WO2022089595A1

WO2022089595A1 - Biomarkers for ige-mediated diseases

Info

Publication number: WO2022089595A1
Application number: PCT/CN2021/127482
Authority: WO
Inventors: Yu-Chen Yang; Nien-Yi Chen; Ming-Liang Kuo
Original assignee: Oneness Biotech Co., Ltd.
Priority date: 2020-10-30
Filing date: 2021-10-29
Publication date: 2022-05-05

Abstract

Provided herein are methods for determining whether a subject will respond to an anti-IgE therapy for treating a disorder associated with immunoglobulin E (IgE) by measuring IgG4 levels in a sample of the subject. A low level of IgG4 antibodies indicates the subject will respond to the therapy.

Description

Biomarkers for IgE-Mediated Diseases

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S. Provisional Application No. 63/107,430, filed October 30, 2020, the entire contents of which are incorporated by reference herein.

Background of the Invention

IgE plays a central role in mediating type I hypersensitivity reactions that are responsible for causing allergic diseases, including allergic asthma, allergic rhinitis, atopic dermatitis, and others. Allergic reactions are the responses of the immune system toward harmless environmental substances, such as dust mites, tree and grass pollens, certain food and drugs, and bee and fire ant bites. In such reactions, the binding of an allergen to IgE on the surface of basophils and mast cells causes the cross-linking of IgE and the aggregation of the underlying receptors of IgE. Fc, the type I IgE. Fc receptors, or FcεRI. This receptor aggregation subsequently activates the signaling pathway leading to the exocytosis of granules and the release of pharmacologic mediators, such as histamine, leukotrienes, tryptase, cytokines and chemokines. The release of those mediators from mast cells and basophils causes the various pathological manifestations of allergy.

There are two types of IgE molecules, free (or soluble) IgE and membrane-bound IgE (mIgE) . Free IgE molecules circulate in the blood and interstitial fluid. mIgE are expressed on the surface of B lymphoblasts and memory B cells. Targeting mIgE is believed to be effective in inhibiting the production of antigen-specific IgE and thus suppressing IgE-medicated immune responses.

Summary of the Invention

The present disclosure is based on the unexpected discovery that atopic dermatitis (AD) patients having a high serum level of IgG4 do not respond to an anti-IgE therapy, while AD patients having a lower serum level of IgG4 showed responsiveness to the anti-IgE therapy. This unexpected discovery indicates that the level of IgG4 could be used as a reliable biomarker for predicting a patient’s responsiveness to an anti-IgE therapy (e.g., a therapy involving an anti-IgE antibody such as FB825) and/or for identifying patients who are likely to respond to the anti-IgE therapy.

Accordingly, one aspect of the present disclosure features a method for assessing responsiveness of a human subject (e.g., an adult patient) to an anti-immunoglobulin E (IgE) therapy, the method comprising: (i) measuring a level of IgG4 in a biological sample of a human subject; and (ii) identifying the human subject as likely to respond to the anti-IgE therapy, if the level of IgG4 in the biological sample from the human subject is not greater than a predetermined reference value (e.g., about 240 mg/dL or about 200 mg/dL) . In some embodiments, the biological sample can be a blood sample, a serum sample, or a plasma sample.

For example, a human subject having a serum IgG4 level ≤ 240 mg/dL may be identified as a responder to an anti-IgE therapy (e.g., a therapy comprising an anti-IgE antibody such as FB825 or a functional variant thereof) . In other examples, a human subject having a serum IgG4 level ≤ 200 mg/dL may be identified as a responder to an anti-IgE therapy (e.g., a therapy comprising an anti-IgE antibody such as FB825 or a functional variant thereof) .

In some embodiments, the human subject has a disorder associated with IgE. In some embodiments, the human subject is suspected of having a disorder associated with IgE. Exemplary disorders associated with IgE include, but are not limited to, allergic asthma, allergic rhinitis, atopic dermatitis, and hyper IgE syndrome. In some examples, the human subject has atopic dermatitis. In other examples, the human patient is suspected of having atopic dermatitis.

Any of the method disclosed herein may further comprise administering to a human an antibody that binds an IgE polypeptide, wherein the subject is identified as likely to respond to the anti-IgE therapy. In some examples, the antibody that binds the IgE polypeptide is FB825 or a functional variant thereof (e.g., those disclosed herein) .

In another aspect, the present disclosure provides a method for treating a disorder associated with immunoglobulin E (IgE) , the method comprising administering to a human subject in need thereof an effective amount of an antibody that binds an IgE polypeptide, wherein the human subject has an IgG4 level not greater than a predetermined reference value. In some instances, the IgG4 level is a blood level of IgG4 of the human subject. In some example, a human subject having a serum IgG4 level ≤ 240 mg/dL may be identified as a responder to an anti-IgE therapy (e.g., a therapy comprising an anti-IgE antibody such as FB825 or a functional variant thereof) . In other examples, a human subject having a serum IgG4 level ≤ 200 mg/dL may be identified as a responder to an anti-IgE therapy (e.g., a therapy comprising an anti-IgE antibody such as FB825 or a functional variant thereof) .

In yet another aspect, provided herein is a method for treating a disorder associated with immunoglobulin E (IgE) , the method comprising: (i) measuring a level of IgG4 in a biological sample of a human subject having or suspected of having a disorder associated with IgE; (ii) comparing the level of IgG4 determined in step (i) with a predetermined reference value; and (iii) administering an effective amount of an antibody specific to an IgE polypeptide to the human subject, whose IgG4 level is not greater than the predetermined reference value.

In any of the methods disclosed herein, the disorder associated with IgE can be allergic asthma, allergic rhinitis, atopic dermatitis, or hyper IgE syndrome. In specific examples, the human subject has or is suspected of having atopic dermatitis.

In any of the methods disclosed herein, the antibody specific to human IgE may bind a CεmX domain on human B lymphocytic cells expressing membrane-bound IgE (mIgE) . In some examples, the antibody binds GLAGGSAQSQRA (SEQ ID NO: 1) . In some embodiments, the antibody binds the same epitope as antibody FB825 or competes against antibody FB825 from binding to the CεmX domain of mIgE. Such an antibody may comprise the same heavy chain complementarity determining regions (CDRs) as antibody FB825 and/or the same light chain CDRs as antibody FB825. For example, the anti-IgE antibody used in any of the methods disclosed herein may comprise the same heavy chain variable region (V _H) and/or the same light chain variable region (V _L) as antibody FB825.

The anti-IgE antibody for use in any of the methods disclosed herein may be a human antibody. Alternatively, it may be a humanized antibody. In some examples, the antibody is a full-length antibody, which optionally is an IgG1 molecule. In one specific example, the antibody is FB825.

In any of the treatment methods disclosed herein, the effective amount of the anti-IgE antibody (e.g., FB825) can be about 5 mg/kg. In some instances, the antibody can be administered by intravenous injection. In some embodiments, the human subject can be administered only one dose of the antibody that binds IgE. In other embodiments, the human subject can be administered multiple doses of the antibody that binds IgE. For example, two consecutive doses of the antibody may be administered to the subject at least 2 weeks apart, e.g., at least three weeks apart, at least four weeks part, or at least two months apart.

