Highly concentrated antibody formulation

Abstract

The high concentration antibody formulation comprises an antibody at a concentration of about 120-200 mg / ml, a histidine buffer at a concentration of about 5-15 mM, an amino acid at a concentration of about 1-1.5% (w / v), a tonicity adjuster such as sodium chloride at a concentration of about 50-100 mM, and a non-ionic surfactant such as polysorbate 80 at a concentration of about 0.005-0.02% (w / v). In some examples, the high concentration antibody formulation may have a pH of about 5.5-6.5.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 170,042, filed April 2, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] High concentrations of formulations containing biomolecules such as recombinant proteins and monoclonal antibodies are usually required for certain drug delivery routes such as subcutaneous injection. Although high-concentration antibody formulations have been developed for certain antibody drugs, it remains difficult to produce high-concentration antibody formulations (e.g., >100 mg / ml), due to irreversible aggregation, irreversible precipitation, and / or high viscosity. Summary of the Invention

[0003] The present disclosure is based, at least in part, on the development of a highly concentrated antibody formulation that exhibits excellent stability under a variety of conditions and over a two-year storage period, and excellent injectability.

[0004] Accordingly, one aspect of the present disclosure features an antibody formulation that includes: (a) an antibody that binds to the CεmX domain of membrane-bound IgE at a concentration of about 120-200 mg / ml; (b) histidine at a concentration of about 5-15 mM; (c) an amino acid (wherein the amino acid is arginine or threonine) at a concentration of about 1-3.0% (w / v); (d) sodium chloride at a concentration of about 50-100 mM; and (e) polysorbate 80 at a concentration of about 0.005-0.02%. Such an antibody formulation may have a pH of about 5.5-6.5. In some embodiments, the antibody formulation consists essentially of the components (a)-(e). Alternatively or additionally, the antibody formulation does not include a sugar or sugar alcohol-based stabilizer. Examples include sucrose, sorbitol, or trehalose.

[0005] In some embodiments, the antibody formulation may comprise about 150 mg / ml antibody, about 10 mM histidine, about 1.25% amino acid, about 75 mM sodium chloride, and about 0.01% polysorbate 80. In some examples, the amino acid in the antibody formulation may be arginine, and the pH of the antibody formulation is about 6.0-6.5. In another example, the amino acid in the antibody formulation is threonine, and the pH of the antibody formulation is about 6.0.

[0006] In any of the antibody formulations disclosed herein, the antibody that binds to the CεmX domain of membrane-bound IgE contained therein may comprise the same heavy chain complementarity determining regions (CDRs) as antibody FB825, and / or the same light chain complementarity determining regions (CDRs) as antibody FB825. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the antibody is a full-length antibody.

[0007] In some embodiments, the antibody comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:9, and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:10. In some examples, the antibody comprises a VH of SEQ ID NO:9, and a VL of SEQ ID NO:10. In some examples, the antibody is an IgG1 molecule. In particular examples, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:11, and a light chain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:12.

[0008] In another aspect, provided herein is a pre-filled syringe containing any of the high concentration antibody formulations disclosed herein. In some embodiments, the amount of antibody formulation in the pre-filled syringe is about 1-2 ml.

[0009] In yet another aspect, the disclosure features a method for treating an immunoglobulin E (IgE)-associated disease, the method comprising administering to a subject in need thereof an effective amount of any of the antibody formulations disclosed herein. In some embodiments, the subject is administered the antibody formulation once. In some embodiments, the subject is administered the antibody formulation at least twice. In some examples, the two consecutive doses are administered to the subject at least three months apart. In any of the methods disclosed herein, the antibody formulation is administered subcutaneously.

[0010] In some embodiments, the subject is a human patient having or suspected of having allergic asthma, allergic rhinitis, atopic dermatitis, or hyper-IgE syndrome. In some examples, the human patient has atopic dermatitis.

[0011] Also within the scope of the disclosure is any of the antibody formulations disclosed herein for treating diseases associated with immunoglobulin E (IgE), such as those disclosed herein, and the use of the antibody formulations for the manufacture of a medicament for treating a target disease.

[0012] The details of one or more embodiments of the invention are set forth below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, and from the appended claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In atopic humans, who are at high risk of developing allergies, IgE concentrations in the circulation can be elevated, for example, to levels at least 10-fold higher than normal levels. The concentration of allergen-specific IgE antibodies correlates closely with clinical symptoms and can be more than 1000-fold higher in patients with allergic disease compared to healthy humans. Immunoglobulin E sensitizes effector cells, such as basophils, mast cells, and activated eosinophils, by occupying the high-affinity IgE receptor FcεRI, which is expressed by these cells. In type I hypersensitivity, allergens crosslink IgE molecules bound to FcεRI, which subsequently triggers degranulation of effector cells and release of proinflammatory mediators such as histamine and leukotrienes. IgE-mediated allergy pathways, which result in mediator-related allergic symptoms, trigger immune activity locally or systemically. Basophils and mast cells also release a wide range of proinflammatory cytokines and chemokines that not only directly cause clinical symptoms but also activate and recruit various cell types to increase the inflammatory state. Thus, anti-IgE therapy can attenuate both IgE-mediated pathways and the inflammatory state.

[0014] Several anti-IgE antibodies have been developed to treat IgE-related allergic diseases. However, it remains difficult to develop highly concentrated antibody formulations for delivery of such antibodies via non-intravenous infusion routes, e.g., via subcutaneous injection.

[0015] The present disclosure is based, at least in part, on the development of a highly concentrated antibody formulation, including an exemplary anti-IgE antibody (FB825), that exhibits excellent stability under various conditions and over a two-year shelf life, and also exhibits excellent injectability. Accordingly, the present disclosure provides highly concentrated antibody formulations and uses thereof for treating IgE-related allergic disorders.

[0016] I. Highly concentrated antibody formulations In some aspects, provided herein are high concentration antibody formulations comprising any of the anti-IgE antibodies disclosed herein. As used herein, a "high concentration formulation" refers to a formulation comprising a biomolecule, such as an antibody, at a concentration of at least 100 mg / ml. High concentration antibody formulations may include, in addition to the anti-IgE antibody, a suitable buffer, a suitable amino acid excipient, a suitable tonicity adjusting agent, and a suitable non-ionic surfactant. Such high concentration antibody formulations may have a pH of about 5.5 to 6.5.

[0017] The highly concentrated antibody formulations disclosed herein may have one or more of the following superior characteristics: (a) they are stable under conditions such as agitation (e.g., 4 hours at room temperature), freeze / thaw (e.g., 5 or more consecutive cycles), and UV light exposure; (b) they are stable under short-term storage (e.g., 8 weeks at 40° C., 2 weeks at 45° C., and / or 1 week at 55° C.) and / or long-term storage (e.g., 2 years under various temperature conditions); and (c) they are stable under turbidity (e.g., A 650 <0.01, e.g., about 0.006-0.008), osmolality (e.g., isotonic, about 300 mOsm), viscosity, and / or injectability (e.g., 2 lb to expel 1 ml of formulation in 5-10 seconds). f Need less than 3-5lb f (less than 5 seconds is required to deliver 1 ml of formulation using a SEC-HPLC) In some instances, stability is demonstrated by the percentage change of the main peak, low molecular weight peak, and / or high molecular weight peak when analyzed by SEC-HPLC, and the percentage change of the main peak, acidic peak, and basic peak when analyzed by SCX-HPLC, according to routine practice and / or the disclosure provided in the Examples below.

