Methods for treating antibody-mediated disorders with fcrn antagonists

Abstract

To provide methods for treating antibody-mediated disorders (e.g., autoimmune diseases).SOLUTION: The disclosure provides a method for treating an antibody-mediated disorder (e.g., autoimmune disease) by using an FcRn antagonist comprising a modified Fc domain, for example, a binding polypeptide (e.g., antibody and / or immunoadhesin). The disclosure also provides a method for enhancing diagnostic imaging using an FcRn antagonist disclosed herein. Further, the disclosure provides a method for reducing exposure of normal tissue to a radiolabeled antibody during diagnostic imaging using an FcRn antagonist disclosed herein. Also provided is a method for administering to a subject a therapeutically effective amount of an FcRn antagonist disclosed herein.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of U.S. Provisional Patent Application No. 62 / 878,541, filed on July 25, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format, the entire disclosure of which is incorporated herein by reference. The ASCII copy created on July 23, 2020 is named 022548_WO062_SL.txt and is 8,193 bytes in size.

Background Art

[0003] The interaction of antibodies with the neonatal Fc receptor (FcRn) is a determinant in maintaining and extending the serum half - life of antibodies and Fc - derived therapeutic agents. FcRn is a heterodimer of an MHC class - I - like α - domain and a β2 - microglobulin (β2 - m) subunit, and recognizes a region of the antibody Fc domain that is distinct from other Fcγ receptors (FcγR). FcRn is expressed in various tissues but is thought to act mainly in vascular endothelium, the kidney, and the blood - brain barrier.

[0004] Antibody binding to FcRn is highly pH - dependent, and the interaction occurs with high affinity (high nanomolar - low micromolar) only at low pH (pH < 6.5) and not at physiological pH (approximately pH 7.4). Acidification of the endosome to a pH below 6.5 makes the interaction between IgG and FcRn very favorable, directly causing inhibition of the degradation of FcRn - bound antibody and promoting recycling to the cell surface. An increase in pH weakens the interaction and promotes the release of the antibody into the bloodstream.

[0005] Enhanced binding likely results in increased efficacy and decreased dosing frequency for therapeutic antibodies as a direct result of an extended serum half-life compared to wild-type IgG antibodies, so Fc engineering using high-throughput mutagenesis approaches has been widely explored to identify variants that enhance FcRn binding affinity. However, variants that enhance FcRn binding affinity can have unanticipated consequences. For example, certain IgG variants that exhibit a substantial increase in FcRn affinity at pH 6.0, such as the N434W or P257I / Q311I substitutions in particular, have wild-type or severely decreased serum half-lives in transgenic mice with respect to cynomolgus and human FcRn (hFcRn) (see, e.g., Kuo et al., Mabs. (2011) 3(5):422-30; Datta-Mannan et al., J Biol Chem. (2007) 282:1709-17; and Datta-Mannan et al., Metab Dispos. (2007) 35:86-94). The T250Q / M428L (QL) variant showed Fv-specific effects in animal models (see, e.g., Datta-Mannan, 2007, supra; and Hinton et al., J Immunol. (2006) 176:346-56). The M252Y / S254T / T256E (YTE, EU numbering) variant showed a 10-fold enhancement in vitro but exhibited decreased antibody-dependent cell-mediated cytotoxicity (ADCC) in vivo due to a 50% decrease in affinity for the FcγRIIIa receptor (see, e.g., Dall’Acqua et al., J Immunol. (2002) 169(9):5171-80).

[0006] Fc variants that bind FcRn with enhanced affinity and reduced pH-dependence can find use in increasing the clearance of IgG from the bloodstream. FcRn-mediated IgG recycling has been found to be the dominant process for maintaining IgG plasma concentrations in humans (Xiao, J Biomed Biotechnol. (2012) 2012:282989). Increasing the clearance of IgG from the bloodstream would be desirable, for example, in autoimmune diseases such as systemic lupus erythematosus (a condition caused by circulating auto-reactive antibodies) and in situations where IgG-complexed toxins, drugs, or diagnostic agents need to be rapidly eliminated from the body. Increased clearance of unwanted antibodies can be achieved by using FcRn antagonists, such as antibodies with engineered Fc that binds FcRn with high affinity and does not dissociate rapidly at near-neutral pH. Such engineered antibodies are called Abdeg since they are antibodies that enhance IgG catabolism (Swiercz et al., J Nucl Med. (2014) 55(7):1204-7). Such FcRn antagonists are not released from cells but instead are predicted to remain bound to FcRn and block the binding of other, lower affinity IgG. As a result, FcRn function is blocked and endogenous or unwanted IgG will be directed to the lysosomal pathway for degradation. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] There is a need for a method of treating IgG-mediated diseases by using Fc variants that have an enhanced binding affinity for FcRn and exhibit a loss of pH-dependence in their binding to FcRn. MEANS FOR SOLVING THE PROBLEMS

[0008] The present disclosure provides a method of treating an antibody-mediated (e.g., IgG-mediated) disorder in a subject in need thereof, the method comprising administering to the subject (e.g., a human) a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain. The therapeutically effective amount of the FcRn antagonist increases the rate of serum IgG clearance in the subject being treated.

[0009] In some embodiments, the modified Fc domain: comprises aspartic acid (D) or glutamic acid (E) at amino acid position 256, and tryptophan (W) or glutamine (Q) at amino acid position 307, wherein amino acid position 254 is not threonine (T), and: further comprises phenylalanine (F) or tyrosine (Y) at amino acid position 434; and tyrosine (Y) at amino acid position 252, and comprises a combination of at least four amino acid substitutions. Unless otherwise indicated, all Fc residue positions described herein follow the EU numbering system.

[0010] In some embodiments, the modified Fc domain: a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; d) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434; e) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256; tryptophan (W) at amino acid position 307, and amino acid position 4 Tyrosine (Y) at position 34; f) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and phenylalanine (F) at amino acid position 434; g) tyrosine (Y) at amino acid position 252 and tyrosine (Y) at amino acid position 434; h) tyrosine (Y) at amino acid position 252, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; i) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, and tyrosine (Y) at amino acid position 434; or j) a combination of amino acid residues selected from the group consisting of aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434.

[0011] In certain embodiments, the modified Fc domain comprises a combination of amino acid substitutions selected from the group consisting of M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, M252Y / T256D / T307W / N434Y, M252Y / T256D / T307W / N434F, M252Y / N434Y, M252Y / T307W / N434Y, M252Y / T256D / N434Y, and T256D / 307W / N434Y.

[0012] In certain embodiments, the modified Fc domain is a modified human IgG Fc domain, such as a modified human IgG1, IgG2, IgG3, or IgG4 Fc domain, or an Fc domain derived from one or more human Fc domains.

[0013] In certain embodiments, the modified Fc domain has binding affinity for FcRn from one or more species, such as human, cynomolgus monkey, mouse, and rat. For example, the modified Fc domain has binding affinity for both human FcRn (hFcRn) and rat FcRn (rFcRn).

[0014] In certain embodiments, the modified Fc domain has enhanced FcRn binding affinity (e.g., enhanced binding affinity for hFcRn) compared to the wild-type Fc domain. In certain exemplary embodiments, the modified Fc domain has enhanced FcRn binding affinity at acidic pH (e.g., about 6.0) compared to the wild-type Fc domain. In certain exemplary embodiments, the modified Fc domain has enhanced FcRn binding affinity at acidic pH compared to an Fc domain having the quintuple mutation M252Y / S254T / T256E / H433K / N434F (“YTEKF”).

[0015] In certain embodiments, the modified Fc domain has enhanced FcRn binding affinity at non-acidic pH (e.g., about 7.4) compared to the wild-type Fc domain. In certain exemplary embodiments, the modified Fc domain has enhanced FcRn binding affinity at non-acidic pH compared to an Fc domain having the YTEKF mutation.

[0016] In certain embodiments, the modified Fc domain has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to the wild-type Fc domain. In certain exemplary embodiments, the modified Fc domain has enhanced FcRn binding affinity at acidic pH and enhanced FcRn binding affinity at non-acidic pH compared to an Fc domain having the YTEKF mutation.

[0017] In certain embodiments, the modified Fc domain has decreased FcγRIIIa binding affinity compared to the wild-type Fc domain.

[0018] In certain embodiments, the FcRn antagonist is or comprises a binding polypeptide, such as an antibody or an antigen-binding fragment thereof (e.g., Fv fragment, single-chain antibody (ScFv), Fab, Fab-H, Fab’, and F(ab’)2). The binding polypeptide may be one or more targets other than FcRn.

[0019] In some embodiments, the IgG-mediated disorder treated by the method is an autoimmune disease. In certain exemplary embodiments, the autoimmune disease is selected from the group consisting of graft-versus-host disease (GVHD), systemic lupus erythematosus (SLE), myasthenia gravis, systemic sclerosis (SSc) / scleroderma, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, diabetes, multiple sclerosis, pemphigus vulgaris, atopic dermatitis, psoriasis, asthma, allergy, idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic purpura (ITP), and hidradenitis suppurativa.

[0020] In certain aspects, the present disclosure provides a method of enhancing diagnostic imaging, the method comprising administering to a subject a therapeutically effective amount of an FcRn antagonist as described herein. In certain embodiments, the method further comprises administering to the subject an effective amount of a radiolabeled antibody. In further embodiments, the FcRn antagonist is administered after the radiolabeled antibody and enhances the contrast against the radiolabeled antibody.

[0021] In certain aspects, the present disclosure provides a method of reducing exposure of non-target tissue to a radiolabeled antibody during diagnostic imaging, the method comprising administering to a subject a therapeutically effective amount of an FcRn antagonist as described herein.

[0022] Also provided is the use of the FcRn antagonist herein for the manufacture of a medicament for use in the methods of treatment and diagnosis and for use in the methods of treatment and diagnosis.

[0023] The present disclosure also provides nucleic acids encoding FcRn antagonist polypeptides, recombinant expression vectors and host cells for producing the polypeptides, and pharmaceutical compositions comprising the FcRn antagonists disclosed herein. Methods of using the FcRn antagonists of the present disclosure for treating diseases are also provided.

[0024] The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of the exemplary embodiments, taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0025]

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Modes for Carrying Out the Invention

[0026] The present disclosure provides polypeptides (e.g., monoclonal antibodies or fragments thereof) having an altered FcRn binding affinity. These polypeptides contain a modified IgG Fc domain, which has an enhanced FcRn binding affinity under both acidic and non-acidic conditions as compared to the wild-type IgG (e.g., human IgG1, IgG2, IgG3, or IgG4) Fc domain. Thus, unlike the wild-type IgG Fc domain, the present polypeptides bind to FcRn in a less pH-dependent manner. These polypeptides remain bound to FcRn while FcRn is being transported between the cell surface and the cytoplasm having different pH conditions. As a result, other IgG molecules having a wild-type (lower) FcRn binding affinity (e.g., serum IgG) are blocked from binding to FcRn and are directed to the lysosomal pathway for degradation at an increased rate, resulting in faster clearance of IgG molecules from the body.

[0027] Due to disrupting the normal function of their FcRn in mediating IgG recycling and maintaining serum IgG levels, these polypeptides are referred to as "FcRn antagonists". The FcRn antagonists of the present disclosure may be antibodies, immunoadhesins or another form of Fc fusion protein, or Fc fragments or portions thereof. The FcRn antagonist may be, for example, a monomeric protein or a dimeric (hetero- or homodimeric) protein. The FcRn antagonist is used to enhance serum IgG clearance from the body, and they can be used to treat patients suffering from IgG-mediated diseases or disorders (e.g., autoimmune diseases), or to remove unwanted IgG from the body (e.g., therapeutic or diagnostic antibodies that are no longer needed or beneficial).

[0028] The methods described in this disclosure are not limited to specific methods, and it is understood that the experimental conditions disclosed herein as such methods and conditions may vary. It is also understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0029] Furthermore, the experiments described herein use conventional molecular and cell biological and immunological techniques within the scope of those skilled in the art, unless otherwise indicated. Such techniques are well known to those skilled in the art and are described in sufficient detail in the literature. For example, see Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987 - 2008), including all supplements; Green et al., Eds., Molecular Cloning: A Laboratory Manual (Fourth Edition); Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2 nd edition).

[0030] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. In the event of potential ambiguity, the definitions provided herein shall prevail over those in a dictionary or external definition. Unless the context requires otherwise, the singular forms shall include the plural, and the plural forms shall include the singular. The use of "or" shall mean "and / or" unless otherwise stated. The use of terms in other forms such as "including", "includes", and "included" is not limiting. Variations of terms such as "have", "comprise", "has", "having", "comprises", or "comprising" are understood to mean including the recited integer or group of integers, but not excluding other integers or groups of integers.

[0031] Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and, unless otherwise indicated, are as described in various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, as commonly practiced in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and delivery, and for the treatment of patients. All publications and other references mentioned herein are incorporated by reference in their entirety. For the sake of easier understanding of the present disclosure, selected terms are defined below.

[0032] The term "polypeptide" refers to a polymeric chain of amino acids and, where the context is not inconsistent, includes natural or artificial proteins, polypeptide analogs, or variants of protein sequences , or fragments thereof. A polypeptide may be monomeric or polymeric; that is, the term includes proteins having one or more polypeptide chains coupled covalently or noncovalently. Polypeptide fragments include, for example, at least about 5 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids.

[0033] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that is not associated with the natural, associated components that accompany it in its natural state due to its origin or source; does not substantially contain other proteins from the same species; is not expressed by cells from a different species; or does not exist in nature. Thus, a protein or polypeptide that is chemically synthesized or synthesized in a cell line different from the cell from which it is naturally derived is "isolated" from its natural, associated components. A protein or polypeptide can also be made substantially free of its natural, associated components by isolation using protein purification techniques well known in the art.

[0034] As used herein, the term "binding protein" or "binding polypeptide" shall refer to a protein or polypeptide (e.g., an antibody or an immunoadhesin) that contains at least one binding site that is responsible for selectively binding to a target antigen of interest (e.g., a human target antigen). Exemplary binding sites include antibody variable domains, receptor ligand-binding sites, or ligand receptor-binding sites. In certain embodiments, a binding protein or binding polypeptide includes multiple (e.g., two, three, four, or more) binding sites. In certain embodiments, a binding protein or binding polypeptide is not a therapeutic enzyme.

[0035] The term "ligand" refers to any substance that can bind to or receive binding from another substance. Similarly, the term "antigen" refers to any substance against which an antibody can be generated. "Antigen" is generally used with reference to an antibody-binding substrate, and "ligand" is often used when referring to a receptor-binding substrate, but these terms are not distinguished from each other and encompass a wide range of overlapping chemical entities. To avoid doubt, antigens and ligands are used interchangeably throughout this specification. An antigen / ligand can be a peptide, polypeptide, protein, aptamer, polysaccharide, sugar molecule, carbohydrate, lipid, oligonucleotide, polynucleotide, synthetic molecule, inorganic molecule, organic molecule, and any combination thereof.

[0036] As used herein, the term "specifically binds" refers to the ability of an antibody or immunoadhesin to bind to an antigen with a dissociation constant (Kd) of at most about 1×10 -6 M, about 1×10 -7 M, about 1×10 -8 M, about 1×10 -9 M, about 1×10 -10 M, about 1×10 -11 M, about 1×10 -12 M or less, and / or to bind to an antigen with an affinity that is at least about two-fold greater than its affinity for a non-specific antigen.

[0037] As used herein, the term "antibody" refers to an assembly (e.g., an intact antibody molecule, an immunoadhesin, or a variant thereof) having a significant known specific immunoreactive activity against an antigen of interest. Antibodies and immunoglobulins include light and heavy chains that may or may not have interchain covalent bonds between them. The basic immunoglobulin structure in the vertebrate system is relatively well understood.

[0038] As will be discussed in more detail below, the general term "antibody" includes five different classes of antibodies that can be biochemically distinguished. Although all five classes of antibodies are clearly within the scope of the present disclosure, the discussion herein generally relates to immunoglobulin molecules of the IgG class. With respect to IgG, an immunoglobulin comprises two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000 - 70,000. The four chains are joined by disulfide bonds in a "Y" configuration, where the light chains are attached to the heavy chains, starting at the mouth of the "Y" and continuing through the variable regions.

[0039] The light chains of immunoglobulins are classified into either kappa (κ) or lambda (λ). Each heavy chain class can bind to either a kappa or a lambda light chain. Generally, the light and heavy chains are covalently bound to each other, and the "tail" portions of the two heavy chains are bound to each other by disulfide covalent bonds or non-covalent bonds (when the immunoglobulin is produced by any of a hybridoma, B cell, or genetically engineered host cell). In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the "Y" configuration to the bottom of each chain at the C-terminus. As will be readily appreciated by those skilled in the art, the heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) (among which there are several subclasses (e.g., γ1 - γ4)). It is this nature of the chain that determines the "class" or "isotype" of the antibody to be IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin isotype subclasses or subtypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well-characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily recognizable to those skilled in the art in view of the present disclosure and are thus within the scope of the present disclosure.

[0040] Both the light and heavy chains are divided into regions of structural and functional homology. The term "region" refers to a part or portion of an immunoglobulin or antibody chain and includes the constant or variable regions as well as further distinct parts or portions of said regions. For example, the light chain variable region includes "complementary determining regions" or "CDRs" incorporated between "framework regions" or "FRs" as defined herein.