In any of the methods disclosed herein, the human subject (e.g., a human adult) may have one or more of the following features:

(i) having an eczema area and severity index (EASI) score ≥ 16;

(ii) having an investigator’s global assessment (IGA) score ≥ 3;

(iii) having ≥10%body surface area (BSA) of atopic dermatitis involvement;

(iv) having inadequate response to topical corticosteroids and/or calcineurin inhibitors;

(v) applying stable doses of emollient for at least 7 days; and

(vi) having normal cardiac conduction parameters.

Alternatively or in addition, the human subject (e.g., a human adult) may be free of one or more of the following:

(i) heart arrhythmias;

(ii) positive for hepatitis B surface antigen, hepatitis C virus antibody, or human immunodeficiency virus antibodies;

(iii) history of anaphylactic reaction or malignancy;

(iv) received any immunoglobulin product or blood product within 3 months prior to the administration of the antibody that binds IgE;

(v) received a biologic product, an immunotherapy, a phototheray, a live vaccine, or a combination thereof;

(vi) one or more laboratory abnormalities;

(vii) received one or more of systemic corticosteroids, leukotriene modifiers, immunosuppressants, immunomodulating drugs, other anti-IgE therapeutics, allergen immunotherapy, and orally inhaled corticosteroids; and

(viii) high risk of parasite infection.

Also within the scope of the present disclosure is an anti-IgE antibody (e.g., those disclosed herein such as FB825) or a pharmaceutical composition comprising such for use in treating a disorder associated with IgE (e.g., those disclosed herein such as AD) in a subject, wherein the subject has a IgG4 level (e.g., a serum IgG4 level) not greater than a predetermined value (e.g., about 240 mg/dL or about 200 mg/dL) . In addition, provided herein is the use of the anti-IgE antibody or the pharmaceutical composition comprising such for manufacturing a medicament for use in treating an IgE-associated disorder (e.g., AD) in a human patient having an IgG4 level not greater than the predetermined value.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also form the appended claims.

Brief Description of the Drawings

FIGS. 1A-1C are graphs showing that the level of IgG4 correlates with AD patients’ responsiveness to a treatment involving an anti-IgE antibody FB825. FIG. 1A shows correlation between reduction of EASI disease score and reduction of IgE+ memory B cells in patients treated with anti-IgE antibody FB825. FIG. 1B shows that the level of IgG4 can be used as a biomarker for prediction of FB825 responders. Atopic dermatitis patients received a single dose of FB825 showed significant improvement in EASI score evaluated during the treatment, which was found to correlate with reduction of IgE+ memory B cells and plasmablasts. FIG. 1C shows EASI scores in patients treated by FB825 on Day 1 and Day 29 and categorized by the level of the IgG4 biomarker. The proposed biomarker could break down two groups: poor responders and responders. After receiving a single dose of FB825, the responder group showed significant improvement in Day 29. To compare these two groups is showed p value=0.002 via unpair t-test, two tailed analysis.

FIGs. 2A-2B include graphs showing the correlation between serum IgG4 levels and patient responsiveness to FB825 treatment. FIG. 2A is a graph showing that IgG4 can serve as a biomarker for predicting responders to an anti-IgE therapy. Patients with lower biomarker level have better responses in EASI reduction after dosed of FB825. FIG. 2B is a graph showing an exemplary cutoff threshold of serum IgG4 levels (200 mg/dL) for identifying responders to anti-IgE therapy. Target population: N=14 with IgG4 < 200 mg/dL; accuracy 13/14 (92.9%) .

FIGs. 3A-3B include graphs showing correlation between EASI disease scores in patients treated by FB825 and IgG4 levels. FIG. 3A: correlation between IgG4 levels and patients EASI scores (using 16 as the cutoff value) . FIG. 3B: correlation between IgG4 levels and patients EASI scores on Day 29.

Detailed Description of the Invention

Provided herein are methods and compositions for diagnosing and treating IgE-associated disorders, including allergic diseases, by measuring levels of IgG4 in a subject, for example, serum levels of IgG4 in the subject. A low level of IgG4 (e.g., ≤240 mg/dL or ≤200 mg/dL) in the patient indicates the patient is likely to be a good responder to the treatment. In embodiments, the treatment comprises an antibody that binds IgE.

IgE-associated diseases, which include atopic dermatitis, allergic asthma/rhinitis, food allergies, and other type I hypersensitivity inflammatory disorders are among the fastest growing chronic diseases in the industrialized countries, and afflict 20%-40%of the population worldwide. Although some of these allergic disorders can be treated by medicines that directly easing the symptoms, many moderate-to-severe diseases remain poorly controlled, which not only exerts a huge impact on the life quality, but also high economic cost to the individual, family, and society. It is recognized that Immunoglobulin E (IgE) plays an important role in mediating hypersensitivity reactions responsible for most of the allergic diseases. IgE is produced by IgE-secreting plasma cells that are generated from continuous differentiation of B lymphocytes expressing membrane-bound IgE (mIgE) on the cell surface (Murphy 2011) . The pharmaceutical development of anti-IgE omalizumab has validated the IgE pathway as an effective therapeutic target for treating IgE-mediated allergic diseases (Chang 2000) . Omalizumab efficiently neutralizes free IgE, which down-regulates FcεRI on basophils, dendritic cells, and mast cells, thus successfully modulating allergic asthma.

In “atopic” individuals who are at increased risk of developing allergies, IgE concentration in the circulatory system may reach over 10 times the normal level. The concentrations of allergen-specific IgE antibody are closely correlated with clinical symptoms and may be over 1000 times higher in patients with allergic diseases than in those found in healthy individuals. IgE sensitizes effector cells such as basophils, mast cells, and activated eosinophils by occupying the high-affinity IgE receptor, FcεRI, on which they are expressed. In type I hypersensitivity, allergens cross-link IgE molecules bound by FcεRI and subsequently triggers the degranulation of effector cells, releasing pro-inflammatory mediators, such as histamines and leukotrienes. The IgE-mediated allergic pathway, which generates mediator-related allergic symptoms, initiates the immune activities locally or systemically. Basophils and mast cells also release a wide spectrum of inflammatory cytokines and chemokines that not only directly cause clinical symptoms but also activate and recruit various cell types to augment the inflammatory status. Hence, anti-IgE therapy can attenuate both the IgE-mediated pathway and the inflammatory conditions. The neutralization of IgE by humanized IgG antibodies has been studied in many clinical trials of allergic disorders (Vichyanond 2011) ; one of the anti-IgE IgG antibodies, omalizumab, (

Genentech USA, Inc and Novartis Pharmaceuticals Corporation) has been approved for the treatment of allergic asthma and chronic idiopathic urticaria. The success of anti-IgE therapy has confirmed the role of IgE in the pathogenesis of asthma and gradually establishes the concept “allergic asthma” .