[0018] A. Antibodies capable of binding to the CεmX domain of membrane-bound IgE In some examples, the anti-IgE antibody in the high concentration antibody formulation disclosed herein may be an antibody that binds to the CεmX domain of a membrane-bound IgE molecule. CεmX is a 52 amino acid segment located between the CH4 domain and the C-terminal membrane anchoring segment of the human membrane-bound ε chain (mε). The amino acid sequence of an exemplary CεmX fragment of human mIgE is provided below (SEQ ID NO: 6). GLAGGSAQSQ RAPDRVLCHS GQQQGLPRAA GGSVPHPRCH CGAGRADWPG PP

[0019] The antibodies described herein can bind to mIgE, e.g., the CεmX domain of mIgE expressed on the surface of B cells. Such antibodies can induce cell death of B cells expressing mIgE, e.g., via antibody-dependent cellular cytotoxicity and / or cell apoptosis, thereby depleting the B cells and resulting in reduced production of free IgE. Thus, the anti-CεmX antibodies described herein can reduce the level of total IgE in a subject (e.g., a human patient) treated with the antibody.

[0020] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, and polypeptides through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, etc.), single chain (scFv), variants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified form of immunoglobulin molecule that contains an antigen recognition site of a desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the constant domain of the heavy chain of the antibody, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM), and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0021] The antibodies used in the methods described herein may be murine, rat, human, or of any other origin, including chimeric or humanized antibodies.

[0022] Any of the antibodies described herein can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a population of homogeneous antibodies, whereas a "polyclonal antibody" refers to a population of heterogeneous antibodies. The two terms do not limit the source of the antibody or the method by which the antibody is made.

[0023] 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 certain chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In many cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity 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 performance. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not present in the recipient antibody or in the introduced CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody comprises at least one, and usually almost all of two, variable domains, in which all or almost all of the CDR regions correspond to those of a non-human immunoglobulin, and all or almost all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody also optimally comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, and are also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody. A humanized antibody may also be affinity matured.

[0024] In another example, the antibodies described herein are chimeric antibodies that may contain heavy and light chain constant regions derived from a human antibody. Chimeric antibodies refer to antibodies that have a variable region or a portion of a variable region derived from a first species and a constant region derived from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of an antibody derived from one mammal (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portion is homologous to the sequence of an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications can be made in the variable and / or constant regions.

[0025] In some examples, the antibodies disclosed herein specifically bind to the CεmX domain of membrane-bound IgE that may be expressed on the surface of B cells. An antibody that "specifically binds" (used interchangeably herein) to a target or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or binds more frequently, more rapidly, for a longer period, and / or with a higher affinity to a particular target antigen than to another target. An antibody "specifically binds" to a target antigen if it binds with higher affinity, avidity, more readily, and / or for a longer period than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to a CεmX domain epitope is one that binds to that CεmX domain epitope with higher affinity, avidity, more readily, and / or for a longer period than it binds to other CεmX domain epitopes or non-CεmX domain epitopes. By reading this definition, it is understood 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. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Usually, but not necessarily, reference to binding refers to preferential binding.

[0026] The binding affinity of the anti-CεmX antibodies 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 any of about 50 pM to about 2 pM. D or the dissociation constant, K D This corresponds to a decrease in the binding affinity of the antibody to CεmX. One way to determine the binding affinity of an antibody to CεmX is to measure the binding affinity of the monofunctional Fab fragment of the antibody. To obtain the monofunctional Fab fragment, an antibody (such as an IgG) can be cleaved with papain or expressed recombinantly. The affinity of the anti-CεmX Fab fragment of the antibody can be determined by surface plasmon resonance (BIAcore3000TM Surface Plasmon Resonance (SPR) System, BIAcore, INC, Piscaway NJ). The kinetic binding rate (k on ) and the kinetic dissociation rate (k off ) (usually measured at 25 °C) and the equilibrium dissociation constant (K D ) value is calculated as koff / kon.

[0027] In some embodiments, the antibody binds to the CεmX domain of human IgE but does not significantly bind to IgE of another mammalian species. In some embodiments, the antibody binds not only to human IgE but also to one or more IgE from another mammalian species. The epitope(s) to which the antibody binds may be continuous or discontinuous.

[0028] In some embodiments, the anti-CεmX antibody described herein binds to the N-terminal portion of the CεmX domain, for example, GLAGGSAQSQRAPDRVL (SEQ ID NO: 1) or GLAGGSAQSQRA (SEQ ID NO: 7). Such an antibody may have the same heavy and / or light chain CDRs as antibody FB825. See also US Patent Application No. 8,460,664, the relevant disclosures of which are incorporated herein by reference. The anti-CεmX antibody may be a humanized antibody. In some examples, the anti-CεmX antibody used in the methods described herein is FB825 or a functional variant thereof. See also US Patent Application No. 8,460,664 and US20200297815, the relevant disclosures of which are incorporated herein by reference.

[0029] Same V H and / or V L Two antibodies that have CDRs mean that their CDRs are the same when measured by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach known in the art; see, e.g., bioinf.org.uk / abs / ).

[0030] A functional variant (equivalent) of FB825 has essentially the same epitope binding specificity as FB825 and exhibits substantially similar biological activity as FB825, including the activity of depleting mIgE-expressing B cells to reduce total IgE levels in a subject. In some embodiments, a functional variant of FB825 contains the same regions / residues as FB825 involved in antigen binding, such as the same specificity determining residues in the CDRs or across the CDRs. In another embodiment, a functional variant of FB825 has the same epitope binding specificity as the corresponding VL of FB825. H V that is at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the CDR H CDR1, V H CDR2, and V H V containing CDR3 H Chain and corresponding V of FB825 HV that is at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the CDR L CDR1, V L CDR2, and V L V containing CDR3 L For example, a functional variant of FB825 includes the V H Compared to CDR, V H CDR area (V H V containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid residue mutations in CDR1, CDR2, and / or CDR3 (total). H Chain and / or V of FB825 H Compared to CDR, V L CDR area (V L V containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid residue mutations in CDR1, CDR2, and / or CDR3 (total). L Includes chains.

[0031] Alternatively, the functional mutant of FB825 is the V H V that is at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the H Chain, and FB825 V L V that is at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the L The amino acid sequence mutations are V H and / or V L It may occur only in one or more of the framework regions.

[0032] The "percent identity" of two amino acid sequences is measured 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 program of Altschul, et al. J. Mol. Biol. 215:403-10, 1990 and the XBLAST program (version 2.0). BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the 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 corresponding programs (e.g., XBLAST and NBLAST) can be used.

[0033] Alternatively or additionally, the amino acid residue variation may be a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution occurs. Variants can be prepared according to methods for altering polypeptide sequences well known to those skilled in the art, such as those described in references that collect such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made within the following amino acids groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N, and (g) E, D.