[0041] Regions of an immunoglobulin heavy or light chain can be defined as "constant" (C) or "variable" (V) regions based on relatively little sequence variation within the regions of various class members in the case of "constant regions", or based on significant variation within the regions of various class members in the case of "variable regions". The terms "constant region" and "variable region" can also be used functionally. In this context, it is of course the case that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody confers important effector functions such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. The subunit structure and three-dimensional arrangement of the constant regions of the various immunoglobulin classes are well known.

[0042] The constant and variable regions of immunoglobulin heavy and light chains fold into domains. The term "domain" refers to a globular region of a heavy or light chain that includes, for example, β-pleated sheets and / or peptide loops stabilized by intrachain disulfide bonds (e.g., including 3 - 4 peptide loops). The constant region domain of the light chain of an immunoglobulin is interchangeably referred to as the "light chain constant region domain", the "CL region" or the "CL domain". The constant domains of the heavy chain (e.g., the hinge, CH1, CH2 or CH3 domains) are interchangeably referred to as the "heavy chain constant region domains", the "CH" region domains or the "CH domains". The variable domain of the light chain is interchangeably referred to as the "light chain variable region domain", the "light chain variable region", the "VL region domain" or the "VL domain". The variable domain of the heavy chain is interchangeably referred to as the "heavy chain variable region domain", the "heavy chain variable region", the "VH region domain" or the "VH domain".

[0043] By convention, the amino acid numbering of the variable constant region domains increases as they are further from the antigen binding site or the amino terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light chain immunoglobulin chain is the variable region, and the C-terminus is the constant region. The CH3 and CL domains include the carboxy termini of the heavy and light chains, respectively. Thus, the domains of the light chain immunoglobulin are arranged in a VL-CL orientation, while the domains of the heavy chain are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0044] The assignment of amino acids to each variable region domain follows the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used conventional numbering method (Kabat numbering), in which corresponding residues between different heavy chain variable regions or different light chain variable regions are assigned the same number. CDR1, 2, and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, or LCDR1, LCDR2, and LCDR3, respectively. CDR1, 2, and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, or HCDR1, HCDR2, and HCDR3, respectively. When shown as such, the assignment of CDRs follows IMGT® (Lefranc et al., Dev Comp Immunol. (2003) 27:55-77) instead of Kabat. The numbering of the heavy chain constant region follows the EU index as described in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0045] As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain.

[0046] As used herein, the term "CH1 domain" includes, for example, the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain extending from approximately positions 114 to 223 (EU positions 118 to 215) in the Kabat numbering system. The CH1 domain is adjacent to the VH domain, is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule, and does not form part of the Fc region of the immunoglobulin heavy chain.

[0047] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues, is flexible, and thus allows the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol. (1998) 161:4083).

[0048] As used herein, the term "CH2 domain" includes, for example, a portion of the heavy chain immunoglobulin molecule extending from approximately positions 244 to 360 (EU positions 231 to 340) in the Kabat numbering system. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched hydrocarbon chains are inserted between the two CH2 domains of an intact native IgG molecule. In one embodiment, the binding polypeptide of the present disclosure includes a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0049] As used herein, the term "CH3 domain" includes a portion of the heavy chain immunoglobulin molecule extending approximately 110 residues from the N-terminus of the CH2 domain, for example, approximately positions 361 to 476 (EU positions 341 to 447) of the Kabat numbering system. The CH3 domain is Typically forms the C-terminal portion of an antibody. However, in some immunoglobulins, additional domains can extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the ε chain of IgE). In one embodiment, the binding polypeptide of the present disclosure comprises a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0050] As used herein, the term “CL domain” includes the constant region domain of an immunoglobulin light chain that extends, for example, from Kabat position approximately 107A to Kabat position approximately 216. The CL domain is adjacent to the VL domain. In one embodiment, the binding polypeptide of the present disclosure comprises a CL domain derived from a kappa light chain (e.g., a human kappa light chain).

[0051] As used herein, the term “FcRn antagonist” refers to any agent that specifically binds to FcRn via the Fc region and inhibits the binding of an immunoglobulin to FcRn, and includes an Fc region (e.g., a variant Fc region disclosed herein).

[0052] As used herein, the terms “Fc,” “Fc domain,” “Fc region,” or “Fc fragment” are used interchangeably and are defined as part of the heavy chain constant region that starts at the hinge region immediately N-terminal to the papain cleavage site (i.e., residue 216 of IgG, with the first residue of the heavy chain constant region being 114) and ends at the C-terminus of the heavy chain. Thus, a full Fc, Fc domain, Fc region, or Fc fragment includes at least the hinge region, CH2 domain, and CH3 domain. An example of a sequence alignment of portions of human IgG1, IgG2, IgG3, and IgG4 Fc domains (CH2 and CH3 domains, residues 231 to 447, Eu numbering) is shown in FIG. 32. The terms encompass the native / wild-type Fc and Fc variants described herein and include molecules in monomeric or multimeric (e.g., dimeric) forms, whether digested from whole antibodies or produced by other means, such as recombinant techniques. See, e.g., Ying et al., JBC (2013) 288:25154-164; and Yang et al., JBC (2019) 294:10638-48. In some embodiments, the modified Fc of the disclosure comprises the entirety of SEQ ID NO: 1, 2, 3, or 4 shown in FIG. 32 or the FcRn-binding portion (or a naturally occurring variant thereof), wherein the sequence is modified to include the amino acid substitutions described herein.

[0053] The original immunoglobulin source of native Fc is typically of human origin and can be any immunoglobulin, such as IgG1 and IgG2. Native Fc molecules are composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent bonds to form dimers or multimers. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgA2). An example of native Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. As used herein, the term “native Fc” is a general term for monomeric, dimeric, and multimeric forms.

[0054] As used herein, the terms "Fc variant", "modified Fc", or "modified Fc domain" refer to a molecule or sequence that has been modified from native / wild-type Fc but still contains a binding site for FcRn. An Fc variant or modified Fc domain may also be shorter or longer than native Fc (e.g., shorter or longer than the sequence spanning residues 216 to 447 of human IgG in Eu numbering); for example, an Fc variant or modified Fc may lack certain N-terminal and / or C-terminal amino acid residues of native Fc, or may contain additional amino acid residues at the N-terminal and / or C-terminal compared to native Fc. The modified Fc domain itself does not include the antigen-binding domain of an antibody or antibody variant, or the target-binding domain of an immunoadhesin, although the modified Fc domain may be linked to such a domain to form an FcRn antagonist that also binds to a non-FcRn target. The terms include molecules or sequences that have been humanized from non-human native Fc. Furthermore, native Fc contains regions that can be removed because they do not provide structural features or biological activities necessary for the FcRn antagonists described herein (e.g., antibody-like binding polypeptides). Thus, the terms include: (1) molecules or sequences lacking one or more native Fc sites or residues that affect or are involved in disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor (FcγR), or (7) antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or molecules or sequences in which one or more Fc sites or residues have been modified. (1) Disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor (FcγR), or (7) antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or molecules or sequences lacking one or more native Fc sites or residues that affect or are involved in these, or molecules or sequences in which one or more Fc sites or residues have been modified.

[0055] As shown above, the variable regions of an antibody enable the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of the antibody combine to form a variable region (Fv) that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites present at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) on each of the variable regions of the heavy and light chains. As used herein, the term "antigen-binding site" includes a site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble antigen). The antigen-binding site includes immunoglobulin heavy and light chain variable regions, and the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by variable regions that vary for each antibody. An FcRn-antagonizing binding polypeptide, e.g., an antibody of the present disclosure, includes at least one antigen-binding site.

[0056] In certain embodiments, the binding polypeptide of the present disclosure includes at least two antigen-binding domains that provide for the association of the binding polypeptide with a selected antigen. The antigen-binding domains need not necessarily be derived from the same immunoglobulin molecule. In this context, the variable regions can be derived from or be of any type of animal that can be introduced to initiate a humoral response and generate immunoglobulins against a desired antigen. As such, the variable regions of the binding polypeptide can be of mammalian origin, e.g., human, mouse, rat, goat, sheep, non-human primates (e.g., cynomolgus monkey, rhesus monkey, macaque monkey, etc.), lupine, or camelid (e.g., camel, llama, and related species).

[0057] In naturally occurring antibodies, assuming that the antibody adopts its three-dimensional configuration in an aqueous environment, the six CDRs present in each antibody are short discontinuous sequences of amino acids that are specifically positioned to form the antigen-binding site. The remaining parts of the variable domains of the heavy and light chains show lower inter-molecular variability in the amino acid sequence and are called framework regions. The framework regions mostly adopt a β-sheet conformation, and the CDRs form loops that connect the β-sheet structures and in some cases form part of the β-sheet structure. Thus, these framework regions act to form a scaffold that provides the correct orientation of the six CDRs by inter-chain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines the surface complementarity to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to the immunoreactive antigen epitope.

[0058] Exemplary binding polypeptides include antibody variants. As used herein the term "antibody variant" includes synthetic and engineered forms of antibodies that have been altered so that they do not occur naturally, e.g., antibodies that contain at least two heavy chain portions but do not contain two full heavy chains (e.g., domain-deleted antibodies or minibodies); antibodies in a multispecific form that have been altered to bind to two or more different antigens or to different epitopes on a single antigen (e.g., bispecific, trispecific, etc.); heavy chain molecules conjugated to scFv molecules, and the like. Further, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four or more copies of the same antigen).

[0059] As used herein, the term "valence" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on a target molecule. If a polypeptide contains more than one target binding site, each target binding site may specifically bind to the same or different molecules (e.g., different ligands or different antigens, or different epitopes on the same antigen). The binding polypeptide of the subject typically has at least one binding site specific for a human antigen molecule.

[0060] The term "specificity" refers to the ability to specifically bind (e.g., immunoreact) to a given target antigen (e.g., a human target antigen). A binding polypeptide can be monospecific, containing one or more binding sites that specifically bind to a target, or the polypeptide can be multispecific, containing two or more binding sites that specifically bind to the same or different targets. In certain embodiments, the binding polypeptide is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the binding polypeptide is specific for more than one target. Binding polypeptides (e.g., antibodies) containing antigen binding sites that bind to an antigen are known in the art, and one or more CDRs from such antibodies can be included in the antibodies described herein.

[0061] As used herein, the term "antigen" or "target antigen" refers to a molecule or a portion of a molecule capable of being bound by a binding site of a binding polypeptide. A target antigen can have one or more epitopes.

[0062] The term "about" or "approximately" means within about 20% of a given value or range, e.g., within about 10%, within about 5%, or within about 1% or less.

[0063] As used herein, "administer" or "administration" refers to the act of introducing or otherwise physically delivering a substance that exists outside the body (e.g., an FcRn antagonist provided herein) to a patient by, but not limited to, for example, the lungs (e.g., inhalation), mucosa (e.g., intranasal), intradermal, intravenous, intramuscular, subcutaneous delivery, and / or any other method of physical delivery described herein or known in the art. When a disease or its symptoms are being managed or treated, administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or its symptoms are being prevented, administration of the substance typically occurs before the onset of the disease or its symptoms and may be continued for a long time to delay or reduce the appearance or magnitude of disease-related symptoms.

[0064] As used herein, the term "composition" is intended to encompass a product that contains a specified ingredient (e.g., an FcRn antagonist provided herein), optionally in a specified amount, and any product that results directly or indirectly from a combination of the specified ingredients, optionally in a specified amount.

[0065] "Effective amount" means an amount of an active agent (e.g., an FcRn antagonist of the present disclosure) sufficient to achieve a desired physiological result in an individual in need of such agent. The effective amount can vary among individuals depending on the health and physical condition of the individual being treated, the formulation of the composition, the assessment of the individual medical condition, and other relevant factors.

[0066] As used herein, the terms "subject" and "patient" are used interchangeably. A subject as used herein is a mammal, e.g., a non-primate (e.g., mouse, rat, cow, pig, horse, cat, dog, etc.) or a primate (e.g., monkey and human). In a preferred embodiment, the subject is a human.

[0067] As used herein, the term "treatment" refers to any protocol, method and / or agent that can be used to prevent, manage, treat, and / or alleviate a disease or a symptom associated therewith. In some embodiments, the term "treatment" refers to any protocol, method and / or agent that can be used to modulate an IgG-mediated disease (e.g., an autoimmune response) or a condition of a subject or a symptom associated therewith. In some embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies useful in preventing, managing, treating, and / or alleviating a disease or a symptom associated therewith known to those of ordinary skill in the art such as medical personnel. In other embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies useful in modulating an immune response or a symptom associated therewith in a subject known to those of ordinary skill in the art such as medical personnel.

[0068] As used herein, the terms "treating", "treatment" and "treatment thereof" refer to a decrease or alleviation in the progression, severity and / or duration of a disease or a symptom associated therewith that results from the administration of one or more treatments (including, but not limited to, the administration of one or more prophylactic or therapeutic agents such as the FcRn antagonists provided herein). The term "treatment thereof" as used herein may also refer to altering the disease course of a subject being treated. Therapeutic effects of treatment include, without limitation, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing direct or indirect pathological consequences of a disease, reducing the rate of disease progression, alleviating or ameliorating a disease state, and remission or improved prognosis.

[0069] FcRn antagonist The FcRn antagonists of the present invention include (including consisting of or consisting essentially of) a modified Fc domain and may be, for example, an antibody, an antibody variant or fragment, such as an Fc fragment, an immunoadhesin, and an Fc fusion protein. The FcRn antagonist may include a monomeric or dimeric Fc fragment.

[0070] The FcRn of the present disclosure can be generated using the Fc domain from any IgG subtype. In some embodiments, the modified Fc is derived from a human IgG1, IgG2, IgG3, or IgG4 Fc domain and includes the substitutions described herein compared to its wild-type origin. In certain other embodiments, the modified Fc is an artificial Fc derived from more than one IgG subtype. In other embodiments, the Fc domain includes a chimeric hinge (i.e., a hinge containing a hinge portion derived from hinge domains of different antibody isotypes, such as the upper hinge domain from an IgG4 molecule and the IgG1 middle hinge domain).

[0071] In certain embodiments, the modified Fc domain is derived from a human IgG1 Fc domain. In other embodiments, the modified Fc domain is derived from a human IgG4 Fc domain. For Fc domains of other subtypes, it will be apparent to those skilled in the art that any of the amino acid substitutions described herein can be suitably adapted (see Figure 32). Unless otherwise indicated, the Fc residue positions described herein follow the EU Ig numbering system. As seen in Figure 32, the human IgG CH2 and CH3 domains are highly conserved among the four different subtypes. The Eu numbering system applies to all subtypes.

[0072] In some embodiments, the modified Fc domain may include amino acid substitutions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, or Y436, and any combination thereof. In some embodiments, the modified Fc domain may include double amino acid substitutions at any two amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436. In some embodiments, the modified Fc domain may include triple amino acid substitutions at any three amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436. In some embodiments, the modified Fc domain may include quadruple amino acid substitutions at any four amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436. In some embodiments, it may be desirable for the modified Fc domain to include amino acid substitutions at any amino acid position selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, or Y436, and any combination thereof, where the amino acid position N434 is not substituted (i.e., the amino acid position N434 is wild-type).

[0073] In some embodiments, the modified Fc domain may comprise amino acid substitutions selected from M252Y (i.e., tyrosine at amino acid position 252), T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, N434F, N434P, N434Y, Y436H, Y436N, or Y436W, and any combination thereof. In some embodiments, the modified Fc domain may comprise double amino acid substitutions selected from M252 [where the substitution is M252Y]; T256 [where the substitution is T256D, or T256E]; K288 [where the substitution is K288D, or K288N]; T307 [where the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W]; E380 [where the substitution is E380C]; N434 [where the substitution is N434F, N434P, or N434Y]; Y436 [where the substitution is Y436H, Y436N, or Y436W]. In some embodiments, the modified Fc domain may comprise triple amino acid substitutions selected from M252 [where the substitution is M252Y]; T256 [where the substitution is T256D, or T256E]; K288 [where the substitution is K288D, or K288N]; T307 [where the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W]; E380 [where the substitution is E380C]; N434 [where the substitution is N434F, N434P, or N434Y]; Y436 [where the substitution is Y436H, Y436N, or Y436W].In some embodiments, the modified Fc domain may comprise a quadruple amino acid substitution selected from M252 [where the substitution is M252Y]; T256 [where the substitution is T256D, or T256E]; K288 [where the substitution is K288D, or K288N]; T307 [where the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W]; E380 [where the substitution is E380C]; N434 [where the substitution is N434F, N434P, or N434Y]; Y436 [where the substitution is Y436H, Y436N, or Y436W]. In some embodiments, the modified Fc. domain may desirably or optionally contain an amino acid substitution at any of the amino acid positions selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, and the amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, the modified Fc domain may desirably or optionally contain an amino acid substitution at any of the amino acid positions selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, and the amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, the modified Fc domain may desirably or optionally contain an amino acid substitution at any of the amino acid positions selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N, or Y436W, and any combination thereof, and the amino acid position N434 is not substituted (i.e., the amino acid position N434 is wild-type).