IgE is biologically expressed in secretory form or membrane-bound form. Membrane-bound IgE forms the core unit of B-cell antigen receptors (BCRs) which determine antigen specificity and are essential for survival and functions of B cells. As a general rule in B-cell activation, besides crosslinking BCRs by specific antigens as the first signal, a second signal of co-stimulatory cytokines and receptor-ligand connection from cognate helper T cells is required. Without the appropriate co-stimulatory signal, a B cell who has its antigen receptors cross-linked will step on processes of energy, a state that is no longer responsive to antigens, or apoptosis.

I. IgG4 as a Biomarker for Predicting Responsiveness to Anti-IgE Therapy

One aspect of the present disclosure features methods for predicting a patient’s responsiveness to an anti-IgE therapy (e.g., a treatment involving an anti-IgE antibody such as FB825) and/or identifying suitable patients for such treatment based on the IgG4 level in that patient, whose may be a human patient having or suspected of having a disease associated with IgE, for example, atopic dermatitis (AD) . In some examples, a biological sample (e.g., a body fluid sample such as a blood sample, a serum sample, or a plasma sample) can be collected from a patient and the IgG4 level in the biological sample can be measured by a routine approach or a method disclosed herein. The IgG4 level of the patient may then be compared with a predetermined value. If the IgG4 level of the patient is higher than the predetermined value, that patient is likely a non-responder to the anti-IgE therapy. On the other hand, if the IgG4 level of the patient is no greater than the predetermined value, the patient is identified as a responder to the anti-IgE therapy. Such a patient may be subject to an anti-IgE therapy as disclosed herein. If desired, IgE levels can be measured along with IgG4 levels.

As used herein, “biomarker” refers to a distinctive biological or biologically derived indicator of a process, event or conditions. In certain embodiments, the biomarker is a gene or gene product (i.e., a polypeptide) .

IgG4 in bodily fluids such as serum can be detected using methods known in the art, e.g., quantitative assays discussed in WO2019/089978, including an enzyme-linked immunosorbent assay (ELISA) ; an alkaline phosphatase immunoassay auto-analyzer, such as an

system (Siemens Healthcare Diagnostics, Erlangen, Germany) ; a radioallergosorbent test (RAST) , or a fluoroenzyme immunoassay auto-analyzer, such as the

system (Thermo Fisher Scientific/Phadia, Uppsala, Sweden) . Additional suitable methods include a fluorescence enzyme immunoassay (FEIA) auto-analyzer (e.g.,

system) . Another technique may be used as the level of antibody (e.g., IgE or IgG4) determined by that technique may be normalized to a measurement by a fluorescence enzyme immunoassay auto-analyzer. That is, a level of antibody (e.g., IgE or IgG4) can be determined by a technique, and can correspond to a level as measured by a fluorescence enzyme immunoassay auto-analyzer. The level of the biomarker is preferably determined in vitro.

The IgG4 level obtained from a patient candidate may be compared with a predetermined value (cutoff value) , which represents the IgG4 level that distinguishes patients responsive to an anti-IgE therapy relative to those that have poor responsiveness to the therapy. Such a predetermined value may be set forth based on analysis of representative IgG4 levels in anti-IgE therapy responders versus anti-IgE therapy non-responders. The predetermined value may take into consideration matched physiological features as the subject, for example, age, gender, ethnic background, etc. Thus, in some instances, the predetermined value may be determined by representative IgG4 levels in anti-IgE therapy responders versus anti-IgE therapy non-responders in a specific patient subpopulation, for example, ethnicity, gender, age, disease severity, etc. The predetermined value may vary among different patient subpopulations. The cutoff value in association with a particular patient subpopulation could be determined following guidance provided herein (see, e.g., Example 1 below) and routine practice.

In some instances, the predetermined value can be a serum IgG4 level of about 200 mg/dL to about 240 mg/dL. In some examples, a human subject having a serum IgG4 level ≤ 240 mg/dL is identified as a responder for an anti-IgE therapy such as those disclosed herein (e.g., comprising FB825) . In some examples, a human subject having a serum IgG4 level ≤ 230 mg/dL is identified as a responder for an anti-IgE therapy such as those disclosed herein (e.g., comprising FB825) . In some examples, a human subject having a serum IgG4 level ≤ 220 mg/dL is identified as a responder for an anti-IgE therapy such as those disclosed herein (e.g., comprising FB825) . In some examples, a human subject having a serum IgG4 level ≤ 210 mg/dL is identified as a responder for an anti-IgE therapy such as those disclosed herein (e.g., comprising FB825) . In yet other examples, a human subject having a serum IgG4 level ≤ 200 mg/dL is identified as a responder for an anti-IgE therapy such as those disclosed herein (e.g., comprising FB825) . Using a lower predetermined value in any of the methods disclosed herein may confer higher accuracy in assessing a patient’s responsiveness to the anti-IgE therapy such as those disclosed herein (e.g., comprising an anti-IgE antibody such as FB825) .

The results regarding responsiveness to the anti-IgE therapy (e.g., an anti-IgE antibody therapy such as one involving FB825 or a functional variant such as those disclosed herein) can be relied on to determine treatment approaches to the patients. For example, if the patient is responsive to the anti-IgE therapy, such a therapy can be performed on the patient or continued if the patient is already on the therapy. Thus, any of the methods for assessing anti-IgE therapy responsiveness may further comprise subjecting the patient to an anti-IgE therapy or continuing the anti-IgE therapy, for example, administering an effective amount of an anti-IgE antibody (e.g., those disclosed herein such as FB825) to the patient.

On the other hand, if the patient is not responsive to the anti-IgE therapy, then the methods may further comprise subjecting the patient to a therapy for a disease associated with IgE, which does not involve an anti-IgE therapeutic.

II. Anti-IgE Therapy for Disorders Associated with IgE

In some aspects, provided herein are methods for treating a disorder associated with IgE (e.g., those disclosed herein such as AD) comprising administering to a subject in need of the treatment (e.g., a human patient having or suspected of having the disorder such as AD) an effective amount of an agent that targets IgE (anti-IgE therapeutic) , for example, an antibody specific to IgE such as human IgE. The subject to be treated by any of the methods disclosed herein may have a low IgG4 level (e.g., a serum IgG4 level not greater than a predetermined value, for example, ≤ 240 mg/dL, ≤ 230 mg/dL, ≤ 220 mg/dL, ≤ 210 mg/dL, or ≤ 200 mg/dL) . The subject may be a human patient identified by any of the methods disclosed herein as a responder to an anti-IgE therapy.

(a) Anti-IgE Antibodies

In some embodiments, the anti-IgE therapeutic may be an anti-IgE antibody. As used herein, the term “antibody” encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F (ab') ₂, Fv) , single chain (scFv) , mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof) , and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes) , e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The antibodies to be used in the methods described herein can be murine, rat, human, or any other origin (including chimeric or humanized antibodies) .

