FcRn antagonists and their uses

Abstract

The present disclosure relates to antagonists of FcRn with extended half-life, comprising an immunoglobulin Fc domain and at least one albumin.

Description

[Technical Field]

[0001] Related application data This application claims priority to Australian Patent Application No. 2022903917, entitled "FcRn Antagonists and Uses Thereof," filed on 20 December 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to antagonists of FcRn with extended half-lives. [Background technology]

[0004] Immunoglobulin gamma (IgG) plays an important role in the pathology of many diseases, including autoimmune diseases, inflammatory diseases, and diseases whose pathology is characterized by overexpression of IgG (e.g., hypergammaglobulinemia) (see, e.g., Junghans, Immunol Res. 16:29 (1997)).

[0005] The half-life of IgG in serum is extended compared to the serum half-lives of other plasma proteins. This long half-life is due, in part, to the binding of the Fc domain of IgG to the neonatal Fc receptor (FcRn). In adults, FcRn functions to protect IgG from degradation. FcRn binds pinocytosed IgG and recycles it back to the extracellular compartment, thereby protecting it from trafficking to lysosomes for degradation. This recycling is facilitated by the pH-dependent binding of IgG to FcRn; the IgG / FcRn interaction is stronger at acidic endosomal pH than at extracellular physiological pH.

[0006] When the serum concentration of IgG reaches a level that exceeds the available FcRn molecules, unbound IgG is not protected from degradation, resulting in a shortened serum half-life. Thus, inhibiting IgG binding to FcRn shortens the serum half-life of IgG by preventing endosomal recycling of IgG. Therefore, agents that antagonize IgG binding to FcRn are useful for modulating, treating, or preventing antibody-mediated diseases such as autoimmune diseases.

[0007] Currently, some autoimmune diseases are treated with intravenous infusions of pooled IgG (IVIg) from human donors. Because many of these autoimmune diseases are chronic, affected individuals may require repeated doses of IVIg and / or other appropriate therapies to manage their disease.

[0008] In another approach, blocking antibodies against FcRn have been developed to inhibit IgG binding to FcRn. Peptides that bind to FcRn and antagonize its function have also been identified.

[0009] Recently, an FcRn inhibitor (efgartigimod), a modified form of the human IgG1 Fc domain, has been developed. This compound has been approved for the treatment of myasthenia gravis (MG). The drawback of this compound is its relatively short half-life, which requires regular injections for effective treatment. Summary of the Invention

[0010] In the research leading to the present disclosure, the inventors recognized the relatively short half-life of FcRn antagonists such as efgartigimod as a molecular limitation and a potential problem with patient compliance, and sought to create an FcRn antagonist with an extended half-life. The inventors conjugated the Fc domain of an immunoglobulin, e.g., IgG1, to albumin to extend the half-life of the resulting FcRn antagonist. Unexpectedly, the inventors discovered that linking the C-terminus of the Fc domain to the N-terminus of albumin resulted in a longer half-life and the resulting FcRn antagonist more potently inhibited FcRn than when linked in the opposite direction, i.e., the N-terminus of the Fc domain to the C-terminus of albumin. Most Fc fusion proteins attach the fusion partner to the N-terminus of the protein, i.e., the Fc C H This finding is considered counterintuitive because it involves a domain far from the stable structure formed by the three domains. An FcRn antagonist containing an Fc domain with its C-terminus fused to the N-terminus of albumin resulted in a longer half-life than efgartigimod.

[0011] The present inventors extended their studies using albumin mutants with reduced affinity for FcRn or no binding to FcRn. The inventors hypothesized that using wild-type albumin as an antagonist might affect endogenous albumin recycling via FcRn, potentially resulting in impaired albumin homeostasis and the risk of dyslipidemia or increased cholesterol, which are known to be associated with hypoalbuminemia. On the other hand, a potential drawback of using albumin with reduced affinity for FcRn or no binding to FcRn is that the half-life of the antagonist may not be extended to the same extent. The inventors demonstrated that FcRn antagonists containing albumin with reduced or no binding to FcRn retain the ability to inhibit IgG binding to FcRn and maintain their extended half-life.

[0012] The present inventors further demonstrated that an FcRn antagonist can be administered subcutaneously to reduce circulating IgG levels.

[0013] Based on the above, the present disclosure provides an FcRn antagonist comprising an Fc domain from an immunoglobulin or a fragment thereof, and albumin or a fragment thereof.