[0074] In certain embodiments, the modified Fc domain can include amino acid substitutions selected from M252, T256, T307, or N434, and any combination thereof. In certain embodiments, the modified Fc domain can include double amino acid substitutions at any two amino acid positions selected from M252, T256, T307, and N434. In certain embodiments, the modified Fc domain can include triple amino acid substitutions at any three amino acid positions selected from M252, T256, T307, and N434. In certain embodiments, the modified Fc domain can include quadruple amino acid substitutions at amino acid positions M252, T256, T307, and N434. In some embodiments, it may be desirable for the modified Fc domain to include amino acid substitutions selected from M252, T256, or T307, and any combination thereof, where amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0075] In an exemplary embodiment, the modified Fc domain may comprise amino acid substitutions selected from M252 [where the substitution is M252Y]; T256 [where the substitution is T256D or T256E]; T307 [where the substitution is T307Q or T307W]; or N434 [where the substitution is N434F or N434Y], and any combination thereof. In certain embodiments, the modified Fc domain may comprise double amino acid substitutions at any two amino acid positions selected from M252 [where the substitution is M252Y]; T256 [where the substitution is T256D or T256E]; T307 [where the substitution is T307Q or T307W]; or N434 [where the substitution is N434F or N434Y]. In certain embodiments, the modified Fc domain may comprise triple amino acid substitutions at any three amino acid positions selected from M252 [where the substitution is M252Y]; T256 [where the substitution is T256D or T256E]; T307 [where the substitution is T307Q or T307W]; or N434 [where the substitution is N434F or N434Y]. In certain embodiments, the modified Fc domain may comprise quadruple amino acid substitutions at amino acid positions selected from M252 [where the substitution is M252Y]; T256 [where the substitution is T256D or T256E]; T307 [where the substitution is T307Q or T307W]; or N434 [where the substitution is N434F or N434Y]. In some embodiments, it may be desirable for the modified Fc domain to comprise amino acid substitutions selected from M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, where amino acid position N434 is ph It is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to include amino acid substitutions selected from M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, where amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to include amino acid substitutions selected from M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, where amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0076] In certain embodiments, the modified Fc domain may include an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, and may further include an amino acid substitution selected from N434F, or N434Y, or M252Y. In some embodiments, it may be desirable for the modified Fc domain to include an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, and further include the amino acid substitution M252Y, where amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to include an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, and further include the amino acid substitution M252Y, where amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, it may be desirable for the modified Fc domain to include an amino acid substitution selected from T256D, or T256E, and / or T307W, or T307Q, and further include the amino acid substitution M252Y, where amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0077] In some embodiments, the modified Fc domain can include the amino acid substitutions shown in FIG. 33. For example, the modified Fc domain can include the double amino acid substitution M252Y / N434Y (YY); or the triple amino acid substitution selected from M252Y / T307W / N434Y (YWY), M252Y / T256D / N434Y (YDY), and T256D / 307W / N434Y (DWY).

[0078] In some embodiments, the modified Fc domain can include the quadruple amino acid substitution selected from M252Y / T256D / T307Q / N434F (YDQF), M252Y / T256D / T307W / N434F (YDWF), M252Y / T256D / T307Q / N434Y (YDQY), M252Y / T256E / T307Q / N434Y (YEQY), M252Y / T256D / T307W / N434Y (YDWY), and M252Y / T256E / T307W / N434Y (YEWY).

[0079] In some embodiments, the modified Fc domain has one or more, such as two or more, three or more, or four or more amino acid substitutions disclosed herein.

[0080] The modified Fc has enhanced FcRn binding affinity at both acidic pH (e.g., less than about 7.0, less than or equal to about 6.5, or less than or equal to about 6.0) and non-acidic pH (e.g., greater than or equal to about 7.0, or greater than or equal to about 7.4) compared to its wild-type counterpart.

[0081] As used herein, the term "enhanced" refers to a 10% increase in a given parameter, at least a 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, 95% increase, 97% increase, 99% increase, or even a 1 It may include a 00% increase. The term "enhanced" may refer to an increase of at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold of a given parameter relative to a control, baseline, or previous in-time value. In certain embodiments, the term "enhanced" refers to an increase compared to a wild-type standard, e.g., a wild-type Fc domain. In other embodiments, the term "enhanced" refers to an increase compared to an FcRn antagonist, e.g., an FcRn antagonist comprising M252Y / S254T / T256E / H433K / N434F ("YTEKF hIgG1"; see, e.g., Swiercz et al., supra).

[0082] In some embodiments, the FcRn antagonist may exhibit species-specific FcRn binding affinity. In some embodiments, the FcRn antagonist may exhibit both human and cynomolgus (cyno) FcRn binding affinity. In one embodiment, the binding polypeptide may exhibit rat and / or mouse FcRn binding affinity. In some embodiments, the binding polypeptide may exhibit cross-species FcRn binding affinity. Such binding polypeptides are said to be cross-reactive across one or more different species. In one embodiment, the binding polypeptide may exhibit both human and rat FcRn binding affinity.

[0083] The neonatal Fc receptor (FcRn) interacts with the Fc region of an antibody and promotes recycling by rescue from normal lysosomal degradation. This process occurs in endosomes at acidic pH (e.g., pH less than 6.5), but not under physiological pH conditions of the bloodstream (e.g., non-acidic pH), and is a pH-dependent process. The FcRn antagonists of the present disclosure have enhanced FcRn binding affinity or slower FcRn dissociation rates under both acidic and non-acidic conditions compared to polypeptides containing the wild-type Fc domain. Acidic pH is a pH less than about 7.0, such as about pH 6.5, about pH 6.0, about pH 5.5, about pH 5.0. Elevated non-acidic pH is a pH of about 7 or greater, such as about pH 7.4, about pH 7.6, about pH 7.8, about pH 8.0, about pH 8.5, or about pH 9.0. In certain embodiments, the polypeptides containing the modified Fc domains described herein exhibit a loss of pH-dependence in FcRn binding; that is, unlike wild-type Fc, the polypeptide does not have a dramatically lower binding affinity for FcRn under non-acidic conditions compared to acidic conditions. Thus, such polypeptides are useful as FcRn antagonists that promote clearance of IgG having wild-type FcRn binding properties from circulation, because the FcRn antagonist binds tightly to FcRn in a pH-independent or lower pH-dependent manner, disrupting FcRn-mediated recycling of IgG having wild-type (i.e., lower) FcRn binding affinity.

[0084] The Fc domain of an immunoglobulin is involved in non-antigen-binding functions and has several effector functions mediated by binding to Fc receptors, such as binding to FcRn. As illustrated in FIG. 1A, the Fc domain is composed of a CH2 domain and a CH3 domain. Most of the Fc residues involved in interaction with FcRn are located in a loop that is directly adjacent to the CH2-CH3 interface (FIG. 1A, dotted line) and on the opposite side of the glycosylation site. FIG. 1B shows a surface depiction of the IgG1 Fc crystal structure (pdb: 5d4q) and indicates residues in the CH2 and CH3 domains that constitute the FcRn binding interface.

[0085] In certain embodiments, the FcRn antagonist of the invention can include a modified Fc domain that includes one or more amino acid mutations (e.g., substitutions), which, compared to the corresponding wild-type molecule, such as a molecule having the same structure as the FcRn antagonist except having a wild-type Fc domain, changes the effector function of the Fc domain (e.g., ADCC or CDC function). For an antibody FcRn antagonist, the corresponding wild-type molecule may be the unmodified antibody as a whole that includes an antibody of approximately the same immunogenicity. For example, an FcRn antagonist having a reduced ADCC and / or CDC effector function (e.g., one containing the LALA mutation) is less likely to cause undesirable side effects when administered to a patient.

[0086] In certain embodiments, the FcRn antagonist of the invention can include a modified Fc domain that includes one or more amino acid mutations (e.g., substitutions), which, compared to the corresponding wild-type molecule, changes (e.g., increases or decreases) the circulating half-life (e.g., serum half-life) of the FcRn antagonist. For example, a decrease in serum half-life can be advantageous when it is not desired for the FcRn antagonist to be present in the patient's body for an extended period.

[0087] In certain embodiments, the FcRn antagonist of the invention can comprise a modified Fc domain that includes one or more mutations (e.g., substitutions), which provides one or more desired biochemical characteristics as compared to the corresponding wild-type molecule, such as the ability to remain monomeric, the ability to dimerize non-covalently, the ability to localize to target sites, and an increased ability to localize to glycosylation patterns. For example, the modified Fc domain can have reduced glycosylation (e.g., N- or O-linked glycosylation). Examples of amino acid substitutions that confer reduced or altered glycosylation are disclosed in International PCT Publication No. WO2005 / 018572. In some embodiments, the Fc domain is modified to remove glycosylation (e.g., an "agly" antibody). An "agly" FcRn antagonist can have an improved safety and stability profile in vivo. A number of methods recognized in the art are available for producing an "agly" antibody or an antibody with an altered glycan. For example, such antibodies can be produced using genetically engineered host cells (e.g., modified yeast, such as Pichia, or CHO cells) having a modified glycosylation pathway (e.g., a glycosyltransferase deletion).

[0088] In certain embodiments, the Fc domain can be mutated to reduce effector function using techniques known in the art. In some embodiments, the modified Fc herein also has an altered binding affinity for the Fc-gamma receptor (FcγR). FcγR belongs to a family that includes several members, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. In some embodiments, the modified Fc herein has an enhanced FcRn binding affinity and a reduced FcγRIIIa binding affinity as compared to the wild-type Fc domain.

[0089] In some embodiments, the FcRn antagonist is an antibody. Suitable antibodies include, without limitation, human antibodies, humanized antibodies, or chimeric antibodies, and may be full-length antibodies or single-chain antibodies. The antibody may target soluble targets associated with a disease state (e.g., cytokines and secreted circulating proteins).

[0090] Proteins with low thermodynamic stability, including antibodies, tend to have an increased tendency to misfold and aggregate, which may limit or impede the activity, efficacy, and potential of the protein as a useful therapeutic agent. In certain embodiments, the FcRn antagonist has a thermal stability approximately the same as or comparable to that of a polypeptide comprising a wild-type Fc domain or a modified Fc domain having the triple amino acid substitution M252Y / S254T / T256E (YTE).

[0091] The resulting physiological profile, bioavailability, and other biochemical effects, e.g., localization, in vivo distribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0092] In certain embodiments, the FcRn antagonist of the present disclosure is an isolated binding polypeptide comprising an antigen-binding fragment derived from an antibody fused to a modified Fc domain. The term "antigen-binding fragment" refers to an immunoglobulin or polypeptide fragment of an antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which it is derived) for antigen binding (i.e., specific binding).

[0093] In some embodiments, the binding polypeptide comprises a single-chain variable region sequence (ScFv) fused to a modified Fc of the present specification. The single-chain variable region sequence comprises a single polypeptide having one or more antigen-binding sites, for example, a VL domain linked to a VH domain by a flexible linker. The ScFv molecule can be constructed in a VH-linker-VL orientation or a VL-linker-VH orientation. The flexible hinge that links the VL and VH domains that make up the antigen-binding site contains about 10 to about 50 amino acid residues. Peptide linkages are known in the art.

[0094] In some embodiments, the binding polypeptide of the present disclosure is a multivalent (e.g., tetravalent) antibody produced by fusing a DNA sequence encoding an antibody having a ScFv molecule (e.g., a modified ScFv molecule). For example, in one embodiment, these sequences are combined such that a ScFv molecule (e.g., a modified ScFv molecule) is linked to the Fc fragment of the antibody via a flexible linker (e.g., a gly / ser linker) at its N-terminus or C-terminus. In another embodiment, the tetravalent antibody of the present disclosure can be made by fusing a ScFv molecule to a connecting peptide and fusing this to a modified Fc domain to construct a ScFv-Fab tetravalent molecule.

[0095] In another embodiment, the binding polypeptide of the present disclosure is a modified minibody. The modified minibody of the present disclosure is a dimer molecule composed of two polypeptide chains each containing a ScFv molecule, which is fused to a modified Fc domain via a connecting peptide. The minibody can be made by constructing the ScFv component and connecting the peptide components using methods described in the art (see, for example, U.S. Patent No. 5,837,821 or WO94 / 09817). In another embodiment, a tetravalent minibody can be constructed. The tetravalent minibody can be constructed in the same manner as the minibody except that two ScFv molecules are linked using a flexible linker. The linked scFv-scFv construct is then joined to a modified Fc domain.

[0096] In another embodiment, the binding polypeptide of the present disclosure comprises a diabody. A diabody is a dimeric tetravalent molecule, each having a polypeptide similar to an scFv molecule such that the VL and VH domains of the same polypeptide chain cannot interact, but having a usually short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting both variable domains. Instead, the VL and VH domains of one polypeptide chain interact with the (respective) VH and VL domains of a second polypeptide chain (see, e.g., WO02 / 02781). The diabody of the present disclosure comprises an scFv-like molecule fused to a modified Fc domain.

[0097] In other embodiments, the binding polypeptide comprises a multispecific or multivalent antibody, such as a tandem variable domain (TVD) polypeptide, that comprises one or more variable domains in series on the same polypeptide chain. An exemplary TVD polypeptide includes the "double head" or "dual-Fv" arrangement described in U.S. Patent No. 5,989,830. In the dual-Fv arrangement, the variable domains of two different antibodies are on two separate chains (one expressed in tandem orientation in a single heavy chain and one light chain), where one polypeptide chain has two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected consecutively by a peptide linker (VL1-linker-VL2). In a crossover double-head configuration, the variable domains of two different antibodies are expressed in tandem orientation in two separate polypeptide chains (one heavy chain and one light chain), where one polypeptide chain has two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected consecutively in the reverse orientation by a peptide linker (VL2-linker-VL1). Further antibody variants based on the "Diabody" format include Dual-Variable-Domain IgG (DVD-IgG) bispecific antibodies (see U.S. Patent No. 7,612,181) and the TBTI format (see US2010 / 0226923A1). In some embodiments, the binding polypeptide comprises a multispecific or multivalent antibody comprising one or more variable domains consecutively in the same polypeptide chain fused to a modified Fc domain.

[0098] In another exemplary embodiment, the binding polypeptide comprises a crossover double variable domain IgG (CODV-IgG) bispecific antibody based on the "double-head" configuration (see US20120251541A1).

[0099] In another exemplary embodiment, the binding polypeptide is an immunoadhesin. As used herein, "immunoadhesin" refers to a binding polypeptide (e.g., derived from a receptor, ligand, or cell adhesion molecule) comprising one or more binding domains linked to an immunoglobulin constant domain (i.e., the Fc region) (e.g., Ashkenazi See et al., Methods (1995) 8(2):104-15, and Isaacs, Brit J Rheum. (1997) 36:305-7, which are hereby incorporated by reference in their entirety). Examples of immunoadhesins are chimeric molecules between the ligand-binding domain of a receptor and the IgG Fc domain. In some embodiments, the FcRn antagonist is an immunoadhesin that binds to and sequesters cytokines involved in inflammation.

[0100] Immunoadhesins are identified by the suffix "-cept" at the end in their International Nonproprietary Names (INNs). Similar to antibodies, immunoadhesins have a long circulating half-life, are easily purified by affinity-based methods, and have the advantage of avidity conferred by bivalency. Examples of commercially available therapeutic immunoadhesins include etanercept (ENBREL®), abatacept (ORENCIA®), rilonacept (ARCALYST®), aflibercept (ZALTRAP® / EYLEA®), and belatacept (NULOJIX®).

[0101] In certain embodiments, the isolated binding polypeptide is an antibody mimetic, e.g., an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, and nanoCLAMPs fused to a modified Fc domain as described herein.

[0102] In certain embodiments, the binding polypeptide comprises an immunoglobulin-like domain. Suitable immunoglobulin-like domains include, without limitation, fibronectin domains (e.g., Koide et al., Methods Mol Biol. (2007) See 352:95-109, which is hereby incorporated by reference in its entirety), DARPins (see, e.g., Stumpp et al., Drug Discov Today (2008) 13(15-16):695-701, which is hereby incorporated by reference in its entirety), the Z domain of protein A (see, e.g., Nygren et al., FEBS J. (2008) 275(11):2668-76, which is hereby incorporated by reference in its entirety), lipocalins (see, e.g., Skerra et al. (2008) FEBS J. 275(11):2677-83, which is hereby incorporated by reference in its entirety), affilins (see, e.g., Ebersbach et al., J Mol Biol. (2007) 372(1):172-85, which is hereby incorporated by reference in its entirety), affitins (see, e.g., Krehenbrink et al., (2008) J Mol Biol. 383(5):1058-68, which is hereby incorporated by reference in its entirety), avimers (see, e.g., Silverman et al., (2005) Nat Biotechnol. 23(12):1556-61, which is hereby incorporated by reference in its entirety), finomers (see, e.g., Grabulovski et al., J Biol Chem. (2007) 282(5):3196-3204, which is hereby incorporated by reference in its entirety), and Kunitz domain peptides (see, e.g., Nixon et al., (2006) Curr Opin Drug Discov Devel 9(2):261-8, which is hereby incorporated by reference in its entirety).

[0103] The binding polypeptides of the disclosure that include the modified Fc domains described herein can include the CDR sequences or variable domain sequences of known "parent" antibodies. In some embodiments, the parent antibody and the antibodies of the disclosure can share similar or identical sequences except for the modifications to the Fc domains disclosed herein.