Any of the antibodies described herein can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogenous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g. murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc) , typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and/or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat) , while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

The antibodies described herein can bind to the CεmX domain of a mIgE, for example, mIgE expressed on the surface of B cells. Such antibodies may induce cell death of the B cells expressing mIgE via, for example, antibody-dependent cell cytotoxicity and/or cell apoptosis, thereby eliminate the B cells, which would lead to reduced production of free IgE. Accordingly, the anti-CεmX antibodies described herein can reduce the level of total IgE in a subject (e.g., a human patient) being treated with the antibody.

In some embodiments, the antibody disclosed herein specifically binds a Cεmx domain of a membrane-bound IgE, which may be expressed on the surface of a B cell. CεmX is a 52-amino acid segment located between the CH4 domain and the C-terminal membrane-anchoring segment of human membrane-bound ε chain (mε) . The amino acid sequence of an exemplary CεmX fragment of human mIgE is provided below:

An antibody that “specifically binds” (used interchangeably herein) to a target or an epitope is a term well understood in the art, and methods to determine such specific binding are also well known in the art. A molecule is said to exhibit “specific binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody “specifically binds” to a target antigen if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to a CεmX domain epitope is an antibody that binds this CεmX domain epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other CεmX domain epitopes or non-CεmX domain epitopes. It is also understood by reading this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

The binding affinity of an anti-CεmX antibody described herein can be less than about 100 nM, e.g., less than about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM to any of about 2 pM. Binding affinity can be expressed K _D or dissociation constant, and an increased binding affinity corresponds to a decreased K _D. One way of determining binding affinity of antibodies to CεmX is by measuring binding affinity of monofunctional Fab fragments of the antibody. To obtain monofunctional Fab fragments, an antibody (for example, IgG) can be cleaved with papain or expressed recombinantly. The affinity of an anti-CεmX Fab fragment of an antibody can be determined by surface plasmon resonance (BIAcore3000TM surface plasmon resonance (SPR) system, BIAcore, INC, Piscaway N.J. ) . Kinetic association rates (k _on) and dissociation rates (k _off) (generally measured at 25 ℃. ) are obtained; and equilibrium dissociation constant (K _D) values are calculated as koff/kon.

In some embodiments, the antibody binds the CεmX domain of a human IgE, and does not significantly bind an IgE from another mammalian species. In some embodiments, the antibody binds human IgE as well as one or more IgE from another mammalian species. The epitope (s) bound by the antibody can be continuous or discontinuous.

In some embodiments, the anti-CεmX antibody described herein binds an N-terminal portion of the CεmX domain, e.g., GLAGGSAQSQRAPDRVL (SEQ ID NO: 3) or GLAGGSAQSQRA (SEQ ID NO: 1) . Such an antibody may have the same heavy chain and/or light chain CDRs as antibody 4B12/FB825 as described in Figure 4 of US20200297815, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. See also U.S. Patent No. 8,460,664, the relevant disclosures therein are incorporated by reference herein. The anti-CεmX antibody may be a humanized antibody. In some examples, the anti-CεmX antibody for use in the methods described herein is FB825, which is a humanized antibody of 4B12 (Figure 4) , or a functional variant thereof. See also U.S. Patent No. 8,460,664, the relevant disclosures therein are incorporated by reference herein.

Two antibodies having the same V _H and/or V _L CDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach as known in the art. See, e.g., bioinf. org. uk/abs/) .

A functional variant (equivalent) of FB825 has essentially the same epitope-binding specificity as FB825 and exhibits substantially similar bioactivity as FB825, including the activity of eliminating B cells expressing mIgE and reducing the level of total IgE in a subject. In some embodiments, a functional variant of FB825 contains the same regions/residues responsible for antigen-binding as FB825, such as the same specificity-determining residues in the CDRs or the whole CDRs. In other embodiments, a functional variant of FB825 comprises a V _H chain that includes a V _H CDR1, V _H CDR2, and V _H CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the corresponding V _H CDRs of FB825, and a V _L chain that includes a V _L CDR1, V _L CDR2, and V _L CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the corresponding V _H CDRs of FB825. For example, a functional variant of FB825 may comprise a V _H chain that includes up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid residue variations in the V _H CDR regions (V _H CDR1, CDR2, and/or CDR3 in total) as compared to the V _H CDRs of mAb7E, and/or a V _L chain that includes up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid residue variations in the V _L CDR regions (V _L CDR1, CDR2, and/or CDR3 in total) as compared to the V _H CDRs of mAb7E.

Alternatively, the functional variant of FB825 comprises a V _H chain at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V _H chain of FB825 and a V _L chain at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V _L chain of FB825. The amino acid sequence variations may occur only in one or more of the V _H and/or V _L framework regions.

The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87: 2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90: 5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215: 403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25 (17) : 3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, the anti-CεmX antibody for use in the treatment method disclosed herein may have one of the following heavy chain variable regions (CDRs following the Kabat definition are in boldface and underlined) :

The heavy chain CDR1, CDR2, and CDR3 identified in the above heavy chain variable regions are represented by SEQ ID NO: 5, 6, and 7, respectively.

Alternatively or in addition, the anti-CεmX antibody for use in the treatment method disclosed herein may have one of the following light chain variable regions (CDRs following the Kabat definition are in boldface and underlined) :

The light chain CDR1, CDR2, and CDR3 identified in the above light chain variable regions are represented by SEQ ID NO: 11, 12, and 13, respectively.

In some embodiments, the methods use the antibody FB825, which is a humanized monoclonal immunoglobulin G1 (IgG1) targeting the CεmX domain on human B lymphocytic cells expressing membrane-bound IgE (mIgE) . FB825 is disclosed in US20200297815. FB825 can bind to mIgE-expressing human B cells and monoclonal immunoglobulin G1 specifically targeting the CεmX domain on human B lymphocytic cells expressing mIgE. The CεmX domain of 52 amino acid residues, located between the CH4 domain and membrane-anchor segment of human membrane-bound ε chain on mIgE ⁺ B cells, is a unique antigenic site for the immunological targeting of B cells. Monoclonal antibodies specific for CεmX can potentially affect mIgE-expressing B lymphocytes and memory B cells, down-regulate IgE synthesis, and treat IgE-mediated allergic diseases (Chang 2006; Chang et al 2007) . FB825 binds specifically to the CεmX domain of mIgE and does not interact with free IgE; therefore, FB825 is not neutralized by IgE in the circulation and can bind to mIgE-expressing human B cells and induce subsequent apoptosis and/or antibody-dependent cellular cytotoxicity (ADCC) . FB825 also reduces the number of IgE-producing plasma B cells in a culture of human peripheral blood mononuclear cells (PBMCs) (Li et al 2013) . Furthermore, FB825 suppresses the anti-CD40-and interleukin-4-stimulated production of IgE from human PBMCs in a xenograft mouse model (Chou et al 2013) . Based on the aforementioned findings, FB825 is postulated to be capable of blocking IgE production driven by allergens and alleviating airway hypersensitive symptoms like allergic asthma and rhinitis. Accumulating data suggests that decreasing serum IgE levels probably helps alleviate atopic skin conditions like atopic dermatitis and chronic urticaria. If the pharmacological efficacy of FB825 can be proven in human clinical studies, its applications can be expanded to other IgE-mediated disorders.