[0014] For example, the present disclosure provides: (i) an immunoglobulin Fc domain or a fragment thereof capable of binding to FcRn, and (ii) Provided is an FcRn antagonist comprising at least one type of albumin or a fragment thereof, which can extend the half-life of the FcRn antagonist compared to the half-life of an immunoglobulin Fc domain or a fragment thereof.

[0015] The FcRn antagonists of the present disclosure inhibit or reduce the binding of immunoglobulins, e.g., IgG, to FcRn. For example, administration of an FcRn antagonist of the present disclosure to a subject inhibits or reduces the binding of immunoglobulins, e.g., IgG, to FcRn. Such inhibition can be determined by standard methods known in the art, e.g., by administering an antibody (a "tracer antibody") to the subject and measuring the clearance of the tracer antibody from the circulation; an increased and / or more rapid clearance of the tracer antibody from the circulation compared to the level or rate of clearance in a control subject is indicative of an FcRn antagonist.

[0016] In the present disclosure, the Fc domain or a fragment thereof of an FcRn antagonist is not an immunoglobulin. For example, the FcRn antagonist of the present disclosure includes only the Fc domain of an immunoglobulin, or includes the Fc domain and hinge region, but not the C H 1. C L and does not contain a variable domain.

[0017] In one embodiment, the present disclosure provides: (i) a region of an immunoglobulin consisting of an Fc domain or a fragment thereof capable of binding to FcRn, and (ii) Provided is an FcRn antagonist comprising at least one type of albumin or a fragment thereof, which can extend the half-life of the FcRn antagonist compared to the half-life of immunoglobulin Fc or a fragment thereof.

[0018] In one embodiment, the present disclosure provides: (i) an immunoglobulin Fc domain or a fragment thereof capable of binding to FcRn; (ii) at least one albumin or a fragment thereof capable of extending the half-life of the FcRn antagonist compared to the half-life of an immunoglobulin Fc domain or a fragment thereof; and (iii) optionally, a linker positioned between (i) and (ii).

[0019] In one example, the FcRn antagonist has a longer serum half-life than the serum half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the FcRn antagonist has a half-life that is at least about two-fold, three-fold, four-fold, or five-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the FcRn antagonist has a half-life that is at least about two-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the FcRn antagonist has a half-life that is at least about three-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the FcRn antagonist has a half-life that is at least about four-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the FcRn antagonist has a half-life that is at least about five-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone.

[0020] In one embodiment, the FcRn antagonist has a half-life that is at least 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 75 hours, or 80 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 25 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 30 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 35 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 40 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 45 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 50 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 55 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 60 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 65 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 70 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one embodiment, the FcRn antagonist has a half-life that is at least 75 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone, hi one embodiment, the FcRn antagonist has a half-life that is at least 80 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone.

[0021] In one embodiment, the antagonist has an affinity constant (K) of at least 700 nM for human FcRn at neutral pH. D ) and / or binds to human FcRn at acidic pH with a K of at least 100 nM D where the antagonist is immobilized on a solid support and binding of soluble human FcRn to the antagonist is measured using surface plasmon resonance (SPR). In one example, the antagonist binds to human FcRn with a K of at least 650 nM or 600 nM at neutral pH. D and / or binds to human FcRn at acidic pH with a K of at least 80 nM, 70 nM, or 65 nM D In one example, the antagonist binds to human FcRn at a K of at least 600 nM at neutral pH, where the antagonist is immobilized on a solid support and binding of soluble human FcRn to the antagonist is measured using SPR. D and / or binds to human FcRn with a K of at least 65 nM at acidic pH D where the antagonist is immobilized on a solid support and binding of soluble human FcRn to the antagonist is measured using SPR.

[0022] For purposes of clarity, and as will be apparent to one of skill in the art based on the description herein, reference to affinity as "at least about" or "at least" will be understood to mean that the affinity is equal to or greater than the stated value (i.e., the stated value for affinity is lower), i.e., an affinity of 2 nM is greater than an affinity of 3 nM. In other words, the term can refer to an "affinity of less than or equal to X" or an "affinity not greater than X," where X is a value described herein.

[0023] In one embodiment, the antagonist has a K for cynomolgus monkey FcRn of at least 500 nM at neutral pH. D and / or binds to cynomolgus monkey FcRn at acidic pH with a K of at least 100 nM D where the antagonist is immobilized on a solid support and binding of soluble cynomolgus FcRn to the antagonist is measured using surface plasmon resonance (SPR).