[0104] Nucleic Acids and Expression Vectors Polynucleotides encoding the polypeptides disclosed herein are typically inserted into an expression vector for introduction into a host cell that can be used to produce the desired amount of the described polypeptide, such as an antibody, a fragment thereof, or an immunoadhesin. Accordingly, in certain aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0105] The terms "vector" or "expression vector" are used herein for the purposes of the specification and claims to mean a vector used to introduce and express a desired gene into a cell. Such vectors can be readily selected from the group consisting of plasmids, phages, and viruses (such as baculovirus, vaccinia virus, lentivirus, adenovirus, adeno-associated virus, and retrovirus). Generally, a vector includes a selectable marker and appropriate restriction sites for facilitating cloning of the desired gene, as well as the ability of the vector to enter and / or replicate in eukaryotic or prokaryotic cells.

[0106] A number of expression vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), or SV40 virus. Others include the use of polycistronic systems having internal ribosome binding sites. Additionally, cells into which the DNA has been integrated can be selected by introducing one or more markers that enable the selection of transfected host cells. The markers can provide prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be ligated directly to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Additional elements may also be required for optimal synthesis of the mRNA. These elements can include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human genes) synthesized as discussed above. Cells into which the DNA has been integrated can be selected by introducing one or more markers that enable the selection of transfected host cells. The markers can provide prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be ligated directly to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Additional elements may also be required for optimal synthesis of the mRNA. These elements can include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human genes) synthesized as discussed above.

[0107] In other embodiments, the binding polypeptides described herein can be expressed using a polycistronic construct. In such expression systems, multiple gene products of interest, such as the heavy and light chains of an antibody, can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptide to eukaryotic host cells. Suitable IRES sequences are disclosed in U.S. Patent No. 6,193,980.

[0108] More generally, once a vector or DNA sequence encoding an FcRn antagonist of the present disclosure is produced, the expression vector can be introduced into a suitable host cell. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques, such as transfection (including electroporation and electropermeabilization), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and infection with intact virus. The transformed cells are grown under conditions appropriate for the production of the present binding polypeptide and assayed for their synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0109] The host cells used for the expression of the present polypeptide may be of eukaryotic (e.g., mammalian, insect, yeast or plant) or prokaryotic origin. In some embodiments, the host cell line used for the expression of the polypeptide is of mammalian origin; one skilled in the art can determine the specific host cell line most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells (e.g., DG44 and DUXB11, CHO strains, DHFR minus), HELA (human cervical cancer), CVI (monkey kidney line), COS (CVI-derived cells having SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). In one embodiment, the cell line results in altered glycosylation of the expressed binding polypeptide (e.g., antibody), e.g., afucosylation (e.g., PER.C6.RTM. (Crucell) or FUT8-knockout CHO cell line (POTELLIGENT™ cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells can be used. Host cell lines are typically available from commercial services, the American Tissue Culture Collection or published literature. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or continuous stirred reactor, or cell culture immobilized or trapped, for example, in hollow fibers, microcapsules, agarose microbeads or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatography methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose and / or (immuno-)affinity chromatography.

[0110] Treatment method In one aspect, the present disclosure provides a method of treating a subject (e.g., a human patient) in need thereof, the method comprising administering an effective amount of an FcRn antagonist disclosed herein. In certain embodiments, the present disclosure provides a product (e.g., a kit) for use in such treatment methods. The patient has an IgG-mediated disease or disorder and benefits from more rapid clearance of serum IgG by treatment with the FcRn antagonists of the present disclosure. As used herein, the terms "antibody-related," "antibody-mediated," and "antibody-responsive" disorders, conditions, or diseases refer to disorders, conditions, or diseases that may be alleviated by the removal of unwanted antibodies (e.g., certain autoreactive IgG that is no longer needed or beneficial to the patient, or exogenously provided therapeutic or diagnostic IgG).

[0111] In certain embodiments, the IgG-mediated disorder can be an autoimmune disease, such as graft-versus-host disease (GVHD), systemic lupus erythematosus (SLE), myasthenia gravis, systemic sclerosis (SSc) / scleroderma, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, diabetes, multiple sclerosis, pemphigus vulgaris, atopic dermatitis, psoriasis, asthma, allergy, idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic purpura (ITP), and hidradenitis suppurativa.

[0112] In certain embodiments, the IgG-mediated disorder can be an allergy. The allergy can be, for example, a food allergy (e.g., egg allergy, fish allergy, shellfish allergy, fruit allergy, garlic allergy, pepper allergy, rye allergy, meat allergy, milk allergy, peanut allergy, rice allergy, sesame allergy, soybean allergy, sulfite allergy, tartrazine allergy, tree nut allergy, wheat allergy, etc.), a drug allergy (e.g., tetracycline allergy, dilantin allergy, tagretol allergy, penicillin allergy, cephalosporin allergy, sulfonamide allergy, non-steroidal anti-inflammatory agent allergy, intravenous contrast dye allergy, etc.), an environmental allergy (e.g., grass allergy, pollen allergy, cat allergy, dog allergy, insect allergy, mold allergy, fragrance allergy, cosmetic allergy, semen allergy, latex allergy, water allergy, dust mite allergy, nickel allergy, gold allergy, chromium allergy, cobalt chloride allergy, developer allergy, disinfectant allergy, etc.) and a contact allergy (e.g., latex allergy, paraphenylenediamine allergy, glyceryl monothioglycolate allergy, toluenesulfanomide formaldehyde allergy, etc.).

[0113] In certain embodiments, the FcRn antagonists disclosed herein may be used in combination with one or more additional therapeutic agents. In certain embodiments, the combination therapy comprising the FcRn antagonists disclosed herein may target the innate and / or adaptive immune system, for example, by binding to and removing proinflammatory cytokines from circulation. In some embodiments, the FcRn antagonist itself may bind to and remove proinflammatory cytokines, achieving a combined effect of removing autoreactive serum IgG as well as proinflammatory cytokines. In certain embodiments, the combination therapy comprising the FcRn antagonists disclosed herein may include a TLR-inhibitor, for example, an IRAK-4 inhibitor.

[0114] In certain embodiments, FcRn antagonists are used to induce Fc-containing The clearance of Fc-containing agents can reduce the serum level of a useful agent (e.g., a therapeutic or diagnostic agent). Clearance of Fc-containing agents may be desired in cases where the Fc-containing agent is harmful (e.g., toxic) to a subject or where it is only desired that the Fc-containing agent be present in the subject for a certain period of time, such as when the Fc-containing agent is immunogenic or when the Fc-containing agent is an antibody-drug conjugate, respectively. In certain embodiments, clearance of Fc-containing agents reduces the subject's exposure to the Fc-containing agent. The level of any Fc-containing agent in serum can be reduced by using the FcRn antagonists described herein. Thus, in certain embodiments, the FcRn antagonists disclosed herein are used to reduce the serum level of the Fc-containing agent in a subject receiving the Fc-containing agent.

[0115] In another aspect, provided is a method of enhancing image diagnosis, the method comprising administering to a subject a therapeutically effective amount of an FcRn antagonist disclosed herein. Use of the FcRn antagonist disclosed herein can reduce both background levels and systemic exposure to radiolabeled antibodies during image diagnosis. In certain embodiments, image diagnosis includes, without limitation, positron emission tomography (PET), single photon emission computed tomography (SPECT), a combination of PET and computed tomography (CT), and the like.

[0116] In certain embodiments, the subject is administered an effective amount of a radiolabeled antibody, and optionally, the FcRn antagonist is administered after administration of the radiolabeled antibody. In certain embodiments, the contrast of the radiolabeled antibody is enhanced in the subject using the FcRn antagonist disclosed herein, for example, by clearing unbound radiolabeled antibody from circulation, to allow for increased contrast (e.g., decreased background signal) and decreased detrimental effects of exposure to the radiolabeled antibody. Accordingly, in certain embodiments, provided is a method of reducing exposure of normal tissue to a radiolabeled antibody during image diagnosis, the method comprising administering to a subject a therapeutically effective amount of an FcRn antagonist disclosed herein.

[0117] In certain embodiments, the FcRn antagonist disclosed herein can be used to eliminate a therapeutic agent from a subject. For example, a subject administered a therapeutic agent can generate antibodies, such as anti-drug antibodies, that reduce the availability and / or efficacy of the administered therapeutic agent. The presence of anti-drug antibodies in a subject can result in undesirable side effects. Accordingly, the FcRn antagonist disclosed herein removes anti-drug antibodies that develop in a subject.

[0118] One of ordinary skill in the art will be able to determine the effective amount of the FcRn antagonist described herein (e.g., a quadruple variant having enhanced FcRn binding at pH 6.0 and pH 7.4) by routine experimentation. In certain embodiments, the effective amount of the FcRn antagonist is non-toxic. The therapeutically effective amount of the FcRn antagonist can vary depending on a variety of factors. For example, without limitation, the age, gender, disease stage, and medical history of the subject to whom the FcRn antagonist is administered can affect the appropriate determination of the therapeutically effective amount of the FcRn antagonist. An appropriate dosing regimen can be determined to provide an effective therapeutic response in the subject. The dosage can be divided and administered over a particular period (e.g., daily for one week).

[0119] In certain embodiments, the present disclosure provides kits and methods for the diagnosis and / or treatment of disorders, such as IgG-mediated disorders, in mammalian subjects in need of such treatment. In exemplary embodiments, the subject is human.

[0120] Pharmaceutical Compositions and Their Administration Methods of making and administering the present FcRn antagonist are well known to those of ordinary skill in the art and can be readily determined. The route of administration can be oral, parenteral, topical, or inhalation. As used herein, the term "parenteral" includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. All of these administration forms are expressly contemplated as being within the scope of the present disclosure, although the administration form will likely be an injectable solution, particularly for intravenous or intraarterial injection or infusion. Typically, a pharmaceutical composition suitable for injection can include a buffer (e.g., an acetate, histidine, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), a preservative, and the like. In some embodiments, the FcRn antagonist can be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0121] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including physiological saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01 - 0.1 M, for example, 0.05 M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluids and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases may also be present. More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and typically protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0122] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition. Delayed absorption agents such as aluminum monostearate and gelatin can be included in the composition to effect extended absorption of the injectable composition.

[0123] In any case, a sterile injectable liquid can be produced by incorporating the required amount of the active compound (e.g., a modified binding polypeptide, either by itself or in combination with other active agents) in a suitable solvent together with one or a combination of the ingredients listed herein and subsequently, if required, subjecting it to sterile filtration. Generally, a dispersion is produced by incorporating the active compound in a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable liquids, exemplary methods of manufacture include vacuum drying and freeze drying which yield a powder of the active ingredient and any additional desired ingredients from its solution which has been previously sterile filtered. Formulations for injection are processed according to methods known in the art, filled into containers such as ampoules, bags, bottles, syringes or vials and sealed under aseptic conditions. Further, the formulation can be packaged and sold in the form of a kit. Such products typically have a label or package insert indicating that the associated composition is useful for treating subjects suffering from or prone to IgG-mediated disorders.

[0124] The effective dosage of the pharmaceutical composition varies depending on many different factors including the means of administration, the target site, the health status of the patient, medical history, age, gender, and weight. Treatment dosages can be titrated using conventional methods known to those of skill in the art to optimize safety and efficacy.

[0125] This FcRn antagonist can be administered multiple times. The interval between single doses can be weekly, monthly, or yearly. The interval may be irregular, following appropriateness as indicated by measuring the blood levels of IgG in the patient. In some methods, the dosing is adjusted to achieve a plasma FcRn antagonist concentration of about 1 - 1000 μg / ml, and in some methods, a plasma FcRn antagonist concentration of about 25 - 300 μg / ml is achieved. Alternatively, the FcRn antagonist can be administered as a sustained release formulation, in which case the required dosing frequency is lower. For antibodies, the dosage and frequency vary depending on the half-life of the antibody in the patient.

[0126] The pharmaceutical compositions herein can include one or more species of this FcRn antagonist and a pharmaceutically acceptable non-toxic, sterile carrier such as physiological saline, non-toxic buffers, etc. The dosage and dosing frequency can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a pharmaceutical composition containing the FcRn antagonist or a cocktail thereof is administered to a patient who is not yet in a disease state (e.g., a genetically susceptible patient) to enhance the patient's resistance to the development of the disease. Such an amount is defined as a "prophylactically effective dose". In this use, the exact amount also depends on the patient's health and overall immunity in this case, but generally ranges from about 0.1 to about 25 mg per dose, particularly from about 0.5 to about 2.5 mg per dose. Relatively low dosages are administered over a long period at relatively infrequent intervals. Some patients continue treatment for the remainder of their lives. In therapeutic applications, relatively high dosages (e.g., about 1 - 400 mg / kg of antibody per dose, e.g., a dosage of about 5 - 25 mg) may be required at relatively short intervals until the progression of the disease decreases or ends, or until the patient shows partial or complete relief of the disease symptoms. Thereafter, the patient can be administered a prophylactic regimen.

[0127] Exemplary embodiments Non-limiting exemplary embodiments of the present disclosure are provided below. 1. A method for treating an antibody-mediated disorder in a subject in need thereof, comprising aspartic acid (D) or glutamic acid (E) at amino acid position 256, and tryptophan (W) or glutamine (Q) at amino acid position 307 [wherein amino acid position 254 is not threonine (T)], and: phenylalanine (F) or tyrosine (Y) at amino acid position 434; and tyrosine (Y) at amino acid position 252 [wherein the amino acid positions follow EU numbering] administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain comprising a combination of at least four amino acid substitutions further comprising the above. 2. A method for treating an antibody-mediated disorder in a subject in need thereof, a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; d) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434; or e) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434, [wherein the amino acid substitutions follow EU numbering] administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain having a combination of positional amino acid substitutions selected from the group consisting of the above. 3. A method of treating an antibody-mediated disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain comprising a quadruple amino acid substitution selected from the group consisting of M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, and M252Y / T256D / T307W / N434Y, wherein the amino acid substitutions are according to EU numbering. 4. A method of treating an antibody-mediated disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain comprising, according to EU numbering, tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434. 5. A method of treating an antibody-mediated disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain comprising, according to EU numbering, tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434. 6. A method of treating an antibody-mediated disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain comprising, according to EU numbering, tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434. 7. A method of treating an antibody-mediated disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain that, according to EU numbering, comprises tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434. 8. A method of treating an antibody-mediated disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain that, according to EU numbering, comprises tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434. 9. The method according to any of the preceding embodiments, wherein the modified Fc domain is a modified human Fc domain. 10. The method according to any of the preceding embodiments, wherein the modified Fc domain is a modified IgG1 Fc domain. 11. The method according to any of the preceding embodiments, wherein the FcRn antagonist has human FcRn binding affinity. 12. The method according to any of the preceding embodiments, wherein the FcRn antagonist has human and rat FcRn binding affinity. 13. The method according to any of the preceding embodiments, wherein the FcRn antagonist has enhanced FcRn binding affinity compared to an FcRn antagonist comprising a wild-type Fc domain. 14. The method according to any of the preceding embodiments, wherein the FcRn antagonist has enhanced FcRn binding affinity at acidic pH compared to an FcRn antagonist comprising a wild-type Fc domain. 15. The method according to any of the preceding embodiments, wherein the FcRn antagonist has enhanced FcRn binding affinity at acidic pH compared to an FcRn antagonist comprising M252Y / S254T / T256E / H433K / N434F according to EU numbering. 16. The method according to any preceding embodiment, wherein the FcRn antagonist has an enhanced FcRn binding affinity at non-acidic pH as compared to an FcRn antagonist comprising a wild-type Fc domain. 17. The method according to any preceding embodiment, wherein the FcRn antagonist has an enhanced FcRn binding affinity at non-acidic pH as compared to an FcRn antagonist comprising M252Y / S254T / T256E / H433K / N434F according to EU numbering. 18. The method according to any preceding embodiment, wherein the FcRn antagonist has an enhanced FcRn binding affinity at acidic pH and an enhanced FcRn binding affinity at non-acidic pH as compared to an FcRn antagonist comprising a wild-type Fc domain. 19. The method according to any preceding embodiment, wherein the FcRn antagonist has an enhanced FcRn binding affinity at acidic pH and an enhanced FcRn binding affinity at non-acidic pH as compared to an FcRn antagonist comprising M252Y / S254T / T256E / H433K / N434F according to EU numbering. 20. The method according to any preceding embodiment, wherein the acidic pH is about 6.0. 21. The method according to any preceding embodiment, wherein the non-acidic pH is about 7.4. 22. The method according to any preceding embodiment, wherein the FcRn antagonist has a reduced serum half-life as compared to an FcRn antagonist comprising a wild-type Fc domain. 23. The method according to any preceding embodiment, wherein the FcRn antagonist has a reduced serum half-life as compared to an FcRn antagonist comprising M252Y / S254T / T256E / H433K / N434F according to EU numbering. 24. The method according to any preceding embodiment, wherein the FcRn antagonist has a reduced FcγRIIIa binding affinity as compared to an FcRn antagonist comprising a wild-type Fc domain. 25. The method according to any preceding embodiment, wherein the FcRn antagonist has a reduced thermal stability compared to an FcRn antagonist comprising a wild-type Fc domain. 26. The method according to any preceding embodiment, wherein the FcRn antagonist is a binding polypeptide. 27. The method according to any preceding embodiment, wherein the FcRn antagonist is an antibody or a fragment thereof selected from the group consisting of an Fv fragment, a single-chain antibody (ScFv), a Fab, a Fab-H, a Fab’, an F(ab’)2, an Fd, a dAb, and multimers thereof, a single-domain antibody, a maxibody, a minibody, a diabody, a triabody, a tetrabody, a vNAR, and a bis-scFv. 28. The method according to any preceding embodiment, wherein the antibody is a monoclonal antibody. 29. The method according to any preceding embodiment, wherein the antibody is a chimeric, humanized, or human antibody. 30. The method according to any preceding embodiment, wherein the antibody is a full-length antibody. 31. The FcRn antagonist is an affibody, an affilin, an affimer, an affitin , an alphabody, an anticalin, an avimer, a DARPin, a finomer, a Kunitz domain peptide, a monobody, and a method according to any one of embodiments 1 to 25, which is an antibody mimic selected from the group consisting of nanoCLAMPs. 32. The method according to embodiment 31, wherein the FcRn antagonist is an affibody. 33. The method according to any preceding embodiment, wherein the FcRn antagonist further comprises an albumin-binding domain. 34. The method according to any preceding embodiment, wherein the FcRn antagonist specifically binds to one or more targets. 35. The method according to any preceding embodiment, wherein the antibody-mediated disorder is an autoimmune disease. 36. The method according to embodiment 35, wherein the autoimmune disease is selected from the group consisting of graft-versus-host disease (GVHD), systemic lupus erythematosus (SLE), myasthenia gravis, systemic sclerosis (SSc) / scleroderma, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, diabetes, multiple sclerosis, pemphigus vulgaris, atopic dermatitis, psoriasis, asthma, allergy, idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic purpura (ITP), and hidradenitis suppurativa. 37. A method for enhancing image diagnosis, comprising administering to a subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain comprising a quadruple amino acid substitution selected from the group consisting of M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, and M252Y / T256D / T307W / N434Y [wherein the amino acid substitutions are according to EU numbering]. 38. The method according to embodiment 37, further comprising administering to a subject an effective amount of a radiolabeled antibody. 39. The method according to embodiment 38, wherein the FcRn antagonist is administered after the radiolabeled antibody. 40. The method according to embodiment 37 or 38, which enhances the contrast to the radiolabeled antibody. 41. A method for reducing the exposure of non-target tissues to a radiolabeled antibody during image diagnosis, comprising administering to a subject a therapeutically effective amount of an FcRn antagonist comprising a modified Fc domain comprising a quadruple amino acid substitution selected from the group consisting of M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, and M252Y / T256D / T307W / N434Y [wherein the amino acid substitutions are according to EU numbering].