[0034] In some embodiments, an anti-CεmX antibody for use in the therapeutic methods disclosed herein may have one of the following heavy chain variable regions (CDRs according to the Kabat definition are bold and underlined. SEQ ID NOs: 13-15 are heavy chain CDRs 1-3, respectively): [ka] [ka] [ka]

[0035] Alternatively or additionally, an anti-CεmX antibody for use in the therapeutic methods disclosed herein may have one of the following light chain variable regions (CDRs according to the Kabat definition are bold and underlined; SEQ ID NOs: 16-18 are light chain CDRs 1-3, respectively): [ka] [ka]

[0036] In some embodiments, the heavy chain of any of the anti-IgE antibodies described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In some examples, the heavy chain constant region is of human origin. Alternatively or additionally, the light chain of the anti-IgE antibody may further comprise a light chain constant region (CL), which may be any CL known in the art, e.g., a human light chain constant region. In some examples, the CL is a kappa light chain. In another example, the CL is a lambda light chain. Antibody heavy and light chain constant regions are known in the art, e.g., as provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0037] In some examples, the anti-IgE antibody is FB825, which is an IgG1 molecule having heavy and light chains as provided below, each of which is represented by SEQ ID NO:2: H and V of SEQ ID NO:3 L The amino acid sequences of the heavy (top) and light (bottom) chains of FB825 in full-length form are shown below (including the N-terminal signal peptide sequence in italics): [ka] [ka]

[0038] SEQ ID NO:4 represents the amino acid sequence of the mature heavy chain of FB825 (without the N-terminal signal peptide sequence) and SEQ ID NO:5 represents the amino acid sequence of the mature light chain of FB825 (without the N-terminal signal peptide sequence).

[0039] In some examples, the FB825 antibody may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5. Alternatively, the heavy and / or light chains of FB825 may comprise an N-terminal signal peptide, such as those described above. In some examples, the FB825 antibody may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0040] B. Antibody Preparation Antibodies capable of binding to the CεmX domain of membrane-bound IgE described herein can be produced by any method known in the art, see, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0041] In some embodiments, an antibody specific for a target antigen (e.g., the CεmX domain of mIgE, such as human mIgE) can be produced by conventional hybridoma technology. If necessary, the antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, which can then be propagated and frozen for future use. The polynucleotide sequence can also be used for genetic engineering to "humanize" the antibody or to improve the affinity (affinity maturation) or other properties of the antibody. For example, if the antibody is to be used in human clinical trials and treatments, the constant region can be adjusted to be more similar to the human constant region to avoid immune responses. It may be desirable to genetically engineer the antibody sequence to increase its affinity for the target antigen and to increase its effectiveness 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 an antibody and still maintain its binding specificity for the target antigen.

[0042] In another embodiment, fully human antibodies can be obtained using commercially available mice engineered to express certain human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or stronger immune response can also be used to generate humanized or human antibodies. Examples of such technology include XenomouseR™ from Amgen, Inc. (Fremont, Calif.), and HuMAb-MouseR™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ). Antibodies can also be recombinantly produced by phage display technology. See, for example, U.S. Patent 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, 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.

[0043] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared routinely. For example, F(ab')2 fragments can be generated by pepsin digestion of the antibody molecule and Fab fragments can be produced by reducing the disulfide bridges of the F(ab')2 fragment.

[0044] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into one or more expression vectors and then transfected into host cells that do not produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. See, for example, PCT Publication No. WO87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, engineered antibodies, such as "chimeric" or "hybrid" antibodies, can be prepared that have the binding specificity of a target antigen.

[0045] 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).

[0046] 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, the VH and V L The variable regions of the human VV are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Framework amino acid residues predicted to be important for the formation of the correct CDR structure are then identified using the same molecular modeling analysis. At the same time, a human VVV having an amino acid sequence homologous to the amino acid sequence of the parent non-human antibody is identified. H and V L The chain is the parent V H and V L The sequence is used as a search query to identify the human V H and V L An acceptor gene is selected.

[0047] The CDR regions in the selected human acceptor gene can be replaced with the CDR regions from the parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain predicted to be important in interactions with the CDR regions (see above) can be used to replace the corresponding residues in the human acceptor gene.

[0048] Alternatively, antibodies (IgE molecules or fragments thereof) capable of binding to the target antigens described herein can be isolated from a suitable antibody library by routine practice. The antibody library can be used to identify proteins that bind to the target antigen (e.g., human IgE, e.g., CεmX fragments thereof) by routine screening processes. In the selection process, the polypeptide components are probed with the target antigen or fragments thereof, and if the polypeptide components bind to the target, the members of the antibody library are identified, typically by being retained on the support. The retained display library members are recovered from the support and analyzed. The analysis can include amplification and subsequent selection under similar or different conditions. For example, positive and negative selections can be alternated. The analysis can further include determining the amino acid sequence of the polypeptide components, and purifying the polypeptide components for detailed characterization.

[0049] There are many routine methods known in the art for identifying and isolating antibodies capable of binding to the target antigens described herein, including phage display, yeast display, ribosome display, or mammalian display technologies.

[0050] The antibodies obtained according to methods 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". Many methods are known in the art for mapping and characterizing the location of epitopes in proteins, including, for example, solving the crystal structure of the antibody-antigen complex, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, competitive assays, gene fragment expression assays, and synthetic peptide-based assays. In a further example, epitope mapping can be used to determine the sequence to which the antibody binds. The epitope can be a linear epitope (i.e., contained in a single stretch of amino acids) or a conformational epitope (linear sequence of primary structure) formed by three-dimensional interactions of amino acids that are not necessarily contained in a single stretch. Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in binding assays with antibodies. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to a gene fragment expression assay, an open reading frame encoding a target antigen is fragmented by random or specific gene construction and the reactivity of the expressed antigen fragments with a test antibody is determined. Gene fragments can be produced, for example, by PCR and then transcribed in vitro in the presence of radioactive amino acids and translated into proteins. Binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by large libraries of random peptide sequences (phage libraries) displayed on the surface of phage particles.Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In a further example, mutagenesis of the 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 mutants of the target antigen in which various fragments of the IgE polypeptide are replaced (swapped) with sequences of a closely related but antigenically distinct protein, such as another member of the immunoglobulin protein family. By assessing the binding of the antibody to the mutant immunoglobulins, the importance of specific antigen fragments to antibody binding can be assessed.

[0051] Alternatively, competition assays can be performed with other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibody. Competition assays are well known to those of skill in the art.

[0052] In some examples, the anti-IgE antibodies disclosed herein, such as FB825, can be prepared by recombinant techniques, as exemplified below.

[0053] The nucleic acids encoding the heavy and light chains of the anti-CεmX antibody described herein can be cloned into one expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter, such that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the sequences encoding the heavy and light chains.

[0054] In some instances, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same cell or different cells. If the two chains are expressed in different cells, each of them can be isolated from a host cell expressing such chain, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions to allow the formation of antibodies.

[0055] Usually, the nucleic acid sequence encoding one or all chains of the antibody can be cloned into a suitable expression vector operably linked to a suitable promoter using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to form complementary ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, synthetic nucleic acid linkers can be attached to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter depends on the type of host cell for the production of the antibody.

