTrials gov identifier: NCT 04296890).

Any suitable anti-fra antibody, e.g., an anti-fra IgG antibody (polyclonal or monoclonal), may be used in such methods. Various anti-FR alpha antibodies suitable for immunohistochemistry are available from commercial sources, for example, thermo filter/Invitrogen (catalog No. PA 5-42004), leica Biosystems (e.g., BN 3.2), enzo Life Sciences (e.g., clone 548908, catalog No. ENZ-ABS 378-0100), abcam (e.g., ab 67422) and Immunogen (353.2.1, FOLR1-2.1). Preferably, the anti-fra antibody used to detect fra is BN3.2, for example as described in the following documents: smith et al, "A novel monoclonal antibody for detection of folate receptor alpha in paraffin-impregnated tissue", hybridoma (Larchmt).2007 Oct;26 (5) 281-8, doi:10.1089/hyb.2007.0512.

"Low FR alpha expressing tumor" generally refers to less than 50% of the tumor cells of a subject expressing FR alpha. Preferably, less than 40%, 30%, 25%, 20% or 10% of the tumor cells of the subject express fra. These expression levels generally refer to membrane froc expression detectable by immunohistochemistry, for example using the methods described above.

As described in the publications cited above, the fra expression can be classified according to the percentage of cells in the tissue sample that exhibit membrane fra expression and fra staining intensity. In some cases, the fra staining intensity can be classified on a scale from 0 (no detectable fra on isotype control) to 3 (high/strong fra staining intensity). Generally, 1 indicates a low/weak fra staining intensity, and 2 indicates a medium fra staining intensity. Classification according to fra staining intensity may also be combined with determination of the percentage of fra positive tumor cells (e.g. from 0 to 100%).

For example, in some cases, less than 50%, 40%, 30%, 25%, 20%, or 10% of the tumor cells of the subject exhibit medium or high (2+) intensity membrane fra staining. For example, in one embodiment, less than 25% of tumor cells exhibit medium or high (i.e., 2+) strength membrane fra staining. Thus, in one embodiment, the fra expression levels can be classified using a "PS2+" scoring method, for example as described in the following documents: martin et al, gynecol Oncol.2017 Nov;147 (2) 402-407; cristea et al, journal of Clinical Oncology 2021 39:15_suppl,5542; the clinicalTrials. Gov identifier: NCT02996825; and 29 th.2019, immunoGen Presents Full Data from Phase 3. 3FORWARD I Study of Mirvetuximab Soravtansine in Ovarian Cancer at ESMO available from https:// inventor. Imunogen.com/news-release-details/imunogen-presentations-full-data-phase-3-forward-i-student or WO 2018/213260).

Preferably, the overall tumor FR alpha expression score of the subject is determined. The overall tumor froc expression score can be defined as the product of the subject's membrane froc staining intensity score (i.e., 0 to 3) and the percentage of froc positive (e.g., at least 1+) tumor cells (0 to 100). This resulted in a highest overall score of 300 points. An overall score of 201 to 300 may be defined as high expression, 101 to 200 as medium expression, 100 or below as low expression. Thus, in some embodiments, the overall tumor froc expression score of the subject 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.

In other embodiments, an "H score" is calculated, for example as described in the following documents: altwerger et al Mol Cancer Ther.2018May;17 (5):1003-1011. The H score is calculated by determining the level of expression of FR alpha (i.e., the intensity level of membrane staining on each cell (0-3, 0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the percentage of cells at each staining intensity (0-100%) in a representative field of view as [1× (% cell 1+) +2× (% cell 2+) +3× (% cell 3+) ]).

In alternative embodiments, the subject's tumor FR alpha expression can be compared to tumor FR alpha expression in a population of cancer subjects. Typically, a population of cancer subjects refers to other subjects having the same type of cancer. Thus, the relative level of tumor fα expression in a subject can be determined as compared to other subjects having the same type of cancer. Since this method does not require absolute quantification of the level of FR alpha expression, but only a relative assay that is compared to other cancer subjects, any systematic deviation due to lack of standardization of expression detection is avoided.

In preferred embodiments, the subject's tumor (e.g., membrane) fα expression is less than at least 50% of cancer subjects. Preferably, the membrane FR alpha expression in tumor cells of a subject is lower than at least 60%, at least 70% or at least 80% of cancer subjects, e.g., subjects suffering from the same form of cancer (e.g., ovarian cancer). More preferably, the subject's tumor membrane expresses less than at least 50%, 60%, 70%, 80%, or at least 90% of the tumors expressing FR (e.g., ovarian tumors expressing FR).