[0128] The contents of the papers, patents, and patent applications mentioned or cited in this specification, as well as all other documents and electronically available information, are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. The Applicants reserve the right to physically incorporate into this application any and all materials and information from any such papers, patents, patent applications, or other physical and electronic documents.

[0129] Although the present invention has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. Other suitable modifications and adaptations of the methods described herein can be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Further, many modifications can be made to adapt a particular situation, material, composition, method, process step, to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the appended claims of this specification. Although specific embodiments have been described in detail, they will be more clearly understood by reference to the following Examples, which are included for illustrative purposes only and are not intended to be limiting.

Examples

[0130] The present invention is further illustrated by the following examples. They should not be construed as further limitations.

Examples

[0131] Materials and Methods Protein Reagents: The following proteins were expressed and isolated: antigen containing an 8x histidine tag at the C-terminus (SEQ ID NO: 5); rat FcRn (rFcRn, UniProt: P1359, p51 subunit: residues 23 - 298; UniProt: P07151, β2-m: residues 21 - 119); biotinylated cynomolgus FcRn (cynoFcRn, UniProt: Q8SPV9, p51 subunit: residues 24 - 297 containing an Avi-tag at the C-terminus; UniProt: Q8SPW0, β2-m: residues 21 - 119); biotinylated human FcRn (hFcRn, UniProt: P55899, p51 subunit: residues 24 - 297 containing an Avi-tag at the C-terminus; UniProt: P61769, β2-m: residues 21 - 119); human CD16a (UniProt: P08637, FcγRIIIa: residues 17 - 208 containing a C-terminal HPC4 tag and valine (V158) at position 158). Heavy chain variants of H435A and H310A / H435Q were obtained from HEK293 conditioned medium. The mAb2 (IgG1) variant was cloned by Evitria and purified from suspension CHO K1 conditioned medium using an mAbSelect™ SuRe™ affinity column (GE Healthcare), and then buffer-exchanged into phosphate-buffered saline (PBS) pH 7.4 for subsequent experiments.

[0132] Construction of a saturated library The heavy and light chains of the WT IgG1 mAb1 (IgG1) antibody with the leader DNA sequence were incorporated into the mammalian expression plasmids pBH6414 and pBH6368, respectively, using the restriction enzyme sites of NcoI and HindIII. A saturation library was generated using the Lightning Site Directed Mutagenesis kit (Agilent) and primers of NNK (N = A / C / G / T, K = G / T) and WWC (W = A / T) from IDT Technologies to introduce all possible amino acids at the following positions: M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436 (EU numbering). The heavy chain DNA sequences of three control variants, AAA (T307A / E380A / N434A), LS (M428L / N434S), and YTE (M252Y / S254T / T256E) in the mAb1 backbone were constructed in the pBH6414 vector from LakePharma.

[0133] In the PCR reaction, a combinatorial saturation library was obtained through site-directed mutagenesis of the mAb1 heavy chain using the Q5 Mutagenesis kit (NEBiolabs) and each primer of T256D, T256E, T307Q, T307W, N434F, and N434Y together with the WT and M252Y templates. Mutagenesis incorporation into the mAb3 backbone was performed using the Q5® Mutagenesis kit (NEBiolabs) together with each primer of M252Y, T256D, T307Q, and T307W. The generation of all Fc variants was confirmed by Sanger Sequencing (Genewiz, Inc.).

[0134] Recombinant Antibody Expression and Purification For conditional medium screening, DNA containing the mutant heavy chain and wild-type light chain of mAb1 was transfected into 1 mL of Expi293 mammalian cells (Invitrogen™) for expression according to the manufacturer's instructions. The cells were incubated in a 2 mL 96-well plate (Greiner Bio-One) at 37 °C with 5% carbon dioxide and 80% humidity while shaking at 900 revolutions per minute (RPM) and sealed with a vented membrane. The conditional medium was collected on the 5th day after transfection and stored at -80 °C until use. Lead variants in the mAb1 and mAb3 frameworks were expressed in a 125 mL flask equipped with a 0.2 μm vented cap (Corning) on a 30 mL scale. The 125 mL culture flask was shaken at 125 RPM throughout the expression period. The conditional medium was collected on the 5th day after transfection, filtered through a 0.22 μm, 50 mL conical filter (Corning), and stored at 4 °C until purification.

[0135] Isolation of mAb1 and mAb3 was performed using a 1 mL mAbSelect™ SuRe™ HiTrap® column (GE Healthcare). Following a washing step with PBS pH 7.4 for 10 column volumes, the antibody was eluted with 0.1 M citric acid pH 3.0 (Sigma) for 5 column volumes and neutralized with 0.5 mL of 1 M Tris base pH 9.0 (Sigma). The eluted antibody was buffer-exchanged against PBS pH 7.4 and then concentrated to >1 mg mL -1 using 30 kDa MWCO Amicon® Concentrators (Millipore®) for subsequent testing. The concentration of the purified antibody was determined from their UV absorbance (UV 280 ) at 280 nm using the appropriate extinction coefficient.

[0136] Octet Conditional Medium Screening and Analysis The screening of the conditioned medium containing the mAb1 variant was performed using an Octet QK 384 (PALL Life Sciences) equipped with a Ni-NTA biosensor. The His-tagged antigen was captured in PBS, 0.1% bovine serum albumin (BSA, Sigma), and 0.01% Tween-20 (Sigma) at pH 7.4 (PBST-BSA 7.4) at 15 μg mL -1 for 300 seconds, followed by washing with PBST-BSA at pH 7.4 for 20 seconds. The antibody was captured for 200 seconds in the conditioned medium diluted 1:1 with PBST-BSA pH 7.4. Following the buffer wash step in the pH 6.0 buffer, FcRn binding kinetics were obtained using 200 nM rFcRn during association and dissociation times of 150 seconds and 200 seconds, respectively, at pH 6.0. The temperature was set at 30 °C under the condition of an oscillation speed of 1000 RPM at all stages of the Octet screening process. The rFcRn binding kinetics profile was corrected relative to the start of the FcRn association phase and modeled using the Octet 7.1 analysis software to a 1:1 binding model.

[0137] FcRn binding kinetics FcRn binding kinetics under multiple pH conditions (pH 6.0, 7.4, 8.0, and 9.0) were measured using a Biacore T200 instrument (GE Healthcare) using a Biotin CAPture kit (GE Healthcare) (see, for example, Abdiche et al., MAbs (2015) 7:331-343; Karlsson et al., Anal. Biochem. (2016) 502:53-63). The Capture reagent was captured on the CAP chip to a surface density of >1500 RU, followed by capturing FcRn at 30 μL min -1 with variable capture times to account for the decreased binding affinity at higher pH. The FcRn concentration and capture time for each pH were as follows: pH 6.0: 0.1 μg mL -1 FcRn for 24 seconds, pH 7.4 and 8.0: 1 μg mL -1 for 60 seconds, pH 9.0: 10 μg mL -1For 60 seconds. The running buffer at all pH states was 20 mM Bis-Tris propane; 150 mM NaCl, 0.05% Tween-20 with the pH appropriately adjusted to pH 6.0, 7.4, 8.0 and 9.0. A concentration series of each antibody variant starting from 1000 nM at 3-fold dilutions was established to cover a wide range of binding affinities. Kinetic measurements were performed between association and dissociation times of 180 seconds and 360 seconds respectively, followed by regeneration of the CAP surface with guanidine hydrochloride and sodium hydroxide in each cycle. Steady-state RU measurements at pH 7.4, 8.0 and 9.0 were obtained in triplicate at 1000 nM. Sensorgrams at each pH were fitted to a 1:1 or bivalent binding model using Biacore T200TM evaluation software. The bivalent model explains the high FcRn capture levels and the avidity effect observed with two FcRn binding sites per IgG. See, for example, Suzuki et al., J Immunol. (2010) 184:1968-1976. Each concentration series was fitted individually to obtain the average association and dissociation rates and binding affinity. Residual binding was determined from the steady-state binding response before the start of the dissociation phase and averaged. The high FcRn capture levels and the avidity effect observed with two FcRn binding sites per IgG were explained by the bivalent model. See, for example, Suzuki et al., J Immunol. (2010) 184:1968-1976. Each concentration series was fitted individually to obtain the average association and dissociation rates and binding affinity. Residual binding was determined from the steady-state binding response before the start of the dissociation phase and averaged.

[0138] FcRn affinity chromatography In one experiment, the FcRn affinity column was prepared from the protocol applied by Schlothauer et al., mAbs (2013) 5:576-586. A 1 mL Streptavidin HP HiTrap® column (GE Healthcare) was equilibrated with binding buffer (20 mM sodium phosphate (Sigma) pH 7.4, 150 mM sodium chloride (NaCl; Sigma)) at 1 mL min -1 for 5 column volumes, followed by injection of 4 milligrams of biotinylated cynoFcRn. The column was washed with binding buffer and stored at 4 °C until use.

[0139] The FcRn affinity column was equilibrated with a low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma) pH 5.5; 150 mM NaCl) against 5 column volumes, and then 300 μg of each antibody was injected. The pH of the antibody solution was adjusted to pH 5.5 using the low pH buffer. Following washing with 10 column volumes of the low pH buffer, the antibody was eluted at 1 mL min -1 over 30 column volumes with a linear pH gradient with a high pH buffer (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bis-tris propane; Sigma) pH 9.5; 150 mM NaCl) while monitoring UV 280 . The FcRn affinity column was then re-equilibrated with 10 column volumes of the low pH buffer for subsequent runs or with the binding buffer for storage. All variants were done in triplicate.

[0140] The FcRn affinity column elution profile for each variant was modeled to a single Gaussian distribution using SigmaPlot 11 (Systat Software, Inc.) and equation 1

Equation

[0141] In another experiment, an FcRn affinity column was applied using biotinylated hFcRn with a 1 mL Streptavidin HP HiTrap® column (GE Healthcare) as described by Schlothauer et al. supra. 300 μg of each antibody in low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma) pH 5.5; 150 mM NaCl) was injected into this column with an AKTA Pure System (AKTA). At 0.5 mL min -1 over 30 column volumes, the antibody was eluted while monitoring absorbance and pH with a linear pH gradient created with low and high pH buffers (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bis-tris propane; Sigma) pH 9.5; 150 mM NaCl). The column was then re-equilibrated with low pH buffer for subsequent runs. Triplicates were performed for all variants. The FcRn affinity column elution profiles were fit to a single Gaussian distribution with Sigmaplot 11 (Systat Software, Inc.) to determine the elution volume and pH at the UV 280 maximum.

[0142] Differential scanning fluorimetry Differential scanning fluorimetry (DSF) experiments were performed with a 20 μL reaction volume on a BioRad CFX96™ real-time thermal cycler (BioRad). Antibody samples and a 5000× stock of Sypro® Orange dye (Invitrogen®) were diluted to 0.4 mg mL -1 and 10× respectively in PBS pH 7.4. 0.2 mg mL of each antibody -1The antibody and Sypro (trademark) Orange were mixed in a 1:1 ratio in a 96-well PCR plate to the final concentration of 5×Sypro (trademark) Orange dye, and sealed with an adhesive microseal (registered trademark) (BioRad). This was done in triplicate for all antibody variants. The thermal cycler program consisted of an equilibration step at 20°C for 2 minutes, followed by a constant temperature ramp rate of 0.5°C / 5 seconds to a final temperature of 100°C. Fluorescence measurements for each well were acquired using FAM excitation wavelength (485 nm) and ROX emission (625 nm) detectors suitable for Sypro (trademark) orange fluorescence (see, for example, Biggar et al., Biotechniques (2012) 53:231-38). The DSF fluorescence intensity profile and the first derivative were exported from BioRad CFX (trademark) Manager and analyzed with Sigmaplot 11. T m was defined as the midpoint of the first transition in the fluorescence intensity profile.

[0143] FcγRIIIa binding kinetics Binding kinetics and affinity were measured using a Biacore (trademark) T200 instrument (GE Healthcare) (Zhou et al., Biotechnol Bioeng. (2008) 99:652-65). The anti-HPC4 antibody (Roche) was coupled to the surface of a CM5 sensor chip at 50 μg mL -1 in acetate pH 4.5 to a final density >20,000 RU using amine chemistry at 10 μl min -1 for 600 seconds. The running buffer for the FcγRIIIa binding kinetics experiment was HEPES buffered saline containing 0.05% Surfactant P-20 (HBS-P+, GE Healthcare) and 2 mM calcium chloride (CaCl2, Fluka) at pH 7.4. Each kinetic trace was started with the capture of 1.25 μg mL -1 of HPC4-tagged FcγRIIIa-V158 for 30 seconds at 5 μl min -1 The association and dissociation kinetics for each variant at 300 nM were at 5 μl min -1At each stage, measurements were taken for 120 - 180 seconds for each variant. As soon as the kinetic measurement was completed, the CM5 chip was regenerated with HBS - P+ buffer supplemented with 10 mM EDTA (Ambion®). Before the next kinetic measurement, the CM5 chip was washed with HBS - P+ containing CaCl2 for 120 seconds.

[0144] In one experiment, the FcγRIIIa kinetic experiment was analyzed in a manner similar to that described for FcRn binding at pH 7.4. For each variant of WT, benchmark, lead single, and lead combination, kinetics were obtained with a series of three - fold dilutions starting from 1000 nM to determine the binding affinity for FcγRIIIa. The steady - state RU at each concentration and in duplicates was determined, plotted as a function of antibody concentration, and fit to the steady - state model as shown in Equation 2

Number

[0145] In another experiment, the FcγRIIIa kinetic experiment was analyzed in a manner similar to that described for FcRn binding at pH 7.4 using the average steady - state binding response. For all variants, the steady - state RU of 300 nM antibody was determined in triplicate and averaged. The fold - change in response (fold - change in response) compared to WT was determined for comparison between variants in each framework.

[0146] Isoelectric focusing The isoelectric point (pI) of the lead variant was determined using capillary electrophoresis on a Maurice C (Protein Simple). To 200 μL of each sample, 0.35% methyl cellulose (Protein Simple), 4% pharmalyte 3-10 (GE Healthcare), 10 mM arginine (Protein Simple), 0.2 mg mL of antibody -1 as well as 4.05 and 9.99 pI markers (Protein Simple) were added. Samples were loaded onto the capillary at 1500 V for 1 minute, followed by a 6-minute separation phase at 3000 V and monitored using tryptophan fluorescence. The pI for each variant was determined using Maurice C software and defined as the pH at the fluorescence maximum for the major species.