The heavy chain (top) and light chain (bottom) amino acid sequence of FB825 in full-length format is provided below (including N-terminal signal peptide sequences) .

(ii) Antibody Preparation

Antibodies capable of binding a CεmX domain of a membrane-bound IgE as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

In some embodiments, antibodies specific to a target antigen (e.g., a CεmX domain of a mIgE such as a human mIgE) can be made by the conventional hybridoma technology. The full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen. The route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256: 495-497 or as modified by Buck, D.W., et al., In Vitro, 18: 377-381 (1982) . Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells may be used to produce the anti-CεmX monoclonal antibodies of the subject invention. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay) .

Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies capable of binding the CεmX domain. Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues) , N-hydroxysuccinimide (through lysine residues) , glutaraldehyde, succinic anhydride, SOCl, or R1N=C=NR, where R and R1 are different alkyl groups, can yield a population of antibodies (e.g., monoclonal antibodies) .

If desired, an antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. In an alternative, the polynucleotide sequence may be used for genetic manipulation to “humanize” the antibody or to improve the affinity (affinity maturation) , or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity to the target antigen and greater efficacy in reducing total IgE. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XenomouseRTM from Amgen, Inc. (Fremont, Calif. ) and HuMAb-MouseRTM and TC MouseTM from Medarex, Inc. (Princeton, N.J. ) . In another alternative, antibodies may be made recombinantly by phage display technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12: 433-455. Alternatively, the phage display technology (McCafferty et al., (1990) Nature 348: 552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody) can be prepared via routine methods. For example, F (ab') 2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F (ab') 2 fragments.

Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant technology. In one example, DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies) . The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., PCT Publication No. WO 87/04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81: 6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, genetically engineered antibodies, such as “chimeric” or “hybrid” antibodies; can be prepared that have the binding specificity of a target antigen.

Techniques developed for the production of “chimeric antibodies” are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314: 452.

Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989) . In one example, variable regions of V _H and V _L of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human V _H and V _L chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent V _H and V _L sequences as search queries. Human V _H and V _L acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage scFv library and scFv clones specific to IgE can be identified from the library following routine procedures.

Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping. ” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence) . Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) . Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of the IgE polypeptide have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein (such as another member of the immunoglobulin protein family) . By assessing binding of the antibody to the mutant immunoglobulin, the importance of the particular antigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.

(iii) Pharmaceutical Compositions

One or more of the above-described anti-CεmX antibodies can be mixed with a pharmaceutically acceptable carrier (excipient) , including buffer, to form a pharmaceutical composition for use in treating a disorder associated with IgE. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover. In one example, a pharmaceutical composition contains more than one anti-CεmX antibodies that recognize different epitopes of the target antigen.

The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes) ; and/or non-ionic surfactants such as TWEEN ^TM, PLURONICS ^TM or polyethylene glycol (PEG) . Pharmaceutically acceptable excipients are further described herein.

In some examples, the pharmaceutical composition described herein comprises liposomes containing the anti-CεmX antibody, which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985) ; Hwang, et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980) ; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE) . Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

The anti-CεmX antibody may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000) .

In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) , or poly (v nylalcohol) ) , polylactides (U.S. Pat. No. 3,773,919) , copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ^TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) , sucrose acetate isobutyrate, and poly-D- (-) -3-hydroxybutyric acid.

In some embodiments, the pharmaceutical composition comprising the anti-CεmX antibody described herein, e.g., FB825 or a functional variant thereof as also described herein, may be an aqueous formulation, which may further comprise a buffer (which may comprise an amino acid such as histidine or arginine) , a salt (e.g., sodium chloride) , and/or a surfactant, such as a nonionic surfactant. For example, the aqueous formulation may comprise the antibody at a concentration of about 10-30 mg/ml, a buffer comprising an amino acid (e.g., histidine or arginine) at a concentration of about 10-30 mM, a surfactant such as polysorbate 80 at a concentration of about 0.01-0.03%, and/or a sodium chloride at a concentration of about 120-160 mM. Such an aqueous formulation may have a pH of about 5-8. In one particular example, the aqueous formulation may comprise antibody FB825 at a concentration of about 20 mg/ml, L-histidine at a concentration of about 20 mM, sodium chloride at a concentration of about 140 mM, polysorbate 80 at a concentration of about 0.02%, and a pH of about 6.5.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1%of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g.,

Tween

^TM 20, 40, 60, 80 or 85) and other sorbitans (e.g.,

Span

^TM 20, 40, 60, 80 or 85) . Compositions with a surface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid ^TM, Liposyn ^TM, Infonutrol ^TM, Lipofundin ^TM and Lipiphysan ^TM. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20%oil, for example, between 5 and 20%.

The emulsion compositions can be those prepared by mixing an anti-CεmX antibody with Intralipid ^TM or the components thereof (soybean oil, egg phospholipids, glycerol and water) .

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

(iv) Use of Anti-IgE Therapeutics for Treating Disorders Associated with IgE

To practice the method disclosed herein, an effective amount of the pharmaceutical composition described above can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, anti-CemX antibodies can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a disorder associated with IgE (e.g., allergic asthma, as well as other disorders known in the art and/or disclosed herein) . A subject having an IgE-associated disorder such as allergic asthma can be identified by routine medical examination, e.g., laboratory tests. A subject suspected of having the IgE-associated disorder might show one or more symptoms of the disorder, e.g., elevated levels of IgE and/or hyper-reactivity to an allergen and/or antigen. A subject at risk for the disorder can be a subject having one or more of the risk factors for that disorder.

Exemplary IgE-associated disorders include, but are not limited to, asthma, allergic rhinitis, hyper IgE syndrome, atopic dermatitis, cold-induced urticaria, chronic urticaria, cholinergic urticaria, chronic rhinosinusitis, systemic mastocytosis, cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, recurrent idiopathic angioedema, and interstitial cystitis, eosinophil-associated gastrointestinal disorders, a food allergy, or a drug allergy.

“An effective amount” as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any) , the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a disorder associated with IgE. Alternatively, sustained continuous release formulations of an anti-CεmX antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one example, dosages for an anti-CεmX antibody as described herein may be determined empirically in individuals who have been given one or more administration (s) of an anti-CεmX antibody. Individuals are given incremental dosages of the anti-CεmX antibody. To assess efficacy of the anti-CεmX antibody, an indicator of a disorder associated with IgE (such as levels of IgE) can be followed.

For the purpose of the present disclosure, the appropriate dosage of an anti-CεmX antibody will depend on the specific anti-CεmX antibody (s) (or compositions thereof) employed, the type and severity of disorder associated with IgE, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. Typically the clinician will administer an anti-CεmX antibody, such as FB825, until a dosage is reached that achieves the desired result. Administration of an anti-CεmX antibody can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.