Preferably, the tumor expresses fra, i.e., the tumor exhibits at least some fra expression. Typically, this means that tumor cells of the subject show detectable membrane froc expression, e.g. by immunohistochemistry. More preferably, at least 1% or at least 5% of the tumor cells of the subject exhibit detectable (e.g., membrane) expression of fra, e.g., detection of fra using immunohistochemistry. In other embodiments, at least 10%, 15% or 20% of the tumor cells of the subject exhibit detectable membrane froc expression.

Preferably, the determination of the expression of fra is performed on a recent tumor biopsy sample, i.e., a biopsy sample obtained from the subject shortly before the onset of anti-fra IgE therapy (rather than an old or archived sample). For example, a biopsy sample may be obtained and/or expression of fra 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-fra IgE therapy.

The invention will now be further described by way of example only with reference to the following non-limiting examples.

Examples

Chimeric MOv18 IgE (MOv 18 IgE)

Chimeric MOv18 IgE (MOv 18 IgE) is an IgE class monoclonal antibody (mAb) to the antifolate receptor alpha (fra). The antibodies have been shown to mediate an effective antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) against a tumor cell line expressing FR alpha in vitro and against a tumor xenograft expressing FR alpha in vivo. The target antigen fα is overexpressed in several solid cancer types, including ovarian, endometrial, and mesothelioma. The antigen has been characterized as having potent tumor specificity, and clinical trials using IgG antibodies to target fra, including chimeric MOv18 IgG1 (MOv 18 IgG 1) have demonstrated good tolerability characteristics. MOv18 IgE is believed to have a mechanism of action involved in binding to blood effector cells (including monocytes, basophils and eosinophils). These IgE-bearing cells enter the tissue and upon encountering cells expressing fra, produce an IgE-mediated immune response against the tumor. MOv18 IgG and IgE antibodies and their properties are described, for example, in Coney, L.R., A.Tomassetti et al, (1991), "Cloning of a tumor-associated antigen: MOv18and MOv19 antibodies recognize a folate-binding protein.," Cancer Res 51 (22): 6125-6132; gould, H.J., G.A.Mackay et al, (1999), "Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carpinoma.," Eur J Immunol 29 (11): 3527-3537; karagiannis, S.N., 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): 1030-1040.

Pharmaceutical formulations and administration

MOv18 IgE is a 168kDa protein having the light and heavy chain amino acid sequences shown in FIGS. 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2). The MOv18 IgE drug product was provided as a sterile, pyrogen-free and particle-free solution of pH 6.5 containing 1mg/mL MOv18 IgE in a 2mL vial with a volume of 1.0mL for dilution prior to infusion. Each MOv18 IgE vial also contained an aqueous solution of excipient 0.1M sodium citrate, 30g/L L-arginine, 50g/L sucrose, 0.02% polysorbate 20 for injection.

MOv18 IgE was administered by Intravenous (IV) infusion after dilution with 0.9% (w/v) saline. In addition, subjects received intradermal [ ID ] administration of MOv18 IgE (histamine and saline control) prior to each IV administration to assess risk of allergic reactions. Alternatively, skin prick tests are used to assess risk of allergic reactions. Patients only performed IV administration of MOv18 IgE without skin response to antibodies.

Blood samples were obtained from subjects prior to administration of therapeutic antibodies and basophil activation tests were performed in the presence of MOv18 IgE (FlowBühlmann Laboratories AG,/>Switzerland)。

Study design

MOv18 IgE was tested in a phase I multiple-increment dose escalation trial of open markers of a fra positive solid tumor. 24 patients with advanced solid tumors expressing FR alpha were enrolled in the study. The eligible subjects had sufficient organ function, no history of severe allergy, no concomitant medication or concomitant disease that could increase the risk of an allergic event.