[0024] In one embodiment, the antagonist has a K for mouse FcRn of at least 20 nM at neutral pH. D and / or binds to mouse FcRn with a K of at least 1 nM at acidic pH D where the antagonist is immobilized on a solid support and binding of soluble mouse FcRn to the antagonist is measured using surface plasmon resonance (SPR).

[0025] In one embodiment, the antagonist has a K for rat FcRn of at least 160 nM at neutral pH. D and / or binds to rat FcRn with a K of at least 2 nM at acidic pH D where the antagonist is immobilized on a solid support and binding of soluble rat FcRn to the antagonist is measured using surface plasmon resonance (SPR).

[0026] In one embodiment, the antagonist has a K for human FcRn of at least 10 nM at neutral pH. D where FcRn is immobilized on a solid support and binding of the FcRn antagonist to FcRn is measured using surface plasmon resonance (SPR). In one example, the antagonist has a K of at least 5 nM, or 4 nM, or 3 nM for human FcRn at neutral pH. D where FcRn is immobilized on a solid support and binding of the FcRn antagonist to FcRn is measured using SPR. In one example, the antagonist has a K D where FcRn is immobilized on a solid support and binding of the FcRn antagonist to FcRn is measured using SPR.

[0027] In one embodiment, a "neutral pH" is about pH 7.3. In one embodiment, an "acidic pH" is about pH 6.

[0028] In one embodiment, the binding of the antagonist to FcRn or K D is conferred by an immunoglobulin Fc domain or a fragment thereof.

[0029] In one embodiment of the present disclosure, the albumin or fragment thereof is a human albumin variant or fragment thereof. In one embodiment, the human albumin variant or fragment thereof is a naturally occurring albumin variant. Those skilled in the art will recognize that in the case of albumin variants, references to "positions corresponding to" a recited amino acid residue may need to be adjusted for any insertions or deletions.

[0030] For example, the albumin variant or fragment thereof binds with reduced affinity to FcRn compared to albumin set forth in SEQ ID NO: 1. For example, the binding affinity is measured at neutral and / or acidic pH.

[0031] In one embodiment, the albumin variant or fragment thereof has a K of greater than 5 μM, or 7.5 μM, or 10 μM at neutral pH or pH 6.0. D In one example, the albumin variant or fragment thereof binds to human FcRn with a K of greater than 10 μM at neutral pH or pH 6.0. D It binds to human FcRn.

[0032] In one example, the albumin variant or fragment thereof does not detectably bind to FcRn on the surface of FcRn-expressing HEK-293 cells.

[0033] In one example, the albumin variant or fragment thereof comprises an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1.

[0034] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 391 of SEQ ID NO:1.

[0035] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 402 of SEQ ID NO:1.

[0036] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 418 of SEQ ID NO:1.

[0037] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 420 of SEQ ID NO:1.

[0038] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 424 of SEQ ID NO:1.

[0039] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 429 of SEQ ID NO:1.

[0040] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 462 of SEQ ID NO:1.

[0041] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 492 of SEQ ID NO:1.

[0042] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 501 of SEQ ID NO:1.

[0043] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 545 of SEQ ID NO:1.

[0044] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 547 of SEQ ID NO:1.

[0045] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 422 of SEQ ID NO:1.

[0046] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 510 of SEQ ID NO:1.

[0047] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 535 of SEQ ID NO:1.

[0048] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 463 of SEQ ID NO:1.

[0049] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 467 of SEQ ID NO:1.

[0050] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 505 of SEQ ID NO:1.

[0051] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 509 of SEQ ID NO:1.

[0052] In one example, the albumin comprises an amino acid substitution at a position corresponding to amino acid 519 of SEQ ID NO:1.