In some embodiments, the anti-CεmX antibody (e.g., FB825) described herein is administered to a subject in need of the treatment at an amount sufficient to reduce the level of the total IgE level by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%or greater) .

As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a disease associated with IgE, a symptom of a disease associated with IgE, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease.

Alleviating a disease associated with IgE includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease (such as a disease associated with IgE) means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a disease associated with IgE includes initial onset and/or recurrence.

To perform the methods as described herein, any of the anti-Cεmx antibodies such as FB825 may be given to a subject in need of the treatment (e.g., a human patient) by a single dose or by multiple doses via a suitable route, for example, intravenous infusion or subcutaneous injection. The dosage of the anti-Cεmx antibody for each administration may range from about 0.5 mg/kg to about 25 mg/kg (e.g., about 1 mg/kg to about 20 mg/kg, about 5 mg/kg to about 15 mg/kg, or about 10 mg/kg to about 20 mg/kg) , depending upon various factors, including those described herein. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a disorder associated with IgE, or a symptom thereof.

The administration of an anti-CεmX antibody (e.g., FB825) may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a disorder associated with IgE. An exemplary dosing regimen comprises administering to a subject in need of the treatment a first dose of an anti-Cemx antibody (e.g., at 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, or 25 mg/kg) , followed by a second dose of the antibody at least 3 months after the first dose (e.g., 4 months, 5 months, or 6 months) . The dosage of the second administration may be higher, the same, or lower than the first administration. Other dosage regimens may be useful depending upon the pattern of pharmacokinetic decay that a practitioner wishes to achieve.

In some embodiments, a subject in need of the treatment can be given a first dose of the antibody at a suitable amount (e.g., at 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, or 25 mg/kg) . The subject is then monitored periodically for symptoms indicative of an IgE-associated disorder, for example, allergic reactions and/or an elevated level of total IgE. A second dose of the antibody may be given to the subject when such a symptom is observed.

Also within the scope of the present disclosure are preventive treatments of an IgE-associated disorder with any of the anti-Cεmx antibodies to reduce the risk for occurrence of such a disorder. Subjects suitable for such a preventive treatment may be human patients having history of an IgE-associated disorder and/or family history of an IgE-associated disorder.

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like) . For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9%saline, Ringer’s solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9%saline, or 5%glucose solution.

In one embodiment, an anti-CεmX antibody is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the anti-CεmX antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisense polynucleotide, expression vector, or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11: 202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed. ) (1994) ; Wu et al., J. Biol. Chem. (1988) 263: 621; Wu et al., J. Biol. Chem. (1994) 269: 542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87: 3655; Wu et al., J. Biol. Chem. (1991) 266: 338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or more can also be used during a gene therapy protocol.

The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1: 51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) 1: 185; and Kaplitt, Nature Genetics (1994) 6: 148) . Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242) , alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247) , Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532) ) , and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655) . Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3: 147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3: 147) ; ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264: 16985) ; eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14: 2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91: 1581.

It is also apparent that an expression vector can be used to direct expression of any of the protein-based anti-CεmX antibodies described herein (e.g., FB825) . For example, other anti-CεmX antibody fragments that are capable of binding CεmX and/or an IgE biological activity are known in the art.

The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject’s medical history. Any of the anti-Cmex antibodies described herein may be used in conjunction with other agents (e.g., other agents for treating IgE-associated disorders) that serve to enhance and/or complement the effectiveness of the agents.

In some embodiments, an anti-CεmX antibody as described herein, for example, FB825, is used for treating atopic dermatitis as follows. Atopic dermatitis, also known as eczema, is a chronical skin condition characterized by redness and/or itchy. It is common in children but can occur at any age. A patient who needs the treatment can be identified by routine medical practice as having one or more symptoms of atopic dermatitis, including dry skin, itching, red to brownish-gray patches, small, raised bumps, which may leak fluid and crust over when scratched, thickened, cracked , scaly skin, and/or raw, sensitive, swollen skin from scratching. In some instances, the total IgE level and the level of allergen-specific IgE of a candidate subject can be examined via routine practice. If the IgE level of the candidate subject (e.g., the total IgE, the allergen-specific IgE, or both) is higher than a normal level (representing the average IgE level in subjects of the same species, e.g., humans, who are free of atopic dermatitis or other allergic disorders associated with IgE) .

A human patient who needs the treatment may be given a first a dose of the antibody, which may range from 3 mg/kg to 8 mg/kg, via a conventional route as described herein. In some instances, the first dose is 5 mg/kg. After the first dose, the total IgE level of the patient can be monitored. If the reduction of the IgE level 3-4 weeks after the first dose is less than 50%, a second dose of the antibody may be given to the patient 3-4 weeks after the first dose. The second dose may be identical to the first dose, or lower than the first dose. In some instances, both the first dose and the second dose are 5 mg/kg and are administered via IV infusion in a 1-2 hour period. Other biomarkers indicating efficacy and/or safety could also be monitored during the course of the treatment. Such biomarkers include, but are not limited to, thymus and activation regulated chemokine (TARC) , Eotaxin-3, thymic stromal lymphopoietin (TSLP) , periostin, IL-1a, IL-4, IL-5, IL-13, IL-16, IL-31, M-CSF, or a combination thereof.

The human patient subject to the above-noted treatment may have chronic atopic dermatitis for at least 3 years as diagnosed by routine medical practice, for example, defined by the Eishenfield revised criteria of Hannifin and Rajka and supported by positive allergen-specific IgE. The patient may have one or more of the following features: (i) eczema area and severity index (EASI) score greater than 16, (ii) Investigator’s Global Assessment (IGA) score greater than 3 (5-point scale) , (iii) greater than 10%body surface area (BSA) , (iv) history of inadequate response to a stable regimen of topical corticosteroids or calcineurin inhibitors for at least one month or at least three months before the treatment. Further, the human patient may be given stable doses of emollient twice daily for at least 7 days before the treatment. See also Example 1 below for characteristic features of patients suitable for the treatment.

In some instances, the anti-CεmX antibody as described herein (e.g., FB825) may be co-used with moisturizers (e.g., at stable doses such as at least twice daily) and/or topical corticosteroid (TCS) . A medium potency TCS may be applied to areas with active lesions and may switch to low potency TCS after the lesions are under control. If lesions reoccur, treatment with a medium potency TCS may resume with a step-down approach. If lesions are persisting or worsening after daily treatment with a medium potency TCS, a high or super-high potency TCS may be used, unless it is deemed unsafe. A low potency TCS may be used on areas of thin skin (e.g., face, neck, intertriginous, genital areas, or areas of skin atrophy) or on areas where continued use of medium potency TCS is considered unsafe.

TCS having low, medium, and high or super-high potency is well known in the art. Exemplary medium potency TCS includes 0.05%fluticasone propionate cream, 0.1s%mometasone furoate cream, or 0.06%betamethasone valerate cream. Exemplary low potency TCS includes 1%hydrocortisone ointment. Exemplary high potency TCS can be 0.05%fluocinonide cream or 0.25%desomimetasone ointment. Exemplary super-high potency TCS can be 0.05%clobetasol propionate ointment.