The FR alpha expression was determined for each patient in tumor biopsy samples using the method described for the anti-FR alpha BN3.2 primary antibodies (available from Leica Biosystems), lawson and Scorr (Lawson, N. And P.Scorr (2010), "Evaluation of Antibody to Folate Receptor-alpha (FR-alpha.)," 16 (21): 5288-5295, available from http:// www.leicabiosystems.com/pathologizers/evaluation-of-anti-body-to-fo late-receptor alpha-FR-%CE% B1 /). Methods for determining expression of fra are also described, for example, in the following documents: ab et al, "IMGN853, a Folate Receptor-a (FR alpha) -Targeting Antibody-Drug Conjugate, exhibits Potent Targeted Antitumor Activity against FR alpha-Expressing Tumors", molecular Cancer Therapeutics 14 (7): 1605-13, july2015. Immunohistochemical (IHC) staining of fra was performed on formalin fixed paraffin embedded tissue of tumor biopsy samples on microscope slides. Slides were baked, deparaffinized and treated with hydrogen peroxide to inactivate endogenous peroxidases. Slides were incubated with anti-fra murine monoclonal antibody BN3.2 or isotype control, specific staining visualized and counterstained with hematoxylin. All tissue slides were evaluated and scored by a qualified pathologist. Fra staining intensity was scored on a scale of 0 to 3 (0 = no specific staining (similar to isotype control), 1 = weak, 2 = medium, 3 = strong staining). The percentage of tumor cells exhibiting expression of fra (i.e., at least 1+ staining intensity) was also determined. Subjects in this study showed expression of fα (i.e., 1+, 2+ or 3+ membrane staining) on at least 5% of tumor cells by immunohistochemistry.

Patients received MOv18IgE once a week at IV infusions for a total of six doses, starting with a fixed dose of 70 μg (flat dose). Dose escalation is performed by defined dosage levels (including 70 μg, 250 μg, 500 μg, 700 μg, 1.5mg and 3 mg) up to a 50mg maximum dose. At this stage, three weeks of treatment are considered one round. Patients who exhibited benefit from MOv18IgE in any cohort, were administered up to three further doses of MOv18IgE at the same dose level (unless dose-limiting toxicity was encountered or the patient developed a progressive disease) at weekly intervals. This extra time is considered as a sustain period.

Initially, patients were included in a single patient cohort (cohorts 1 to 4;70 to 700 μg dose of MOv18 IgE) because the planned dose was very low and considered unlikely to elicit a significant biological response. For cohorts 5 to 10 (MOv 18IgE at doses of 1.5 to 50 mg), three patients were included in each cohort, and if toxicity required, three additional patients were added to the cohort. To further investigate the safety and efficacy of MOv18IgE, the cohort was expanded to 6 patients. There was no further dose escalation after cohort 10 (i.e., 50mg was the highest dose evaluated).

Results

In the initial phase of the study, 10 patients received MOv18IgE, two of which were treated at a dose level of 500 μg. At 6 weeks and/or follow-up after study (greater than 8 weeks), anti-drug antibodies were detected in 3/21 of the evaluable patients (2 patients detected ADA, another 1 patient suspected of ADA). One of the patients treated at a dose level of 500 μg developed a grade 3 allergic response experience shortly after receiving MOv18 IgE. The patient responded to standard allergy treatment according to the regimen and was fully recovered.

The pharmacokinetics (serum concentrations) of MOv18IgE following intravenous administration are shown in figure 5 for subjects of a particular dose cohort. Queue 1:70 μg; queue 2:250 μg; queue 3:500 μg; queue 4:700 μg; queue 5:1.5mg.

Figures 6 and 7 show that administration of MOv18IgE antibodies at a unit dose of 700 μg produced an anti-tumor effect. Figure 6 shows tumor measurements obtained from CT scan images, indicating a decrease in tumor size in ovarian cancer subjects treated with MOv18IgE antibodies at a dose level of 700 μg. Figure 7 shows that serum concentration of ovarian cancer antigen CA125 was significantly reduced during treatment of patients with 6 700 μg weekly doses of MOv18IgE antibody followed by a further 3 2 week interval of 700 μg doses of antibody. Reduction of CA125 during Ovarian cancer treatment has been shown to correlate with positive therapeutic outcomes (see, e.g., yang, z., zhao, B. & Li, l.the significance of the change pattern of serum CA, 125 level for judging prognosis and diagnosing recurrences of epithelial Ovarian cancer j Ovarian Res 9,57 (2016)). According to the Gynecological Oncology Group (GOG) criteria, the decrease in CA125 levels shown in FIG. 7 is above the threshold used to define ovarian cancer chemotherapy responses (see, e.g., rustin et al, defining response of ovarian carcinoma to initial chemotherapy according to serum CA125,Journal of Clinical Oncology1996 14:5,1545-1551).