[0056] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0057] Regulatable promoters can also be used. Such regulatable promoters include promoters that use the lac repressor from E. coli as a transcription regulator to regulate transcription from mammalian cell promoters with lac operators [Brown, M. et al., Cell, 49:603-612 (1987)] and promoters that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astroradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0058] Regulatable promoters containing repressors with operons can be used. In one embodiment, the lac repressor from E. coli can function as a transcription regulator to regulate transcription from mammalian cell promoters with lac operators [M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)], and the tetracycline repressor (tetR) and the transcription activator (VP16) are combined to form a tetR-mammalian cell transcription activator fusion protein, i.e., tTa (tetR-VP16), and combined with a minimal promoter with tetO from the major immediate early promoter of human cytomegalovirus (hCMV) to form a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. The tetracycline repressor (tetR) alone, not the tetR mammalian cell transcription factor fusion derivative, can function as a strong transregulator to regulate gene expression in mammalian cells when the tetracycline operator is appropriately located downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399(2003)). One particular advantage of the tetracycline-induced switch is that it does not require the use of tetracycline repressor-mammalian cell transcription activator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its regulatory effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)).

[0059] The vectors may also contain some or all of the following: a selectable marker gene, such as, for example, a neomycin gene for selection of stable or transient transfectants in mammalian cells, an enhancer / promoter sequence from the human CMV immediate early gene for high levels of transcription, a transcription termination and RNA processing signal from SV40 for mRNA stability, the SV40 polyoma origin of replication and ColE1 for proper episomal replication, an internal ribosome binding site (IRES), a versatile multiple cloning site, and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for generating vectors containing transgenes are well known and available in the art.

[0060] Examples of polyadenylation signals useful in carrying out the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0061] One or more vectors (e.g., expression vectors) containing a nucleic acid encoding any of the antibodies can be introduced into a suitable host cell to produce the antibody. The host cell can be cultured under conditions suitable for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered by the cultured cells (e.g., from the cells or culture supernatant) by conventional methods, such as affinity purification. If desired, the polypeptide chains of the antibody can be incubated under suitable conditions and for a suitable period of time to allow production of the antibody.

[0062] In some embodiments, the method for preparing the antibodies described herein requires a recombinant expression vector encoding both the heavy and light chains of the anti-CεmX antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, for example by calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under appropriate conditions that allow the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or the medium. If necessary, the two chains recovered from the host cells can be incubated under appropriate conditions that allow the formation of the antibody.

[0063] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-IgE antibody and the other encoding the light chain of an anti-IgE antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow the expression of the polypeptide chains of the antibody. If the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or medium. If necessary, the polypeptide chains can be recovered from the host cell or medium and then incubated under suitable conditions that allow the formation of the antibody. If the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or the corresponding medium. The two polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

[0064] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a protein A or protein G-binding matrix.

[0065] Any nucleic acid encoding the heavy chain, light chain or both of the anti-CεmX antibodies described herein, vectors containing same (eg, expression vectors) and host cells containing the vectors are within the scope of the disclosure.

[0066] C. Highly concentrated antibody formulations Any of the anti-IgE antibodies disclosed herein, such as FB825, may be used to prepare a high concentration antibody formulation. In addition to the anti-IgE antibody, the high concentration antibody formulation disclosed herein may further include a suitable buffer (e.g., histidine at a concentration of about 5-15 mM), an amino acid excipient (e.g., arginine or threonine at a concentration of about 1-3% w / v), a tonicity adjusting agent (e.g., sodium chloride at a concentration of about 50-100 mM), and a non-ionic surfactant (e.g., polysorbate 80 at a concentration of about 0.005-0.02%). The antibody formulation may have a pH of about 5.5-6.5. In some examples, the antibody formulation may have a pH value of about 6.0 to about 6.5. In certain examples, the pH value of the antibody formulation is about 6.0.

[0067] The term "about" or "approximately" means within an acceptable error range of a particular value measured by one of ordinary skill in the art, which depends in part on the limitations of how the value is measured or determined, i.e., the measurement system. For example, "about" can mean within an acceptable standard deviation, per practice in the art. Alternatively, "about" can mean within a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1% of any value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of the value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" is implicit, and in this context means within an acceptable error range of the particular value.

[0068] The anti-IgE antibody in the high concentration formulation may be at a concentration of at least 100 mg / ml. In some embodiments, the high concentration formulation contains the antibody at a concentration of about 100 mg / ml to about 250 mg / ml, e.g., about 100 mg / ml to about 150 mg / ml, about 125 mg / ml to about 150 mg / ml, about 150 mg / ml to about 180 mg / ml, about 150 mg / ml to about 200 mg / ml, about 180 mg / ml to about 200 mg / ml, or about 200 mg / ml to about 250 mg / ml. In some examples, the high concentration antibody formulation may contain the antibody (e.g., FB825) at a concentration of about 130 to 165 mg / ml, e.g., about 150 mg / ml.

[0069] The highly concentrated antibody formulations disclosed herein further comprise a buffering agent. A buffering agent refers to a weak acid or weak base that helps maintain the pH of an aqueous solution. In some examples, the buffering agent used in the highly concentrated antibody formulation is histidine, and the concentration can be about 5-15 mM. In some examples, the concentration of histidine is about 5-8 mM, 5-10 mM, 10-15 mM, or 12-15 mM. In certain examples, the concentration of histidine is about 10 mM.

[0070] Further, the high concentration antibody formulation may include an amino acid excipient, the concentration of which may be about 1-3% (w / v). In some embodiments, the amino acid excipient is arginine. In some examples, the concentration of arginine is about 1-1.3% (w / v), e.g., about 1.25% (w / v). In some examples, the concentration of arginine may be about 2-3% (w / v), e.g., about 2.5% (w / v). In some embodiments, the amino acid excipient is threonine. In some examples, the concentration of threonine is about 1-1.3% (w / v), e.g., about 1.25% (w / v). In other examples, the concentration of threonine is about 2-3% (w / v), e.g., about 2.5% (w / v).

[0071] The high concentration antibody formulations disclosed herein also include a tonicity modifier. In terms of a solution, tonicity is a property of a particular membrane (e.g., a cell membrane) that is equal to the sum of the concentrations of solutes in the solution (e.g., an aqueous formulation) and has the ability to exert an osmotic force across the membrane. The tonicity modifier adjusts the tonicity of the formulation. In some embodiments, the tonicity modifier included in the high concentration antibody formulations disclosed herein is sodium chloride, and the concentration can be about 50-100 mM. In some examples, the concentration of the tonicity modifier (e.g., sodium chloride) is about 50-80 mM, about 60-80 mM, or about 70-80 mM. In a particular example, the tonicity modifier is sodium chloride at a concentration of about 75 mM.

[0072] In addition, the high concentration antibody formulations disclosed herein further comprise a non-ionic surfactant. A non-ionic surfactant is a type of surfactant that does not have a charge on the hydrophilic head group and therefore has no net charge in the formulation. In some embodiments, the non-ionic surfactant in the high concentration antibody formulations disclosed herein is polysorbate 80, and the concentration can be about 0.005-0.02% (w / v). In some examples, the concentration of the non-ionic surfactant, such as polysorbate 80, can range from 0.008-0.015%. In a particular example, the non-ionic surfactant is polysorbate 80 at a concentration of about 0.01%.

[0073] In some examples, a high concentration antibody formulation disclosed herein may contain about 150 mg / ml of an anti-IgE antibody (e.g., FB825), about 10 mM histidine, about 1.25% amino acids, about 75 mM sodium chloride, and about 0.01% polysorbate 80.

[0074] In some examples, the high concentration antibody formulations disclosed herein consist essentially of an anti-IgE antibody, a buffer, an amino acid excipient, a tonicity adjusting agent, and a non-ionic surfactant. Such formulations are free of ingredients that do not materially affect the basic and novel properties of the formulation.