[0147] Homogeneous bridging rheumatoid factor (RF) ELISA Antibodies were biotinylated and digoxigenin-labeled using EZ-Link Sulfo-NHS-LC-Biotin and Mix-n-Stain™ Digoxigenin Antibody Labeling Kit (Biotium) according to the manufacturer's instructions. Biotinylated and digoxigenin-labeled antibodies at 4 μg mL -1Stock solutions containing were prepared for each variant and mixed with 300 U / mL RF (Abcam) in a 1:1 ratio. After incubation for 20 hours at room temperature, 100 μL of each antibody-RF mixture was added to a Streptawell™ plate (Sigma-Aldrich) and incubated for 2 hours at room temperature. The plates were washed three times with PBS pH 7.4 containing 0.05% Tween-20, and 100 μL of a 1:2000 dilution of an HRP-conjugated anti-digoxigenin secondary antibody (Abcam) was added to each well. After incubation for 2 hours at room temperature, the wells were washed and treated with 100 μL of TMB substrate (Abcam) for 15 minutes at room temperature. The reaction was stopped using 100 μL of stop solution (Abcam), and the absorbance was measured at 450 nm using a SpectraMax® plate reader. A blank subtraction was provided by wells containing no antibody-RF mixture, and the experiment was repeated three times. Student's t-test was used to determine the P-value.

Example

[0148] Octet Screening of Saturated Point Mutations in Conditioned Medium FcRn is a heterodimer of an MHC class-I-like α-domain and a β2-microglobulin (β2-m) subunit (Figure 1A) and is common to most Fc receptors. FcRn recognizes a region on the antibody Fc heavy chain that is clearly different from other FcγRs (see, for example, Oganesyan et al., J Biol Chem. (2014) 289:7812-24; and Shields et al., J Biol Chem. (2001) 276: 6591-604).

[0149] To identify variants with slower FcRn dissociation rates than the WT antibody, a biolayer interferometry (BLI)-based assay was designed to screen antibody variants in conditioned media in a high-throughput manner (Figure 2A). This assay was developed using several benchmark variants that enhance (AAA, LS, and YTE) or decrease (H435A, H310A / H435Q) affinity for FcRn at pH 6.0 compared to the WT antibody. The NiNTA biosensor captures the his-tagged antigen at pH 7.4 to mimic conditioned media, followed by capture of each antibody variant (Figure 2A). The binding kinetics at pH 6.0 to rat FcRn (rFcRn), which has a dissociation rate reduced to approximately 1 / 25 that of human IgG1 and is more suitable for Octet studies than human FcRn (hFcRn), were measured for each of the variants (Figure 2B). Each of the variants H435A (Figure 2B, long dashed line) and H310A / H435Q (Figure 2B, long dotted line) showed little or no FcRn binding kinetics (see also, e.g., Shields et al., supra; Medesan et al., J Immunol. (1997) 158:2211-2217; and Raghavan et al., Biochemistry (1995) 34(45):14649-14657). Each of the variants AAA (Figure 2B, short dashed line), LS (Figure 2B, short dashed-dotted line), and YTE (Figure 2B, long dashed line) all presented slower dissociation kinetics compared to WT (Figure 2B, solid line), with a reduction to FcRn dissociation rates between 1 / 2 and 1 / 7.3. This demonstrated that Octet screening is suitable for distinguishing between variants with perturbed rFcRn dissociation kinetics.

[0150] The IgG1 antibody, mAb1, served as a model system for creating a saturation mutagenesis library to screen for mutants with a decreased FcRn dissociation rate. Eleven positions in the Fc region of mAb1 were selected based on their proximity to the FcRn interface or direct contribution (FIGS. 1A and 1B) (see, e.g., Oganesyan et al., supra; and Shields et al., supra). All point mutations at these positions were constructed using site-directed mutagenesis and transfected into Expi293 cells for expression. Conditioned media screening was performed on the saturation library mutants as described above. Normalized FcRn-binding Octet sensorgrams (long dashed lines) for a subset of variants are shown in FIG. 2C, along with wild type (FIG. 2C, thick long dashed line) and mock negative control (FIG. 2C, dotted line). The mock showed a lack of observable FcRn binding. Some variants clearly disrupted binding to rFcRn, as little or no signal change was observed in the kinetic profiles (FIG. 2C, long dashed lines, below the dotted line (mock)). A cutoff for variants with an improved FcRn dissociation rate was defined as three standard deviations lower than the mean of the WT antibody. In the subset of mutations shown in FIG. 2C, two (FIG. 2C, solid lines) had a significantly decreased dissociation rate compared to the wild type antibody (FIG. 2C, thick long dashed line), while the remaining variants had similar (FIG. 2C, short dash-dotted line) or faster (FIG. 2C, long dashed line above the dotted line (mock)) rFcRn dissociation rates.

[0151] The rFcRn dissociation rates for all single point mutations are shown in FIGS. 2D and 14, by position and mutation. In FIG. 14, the data are sorted into one of four categories according to the fold change in rFcRn dissociation rate compared to wild type, with wild type species Black represented by squares.

[0152] In Figure 14, the fold change in the rFcRn dissociation rate for all possible substitutions at 11 positions in the saturated library was normalized to the mean of the WT antibody and color-coded. All mutants fall into one of four categories: little or no binding to rFcRn (dark gray), faster rFcRn dissociation rate (gray), WT-like rFcRn dissociation rate (horizontal stripes) and slower rFcRn dissociation rate (grid). Multiple variants had an rFcRn dissociation rate slower than that of the WT antibody (grid).

[0153] The mutants colored dark gray in Figure 14 showed little or no binding to rFcRn in a manner similar to the mock (Figure 2C, dotted line) and were localized in the M252, I253, and S254 loops. The only mutations at I253 were methionine and valine, both of which significantly improved the rFcRn dissociation rate, which further supports the importance of I253 for FcRn interaction. Another 120 variants (Figure 2D and Figure 14, light gray squares) were located in each of the C H 2 and C H 3 domains and destabilized the interaction with rFcRn by approximately 50%. Twenty-five mutants had a WT-like dissociation rate (Figure 2D and Figure 14, white squares), in which case 8 of the 11 positions carried at least one WT-like mutation (Figure 14, white squares). The following mutations had a significantly decreased rFcRn dissociation rate compared to the wild type (Figure 2D and Figure 14, black squares): M252Y, T256D / E, K288D / N, T307A / E / F / M / Q / W, E380C, N434F / P / Y, and Y436H / N / W. Each of the mutations M252Y, N434F, and N434Y had a dissociation rate that decreased to less than one-half that of the WT antibody (Figure 2D). These mutations were expressed and purified using protein A chromatography for further in vitro FcRn kinetic characterization.

Example

[0154] Biacore™ FcRn Binding Kinetics at pH 6.0 Each variant of AAA, LS, and YTE functioned as a positive control in FcRn binding kinetics measurements using Biacore™ for both human FcRn and rat FcRn at pH 6.0. Concentration-dependent binding to FcRn was observed for all variants, including wild-type, benchmark (Figure 3), and lead (Figures 4A and 4B), and the binding profiles for single injections of human FcRn and rat FcRn are shown in Figures 5A and 5B, respectively. The wild-type antibody had binding affinities of 2380 ± 470 nM and 207 ± 43 nM for human FcRn and rat FcRn, respectively (Table 1).

[0155] [Table 1]

[0156] All data shown in Table 1 were obtained using the experimental techniques indicated at the top of each column.

[0157] The dissociation rate of rFcRn by Octet using purified protein was measured by comparing it with the rate constant obtained from screening in conditioned medium. The elution pH was determined by FcRn affinity chromatography in triplicate (n = 3), and the thermal stability was explored by DSF in triplicate (n = 3). The FcRn binding kinetics to human FcRn and rat FcRn were obtained from Biacore™ in duplicate (n = 2) using a series of antibody concentrations and were individually fitted. The steady-state binding responses (RU) of each variant to human FcRn and rat FcRn at pH 7.4 were measured using Biacore™ in triplicate (n = 3) with 1000 nM antibody. The units of each measurement are as follows: Octet pH6.0 rFcRn dissociation rate (×10 -3 s -1 ); elution pH (unitless); DSF T m (°C); Biacore™ pH6.0 hFcRn association rate (×10 4 M -1 s -1 ), dissociation rate (×10 -1 s -1and K D,app (×10 9 M); Biacore (trademark) pH6.0 rFcRn binding rate (×10 4 M -1 s -1 ), dissociation rate (×10 -3 s -1 ) and K D,app (×10 9 M); and Biacore (trademark) pH7.4 steady-state binding response (RU).

[0158] In Figure 5B, each of the variants AAA (dotted line), LS (two-dot chain line) and YTE (one-dot chain line) had an enhanced binding affinity between 1.6-fold and 10.4-fold compared to WT. LS had the tightest affinity for hFcRn, while rFcRn had a tighter affinity for YTE, so the identity of the benchmark variant with the tightest FcRn affinity was species-specific (Table 2A).

[0159]

Table 2

[0160] For both human FcRn and rat FcRn, the overwhelming majority of lead variants (solid lines of various shades in Figures 5A and 5B) had a significantly slower binding rate than WT or benchmark variants (decreased to less than half) (Table 1). The N434F and N434Y mutations were the only variants that presented an enhanced binding rate for both species with respect to FcRn. Without being bound by any theory, the result of the slower association kinetics with hFcRn was that the apparent binding affinity of the lead variants was overall weaker than WT, unlike rFcRn (Figures 5C and 5D, Table 1). The affinity of rFcRn was weaker than that of YTE (diagonal stripes going towards the lower left in Figure 5D, Table 2A).

[0161] Without being bound by any theory, these results indicate that single mutations were not sufficient to enhance affinity over the LS and YTE variants. Ranking of FcRn dissociation rates (by the variants' weak binding affinity to hFcRn) revealed a subset with reduced dissociation rates to both human and rat FcRn: M252Y, N434F / P / Y, T256D / E, and T307A / E / F / Q / W (Table 2A). These variants are of further interest in combination to further improve the FcRn binding ability of the Fc region over the benchmark variants.

[0162] The in vitro characterization parameters of the lead variants are shown in Table 2B.

[0163] [Table 3]

[0164] In Table 2B, all data were obtained using the experimental technique at the top of each column. Affinity chromatography, DSF and FcγRIIIa binding were performed in triplicate (n=3). FcRn binding kinetics for human FcRn and rat FcRn were obtained in quadruplicate and fitted separately. Units: DSF T m (°C); FcγRIIIa binding (fold change compared to WT); Biacore™ pH 6.0 hFcRn binding rate (×10 4 M -1 s -1 ), dissociation rate (×10 -1 s -1 ) and K D,app (×10 9 M);Biacore(TM) pH6.0 rFcRnK D,app (×10 9 M); Biacore™ pH 7.4 hFcRn and rFcRn steady state RU (RU). EXAMPLES

[0165] The combinatorial variant further reduces the FcRn binding dissociation rate Multiple lead mutations were located at a single position, such as T307 and N434 (Figure 14, black squares), where six and three mutations were identified, respectively, that showed slower FcRn dissociation kinetics. Only the mutations with the slowest FcRn dissociation rate for hFcRn at these positions were used to generate combinatorial variants. In this case, T307Q, T307W, N434F, and N434Y were mixed with M252Y, T256D, and T256E, and double, triple, and quadruple variants were obtained using mixed primer PCR and site-directed mutagenesis. In total, the combinatorial library was assumed to consist of 54 variants, including 7 lead single, 18 lead double, 20 lead triple, 8 lead quadruple variants, and the WT antibody. The naming of these variants is as follows: the wild-type background contains M252, T256, T307, and N434, and is given a new name, MTTN. Thus, the triple variant Y T QY contains each mutation of M252 Y , T307 Q and N434 Y , while maintaining the WT threonine at position 256.

[0166] In a single mutation, the combination variants with improved affinity were determined using the FcRn binding kinetics at pH 6.0 using Biacore™. Representative FcRn binding kinetic traces for single (long dashed-dotted line), double (long dashed line), triple (long dotted line), and quadruple (short dotted line) are shown in FIGS. 6A and 6B compared to the benchmark variant with the tightest affinity for each species of WT (dotted line) and FcRn (hFcRn:LS (long dashed-dotted line); rFcRn:YTE (solid line)). By the association and dissociation rates of hFcRn (FIG. 6C), it was revealed that each of the two single, 15 double, 18 triple, and 8 quadruple variants had enhanced binding affinity over the LS variant (FIG. 6C, spots). Similarly, all combinations except one triple variant had a tighter affinity for rFcRn than YTE (diagonal stripes going towards the lower left in FIG. 6D). In the case of hFcRn, further FcRn-enhancing mutations further improved the binding affinity (FIG. 6C). The five combinations with the tightest affinity for hFcRn were all quadruple variants (FIG. 6C, lattice pattern), and the binding affinity was approximately 500-fold greater than that of the wild type. Although the variant with the highest affinity was a double variant (FIG. 6D, horizontal stripes), a similar phenomenon did not occur with rFcRn (FIG. 6D). Triple (FIG. 6D, vertical stripes) and quadruple (FIG. 6D, lattice pattern) variants typically showed only a slight decrease in the dissociation rate (decreasing to less than half), but also presented a decreased association rate (FIG. 6D). Without being bound by any theory, these results suggest that there is probably a lower limit (approximately 0.5 nM) for the apparent binding affinity of FcRn that was reached with rFcRn but not with hFcRn (FIG. 6B). In total, over 40 combination variants had a tighter affinity than the benchmark variant, but further characterization is needed to select combinations with properties favorable for in vivo studies.

Example

[0167] Combination variants retain significant binding at physiological pH As a result of the significantly improved FcRn affinity at pH 6.0, the effect on pH-dependency was investigated using FcRn affinity chromatography and Biacore™ steady state measurements at pH 7.4. In FcRn affinity chromatography, a linear pH gradient was used to directly measure the perturbation of pH-dependency by the mutations. Variants with weak FcRn binding, H435A and H310A / H435Q, did not bind to the column regardless of pH (Figure 8A). WT eluted near physiological pH (pH 7.37 ± 0.05), while higher pH was required for AAA, LS and YTE (Table 2B). All combinatorial variants and seven lead single variants required higher pH than WT to elute from the affinity column (Figures 8A and 8C). The N434F / Y variant eluted at a higher pH than LS (Table 2B), which, without intending to be bound by scientific theory, indicates that the pH-dependency was disrupted by these variants both alone and in combination. Representative chromatograms showed a clear shift to higher elution pH with the number of mutations (Figures 9A and 9B). A strong correlation (R2 = 0.94) between the elution pH and the hFcRn dissociation rate (Figure 9C) indicates that the slower FcRn dissociation rate at pH 6.0 was the direct cause of the increased elution pH for the FcRn variants.

[0168] To measure the residual binding activity under physiological conditions, FcRn binding kinetics experiments were performed at pH 7.4 using Biacore™. Since some variants showed uncertain kinetics and little or no binding at this pH, the steady state RU was used as a measure of the residual FcRn binding affinity. Representative kinetic traces for each of the single (long dashed line), double (long dash-dotted line), triple (long dotted line) and quadruple (short dotted line) variants are shown in FIGS. 7A and 7B compared to LS (solid line in FIG. 7A) and YTE (solid line in FIG. 7B). These two variants exhibited the greatest residual binding to human and rat FcRn at pH 7.4, respectively. The majority of the lead single variants had slightly elevated FcRn binding compared to WT (4.3 ± 1.0 RU), but had less FcRn binding than AAA (13.1 ± 1.7 RU), LS (18.5 ± 2.6 RU) and YTE (13.1 ± 1.6 RU) except for the N434F / Y mutation (Tables 2A and 2B). The combination variants also retained significant residual binding to both FcRns at pH 7.4 to a greater extent than N434F / Y (FIGS. 7A and 7B). Without being bound by any theory, ideal candidates for in vivo studies are variants that have improved FcRn binding at low pH (e.g., each of the AAA, LS and YTE variants), but maintain low levels of binding at elevated pH in the same manner as WT. In the plots shown in FIGS. 7C and 7D, these combinations would occupy the lower left quadrant as specified by the affinity of the LS and YTE variants at each pH for human and rat FcRn, respectively.

Example

[0169] FcRn affinity chromatography The combination variants had a moderate positive correlation between the apparent binding affinity at pH 6.0 and the steady state RU at pH 7.4 (hFcRn:R 2 = 0.69, rFcRn:R 2= 0.71) (Figs. 7C and 7D). Without being bound by any theory, what these results suggest is that a significantly higher affinity at pH 6.0 typically leads to higher residual FcRn binding at pH 7.4. These variants, like the Abdeg mutants with high FcRn affinity, were able to remain bound to FcRn in the bloodstream and had a short serum half-life and / or were able to promote their clearance (see, e.g., Swiercz et al., J Nucl Med. (2014) 55:1204 - 1207; and Vaccaro et al., Nat Biotechnol. (2005) 23:1283 - 1288). Since the IgG-FcRn interaction is pH-dependent and occurs only at low pH (<pH 6.5), the saturation mutations can enhance the interaction by contributions from hydrophobic and / or charge-derived interactions, and these interactions can prevent the deprotonation of key histidine residues (as shown in Fig. 1B) and the attenuation of this interaction at physiological pH. As a result, the FcRn binding interaction is thus less sensitive at the pH of its environment. can enhance the interaction by contributions from charge-derived interactions, and these interactions can prevent the deprotonation of key histidine residues (as shown), and the attenuation of this interaction at physiological pH. As a result, the FcRn binding interaction is thus less sensitive at the pH of its environment.