In some instances, the patient subject to the treatment described herein is free of one or more of the following therapy: (i) topical tacrolimus and pimecrolimus, (ii) systemic treatment of corticosteroids, (iii) leukotriene inhibitors, (iv) allergen immunotherapy, (v) systemic treatment of immunosuppressors or immunomodulators (e.g., cyclosporine, mycophenolate-mofetil, IFN-γ, azathioprine, methotrexate, or biologics) , (vi) live (e.g., attenuated) vaccines, and/or (vii) traditional Chinese medicine. The patient may also be free of any surgical procedures and/or UV procedures.

Any of the methods described herein may further comprise assessing occurrence of decreased hemoglobin, upper respiratory tract infection, urinary tract infection, or a combination thereof in the subject after the first dose. If one or more occurrences are observed, the amount of the anti-Cemx antibody (e.g., FB825) of the second dose may be reduced. Alternatively, the treatment may be stopped.

III. Kits for Use in Treating IgE-Associated Disorders

The present disclosure also provides kits for use in treating IgE-associated disorders and/or that include reagents for detecting IgG4 levels in a patient. Such kits can include one or more containers comprising an anti-Cεmx antibody as described herein (such as FB825) and anti-IgG4 antibodies or other reagents for detecting IgG4.

In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the anti-Cεmx antibody to treat, delay the onset, or alleviate an IgE-associated disorder according to any of the methods described herein. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has, is suspected of having, or is at risk for the disorder. In still other embodiments, the instructions comprise a description of administering anti-Cεmx antibody to a subject in need of the treatment to reduce the risk for developing the IgE-associated disorder.

The instructions relating to the use of an anti-Cεmx antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit) , but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating an IgE-associated disorder. Instructions may be provided for practicing any of the methods described herein.

The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . At least one active agent in the composition is an anti-Cεmx antibody, such as FB825.

Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert (s) on or associated with the container. In some embodiments, the present disclosure provides articles of manufacture comprising contents of the kits described above.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques) , microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984) ; Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987) ; Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc. ) ; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds. ) ; Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987) ; Current Protocols in Molecular Biology (F.M. Ausubel, et al., eds., 1987) ; PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994) ; Current Protocols in Immunology (J.E. Coligan et al., eds., 1991) ; Short Protocols in Molecular Biology (Wiley and Sons, 1999) ; Immunobiology (C.A. Janeway and P. Travers, 1997) ; Antibodies (P. Finch, 1997) ; Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989) ; Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000) ; Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999) ; The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995) .

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

Example 1: IgG4 Level Correlates with Atopic Dermatitis Patients’ Responsiveness To Anti-IgE Treatment

The correlation between levels of IgG4 in patients with atopic dermatitis (AD) and responsiveness to FB825 therapy was examined.

20 AD patients aged from 20 to 65 years old (mean: 28.3 years) and diagnosed as having moderate-to-severe atopic dermatitis were participated in this study. Eczema Area Severity Index (EASI) score and Investigator’s Global Assessment (IGA) score were used to evaluate disease severity (inclusion criteria: EASI ≥16, IGA ≥3; mean: EASI=26.4±9.3, IGA=3.6±0.5) . Furthermore, body surface area (BSA) of AD involvement at the screening and baseline visits should ≥ 10%. All subjects had a physician confirmed diagnosis of chronic atopic dermatitis based on 3-year history of symptoms defined by the Eichenfield revised criteria of Hannifin and Rajka and supported by positive allergen specific IgE at the screening visit. They all had a history of inadequate response to a stable (1 month) regimen of topical corticosteroids or calcineurin inhibitors as treatment for AD within 3 months before the screening visit. Before the baseline visit, patients must be applying stable doses of emollient provided for atopic dermatitis twice daily for at least 7 days.

All patients received a single dose of FB825, 5 mg/kg, by 1 hour IV infusion on Day 1 and returned to the study site on

Days

15, 29 and 57 for the safety and efficacy evaluation. Peripheral blood samples were collected before the start of infusion on Day 1 and at screening visits on

Days

15 and 29. Blood samples (approximately 10 ml of venous blood) were collected from the patients within 2 hours before the FB825 infusion and on

Days

15 and 29 after the infusion. The levels of IgG4 therein were measured using an IgG subclass 1-4 immunoassay kit provided by SIEMENS.

The EASI scoring system were used to assess disease severity in the patients. This system uses a defined process to grade the severity of the signs of eczema and the extent affected. Extent and severity of signs of eczema are evaluated in four body regions and the total score will be the sum of the four regions scores adjusted with multipliers. The EASI score is ranged from 0-72.

Additional assays for assessing disease severity include the pruritus visual analogue scale (VAS) of Severity Scoring of Atopic Dermatitis Index (SCORAD) . The SCORAD (Index) is a validated scoring system in atopic dermatitis (AD) . To measure the extent of AD, the rule of nines is applied on a front/back drawing of the patient’s inflammatory lesions. The extent can be graded from 0 to 100. The intensity part of the SCORAD consists of 6 items: erythema, oedema/papulation, excoriations, lichenification, oozing/crusts and dryness. Each item can be graded on a scale from 0 (absent) to 3 (severe) . The subjective items include daily pruritus and sleeplessness. The SCORAD Index formula is: A/5 + 7B/2 + C. In this formula A is defined as the extent (0–100) , B is defined as the intensity (0–18) and C is defined as the subjective symptoms (0–20) .

IGA allows investigators to assess overall disease severity at one given time point, and it consists of a 5-point severity scale from clear to very severe disease (0= clear, 1 =almost clear, 2 = mild disease, 3 = moderate disease, and 4= severe disease) .

Infusion of the FB825 antibody successfully reduced AD symptoms as assessed using the Eczema Area and Severity Index EASI scoring system, (left y-axis) , and IgE pharmablasts (right y-axis) . The data shown in FIGs. 1A and 1B demonstrate that AD patients receiving a single dose FB825 showed a reduction in IgE+ memory B cells and plasmablasts and a significant improvement in EASI score evaluation during this trial.

Based on the EASI Scores, the patients received FB825 treatment are divided into two groups: a poor responder group (four patients) and a responder group (six patients) . FIG. 1C. All patients in the poor responder group showed a high serum IgG4 level, while all patients in the responder group showed a low serum IgG4 level, indicating that IgG4 can serve as a reliable biomarker for predicting a patient’s responsiveness to the anti-IgE treatment and/or identifying patients who are likely to respond to the treatment. See also FIG. 2A, which shows a plot of IgG4 levels (y-axis) vs. the percentage change from basesline of EASI scores at Day 29 post injection (X-axis) . Patients with lower IgG4 biomarker level (right bottom) had better responses to the therapy than patients with higher IgG4 biomarker levels (left top) .