[0075] In some examples, the highly concentrated antibody formulations are substantially free (e.g., completely free) of sugar-based or sugar alcohol-based stabilizers. For example, the formulations are substantially free (e.g., completely free) of sucrose. Alternatively, the formulations are substantially free (e.g., completely free) of sorbitol. And, in other examples, the formulations are substantially free (e.g., completely free) of trehalose.

[0076] D. Delivery Devices Containing Highly Concentrated Antibody Formulations Any of the high-concentration antibody formulations disclosed herein can be placed in a delivery device, such as a glass vial, a syringe, a pre-filled syringe, a pen-type delivery device, or an auto-injector. In some embodiments, the high-concentration antibody formulation (e.g., comprises or consists essentially of about 150 mg / ml of an anti-IgE antibody such as FB825, about 10 mM histidine, about 1.25% amino acids, about 75 mM sodium chloride, and about 0.01% polysorbate 80) can be contained in a pre-filled syringe. In some examples, the pre-filled syringe is a single-dose pre-filled syringe. In another example, the high-concentration antibody formulation is contained in an auto-injector. In yet another example, the high-concentration antibody formulation is contained in a pen-type delivery device (e.g., a pre-filled pen).

[0077] In some embodiments, the highly concentrated antibody formulations disclosed herein can be delivered subcutaneously, for example, by a standard needle and syringe. In some embodiments, the syringe is a pre-filled syringe. In some embodiments, a pen delivery device or an autoinjector is used to deliver the highly concentrated antibody formulation (e.g., for subcutaneous delivery). The pen delivery device can be reusable or disposable. Typically, a reusable pen delivery device utilizes a replaceable cartridge that contains the highly concentrated antibody formulation. Once the antibody formulation in the cartridge is administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the antibody formulation. The pen delivery device is then reusable. A disposable pen delivery device does not have a replaceable cartridge. In a disposable pen delivery device, the antibody formulation is pre-filled to be contained in a reservoir in the device. Once the reservoir is emptied of the antibody formulation, the entire device is discarded.

[0078] Examples of suitable pen delivery devices and autoinjectors include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), the NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BDM pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN Examples of disposable pen delivery devices applicable to subcutaneous delivery of the highly concentrated antibody formulations disclosed herein include, but are not limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWiKPEN (Eli Lilly), the SURECLICK autoinjector (Amgen, Thousand Oaks, Calif.), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA pen (Abbott Labs, Abbott Park Ill.).

[0079] In some embodiments, the highly concentrated antibody formulations disclosed herein are delivered by a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may be used, see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. Furthermore, in another embodiment, the controlled release system may be placed in close proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0080] Any of the delivery devices that accommodate the highly concentrated antibody formulations disclosed herein are also within the scope of the present disclosure.

[0081] II. Medical applications of high-concentration antibody preparations To practice the methods disclosed herein, an effective amount of any of the high concentration anti-IgE antibody formulations disclosed herein can be administered to a subject (e.g., a human) in need of treatment via an appropriate route, such as subcutaneous or intramuscular injection.

[0082] The subject treated by the methods described herein may 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 in need of treatment may be a human patient having, at risk of, or suspected of having an IgE-related disorder (e.g., allergic asthma, as well as other disorders known in the art and / or disclosed herein). A subject having an IgE-related disorder, such as allergic asthma, may be identified by routine medical examination, e.g., laboratory testing. A subject suspected of having an IgE-related disorder may exhibit one or more symptoms of the disorder, such as, for example, elevated IgE levels and / or hypersensitivity to allergens and / or antigens. A subject at risk of a disorder may be a subject having one or more risk factors for the disorder.

[0083] Exemplary IgE-related diseases include, but are not limited to, asthma, allergic rhinitis, hyper-IgE syndrome, atopic dermatitis, cold urticaria, chronic urticaria, cholinergic urticaria, chronic rhinosinusitis, systemic mastocytosis, cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, recurrent idiopathic angioedema, and interstitial cystitis, eosinophilic gastrointestinal disease, food allergy, or drug allergy. In some examples, the target IgE-related disease is atopic dermatitis.

[0084] As used herein, "effective amount" refers to the amount of each active agent required to provide a therapeutic effect to a subject, alone or in combination with one or more other active agents. As recognized by those skilled in the art, effective amount varies depending on the specific condition being treated, the severity of the condition, the characteristics of the individual patient (including age, health, size, sex, and weight), the duration of treatment, the nature of the concomitant treatment (if any), the specific route of administration, and similar factors within the knowledge and expertise of medical practitioners. Such factors are well known to those skilled in the art and can be addressed with only routine experimentation. In general, it is preferable to use the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment. However, those skilled in the art will understand that a patient may insist on a lower dose or tolerated dose for medical reasons, psychological reasons, or virtually any other reason.

[0085] Empirical considerations such as half-life usually contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment and is usually, but not necessarily, based on the treatment and / or suppression and / or amelioration and / or delay of IgE-related diseases. Alternatively, sustained release formulations of anti-CεmX antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0086] In one example, the dose of the anti-CεmX antibody described herein can be empirically determined in individuals who have been administered one or more doses of the anti-CεmX antibody. Individuals are given increasing doses of the anti-CεmX antibody. To assess the effectiveness of the anti-CεmX antibody, indicators of IgE-related diseases (such as IgE levels) can be tracked.

[0087] For the purposes of this disclosure, the appropriate dose of anti-CεmX antibody depends on the specific anti-CεmX antibody(ies) (or composition thereof) used, the type and severity of the IgE-related disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical 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 dose is reached that produces the desired results. Administration of anti-CεmX antibody can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art.

[0088] In some embodiments, an anti-CεmX antibody described herein (e.g., FB825) is administered to a subject in need of treatment in an amount sufficient to reduce total IgE levels by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0089] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having an IgE-related disease, a symptom of an IgE-related disease, or a predisposition to a disease in order to cure, heal, alleviate, relieve, alter, treat, ameliorate, improve, or affect the disease, symptom of the disease, or predisposition to the disease.

[0090] Alleviating an IgE-related disease includes delaying the onset or progression of the disease, or alleviating the severity of the disease. Alleviating the disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a disease (such as an IgE-related disease) means delaying, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay can be for various lengths of time, depending on the medical history and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or a method of delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of the symptoms within a given time frame, compared to not using the method. Such comparisons are usually based on clinical studies using a sufficient number of subjects to obtain statistically significant results.

[0091] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of a disease. Onset of a disease can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to progression that may not be detectable. For purposes of this disclosure, onset or progression refers to the biological course of a condition. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of an IgE-related disease includes early onset and / or early recurrence.

[0092] To carry out the methods described herein, any of the anti-CεmX antibodies, such as FB825, can be administered to a subject (e.g., a human patient) in need of treatment in a single dose or several doses via an appropriate route, such as intravenous infusion or subcutaneous injection. The dose of anti-CεmX antibody in each administration can 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 on various factors, including those described herein. Depending on the circumstances, in the case of repeated administration over several days or more, treatment is continued until the desired suppression of symptoms occurs or until a therapeutic level sufficient to alleviate the IgE-related disease or its symptoms is achieved.