[0053] Exemplary albumin variants or fragments thereof include: (i) an aspartic acid substituted with asparagine at a position corresponding to amino acid 391 of SEQ ID NO: 1; (ii) a glutamic acid substituted with a lysine at a position corresponding to amino acid 402 of SEQ ID NO: 1; (iii) a methionine substituted with valine at a position corresponding to amino acid 418 of SEQ ID NO: 1; (iv) an alanine substituted for threonine at a position corresponding to amino acid 420 of SEQ ID NO: 1; (v) an isoleucine substituted for isoleucine at a position corresponding to amino acid 424 of SEQ ID NO: 1; (vi) an aspartic acid substituted with asparagine at a position corresponding to amino acid 429 of SEQ ID NO: 1; (vii) a methionine substituted with valine at a position corresponding to amino acid 462 of SEQ ID NO: 1; (viii) a glycine substituted for glutamic acid at a position corresponding to amino acid 492 of SEQ ID NO: 1; (ix) a valine substituted with glutamic acid at a position corresponding to amino acid 501 of SEQ ID NO: 1; (x) glutamic acid substituted with lysine at a position corresponding to amino acid 545 of SEQ ID NO: 1; (xi) an alanine substituted with valine at a position corresponding to amino acid 547 of SEQ ID NO: 1; (xii) glutamine substituted with histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; (xiii) tryptophan substituted with threonine at a position corresponding to amino acid 422 of SEQ ID NO: 1; (xiv) a methionine substituted for threonine at a position corresponding to amino acid 422 of SEQ ID NO: 1; (xv) glutamine substituted with histidine at a position corresponding to amino acid 510 of SEQ ID NO: 1; (xvi) arginine substituted for histidine at a position corresponding to amino acid 510 of SEQ ID NO: 1; (xvii) phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; (xviii) glutamine substituted with histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; (xix) tryptophan substituted with leucine at a position corresponding to amino acid 463 of SEQ ID NO: 1; (xx) a methionine substituted with threonine at a position corresponding to amino acid 467 of SEQ ID NO: 1; (xxi) a lysine substituted with glutamic acid at a position corresponding to amino acid 505 of SEQ ID NO: 1; (xxii) glycine substituted for glutamic acid at a position corresponding to amino acid 505 of SEQ ID NO: 1; (xxiii) arginine substituted with glutamic acid at a position corresponding to amino acid 505 of SEQ ID NO: 1; (xxiv) tryptophan substituted with phenylalanine at a position corresponding to amino acid 509 of SEQ ID NO: 1; (xxv) glutamic acid substituted with lysine at a position corresponding to amino acid 519 of SEQ ID NO: 1, and (xxvi) a combination thereof.

[0054] In one embodiment, the albumin variant or fragment thereof comprises: (i) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO: 1, or (ii) an amino acid substitution at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (iii) an amino acid substitution at a position corresponding to amino acid 422 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1; or (iv) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 535 of SEQ ID NO:1; or (v) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 509 of SEQ ID NO:1; or (vi) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 519 of SEQ ID NO:1; or (vii) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 510 of SEQ ID NO:1; or (viii) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 505 of SEQ ID NO:1; or (ix) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 467 of SEQ ID NO:1; (x) an amino acid substitution at a position corresponding to amino acid 418 of SEQ ID NO:1, an amino acid substitution at a position corresponding to amino acid 420 of SEQ ID NO:1, an amino acid substitution at a position corresponding to amino acid 505 of SEQ ID NO:1, and an amino acid substitution at a position corresponding to amino acid 547 of SEQ ID NO:1; or (xi) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 463 of SEQ ID NO:1.

[0055] Exemplary albumin variants or fragments thereof include: (i) glutamine substituted with histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; (ii) a tryptophan substituted with threonine at a position corresponding to amino acid 422 of SEQ ID NO: 1; (iii) glutamine substituted with histidine at a position corresponding to amino acid 510 of SEQ ID NO: 1; (iv) a phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1, and (v) containing one amino acid substitution selected from the group consisting of combinations thereof.

[0056] In one example, an exemplary albumin variant or fragment thereof comprises a glutamine substituted for a histidine at a position corresponding to amino acid 464 of SEQ ID NO:1.

[0057] In one embodiment, the albumin variant or fragment thereof comprises: (i) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO: 1, or (ii) an amino acid substitution at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (iii) an amino acid substitution at a position corresponding to amino acid 422 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1; or (iv) an amino acid substitution at a position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at a position corresponding to amino acid 535 of SEQ ID NO:1.