Figures 8 to 10 show that most patients treated with low doses of MOv18 IgE antibodies experienced disease stabilization. Figure 8 shows a graph of the variation in RECIST (solid tumor response assessment criteria) scores from the beginning to the end of treatment (weeks 0-12) for individual ovarian cancer subjects treated with MOv18 IgE antibodies. Figures 9 and 10 show the variation in RECIST scores of individual subjects at 6 weeks and 12 weeks of treatment, respectively. A RECIST score change of less than 20% indicates stable disease. After 6 weeks of treatment, 70% (14/20) of the treated patients had stable disease. In patients treated for 12 weeks, 60% (3/5) of the disease remained stable. Let alone that the dosing frequency was reduced from once per week to once every two weeks during the 6-12 weeks.

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

Figure 11 shows that MOv18 IgE is effective in treating subjects with low levels of FR alpha antigen. Inclusion criteria for this study required detection of at least 5% of tumor cells positive for fra by immunohistochemistry. However, in subjects showing disease stabilization at 6 weeks, 6/14 was a low fra expresser (exhibiting an overall fra expression score of 100 or less). In each of these subjects, 50% or less of the tumor cells were positive for fra.

Of 6 subjects with no change, or a decrease, in RECIST score at 6 weeks, 4 were low-FR-alpha-expressive (overall FR-alpha expression score was 100 or less, and 50% or less of FR-alpha positive tumor cells). Furthermore, both best responders in the study (who showed a decrease in RECIST score) were low-FR alpha-expressing. The overall FR expression score of the subjects showing the best response (as shown in fig. 6 and 7) was very low, 20, and only 10% of the FR positive tumor cells.

These results indicate that MOv18 IgE is well suited for anti-cancer therapy and that administration is tolerable in most patients. Most notably, in subjects with low FR alpha expression, antibodies show anti-tumor activity even at very low doses (e.g. 700 μg). These results support for the first time the safety and efficacy of IgE treatment for low FR alpha expressing tumors, including unit doses of less than 50mg (less than 1 mg/kg/week).

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

Sequence listing

<110> London prince College (King's College London)

<120> composition comprising IgE antibody

<130> P6150GBWO

<150> GB2109550.0

<151> 2021-07-01

<150> PCT/EP2021/060749

<151> 2021-04-23

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> MOv18 IgE light chain (L)

<400> 1

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ile Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Ile Tyr Phe Cys Gln Gln Ser Ser Thr Ile Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 2

<211> 549

<212> PRT

<213> artificial sequence

<220>

<223> MOv18 IgE heavy chain (H)

<400> 2

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Asp Tyr Ile Phe Thr Asn Tyr

20 25 30

Asp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Arg Ser Gly Lys Ser Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ser Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Thr Met Tyr Tyr Tyr Gly Ser Ser Pro Pro Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Gln Ser Pro Ser

115 120 125

Val Phe Pro Leu Thr Arg Cys Cys Lys Asn Ile Pro Ser Asn Ala Thr

130 135 140

Ser Val Thr Leu Gly Cys Leu Ala Thr Gly Tyr Phe Pro Glu Pro Val

145 150 155 160

Met Val Thr Trp Asp Thr Gly Ser Leu Asn Gly Thr Thr Met Thr Leu

165 170 175

Pro Ala Thr Thr Leu Thr Leu Ser Gly His Tyr Ala Thr Ile Ser Leu

180 185 190

Leu Thr Val Ser Gly Ala Trp Ala Lys Gln Met Phe Thr Cys Arg Val

195 200 205

Ala His Thr Pro Ser Ser Thr Asp Trp Val Asp Asn Lys Thr Phe Ser

210 215 220

Val Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser

225 230 235 240

Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys

245 250 255

Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu

260 265 270

Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln

275 280 285

Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys

290 295 300

His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly

305 310 315 320

His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg

325 330 335

Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile

340 345 350

Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp Leu Ala Pro Ser

355 360 365

Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val

370 375 380

Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr

385 390 395 400

Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu

405 410 415

Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met

420 425 430

Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr

435 440 445

Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu

450 455 460

Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp

465 470 475 480

Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln

485 490 495

Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu

500 505 510

Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala

515 520 525

Val His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser

530 535 540

Val Asn Pro Gly Lys

545

<210> 3

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> MOv18 IgE light chain variable Domain

<400> 3

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ile Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Ile Tyr Phe Cys Gln Gln Ser Ser Thr Ile Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 4

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> MOv18 IgE heavy chain variable Domain

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Asp Tyr Ile Phe Thr Asn Tyr

20 25 30

Asp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Arg Ser Gly Lys Ser Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ser Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Thr Met Tyr Tyr Tyr Gly Ser Ser Pro Pro Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 5