[0093] The administration of the anti-CεmX antibody (e.g., FB825) can be essentially continuous over a preselected period of time, or can be administered at a series of intervals, for example, before, during, or after the onset of an IgE-related disease. An exemplary dosing regimen includes administering to a subject in need of treatment an initial dose of anti-CεmX antibody (e.g., 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 (e.g., 4 months, 5 months, or 6 months) after the initial dose. The second dose may be greater, equal, or less than the first dose. Other dosing regimens may also be useful, depending on the pattern of pharmacokinetic decay the physician wishes to achieve.

[0094] In some embodiments, a subject in need of treatment (e.g., a human patient having an IgE-related allergic disease such as atopic dermatitis) may be administered an initial dose of an appropriate amount of antibody (e.g., 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-related disease, e.g., allergic reactions and / or elevated total IgE levels. If such symptoms are observed, a second dose of antibody may be administered to the subject. The initial and second doses may be administered to the subject at least 3 months apart, e.g., 3 months apart, 4 months apart, 5 months apart, 6 months apart, 9 months apart, or 12 months apart.

[0095] In some embodiments, a subject in need of treatment (e.g., a human patient with an IgE-related allergic disease such as atopic dermatitis) is administered a single dose of a high concentration antibody formulation. The antibody formulation may include an anti-IgE antibody of FB825.

[0096] Also within the scope of the present disclosure is the prophylactic treatment of IgE-related diseases using any of the anti-CεmX antibodies to reduce the risk of developing IgE-related diseases. Subjects suitable for such prophylactic treatment may be human patients with a medical history of IgE-related diseases and / or a family history of IgE-related diseases.

[0097] Depending on the type of disease or site of disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the art of medicine. The composition can also be administered orally, parenterally, via inhalation spray, topically, rectally, nasally, bucally, intravaginally, or via an implanted reservoir, etc. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via an injectable depot administration route, such as using 1, 3, or 6 month depot injectable or biodegradable materials and methods.

[0098] The particular dosing regimen used in the methods described herein, i.e., dosage, timing and frequency, will depend on the particular subject and the subject's medical history. Any of the anti-CεmX antibodies described herein can be used in combination with other agents (e.g., other agents for treating IgE-related disorders) that serve to enhance and / or complement the effectiveness of the agent.

[0099] In some embodiments, the anti-CεmX antibodies described herein, e.g., FB825, are used to treat atopic dermatitis as follows: Atopic dermatitis, also known as eczema, is a chronic skin disease characterized by redness and / or itching. It is common in children, but can occur at any age. Patients in need of treatment are identified by routine medical practice as having one or more symptoms of atopic dermatitis, including dry, itchy skin, red to brownish-gray patches, small raised bumps that leak fluid or scab when scratched, thickened, cracked, scaly skin, and / or raw, sensitive, swollen skin from scratching. In some examples, the total IgE levels and allergen-specific IgE levels of candidate subjects can be tested by routine practice. For example, the candidate subject's IgE level (e.g., total IgE, allergen-specific IgE, or both) is higher than a normal level (representing the average IgE level of subjects of the same species (e.g., humans) who are free of atopic dermatitis and other IgE-associated allergic disorders).

[0100] A human patient in need of treatment may be administered an initial dose of the antibody via conventional routes as described herein, which may range from 3 mg / kg to 8 mg / kg. In some instances, the initial dose is 5 mg / kg. After the initial dose, the patient's total IgE levels may be monitored. If there is less than a 50% decrease in IgE levels 3-4 weeks after the initial dose, a second dose of the antibody may be administered to the patient 3-4 weeks after the initial dose. The second dose may be the same as the initial dose or may be less than the first dose. In some instances, the initial and second doses are both 5 mg / kg and are administered via IV infusion over a period of 1-2 hours. Other biomarkers indicative of efficacy and / or safety may also be monitored over the course of 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 combinations thereof.

[0101] The human patient to be treated as described above may have chronic atopic dermatitis diagnosed by routine medical practice (e.g., as defined by the revised Eishenfield criteria of Hannifin and Rajka and supported by positive allergen-specific IgE) for at least three years. The patient may have one or more of the following characteristics: (i) Eczema Area and Severity Index (EASI) score greater than 14, (ii) Investigator's Global Assessment (IGA) score greater than 3 (on a 5-point scale), (iii) Body Surface Area (BSA) greater than 10%, (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 prior to treatment. Additionally, the human patient may be administered a stable dose of an emollient twice daily for at least seven days prior to treatment.

[0102] In some embodiments, the subject of the treatment disclosed herein is a human patient with low IgG4 level (e.g., low serum IgG4 level). Optionally, IgE level and IgG4 level are measured simultaneously. The human patient may suffer from or be suspected of suffering from an IgE-related allergic disease, such as atopic dermatitis.

[0103] IgG4 in body fluids such as serum can be detected by methods known in the art, for example, enzyme-linked immunosorbent assay (ELISA), alkaline phosphatase immunoassay autoanalyzers such as the EVEVIULITE® system (Siemens Healthcare Diagnostics, Erlangen, Germany), radioallergosorbent test (RAST), or quantitative assays discussed in WO2019 / 089978, including fluorescent enzyme immunoassay autoanalyzers such as the ImmunoCAP® system (Thermo Fisher Scientific / Phadia, Uppsala, Sweden). Additional suitable methods include fluorescent enzyme immunoassay (FEIA) autoanalyzers (such as the ImmunoCAP® system). Other techniques may be used, and the levels of antibodies (e.g., IgE or IgG4) determined by the techniques may be normalized to measurements by fluorescent enzyme immunoassay autoanalyzers. That is, the level of an antibody (e.g., IgE or IgG4) may be determined by certain techniques and may correspond to the level measured by a fluorescent enzyme immunoassay automated analyzer. Preferably, the level of a biomarker is determined in vitro.

[0104] The IgG4 levels obtained from the potential patient may be compared to a predefined value representing an IgG4 level that distinguishes between patients who respond to anti-IgE therapy and those who are less responsive to the therapy. Such predefined value may be based on an analysis of representative IgG4 levels in anti-IgE therapy responders versus non-anti-IgE therapy responders. The predefined value may take into account physiological characteristics consistent with the subject, such as age, sex, ethnic background, etc.

[0105] In some cases, the anti-CεmX antibody (e.g., FB825) described herein can be used in combination with moisturizers (e.g., at a stable dose, such as at least twice a day) and / or topical corticosteroids (TCS). Medium-potency TCS can be applied to areas with active lesions, and switched to low-potency TCS after the lesions are controlled. If lesions recur, treatment with medium-potency TCS can be resumed in a stepwise approach. If lesions persist or worsen after daily treatment with medium-potency TCS, high-potency or ultra-high-potency TCS can be used unless deemed unsafe. Low-potency TCS can be used in areas with thin skin (e.g., face, neck, intertriginous areas, genital areas, or areas of skin atrophy) or areas where continued use of medium-potency TCS is deemed unsafe.

[0106] TCS with low, medium, high or extra-high potency are well known in the art. Exemplary medium potency TCS include 0.05% fluticasone propionate cream, 0.1% mometasone furoate cream, or 0.06% betamethasone valerate cream. Exemplary low potency TCS include 1% hydrocortisone ointment. Exemplary high potency TCS can be 0.05% fluocinonide cream or 0.25% desomimethasone ointment. Exemplary extra-high potency TCS can be 0.05% clobetasol propionate ointment.