[0058] In one embodiment, the albumin variant or fragment thereof comprises: (i) glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO: 1, or (ii) a phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (iii) glutamine substituted with histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (iv) tryptophan substituted with threonine at the position corresponding to amino acid 422 of SEQ ID NO:1 and glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or (v) glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO:1 and phenylalanine substituted with histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or (vi) a methionine substituted with a threonine at the position corresponding to amino acid 422 of SEQ ID NO:1 and a glutamine substituted with a histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or (vii) a tyrosine substituted with a phenylalanine at a position corresponding to amino acid 509 of SEQ ID NO: 1 and a glutamine substituted with a histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; or (viii) glutamic acid substituted with lysine at the position corresponding to amino acid 519 of SEQ ID NO: 1 and glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO: 1; or (ix) an arginine substituted with a histidine at a position corresponding to amino acid 510 of SEQ ID NO: 1, and a glutamine substituted with a histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; or (x) an arginine substituted with histidine at a position corresponding to amino acid 510 of SEQ ID NO: 1, and a phenylalanine substituted with histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (xi) a lysine substituted with glutamic acid at a position corresponding to amino acid 505 of SEQ ID NO: 1 and a glutamine substituted with histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; or (xii) a methionine substituted with a threonine at the position corresponding to amino acid 467 of SEQ ID NO: 1 and a glutamine substituted with a histidine at the position corresponding to amino acid 464 of SEQ ID NO: 1; or (xiii) comprises a tyrosine substituted with a leucine at a position corresponding to amino acid 519 of SEQ ID NO:1, and a glutamine substituted with a histidine at a position corresponding to amino acid 464 of SEQ ID NO:1.

[0059] In one embodiment, the albumin variant or fragment thereof comprises: (i) glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO: 1, or (ii) a phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (iii) glutamine substituted with histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (iv) tryptophan substituted with threonine at the position corresponding to amino acid 422 of SEQ ID NO:1 and glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or (v) glutamine substituted for histidine at a position corresponding to amino acid 464 of SEQ ID NO:1, and phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO:1.

[0060] In one embodiment of the present disclosure, the immunoglobulin Fc domain or fragment thereof is an Fc domain derived from IgG or a fragment thereof.

[0061] In one embodiment of the present disclosure, the immunoglobulin Fc domain or fragment thereof is an Fc domain derived from IgG1 or a fragment thereof.

[0062] In one embodiment of the disclosure, the immunoglobulin Fc domain or fragment thereof is an Fc domain derived from IgG4 or a fragment thereof. In one embodiment of the disclosure, the immunoglobulin Fc domain or fragment thereof is not derived from IgG4 or a fragment thereof.

[0063] In one embodiment of the present disclosure, the immunoglobulin Fc domain or fragment thereof is an Fc domain variant or fragment thereof, for example, an IgG1 Fc domain variant or fragment thereof, or an IgG4 Fc domain variant or fragment thereof.

[0064] In one example, the IgG1 Fc domain variant or fragment thereof, or the IgG1 Fc domain variant or fragment thereof, binds with increased affinity to human FcRn compared to the IgG1 Fc domain set forth in SEQ ID NO:2.

[0065] For example, binding affinity is measured at neutral and / or acidic pH. Exemplary K D is the K of the antagonist of the present disclosure against FcRn D and shall be deemed to apply mutatis mutandis to the embodiments of the present disclosure.

[0066] In one embodiment, the Fc domain variant or fragment thereof comprises: (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) glutamic acid substituted for asparagine at the position corresponding to amino acid 286 according to the EU numbering system; (v) proline substituted with valine at the position corresponding to amino acid 308 according to the EU numbering system; (vi) a lysine substituted with a histidine at the position corresponding to amino acid 433 according to the EU numbering system; (vii) a tyrosine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (viii) phenylalanine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (ix) a histidine substituted with a tyrosine at the position corresponding to amino acid 436 according to the EU numbering system, and (x) a combination thereof.

[0067] For example, the Fc domain variant or fragment thereof may be (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or (ii) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with a serine at the position corresponding to amino acid 254 according to the EU numbering system, and a glutamic acid substituted with a threonine at the position corresponding to amino acid 256 according to the EU numbering system; or (iii) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a glutamic acid substituted with an asparagine at the position corresponding to amino acid 286 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or (iv) a tyrosine substituted with methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system, a glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system, a lysine substituted with histidine at the position corresponding to amino acid 433 according to the EU numbering system, and a phenylalanine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0068] In one example, the FcRn antagonist of the present disclosure comprises: (i) an Fc domain comprising a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (ii) Albumin containing glutamine substituted with histidine at the position corresponding to amino acid 464 according to the EU numbering system.