<211> 257

<212> PRT

<213> person (Homo sapiens)

<400> 5

Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val

1 5 10 15

Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu

20 25 30

Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly

35 40 45

Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala

50 55 60

Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr

65 70 75 80

Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys

85 90 95

Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn

100 105 110

Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg

115 120 125

Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu

130 135 140

Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp

145 150 155 160

Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln

165 170 175

Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile

180 185 190

Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg

195 200 205

Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu

210 215 220

Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala

225 230 235 240

Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu

245 250 255

Ser

<210> 6

<211> 771

<212> DNA

<213> person (Homo sapiens)

<400> 6

atggctcagc ggatgacaac acagctgctg ctccttctag tgtgggtggc tgtagtaggg 60

gaggctcaga caaggattgc atgggccagg actgagcttc tcaatgtctg catgaacgcc 120

aagcaccaca aggaaaagcc aggccccgag gacaagttgc atgagcagtg tcgaccctgg 180

aggaagaatg cctgctgttc taccaacacc agccaggaag cccataagga tgtttcctac 240

ctatatagat tcaactggaa ccactgtgga gagatggcac ctgcctgcaa acggcatttc 300

atccaggaca cctgcctcta cgagtgctcc cccaacttgg ggccctggat ccagcaggtg 360

gatcagagct ggcgcaaaga gcgggtactg aacgtgcccc tgtgcaaaga ggactgtgag 420

caatggtggg aagattgtcg cacctcctac acctgcaaga gcaactggca caagggctgg 480

aactggactt cagggtttaa caagtgcgca gtgggagctg cctgccaacc tttccatttc 540

tacttcccca cacccactgt tctgtgcaat gaaatctgga ctcactccta caaggtcagc 600

aactacagcc gagggagtgg ccgctgcatc cagatgtggt tcgacccagc ccagggcaac 660

cccaatgagg aggtggcgag gttctatgct gcagccatga gtggggctgg gccctgggca 720

gcctggcctt tcctgcttag cctggcccta atgctgctgt ggctgctcag c 771

<210> 7

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> Fatuzumab VH Domain

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg His Gly Asp Asp Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Pro Val Thr Val Ser Ser

115

<210> 8

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> Fatuzumab VL domain

<400> 8

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Asn

20 25 30

Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp

35 40 45

Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro

85 90 95

Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 9

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> huMOv19 VH Domain

<400> 9

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 10

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> huMOV19 VL domain

<400> 10

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala

20 25 30

Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 11

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> huMOv19 VH-CDR1

<400> 11

Gly Tyr Phe Met Asn

1 5

<210> 12

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> huMOv19 VH-CDR2

<400> 12

Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

Gly

<210> 13

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> huMOv19 VH-CDR3

<400> 13

Tyr Asp Gly Ser Arg Ala Met Asp Tyr

1 5

<210> 14

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> huMOv19 VL-CDR1

<400> 14

Lys Ala Ser Gln Ser Val Ser Phe Ala Gly Thr Ser Leu Met His

1 5 10 15

<210> 15

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> huMOv19 VL-CDR2

<400> 15

Arg Ala Ser Asn Leu Glu Ala

1 5

<210> 16

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> huMOv19 VL-CDR3

<400> 16

Gln Gln Ser Arg Glu Tyr Pro Tyr Thr

1 5

Claims (30)

1. An anti-folate receptor alpha (fra) immunoglobulin E (IgE) antibody for use in treating a low fra expressing tumor in a subject.

2. The IgE antibody of claim 1, wherein less than 50% of tumor cells of the subject express fra; preferably, wherein less than 40%, 30%, 20% or 10% of the tumor cells of the subject express fra.

3. IgE antibody according to claim 1 or claim 2, wherein less than 50%, 30% or 20% of the tumor cells of the subject show detectable membrane fra expression using immunohistochemistry for fra.

4. The IgE antibody of any preceding claim, wherein less than 50%, 30% or 20% of tumor cells of the subject show medium or high (2+) intensity membrane fra staining using immunohistochemistry to detect fra.

5. The IgE antibody of any preceding claim, wherein the subject's tumor cells are classified according to (i) membrane fra staining intensity on a scale of 0 (no detectable fra) to 3 (high fra), and (ii) percentage of fra positive tumor cells (0 to 100%); and wherein the overall tumor FR alpha expression score of the subject is determined as the product of (i) and (ii).

6. The IgE antibody of claim 5, wherein the subject has a total tumor froc expression score of less than 100, preferably less than 50, more preferably less than 30.