Further analysis was performed as described above on additional patients receiving FB825. As shown in FIG. 2B, 13 out of 14 patients (92.9%) having an IgG4 level below about 200 mg/dL, showed responsiveness to FB825. The data further suggest that a cutoff level of IgG4 at about 200 mg/dL or about 240 mg/dL can be used as a threshold for distinguishing responders from poor responders. See also FIGs. 3A and 3B.

Table 1 below shows statistical analysis on the correlation between responsiveness to FB825 (indicated by EASI improvement) and patient serum IgG4 levels on Day 29.

Table 1. Correlation Between Responsiveness to FB825 and Patient Serum Levels of IgG4

Overall, these data demonstrate that IgG4 levels inversely correlate with the responsiveness to FB825 therapy and thus can be used as a biomarker for predicting responsiveness of the treatment.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an, ” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one. ”

The phrase “and/or, ” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B” , when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B) ; in another embodiment, to B only (optionally including elements other than A) ; in yet another embodiment, to both A and B (optionally including other elements) ; etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of, ” or, when used in the claims, “consisting of, ” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both” ) when preceded by terms of exclusivity, such as “either, ” “one of, ” “only one of, ” or “exactly one of. ” “Consisting essentially of, ” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ± 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 %of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein.

As used herein in the specification and in the claims, the phrase “at least one, ” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B, ” or, equivalently “at least one of A and/or B” ) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B) ; in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A) ; in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements) ; etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

A method for assessing responsiveness of a human subject to an anti-immunoglobulin E (IgE) therapy, the method comprising:

(i) measuring a level of IgG4 in a biological sample of a human subject; and

(ii) identifying the human subject as likely to respond to the anti-IgE therapy, if the level of IgG4 in the biological sample from the human subject is not greater than a predetermined reference value.
The method of claim 1, wherein the biological sample is a blood sample, a serum sample, or a plasma sample.
The method of claim 2, wherein the predetermined reference value is about 240 mg/dL.
The method of claim 2, wherein the predetermined reference value is about 200 mg/dL.
The method of any one of claims 1-4, wherein the human subject has or is suspected of having a disorder associated with IgE.
The method of claim 5, wherein the disorder associated with IgE is selected from the group consisting of allergic asthma, allergic rhinitis, atopic dermatitis, and hyper IgE syndrome.
The method of claim 6, wherein the human subject has or is suspected of having atopic dermatitis.
The method of any one of claims 1-7, further comprising administering to the human subject an antibody that binds an IgE polypeptide, wherein the subject is identified in step (ii) as likely to respond to the anti-IgE therapy.
The method of claim 8, wherein the antibody that binds the IgE polypeptide is FB825, an antigen-binding fragment thereof, or a functional variant thereof.
The method of claim 9, wherein the antibody is a functional variant of FB825, which has the same heavy chain complementary determining regions (CDRs) and the same light chain CDRs as FB825.
The method of claim 10, wherein the antibody has the same heavy chain variable region (V _H) and the same light chain variable region (V _L) as FB825.
The method of claim 10 or claim 11, wherein the antibody is an IgG1 molecule.
The method of any one of claims 1-12, wherein the human subject is an adult.
The method of any one of claims 1-13, wherein the human subject is diagnosed as having moderate-to-severe atopic dermatitis.
The method of any one of claims 1-14, wherein the human subject is a patient having atopic dermatitis with an Eczema Area Severity Index (EASI) score ≥ 16, an Investigator’s Global Assessment (IGA) score ≥ 3, a ≥10%body surface area affected by atopic dermatitis, or a combination thereof.
The method of any one of claims 9-15, wherein the antibody is administered to the human subject at about 5 mg/kg.
The method of claim 16, wherein the human subject is administered a single dose of the antibody.
A method for treating a disorder associated with immunoglobulin E (IgE) , the method comprising administering to a human subject in need thereof an effective amount of an antibody that binds an IgE polypeptide, wherein the human subject has an IgG4 level not greater than a predetermined reference value.
The method of claim 18, wherein the IgG4 level is a blood level of IgG4 of the human subject.
The method of claim 19, wherein the predetermined reference value is about 240 mg/dL.
The method of claim 19, wherein the predetermined reference value is about 200 mg/dL.
The method of any one of claims 18-21, wherein the antibody comprises the same heavy chain complementarity determining regions (CDRs) as antibody FB825 and/or the same light chain CDRs as antibody FB825.
The method of claim 22, wherein the antibody is a human antibody or a humanized antibody.
The method of claim 23, wherein the antibody has the same heavy chain variable region (V _H) as antibody FB825 and/or the same light chain variable region (V _L) as antibody FB825.
The method of any one of claims 18-24, wherein the antibody is a full-length antibody.
The method of claim 25, wherein the antibody is an IgG1 molecule.
The method of any one of claims 18-26, wherein the antibody is FB825.
The method of any one of claims 18-27, wherein the effective amount of the antibody is about 5 mg/kg.
The method of any one of claims 18-28, wherein the antibody is administered by intravenous injection.
The method of any one of claims 18-29, wherein the human subject has one or more of the following features:

(i) having an eczema area and severity index (EASI) score ≥ 16;

(ii) having an investigator’s global assessment (IGA) score ≥ 3;

(iii) having ≥10%body surface area (BSA) of atopic dermatitis involvement;

(iv) having inadequate response to topical corticosteroids and/or calcineurin inhibitors;

(v) applying stable doses of emollient for at least 7 days; and

(vi) having normal cardiac conduction parameters.
The method of any one of claims 18-30, wherein the human subject is free of one or more of the following:

(i) heart arrhythmias;

(ii) positive for hepatitis B surface antigen, hepatitis C virus antibody, or human immunodeficiency virus antibodies;

(iii) history of anaphylactic reaction or malignancy;

(iv) received any immunoglobulin product or blood product within 3 months prior to the administration of the antibody that binds IgE;

(v) received a biologic product, an immunotherapy, a phototheray, a live vaccine, or a combination thereof;

(vi) one or more laboratory abnormalities;

(vii) received one or more of systemic corticosteroids, leukotriene modifiers, immunosuppressants, immunomodulating drugs, other anti-IgE therapeutics, allergen immunotherapy, and orally inhaled corticosteroids; and

(viii) high risk of parasite infection.
The method of any one of claims 9-27, wherein the human subject is an adult.
The method of any one of claims 18-32, wherein the human subject is administered one dose of the antibody that binds IgE.
The method of any one of claims 18-32, wherein the human subject is administered multiple doses of the antibody that binds IgE.
A method for treating a disorder associated with immunoglobulin E (IgE) , the method comprising:

(i) measuring a level of IgG4 in a biological sample of a human subject having or suspected of having a disorder associated with IgE;

(ii) comparing the level of IgG4 determined in step (i) with a predetermined reference value; and

(iii) administering an effective amount of an antibody specific to an IgE polypeptide to the human subject, whose IgG4 level is not greater than the predetermined reference value.
A pharmaceutical composition for use in treating a disorder associated with immunoglobulin E (IgE) in a subject, wherein the pharmaceutical composition comprises an antibody that binds IgE, and wherein the subject is a human patient having an IgG4 level not greater than a predetermined value.