[0107] In some cases, patients receiving the treatments described herein have not received one or more of the following therapies: (i) topical tacrolimus and pimecrolimus, (ii) systemic corticosteroid therapy, (iii) leukotriene inhibitors, (iv) allergen immunotherapy, (v) systemic therapy with immunosuppressants or immunomodulators (e.g., cyclosporine, mycophenolate mofetil, IFN-γ, azathioprine, methotrexate, or biologics), (vi) live (attenuated) vaccines, and / or (vii) traditional Chinese medicine. Patients may also not have received any surgical and / or UV treatments.

[0108] Any of the methods described herein may further comprise evaluating the occurrence of hemoglobin reduction, 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 anti-CεmX antibody (e.g., FB825) in the second dose may be reduced. Alternatively, treatment may be discontinued.

[0109] III. Kits for use in treating IgE-related disorders The present disclosure also provides kits for use in treating an IgE-related disorder using any of the high concentration antibody formulations disclosed herein. Such kits may include one or more containers containing a high concentration antibody formulation (e.g., a formulation containing about 150 mg / ml of an antibody such as FB825, about 10 mM histidine, about 1.25% amino acids, about 75 mM sodium chloride, and about 0.01% polysorbate 80).

[0110] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The accompanying instructions may include instructions for administration of a high concentration antibody formulation to treat, delay onset, or alleviate an IgE-related disease according to any of the methods described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has, is suspected of having, or is at risk for the disease. Additionally, in another embodiment, the instructions include instructions for administering a high concentration antibody formulation to a subject in need of treatment to reduce the risk of developing an IgE-related disease.

[0111] Instructions for use of the highly concentrated antibody formulations typically include information regarding the dosage, administration schedule, and route of administration for the intended treatment. The containers may be unit dose, bulk packages (e.g., multi-dose packages), or small unit doses. Instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions recorded on a magnetic or optical memory disk) are also acceptable.

[0112] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or ameliorating an IgE-related disorder. Instructions for use can be provided for practicing any of the methods described herein.

[0113] The kit of the present disclosure is placed in suitable packaging.Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Also contemplated is packaging for use in combination with a specific device, such as an inhaler, a nasal administration device (e.g., an atomizer), or an injection device, such as a minipump.

[0114] The kit may optionally provide additional components such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described above.

[0115] General Technology The practice of the present disclosure 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 described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan 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 practice 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), DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985), Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985>>, Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984>>, Animal Cell Culture (R. I. Freshney, ed. (1986>>, Immobilized Cells and Enzymes (lRL Press, (1986>>, and are fully described in the literature such as B. Perbal, A practical Guide To Molecular Cloning (1984), F. M. Ausubel et al. (eds.).

[0116] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purposes and subject matter referenced herein.

[0117] Example 1: Screening for suitable surfactants FB825 was concentrated to 10mg / ml by 10kDa MWCO centrifugal concentrator, and then various candidate surfactants were added to reach the final desired concentration. The resulting formulation was stirred at room temperature for 4 hours, and then analyzed by SEC-HPLC and Micro-Flow Imaging (MFI). Subvisible particle size measurements and counts were collected by Brightwell Technologies MFI instrument. Digital filters were applied to the MFI data to remove air bubbles and static particles.

[0118] Three surfactants were investigated in this study: polysorbate 80 (P80, 0.01%), polysorbate 20 (P20, 0.01%), and Pluronic F68 (F68, 0.1%). MFI analysis showed that particle accumulation was observed in samples stirred without added surfactant, but particle accumulation was reduced by the addition of surfactant. Polysorbate 80 at 0.01% showed the lowest particle counts.

[0119] Example 2: Screening for suitable buffers and tonicity adjusters In this example, multiple buffers (acetate, histidine and phosphate), pH conditions (pH 4.0, 5.0, 6.0, 7.0 and 8.0), and tonicity modifiers (sodium chloride and sorbitol) were investigated to identify alternative components and concentrations for producing stable FB825 formulations. Table 1 below lists the components of the various F825 formulations tested in this example. [Table 1]

[0120] The formulations listed in Table 1 above were tested for stability by SEC-HPLC analysis under the agitation, freeze / thaw, light sensitivity, and temperature conditions shown in Table 2 below. [Table 2]

[0121] The results show that the formulation containing 10 mM histidine, 150 mM NaCl, 0.01% PS80 at pH 6.0 (H6N) has the best stability under the above test conditions. For example, formulation H6N shows the best stability after 8 weeks storage at 40° C. Table 3. [Table 3]

[0122] Example 3: Screening of Amino Acid Excipients, Stabilizers, and pH Conditions FB825 was concentrated to approximately 200 mg / ml using a 10 kDa MWCO centrifugal concentrator (20 mg / ml FB825 stock solution in 20 mM histidine and 140 mM NaCl, pH 6.5, 0.02% PS80). Concentrated FB825 formulations were dialyzed against 10 mM histidine, 75 mM NaCl, pH 6.0, and 0.01% PS80 or 10 mM histidine, pH 6.0, and 0.01% PS80 for screening of stabilizer and pH effects. Dialyzed samples were concentrated to approximately 200 mg / ml FB825 and used to characterize any amino acid excipients, stabilizers, and pH conditions.

[0123] (i) Amino acid excipient screening Formulations of FB825 each containing one of the 19 amino acids listed in Table 5 below were held at 50° C. for 48 hours and then examined for the physical characteristics also listed in Table 4. [Table 4]

[0124] The results are shown in Table 5 below. [Table 5-1] [Table 5-2]

[0125] As shown in Table 4 above, arginine, glycine, lysine, serine, and threonine exhibit good physical stability in this assay.

[0126] (b) Screening of stabilizers and pH conditions The formulations listed in Table 7 below, containing various stabilizers and pH conditions, were tested for the physical stability characteristics listed in Table 6 below. The results are shown in Tables 7 and 8. [Table 6] [Table 7-1] [Table 7-2]

[0127] According to this study, formulations containing the tested stabilizers (sucrose, sorbitol, or trehalose) showed a lower main peak percentage compared to the corresponding stabilizer-free formulations. Formulations R5.5, T6.0, and R6.5 showed the highest main peak percentage, indicating high stability after incubation at 55° C. for 48 hours. All arginine-containing formulations showed both a high main peak percentage and a low HMW peak percentage, indicating the stability of those formulations (e.g., less degradation and aggregation). [Table 8]

[0128] All formulations showed osmolality values ​​comparable to theoretical values, and most formulations were approximately isotonic with some variation.

[0129] (c) Stress optimization screening High concentration FB825 formulations (approximately 150 mg / ml) were stored at 45° C. for up to two weeks or at 55° C. for one week. These samples were then analyzed by SEC-HPLC. As shown in Table 9 below, formulations stored at 45° C. for up to two weeks showed no significant change in main peak value, while formulations stored at 55° C. for one week showed a larger decrease in the percentage of main peak (approximately 4%). [Table 9]

[0130] Additionally, the formulations listed in Table 10 were subjected to the same stability analysis after storage at 55° C. for 1 week, the results of which are shown in Table 11. [Table 10] [Table 11]

[0131] As shown in Table 11, the arginine-containing formulations showed the highest stability in the stress optimization screening assay. The arginine-containing formulations (R5.5, R6.0, and RR6.0) were clear, showed low turbidity values, and were isotonic. They also showed excellent injectable characteristics in the injectability study.