[0170] In FcRn affinity chromatography, a linear pH gradient was used to directly measure the pH-dependent perturbation of FcRn interaction (see, for example, Schlothauer et al., supra). By FcRn affinity chromatography using each of the variants of AAA, LS, YTE, H435A and H310A / H435Q, it was revealed that H435A (Figure 8A, light gray solid line) and H310A / H435Q (Figure 8A, AQ, dark gray solid line) did not bind to FcRn even at pH 5.5 and eluted in the flow-through fraction. The wild-type antibody eluted near physiological pH (pH 7.37 ± 0.05), while AAA, LS and YTE, which have a dissociation rate slower than that of the wild-type and a tighter FcRn binding affinity than these by Octet (Figures 2B - D) and Biacore™ (Figure 3), required a significantly higher pH (AAA: 7.94 ± 0.06; LS: 8.29 ± 0.03; YTE: 8.14 ± 0.03) to dissociate from the column. The elution profiles revealed that for all variants of the combinatorial library, a higher pH than that of the wild-type was required to elute from the affinity column. Representative chromatograms at the average elution pH for single (long dashed-dotted line), double (long dashed line), triple (long dotted line) and quadruple (short dotted line) variants are shown in Figure 9A. Seven lead single variants (M252Y, T256D, T256E, T307Q, T307W, N434F and N434Y) required a higher pH to dissociate from the column compared to WT (Figure 10A, Table 3), while all those having wild-type-like kinetics for hFcRn (K288D / N, Y436H / H / W) eluted at a pH similar to that of the wild-type.

[0171]

Table 4

[0172] All data were obtained using the experimental techniques at the top of each column. The elution pH was determined in triplicate (n = 3) by FcRn affinity chromatography, and the thermal stability was explored in triplicate (n = 3) by DSF. The FcRn binding kinetics to human FcRn and rat FcRn were obtained from Biacore™ using a series of antibody concentrations (n = 4) and fit individually. The units for each measurement are as follows: elution pH (unitless); DSF T m (°C); Biacore™ pH6.0 hFcRn association rate (×10 4 M -1 s -1 ), dissociation rate (×10 -1 s -1 ) and K D,app (×10 9 M); Biac ore™ pH6.0 rFcRn association rate (×10 4 M -1 s -1 ), dissociation rate (×10 -3 s -1 ) and K D,app (×10 9 M).

[0173] Both N434F / Y variants eluted at higher pH than the LS variant (N434F: 8.30 ± 0.05; N434Y: 8.46 ± 0.02) and showed significant FcRn binding at pH 7.4 (Table 4). These results indicate that only these variants are able to disrupt the pH-dependence. Generally, the average elution pH increased with increasing number of mutations enhancing FcRn binding (Figure 9B). A strong correlation (R 2 = 0.94) emerged between the elution pH and the hFcRn dissociation rate (Figure 9C); without being bound by any theory, this indicates that the disruption of the pH-dependence of the interaction is the direct cause of the slower FcRn dissociation rate observed for the combinatorial library at pH 6.0.

Example

[0174] Thermal stability Most proteins with low thermodynamic stability, including antibodies, have an increased tendency to misfold and aggregate, which is thought to limit or impede their activity, efficacy, and potential as novel therapeutic agents. The thermal stability of each variant was determined using DSF, and the reported melting temperature (T m ) was defined as the midpoint of the first transition in the Sypro™ Orange fluorescence intensity profile. Compared to WT with a T m of 69.0 ± 0.2 °C, the LS variant was WT-like (68.5 ± 0.3 °C), and AAA and YTE were thermally destabilized by approximately 8 °C (AAA: 61.3 ± 0.6 °C; YTE: 61.2 ± 0.3 °C) (Figures 8B, 9B, and 10B; and Tables 2B, 3, and 4). Compared to WT and LS with a T m of 69.0 ± 0.2 °C, the AAA and YTE variants had approximately 8 °C lower thermal stability by DSF.

[0175]

Table 5

[0176] Mutations introduced into the wild-type backbone are underlined. All data were obtained using the experimental techniques indicated at the top of each column. Elution pH and T m were determined in triplicate (n = 3). FcRn binding kinetics to human FcRn and rat FcRn at pH 6.0 were obtained from Biacore™ (n = 4) and fit individually. The steady-state FcRn binding response at pH 7.4 was measured in triplicate at a single antibody concentration using Biacore™. FcγRIIIa binding affinity was determined from a series of antibody concentrations in duplicate using Biacore™. The units for each measurement are as follows: elution pH (unitless); DSF T m (°C); Biacore™ pH 6.0 hFcRn association rate (×10 5 M -1 s -1 ), dissociation rate (×10 -2 s -1 ), and K D,app (×109 M); Biacore (trademark) pH6.0 rFcRn binding rate (×10 5 M -1 s -1 ), dissociation rate (×10 -3 s -1 ) and K D,app (×10 9 M); Biacore (trademark) pH7.4 steady-state binding response (RU) and FcγRIIIa K D,app (×10 9 M).

[0177] Of the 18 lead saturation variants, 12 (each variant of E380C, M252Y, T256D, T256E, K288N, K288D, N434P, T307A, T307W, Y436H, Y436N, Y436W) had a decreased T m compared to the wild type, and some of the T307 mutations (T307E / F / M / Q) showed slight stabilization (Table 4). None of the seven single variants used in the combination (Figure 10B and Table 4) were significantly more stabilized compared to YTE (Figure 8B and Table 5). By adding each double (Figure 9D, horizontal stripes), triple (Figure 9D, vertical stripes) and quadruple (Figure 9D, lattice pattern) mutation to the Fc domain, a further decrease in overall thermal stability was brought about compared to the single mutation (Figure 9D, white circles). Multiple variants exhibited a lower T than AAA or YTE (61.2 ± 0.°C), and in this case, >60% of these variants contained T307W. The quadruple variant (Figure 9D, lattice pattern) showed a distinguishable bimodal distribution with respect to the melting temperature, and in this case, the combination containing T307Q had a further thermal stability of approximately 6°C higher than those carrying T307W (Figure 9D). m Although multiple variants exhibited a lower T

Example

[0178] Fc variants alter the binding interaction with FcγRIIIa In addition to the interaction with FcRn, the Fc region hinge and C HThe 2-domain is responsible for the interaction with other Fc receptors, including FcγRIIIa. Five of the seven single variants used for the construction of the combinatorial saturation library are located within the C H 2-domain. Therefore, their ability to interact with those receptors may be impaired compared to the wild type, despite their variant positions being far from the interaction interface. By using Biacore™ to measure FcγRIIIa binding in a manner similar to FcRn binding at pH 7.4, it was revealed that the YTE (Figure 11A, dark gray) variant showed an approximately 50% decrease in the binding response compared to the wild type (Figure 11A, black). Without being bound by any theory, since only the M252Y variant has a significantly reduced affinity for this receptor, the decrease in FcγRIIIa binding for YTE is the result of the M252Y mutation (Figure 11B, bottom white circle). The other single mutations do not share such a decrease in affinity (Figure 11B, white circles), and only the N434F / Y variant enhanced the binding by 16 - 40%. Such effects were retained in most, if not all, of their corresponding combinations. For example, the M252Y-containing combinations had a decrease in FcγRIIIa binding between 17% and 72% (Table 5).

[0179] [Table 6]

[0180] One variant M DQF (the highest in the triple variant category in Figure 11B) showed a dramatic 140% improvement in FcγRIIIa binding. Therefore, the combinatorial saturation library provided variants with a wide range of Fc receptor functions that can be utilized to target therapeutic antibodies with specific effector functions.

[0181] Figure 11C shows a box-and-whisker plot of the FcγRIIIa binding responses of the seven lead single variants compared to the WT and YTE variants. [Examples]

[0182] The seven lead combinations maintain the balance of the pH-dependence of the FcRn interaction Without being bound by any theory, candidate variants for further in vivo studies occupied the lower left quadrant of the plots shown in FIGS. 7C and 7D. Seven variants met these criteria for hFcRn, including five double combinations and two triple combinations (M DQ N, M DW N, YD TN, YE TN, Y T W N, YDQ N and YEQ N) and did not contain a mutation at position N434 (Table 3). Each of these combinations eluted from the FcRn affinity column between AAA (pH 7.94 ± 0.06) and LS (pH 8.29 ± 0.03), YDQ N eluted at the highest pH of 8.51 ± 0.14 (FIG. 12A, Table 5), indicating only a slight perturbation in pH-dependence at pH 7.4 and higher residual binding (Table 2A). One of the variants (M DQ N) possessed wild-type-like thermal stability and six had similar or decreased T m compared to the YTE variant (FIG. 12B, Table 4). In the FcγRIIIa binding assay, five combinatorial variants showed a similar decrease as YTE (Table 4). Further investigation using single mutations revealed, without being bound by any theory, that M252Y significantly affected FcγRIIIa binding and that this effect was retained in the combinations bearing that mutation. The remaining six single mutations were either WT-like or had slightly improved binding to this receptor.

[0183] Three combinatorial variants were selected for further study based on their FcRn binding properties, thermal stability, and FcγRIIIa binding. DQ (T256D / T307Q), DW (T256D / T307W), and YD (M252Y / T256D) each resulted in optimal FcRn binding properties like the LS variant (Figure 12E) (Table 2B). Each variant enabled diverse thermal stability and FcγRIIIa binding properties that resulted in various functionalities (Figures 12F and 12G, Table 2B). Figure 12H is a plot of homogeneous bridging RF.

[0184] Enhanced apparent binding affinities for both human and rat FcRn at pH 6.0, compared to the LS variant (Figure 13A, thick long dashed line) and the YTE variant (Figure 13B, thick long dashed line) respectively, were a trade-off between the association and dissociation rates (Figures 13A and 13B, Table 4). Typically, combinations with faster dissociation rates also possessed faster association rates, and vice versa. Such observations were maintained between human FcRn and rat FcRn (Table 4). Furthermore, all of these variants had a lower steady-state response to hFcRn at pH 7.4 than the LS variant (Figure 13C, thick long dashed line). These results are for the five M252Y-containing variants, YD TN, YE TN, Y T W N, YDQ N and YEQ N had improved FcRn binding at pH 7.4 compared to YTE (Table 5), but did not match rFcRn. M DQ N and M DWThe N variant was the only combination that was cross-reactive between human FcRn and rat FcRn. Furthermore, these two variants did not disrupt the interaction with FcγRIIIa to the same extent as the M252Y-containing variant (Figures 12C and 12D and Table 5; MDQN: 600 ± 4 nM; MDWN: 512 ± 30 nM; WT: 467 ± 99 nM). Thus, saturation and combinatorial mutagenesis at the key FcRn interaction sites led to the identification of lead variants that could maintain the pH-dependence balance of the interaction, maintain functionality with Fc receptors, enhance FcRn functionality in vivo, and extend the serum half-life of therapeutic antibodies.

Example

[0185] Rheumatoid factor binding characteristics of lead combination variants Since these mutations can alter antibody surface charge and immunogenicity, the isoelectric point and RF binding of the lead variants were investigated. More acidic antibodies were thought to extend antibody pharmacokinetics. Compared with each control of WT and LS, all three leads resulted in a decrease in pI of approximately 0.2 pH units as a result of the T256D substitution. FcRn-enhancing mutations can simultaneously alter binding to host antibodies such as rheumatoid factor (RF) due to overlapping interaction interfaces. Homogeneous bridging ELISA was applied to measure changes in RF binding for lead variants. Interestingly, LS and YTE showed completely opposite shifts in RF binding compared to WT (Figure 12H). LS significantly improved RF binding, while YTE showed a significant decrease (p<0.001). YD (p<0.001) and DW (p<0.01) also significantly decreased RF binding, while DQ produced a response similar to WT. Without being bound by any theory, these results indicate that DQ, DW, and YD can confer immunogenic advantages compared to LS. Each of the YD, DW, and DQ variants represents various key antibody characteristics that can be exploited in combination with improved FcRn binding properties superior to the benchmark YTE and LS variants.

Example

[0186] Lead combination variants can be transferred to other antibodies As shown in Figure 15A, a new binding assay was developed using a CM5 sensor chip. The binding assay includes immobilizing streptavidin on the CM5 sensor chip to approximately 30 RU to capture biotinylated FcRn, which is replenished as needed. Antibody binding kinetics were measured at pH 6.0 and 7.4, and at pH 8.5 for regeneration. Figures 15B and 15C show direct immobilization of FcRn and streptavidin capture of biotinylated FcRn, respectively, using the new binding assay.

[0187] FcRn binding of MAb2 at pH 6.0: For mouse FcRn, the lead mAb2 variant demonstrated a slower dissociation rate compared to the LS variant (dashed line) and wild type (black) (Figure 16A). For human FcRn, all lead variants had a faster binding rate but a dissociation rate similar to LS (dashed line) (Figure 16B).

[0188] FcRn Binding of MAb2 at pH 7.4: All lead variants showed a decrease in human FcRn binding at pH 7.4 compared to LS (dashed line) (Figure 17A). Similar to the mAb1mAb1’ background, each variant of DW(M DW N) and DQ(M DQ N) also showed lower residual binding to mouse (rat) FcRn at pH 7.4 (Figure 17B).

[0189] The lead variants maintained higher binding affinity at pH 6.0 and lower residual binding at pH 7.4 compared to LS (Figure 18). Importantly, it was found that the variants could be transferred between different IgG1 backgrounds with little effect on FcRn binding. As shown in Figure 19, LS had a similar elution pH regardless of the background. WT, DQ, and DW in the mAb2 background showed a higher elution pH than in the mAb1 background, presumably as a result of tighter binding at pH 6.0 in the mAb2 background.

[0190] All mAb2 background variants showed slightly improved thermal stability, as shown in Figure 20.

[0191] As shown in Figure 21, similar to the mAb1 background, YD( YD TN) showed a decrease in the binding response (left) and affinity (right) for FcγRIIIa. DQ (light gray) and DW (dark gray) showed FcγRIIIa binding characteristics similar to WT (black) in the mAb2 background. The effect on FcγRIIIa binding relative to LS was consistent between mAb1 and mAb2.

[0192] Therefore, lead variants in the mAb2 background did not significantly affect FcRn binding, pH-dependence, thermal stability, or FcγRIIIa binding compared to the same lead variants in the mAb1 background.

[0193] In one embodiment, each of the variants DQ (T256D / T307Q), DW (T256D / T307W), and YD (M252Y / T256D) was incorporated into additional IgG1 antibodies and recombinant Fc fragments: mAb2 recognizes an antigen different from mAb1, and mAb3 is an Fc fragment. In each case, the pH-dependent FcRn binding kinetics (Figure 22) were highly similar in addition to the elution pH, thermal stability, and FcγRIIIa binding affinity (Table 2B and Table 6). Without being bound by any theory, what these results indicate is that each of the DQ, DW, and YD variants conferred these improved FcRn binding properties to the protein composed of the Fc domain.

[0194]

Table 7

Example

[0195] The lead variants extended the in vivo plasma antibody elimination half-life The pharmacokinetics (PK) of each of the DQ, DW, and YD variants were examined for their effects on antibody circulation half-life using cynomolgus monkeys and hFcRn transgenic mice (strain Tg32) (see, for example, Avery et al., Mabs (2016) 8:1064-1078) compared to WT controls and LS controls. Cynomolgus monkey FcRn was FcRn binding studies revealed similar binding affinities for hFcRn (Figures 23A - 23B; Table 6). Each animal was injected intravenously with the WT, LS, DQ, DW, or YD variant, and antibody concentrations were quantified by a mass spectrometry approach to determine clearance rates and serum half - lives in monkeys (Figure 24A) and hFcRn transgenic mice (Figure 24B). Clearance rates and serum half - lives were obtained from a non - compartmental model of antibody concentration as a function of time. All three lead variants and LS showed significantly decreased clearance rates compared to WT in both monkeys and mice (p < 0.001). The plasma half - lives of the WT antibody were 9.9 ± 0.5 days and 11.7 days in monkeys and mice, respectively. Furthermore, the LS benchmark and the identified variants showed a significant prolongation of the elimination half - life in both species compared to the wild type (2.5 - fold and 1.7 - fold prolongation in monkeys and mice, respectively) (Table 7). DQ, DW, and YD showed a similar prolongation of the half - life compared to the LS benchmark (Table 7). Each of the DQ, DW, and YD mutations identified herein by saturation mutagenesis demonstrated a significantly prolonged plasma half - life compared to their WT counterparts in both mouse and non - human primate models.

[0196]

Table 8

[0197] In Table 7, clearance rates and plasma half - lives were determined using mAb2. Each clearance rate and half - life was taken as the mean for n = 3 cynomolgus monkeys and as a single evaluation from a pool of n = 6 hFcRn transgenic mice. The fold over WT and fold over LS indicate the relative improvement in serum half - life compared to WT and LS, respectively. * n = 2 for ADA formation, ** n = 2 for the partial subcutaneous route of administration

Example

[0198] Combinatorial variants with enhanced FcRn binding at pH 6.0 and pH 7.4 Based on the Octet screening (BLI-based screening) described in Example 2, various single, double, triple, and quadruple variants were generated and evaluated for their binding to FcRn at pH 6.0 and pH 7.4 (Table 8; substituted residues are underlined).

[0199]

Table 9-1

Table 9-2

[0200] Table 8 shows the binding affinities for FcRn at pH 6.0 and the steady-state binding to FcRn at pH 7.4 for various single, double, triple, and quadruple mutants, as well as benchmark variants (AAA, LS, YTE).

[0201] These values were plotted in Figure 25. Figure 25 shows a comparison of the binding affinity at pH 6.0 and RU at pH 7.4. As shown, the benchmark variant LS had the tightest binding affinity at pH 6.0 and the greatest residual binding at pH 7.4 among the benchmark variants (AAA, LS, YTE) tested.