7. The IgE antibody of any preceding claim, wherein the subject has a tumor froc expression of less than at least 50% of cancer subjects; preferably, wherein the membrane fra expression in tumor cells of the subject is less than at least 60%, at least 70% or at least 80% of subjects with the same form of cancer; more preferably, wherein the subject's tumor FR alpha expression is lower than at least 50%, at least 70% or at least 90% of the FR alpha expressing tumors (preferably FR alpha expressing ovarian tumors).

8. The IgE antibody of any preceding claim, wherein the tumor expresses fra; preferably, wherein the tumor cells of the subject exhibit detectable membrane fra expression by immunohistochemistry.

9. The IgE antibody of any preceding claim, wherein at least 1% of tumor cells of the subject exhibit detectable membrane fra expression using immunohistochemistry to detect fra; preferably, wherein at least 5%, 10%, 15% or 20% of the tumor cells of the subject exhibit detectable membrane fra expression.

10. The IgE antibody for use according to any preceding claim, wherein the antibody is a MOv18 IgE antibody.

11. The IgE antibody for use according to any preceding claim, for use in treating cancer in the subject and/or delaying the progression of cancer in the subject.

12. The IgE antibody for use according to any preceding claim, wherein the tumor or cancer is ovarian tumor or ovarian cancer.

13. IgE antibody for use according to any preceding claim, wherein said antibody does not have a cytotoxic moiety, preferably wherein said antibody is not an antibody-drug conjugate (ADC).

14. The IgE antibody for use according to any preceding claim, wherein the maximum weekly dose of IgE antibody administered to the subject is 50mg, 25mg, 10mg, 3mg or 1mg.

15. The IgE antibody for use according to claim 14, wherein the IgE antibody is at a weekly dose of 10 μg to 50mg, 70 μg to 30mg, 70 μg to 3mg, 500 μg to 1mg or about 700 μg.

16. The IgE antibody for use according to any preceding claim, wherein the IgE antibody is administered to the subject once weekly or once biweekly.

17. The IgE antibody for use of claim 16, wherein the IgE antibody is administered to the subject for up to 12 weeks.

18. The IgE antibody of claim 17, wherein the IgE antibody is administered to a subject (i) once per week for 6 weeks; followed by (ii) once every two weeks for 6 weeks.

19. The IgE antibody for use according to any preceding claim, wherein the IgE antibody is administered to the subject at a dose of less than 1mg/kg, less than 0.1mg/kg, or less than 0.03mg/kg per administration.

20. The IgE antibody for use according to any preceding claim, wherein 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.

21. A method for treating cancer and/or delaying progression of cancer in a subject having a tumor with low FR alpha expression, the method comprising the step of administering an anti-folate receptor alpha (FR alpha) immunoglobulin E (IgE) antibody as defined in any preceding claim to the subject in a therapeutically effective amount.

22. A pharmaceutical composition comprising an anti-folate receptor alpha (fra) immunoglobulin E (IgE) antibody as defined in any one of claims 1 to 20 and one or more pharmaceutically acceptable excipients, carriers or diluents for use in the treatment of a low fra expressing tumor in a subject.

23. The pharmaceutical composition for use according to claim 22, wherein the composition comprises less than 50mg of the IgE antibody.

24. The pharmaceutical composition for use according to claim 23, comprising less than 30mg, less than 25mg, less than 10mg, less than 5mg, less than 3mg or less than 1mg of the IgE antibody.

25. The pharmaceutical composition for use according to claim 23 or claim 24, comprising 10 μg to 50mg, 70 μg to 30mg, 70 μg to 3mg, 500 μg to 1mg or about 700 μg of the IgE antibody.

26. The pharmaceutical composition for use according to any one of claims 22 to 25, wherein the composition is in liquid form.

27. The pharmaceutical composition for use according to any one of claims 22 to 26, comprising an aqueous solution of the IgE antibody at a concentration of 0.1mg/ml to 10mg/ml, 0.5mg/ml to 2mg/ml or about 1 mg/ml.

28. The pharmaceutical composition for use according to any one of claims 22 to 27, wherein the pharmaceutically acceptable excipient is selected from sodium citrate, L-arginine, sucrose, polysorbate 20 and/or sodium chloride.

29. The pharmaceutical composition for use according to any one of claims 22 to 28, wherein the composition is suitable for intravenous injection.

30. The composition of claim 29, wherein the composition is suitable 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.