[0132] Example 4: Accelerated stability evaluation of high concentration formulations Formulations C150, R5.5, R6.0, and RR6.0 were further investigated in an accelerated stability assay with conditions shown in Table 12. [Table 12]

[0133] SEC-HPLC analysis shows that no significant changes were observed after the formulations were treated by agitation and freeze / thaw. Furthermore, no significant differences were observed among the four formulations after UV light irradiation. Furthermore, formulations R5.5, R6.0, and RR6.0 showed excellent physical stability, and no significant differences in purity were observed among the three formulations.

[0134] Example 5: Evaluation of long-term stability As shown in Table 13, formulations C10, R6.0, and RR6.0 were analyzed by visual inspection, protein concentration (A 280 ), pH, osmolality, and long-term stability analyses by SEC-HPLC, SCX-HPLC, and SDS-PAGE. [Table 13]

[0135] The SEC-HPLC method was carried out under the following conditions. Column: Tosoh TSK-Gel G3000W XL , 7.8mm ID X 30cm, 5μm P / N08541 Mobile phase: 4X Dulbecco's PBS (pH 7.3) Instrument: Agilent 1100 HPLC system · Flow rate: 0.4ml / min Column temperature: Ambient temperature Detection: 280nm Sample load: 20μg (diluted with mobile phase) Total Run Time: 30 minutes

[0136] The SCX-HPLC method was carried out under the following conditions. Column: Dinoex MabPac SCX-10, 4 x 250mm, 10μm P / N074625 Mobile phase A: 20 mM Tris (pH 7.2) Mobile phase A: 20 mM Tris and 0.2 M NaCl (pH 7.2) Instrument: Agilent 1100 HPLC system · Flow rate: 0.5ml / min Column temperature: 40 Autosampler: 4 Detection: 215nm Sample load: 50μg (diluted with mobile phase A) Total running time: 90 minutes

[0137] Table 14 below shows the SEC-HPLC and SCX-HPLC results for formulations C10, R6.0, and RR6.0 over a two year period. The high concentration formulations R6.0 and RR6.0 showed excellent stability over a two year storage period. [Table 14]

[0138] Alternative Embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein 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.

[0139] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to adapt the present invention to various applications and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the claims.

[0140] Equivalent While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention 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 on the particular application or applications utilizing the teachings of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it will be understood that the above embodiments are provided by way of example only, and that within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0141] All definitions and those used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0142] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are cited, and in some cases may include the entire document.

[0143] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0144] The term "and / or" as used herein and in the claims should be understood to mean "one or both" of the elements so connected, i.e., conjunctively indicated in some instances and disjunctively indicated in other instances. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so connected. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used with an open-ended term such as "comprising," can mean in one embodiment only A (optionally including elements other than B), in another embodiment only B (optionally including elements other than A), in yet another embodiment both A and B (optionally including other elements), and so forth.

[0145] "Or" as used in the specification and claims should be understood to have the same meaning as "and / or" defined above. For example, when separating elements of a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of some of the elements or list (including two or more of them as well) and optionally other unlisted items. Only when a term clearly indicates the contrary, such as "only one of" or "exactly one of," or "consisting of" as used in the claims, is meant to include some of the elements or exactly one element of the list. In general, the term "or" as used herein should be interpreted to indicate exclusive alternatives (i.e., "one or the other but not both") only when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, should have its ordinary meaning as used in the field of patent law.

[0146] As used in this specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to refer to 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 element specifically listed in the list of elements, and not necessarily excluding combinations of elements in the list of elements. This definition also permits the optional presence of elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the 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") refers to, in one embodiment, at least one A (or optionally more than one), where B is absent (and optionally including elements other than B), and at least one B (or optionally more than one), where A is absent (and optionally including elements other than A), and at least one B (or optionally more than one), and in yet another embodiment, at least one A (or optionally more than one), and at least one B (or optionally more than one), (and optionally including other elements).

[0147] It is also to be understood that, unless expressly indicated to the contrary, in any method claimed in the present invention that includes multiple steps or acts, the order of the method steps or acts is not necessarily limited to the recited order of the method steps or acts.

Claims

1. 1. An antibody formulation comprising: (a) an antibody that binds to the CεmX domain of membrane-bound IgE at a concentration of about 120-200 mg / ml; (b) histidine at a concentration of about 5-15 mM; and (c) an amino acid at a concentration of about 1-3.0% (w / v), where the amino acid is arginine or threonine; (d) sodium chloride at a concentration of about 50-100 mM; (e) polysorbate 80 at a concentration of about 0.005-0.02%; and Including, wherein the antibody formulation has a pH of about 5.5 to 6.

5.

2. 2. The antibody formulation of claim 1, wherein the formulation comprises about 150 mg / ml of the antibody, about 10 mM of the histidine, about 1.25% of the amino acids, about 75 mM of the sodium chloride, and about 0.01% of the polysorbate 80.

3. 2. The antibody formulation of claim 1, wherein the amino acid is arginine and the pH of the antibody formulation is about 6.0 to 6.

5.

4. 2. The antibody formulation of claim 1, wherein the amino acid is threonine and the pH of the antibody formulation is about 6.

0.

5. The antibody formulation of claim 1, wherein the antibody formulation consists essentially of (a) to (e).

6. The antibody formulation of claim 1, wherein the antibody formulation does not include a sugar or sugar alcohol based stabilizer.

7. 7. The antibody formulation of claim 6, wherein the sugar or sugar alcohol based stabilizer is sucrose, sorbitol, or trehalose.

8. The antibody formulation of claim 1, wherein the antibody that binds to the CεmX domain of membrane-bound IgE comprises the same heavy chain complementarity determining regions (CDRs) as antibody FB825 and / or the same light chain complementarity determining regions (CDRs) as antibody FB825.

9. The antibody formulation of claim 8 , wherein the antibody is a human antibody or a humanized antibody.

10. The antibody formulation of claim 8 , wherein the antibody is a full-length antibody.

11. 9. The antibody formulation of claim 8, wherein the antibody comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:9, and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

10.

12. The antibody formulation of claim 11 , wherein the antibody comprises a VH of SEQ ID NO:9 and a VL of SEQ ID NO:

10.

13. The antibody formulation of claim 8 , wherein the antibody is an IgG1 molecule.

14. The antibody formulation of claim 13, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:11, and a light chain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:

12.

15. A pre-filled syringe comprising the antibody formulation of any one of claims 1 to 14.

16. The pre-filled syringe of claim 15, wherein the amount of the antibody formulation in the pre-filled syringe is about 1 to 2 ml.

17. A pharmaceutical composition for treating an immunoglobulin E (IgE) associated disease in a subject, the pharmaceutical composition being an antibody formulation described in any one of claims 1 to 14.

18. The pharmaceutical composition of claim 17 , wherein the subject receives a single dose of the antibody formulation.

19. 20. The method of claim 17, wherein the subject receives at least two doses of the antibody formulation, the two consecutive doses being separated by at least three months.

20. The pharmaceutical composition of claim 17 , wherein the antibody formulation is administered subcutaneously.

21. 18. The pharmaceutical composition of claim 17, wherein the subject is a human patient having or suspected of having allergic asthma, allergic rhinitis, atopic dermatitis, or hyper-IgE syndrome.

22. 22. The pharmaceutical composition of claim 21, wherein the human patient has atopic dermatitis.

23. The pharmaceutical composition described in claim 22, wherein the human patient has a low serum IgG4 level.