[0202] It was determined that several combinatorial variants shown in Figure 25 exhibited enhanced FcRn binding affinity at pH 6.0 and pH 7.4. To examine which of the combinatorial variants showed tighter binding than the MST-HN variant (referred to herein as the "YTEKF benchmark", containing mutations from Met252, Ser254, Thr256, His433 and Asn434 to Tyr252, Thr254, Glu256, Lys433 and Phe434) at both pH 6.0 and pH 7.4, the following methodology was performed. The capture of biotinylated human FcRn and cynomolgus FcRn was performed by the Biotin CAPture method (see Figure 26 for a schematic). For pH 6.0, concentration series (5 pts) from 1000 nM were performed in duplicate. For pH 7.4 and 8.0, in addition to concentration series from 1000 nM being performed in duplicate, injections at a single concentration (1000 nM) were performed in triplicate. The experiments at pH 7.4 and 8.0 were performed at 10-fold elevated FcRn capture levels compared to pH 6.0 to observe binding at these pHs. For pH 9.0, injections at a single concentration (1000 nM) were performed in triplicate. The experiment at pH 9.0 was performed at 100-fold elevated FcRn capture levels compared to pH 6.0 to observe binding at this pH. For pH 6.0, concentration series (5 pts) from 1000 nM were performed in duplicate. For pH 7.4, injections at a single concentration (1000 nM) were performed in triplicate (the capture levels of each FcRn were increased 10-fold to observe binding at this pH). Association: 180 seconds; Dissociation: 300 seconds.

[0203] Binding kinetic sensorgrams of human FcRn and cyno FcRn at pH 6.0 (human: Figure 27A, Cyno: Figure 27B), pH 7.4 (human: Figure 27C, Cyno: Figure 27D), and pH 8.0 (human: Figure 27E, Cyno: Figure 27F) for wild-type, YTEKF benchmark, and combinatorial variants of 10 FcRn antagonists shown in Figures 27A - G. All variants examined showed two-digit tighter affinity and observable dynamics at higher pH for both human and cyno FcRn compared to WT at pH 6.0. The variants examined had enhanced affinity for FcRn and were thus grouped into each combination of six quadruples (YDQF, YDQY, YDWF, YDWY, YEQY, YEWY), three triples (MDWY, YDTY, YTWY), and one double (YY) (Table 8). Two additional quadruple variants, YEQF and YEWF (Figure 27G), were not selected as they had weaker affinity for human FcRn and rat FcRn.

[0204] Figures 28A - H and 29A - H show the binding kinetics of 10 FcRn antagonist variants (black solid lines) to human FcRn and cyno FcRn, compared to the YTEKF benchmark (dotted line) and WT (dashed line). In Figures 28A and 29A, 8 out of 10 of the variants exhibited a slower dissociation rate than the YTEKF benchmark and showed similar binding rates to human FcRn and cyno FcRn at pH 6.0 (Tables 9 and 10). All variants showed significant FcRn binding to both human (Figures 28B and 28D) and cyno (Figures 29C and 29D) FcRn at pH 7.4 and pH 8.0 (Tables 9 and 10). Five quadruple variants, YDQF, YDQY, YDWY, YEQY, and YEWY, showed a higher binding signal than the YTEKF benchmark, indicating enhanced affinity at this pH. Normalization of the sensorgrams at pH 7.4 (Figures 28C and 29C) and pH 8.0 (Figures 28E and 29E) for human FcRn and cyno FcRn respectively showed that these 5 variants exhibited significant FcRn binding at these pHs with a slower dissociation rate than the YTEKF benchmark. In Figures 28F and 29F, at pH 9.0, human FcRn and cyno FcRn binding decreased to levels similar to the WT and YTEKF variants. Figures 28G and 29G show isothermal affinity plots at three pHs (pH 6.0, black; pH 7.4, dark gray; pH 8.0, gray) for the binding of human FcRn (28G) and cyno FcRn (Figure 29G), compared to the YTEKF benchmark (white squares) and WT (black, pH 6.0 only). The YTEKF benchmark and lead combination variants have binding affinities for human FcRn and cyno FcRn that are 2 - 3 orders of magnitude greater than WT at pH 6.0. The FcRn binding affinity decreased as the pH increased, and the benchmark and combination variants presented similar affinities at all pHs examined. Three variants, YDQY, YDWY, and YEWY, showed greater binding to human FcRn and cyno FcRn at all pHs (Tables 9 and 10).When comparing the FcRn binding affinities for human FcRn (Figure 28H) and cyno FcRn (Figure 29H) as a function of pH, it was shown that for all the combinatorial variants examined, the FcRn affinity decreased by a factor of 10 per pH unit.

[0205] Figures 33 and 34 show the association rates, dissociation rates, binding affinities, and steady-state binding for human FcRn (Figure 33) and cyno FcRn (Figure 34) at pH 6.0, 7.4, 8.0, and 9.0 for selected combinatorial variants, the YTEKF benchmark, and WT. Although all variants exhibited measurable binding kinetics (association rate, dissociation rate, and binding affinity) at pH 6.0, the steady-state binding levels at this pH have not been reported. Since the FcRn binding affinity of all variants decreased significantly at pH 9.0, only the steady-state binding levels are shown as a measure of residual binding at this pH. The inserted mutations are in boldface and underlined in the WT sequence (MTTN: M252 / T256 / T307 / N434). For example: MDWY contained the T256D / T307W / N434Y mutation and the WT residue at position 252.

[0206] Other characterization parameters of the FcRn antagonist variants, the YETK benchmark, and WT, such as binding to FcγRIIIa, thermal stability, and elution pH, were determined and shown in Table 9. The inserted mutations are in boldface and underlined in the WT sequence (MTTN: M252 / T256 / T307 / N434). For example: M DWY contained the T256D / T307W / N434Y mutation and the WT residue at position 252.

[0207]

Table 10

[0208] As shown in Table 9, all FcRn antagonists were thermally unstable and eluted from the FcRn column at significantly higher pH compared to WT. FcγRIIIa binding was highly variable, but in this case, steady-state binding was similar or decreased compared to WT. The inserted mutations are underlined in bold in the WT sequence (MTTN: M252 / T256 / T307 / N434). For example: MDWY contained the T256D / T307W / N434Y mutation and the WT residue at position 252.

Example

[0209] FcRn antagonists promote IgG degradation FcRn antagonists were co-administered with human IgG to determine IgG depletion. Experiments were performed using human FcRn homozygous transgenic mice (Tg32; e.g., Jackson Laboratory stock #014565, M01 genotype), which allows for scaling to predict human pharmacokinetics.

[0210] The effect of FcRn antagonists (e.g., any of the quadruple variants described in FIGS. 33 and 34; e.g., YDQY, YEWY, YEQY, YDQF, and YDWY) on serum IgG levels was determined in Tg32 mice. Tg32 mice were administered non-target human IgG1 5 mg / kg by intravenous bolus injection. Six hours later, the mice were administered either human IgG1 containing an FcRn antagonist (e.g., as described in FIGS. 33 and 34; e.g., YDQY, YEWY, YEQY, YDQF, and YDWY), or a targeting antibody containing an FcRn antagonist (e.g., as described in FIGS. 33 and 34; e.g., YDQY, YEWY, YEQY, YDQF, and YDWY), or a vehicle control. The animals were administered according to the dosing schedule described in Table 10.

[0211]

Table 11

[0212] Blood samples were collected according to the bleeding schedule shown in Table 11. The levels of FcRn antagonist IgG1 (FcRn-A; a targeting antibody containing human IgG1 and quadruple variants), non-targeting human IgG1 (hIgG), endogenous mouse IgG (murIgG), and mouse serum albumin (murAlb) were determined by quantitative LC-MS / MS and ELISA.

[0213] [Table 12] Examples

[0214] FcRn antagonist enhances IgG degradation This example describes a study showing that the clearance of co-administered IgG was enhanced by an exemplary FcRn antagonist. In this study, mAb1 YDQY (hIgG1 variant) was administered to wild-type mAb1 together with hFcRn homozygous transgenic mice (Tg32; Jackson Laboratory stock #014565, M01 genotype), which enabled prediction of scaling to humans in terms of pharmacokinetics. As part of the study, PK measurements were obtained for both YDQY hIgG1 and WT hIgG1. Albumin measurements further suggest that FcRn inhibition does not interfere with albumin binding sites.

[0215] More specifically, 5 mg / kg of WT hIgG1 ("tracer IgG") was given to Tg32 mice by intravenous bolus injection. Six hours later, either YTEKF hIgG1 (ABDEG™) or YDQY hIgG1 was administered to the mice according to the dosing schedule shown in Table 12. All doses were administered at a dosing volume of 10 mL / kg.

[0216] [Table 13]

[0217] As shown by the data, three 20 mg / kg doses of YDQY hIgG1 (at 6, 24, and 48 hours) significantly decreased the tracer WT hIgG1 concentration (Group 6; Figures 30A and 30B; Table 13), resulting in a significantly improved outcome compared to YTEKF hIgG1. A single dose of YDQY hIgG1 or YTEKF hIgG1 did not appear to cause a robust decrease in tracer WT IgG (Figure 30C; Table 13).

[0218]

Table 14

[0219] The clearance of YDQY hIgG1 (approximately 2.6 mL / h / kg) was faster than that of typical IgG in Tg32 mice (approximately 0.1 - 1.0 mL / h / kg) or YTEKF hIgG (approximately 1.1 mL / h / kg) (Figures 31A and 31B), but it was a sustained exposure throughout the study period. As the results show, this FcRn antagonist was able to remain in the host for a sufficient period to decrease FcRn-mediated recycling of WT IgG and promote WT IgG clearance.

Claims

1. 1. A pharmaceutical composition comprising an FcRn antagonist for use in treating an IgG-mediated disorder in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist is selected from the group consisting of: a) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; b) a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; c) a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; d) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a phenylalanine (F) at amino acid position 434; e) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; and f) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a phenylalanine (F) at amino acid position 434. The pharmaceutical composition comprising a modified human IgG Fc domain comprising a combination of amino acid residues selected from the group consisting of:

2. The modified Fc domain comprises: M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, M252Y / T256D / T307W / N434Y, and M252Y / T256D / T307W / N434F 2. The pharmaceutical composition of claim 1, comprising a quadruple amino acid substitution selected from the group consisting of:

3. 1. A pharmaceutical composition comprising an FcRn antagonist for use in treating an IgG-mediated disorder in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist comprises a modified human IgG Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434, wherein the therapeutically effective amount of the FcRn antagonist increases serum IgG clearance in the subject.

4. 1. A pharmaceutical composition comprising an FcRn antagonist for use in treating an IgG-mediated disorder in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, the FcRn antagonist comprising a modified human IgG Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434, wherein the therapeutically effective amount of the FcRn antagonist increases serum IgG clearance in the subject.

5. 1. A pharmaceutical composition comprising an FcRn antagonist for use in treating an IgG-mediated disorder in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist comprises a modified human IgG Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434, wherein the therapeutically effective amount of the FcRn antagonist increases serum IgG clearance in the subject.

6. 1. A pharmaceutical composition comprising an FcRn antagonist for use in treating an IgG-mediated disorder in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist comprises a modified human IgG Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a phenylalanine (F) at amino acid position 434, wherein the therapeutically effective amount of the FcRn antagonist increases the clearance of serum IgG in the subject.

7. 1. A pharmaceutical composition comprising an FcRn antagonist for use in treating an IgG-mediated disorder in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist comprises a modified human IgG Fc domain comprising, according to EU numbering, a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434, wherein the therapeutically effective amount of the FcRn antagonist increases serum IgG clearance in the subject.

8. 1. A pharmaceutical composition comprising an FcRn antagonist for use in treating an IgG-mediated disorder in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist is a FcRn antagonist comprising a combination of the following amino acid residues according to EU numbering: a) a tyrosine (Y) at amino acid position 252 and a tyrosine (Y) at amino acid position 434; b) a tyrosine (Y) at amino acid position 252, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; c) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, and a tyrosine (Y) at amino acid position 434, or d) an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; wherein a therapeutically effective amount of an FcRn antagonist increases clearance of serum IgG in a subject.

9. The modified Fc domain comprises: M252Y / N434Y, M252Y / T307W / N434Y, M252Y / T256D / N434Y, and T256D / 307W / N434Y 9. The pharmaceutical composition of claim 8, comprising a combination of amino acid substitutions selected from the group consisting of:

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the IgG-mediated disorder is an autoimmune disease.

11. 11. The pharmaceutical composition of claim 10, wherein the autoimmune disease is selected from the group consisting of graft-versus-host disease (GVHD), systemic lupus erythematosus (SLE), myasthenia gravis, systemic sclerosis (SSc) / scleroderma, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, diabetes, multiple sclerosis, pemphigus vulgaris, atopic dermatitis, psoriasis, asthma, allergy, idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic purpura (ITP), and hidradenitis suppurativa.

12. 1. A pharmaceutical composition comprising an FcRn antagonist for use in enhanced diagnostic imaging in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist is selected from the group consisting of: M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, M252Y / T256D / T307W / N434Y, and M252Y / T256D / T307W / N434F, The pharmaceutical composition comprising a modified human IgG Fc domain comprising a combination of amino acid substitutions selected from the group consisting of:

13. 1. A pharmaceutical composition comprising an FcRn antagonist for use in enhanced diagnostic imaging in a subject in need thereof, wherein the composition is administered to the subject in a therapeutically effective amount of the FcRn antagonist, wherein the FcRn antagonist is selected from the group consisting of: M252Y / N434Y, M252Y / T307W / N434Y, M252Y / T256D / N434Y, and T256D / 307W / N434Y The pharmaceutical composition comprising a modified human IgG Fc domain comprising a combination of amino acid substitutions selected from the group consisting of:

14. 14. The pharmaceutical composition of claim 12 or 13, wherein the subject is further administered a radiolabeled IgG antibody.

15. 15. The pharmaceutical composition of claim 14, wherein the FcRn antagonist is administered after the radiolabeled antibody, thereby enhancing contrast to the radiolabeled antibody.

16. 1. A pharmaceutical composition comprising an FcRn antagonist for use in reducing exposure of non-target tissue to a radiolabeled antibody during diagnostic imaging in a subject in need thereof, wherein the composition is administered to the subject in an effective amount of the FcRn antagonist, wherein the FcRn antagonist is an FcRn antagonist selected from the group consisting of: M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, M252Y / T256D / T307W / N434Y, and M252Y / T256D / T307W / N434F, The pharmaceutical composition comprising a modified human IgG Fc domain comprising a combination of amino acid substitutions selected from the group consisting of:

17. 1. A pharmaceutical composition comprising an FcRn antagonist for use in reducing exposure of non-target tissue to a radiolabeled antibody during diagnostic imaging in a subject in need thereof, wherein the composition is administered to the subject in an effective amount of the FcRn antagonist, wherein the FcRn antagonist is an FcRn antagonist selected from the group consisting of: M252Y / N434Y, M252Y / T307W / N434Y, M252Y / T256D / N434Y, and T256D / 307W / N434Y The pharmaceutical composition comprising a modified human IgG Fc domain comprising a combination of amino acid substitutions selected from the group consisting of:

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the modified Fc domain is a modified human IgG1 Fc domain.

19. 19. The pharmaceutical composition of any one of claims 1 to 18, wherein the altered Fc domain has enhanced FcRn binding affinity at acidic and non-acidic pH compared to the wild-type Fc domain.

20. 20. The pharmaceutical composition of any one of claims 1 to 19, wherein the modified Fc domain is a modified human IgG Fc domain and has enhanced FcRn binding affinity at acidic and / or non-acidic pH compared to a human IgG Fc domain of the same subtype comprising the amino acid substitutions M252Y / S254T / T256E / H433K / N434F.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein the altered Fc domain has reduced FcγRIIIa binding affinity compared to the wild-type Fc domain.

22. 22. The pharmaceutical composition of any one of claims 1 to 21, wherein the FcRn antagonist comprises the amino acid sequence SEQ ID NO: 1, 2, 3, or 4, or an FcRn-binding portion thereof, having an amino acid substitution.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the FcRn antagonist is a binding polypeptide that binds to one or more targets that are not FcRn.

24. The pharmaceutical composition of any one of claims 1 to 22, wherein the FcRn antagonist is an antibody or comprises an antigen-binding fragment thereof.

25. 25. The pharmaceutical composition of claim 24, wherein the FcRn antagonist is a monoclonal antibody.

26. 26. The pharmaceutical composition of claim 25, wherein the antibody is a chimeric, humanized, or human antibody.

27. The pharmaceutical composition of any one of claims 1 to 22, wherein the FcRn antagonist is an Fc fusion protein.

28. 28. The pharmaceutical composition of claim 27, wherein the Fc fusion protein is an immunoadhesin.

29. The pharmaceutical composition of any one of claims 1 to 22, wherein the FcRn antagonist is an Fc fragment.

30. An isolated polypeptide consisting of a modified human IgG Fc domain comprising a combination of amino acid substitutions of the FcRn antagonist of any one of claims 1 to 10.

31. 31. The isolated polypeptide of claim 30, wherein the modified Fc domain comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, or an FcRn-binding portion thereof, with the indicated substitutions.

32. The pharmaceutical composition of any one of claims 1 to 29, comprising a pharmaceutically acceptable carrier.

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