[0069] In one example, the Fc domain or fragment thereof comprises a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with a serine at the position corresponding to amino acid 254 according to the EU numbering system, a glutamic acid substituted with a threonine at the position corresponding to amino acid 256 according to the EU numbering system, a lysine substituted with a histidine at the position corresponding to amino acid 433 according to the EU numbering system, and a phenylalanine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0070] In one example, the Fc domain or fragment thereof comprises a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0071] In one embodiment, the Fc domain or fragment thereof is linked to one albumin. In some embodiments, the Fc domain can form a dimer of two Fc domains, and the antagonist of the present disclosure can comprise an Fc domain dimer, where each Fc domain is linked to an albumin, i.e., the antagonist comprises two albumins.

[0072] In another embodiment, one of the Fc domains in the Fc domain dimer is linked to albumin or a fragment thereof, and the other Fc domain is not linked to albumin or a fragment thereof.

[0073] In one example, each Fc domain of the FcRn antagonist is linked to two or more albumins or fragments thereof.

[0074] In one example, each Fc domain is linked to one albumin or fragment thereof.

[0075] In one embodiment, the Fc domain or fragment thereof is indirectly linked to albumin or a fragment thereof, e.g., via a linker. For example, the antagonist includes a linker disposed between the Fc domain or fragment thereof and albumin or a fragment thereof. In one embodiment, the linker is a peptide or polypeptide. For example, the linker is a peptide linker comprising 2 to 31 amino acids in length. For example, the linker comprises glycine or glycine and serine. For example, the linker comprises one or more repeats of Gly4Ser.

[0076] In another embodiment, the Fc domain or fragment thereof is linked directly to albumin or a fragment thereof, eg, without an intervening linker.

[0077] In an exemplary embodiment of the present disclosure, the C-terminus of the Fc domain or fragment thereof is linked, indirectly or directly, to the N-terminus of albumin or a fragment thereof.

[0078] In another aspect of the present disclosure, the C-terminus of albumin or a fragment thereof is linked, indirectly or directly, to the N-terminus of the Fc domain or a fragment thereof.

[0079] In another aspect of the present disclosure, the C-terminus of the Fc domain or fragment thereof is linked, indirectly or directly, to the C-terminus of albumin or a fragment thereof.

[0080] In another aspect of the present disclosure, the N-terminus of the Fc domain or fragment thereof is linked, indirectly or directly, to the N-terminus of albumin or a fragment thereof.

[0081] In one embodiment, the Fc domain or fragment thereof is a monomeric Fc domain or fragment thereof. In another embodiment, the albumin or fragment thereof is a monomeric albumin or fragment thereof. For example, a monomeric Fc domain is fused to a monomeric albumin.

[0082] In another embodiment, the FcRn antagonist comprises a single albumin or fragment thereof. In such an embodiment, the FcRn antagonist is a heterodimer. For example, a heterodimeric FcRn antagonist comprises a single albumin or fragment thereof fused to a dimeric Fc domain or fragment thereof.

[0083] In one embodiment, the FcRn antagonist comprises two types of albumin or fragments thereof, e.g., the FcRn antagonist is a homodimer.

[0084] The present disclosure further comprises: (i) an immunoglobulin Fc domain or fragment thereof comprising: a tyrosine substituted with methionine at the position corresponding to amino acid 252 according to the EU numbering system; a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; a glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; a lysine substituted with histidine at the position corresponding to amino acid 433 according to the EU numbering system; and a phenylalanine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (ii) providing an FcRn antagonist comprising albumin or a fragment thereof comprising glutamine substituted with histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; The C-terminus of the Fc domain or a fragment thereof is indirectly or directly linked to the N-terminus of albumin or a fragment thereof.

[0085] The present disclosure further provides an FcRn antagonist comprising the sequence set forth in SEQ ID NO:8.

[0086] The present disclosure further comprises: (i) an immunoglobulin Fc domain or fragment thereof comprising a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; and (ii) an FcRn antagonist comprising albumin or a fragment thereof comprising glutamine substituted with histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1, The present invention provides an FcRn antagonist in which the C-terminus of an Fc domain or a fragment thereof is indirectly or directly linked to the N-terminus of albumin or a fragment thereof.

[0087] The present disclosure further provides an FcRn antagonist comprising the sequence set forth in SEQ ID NO:11.

[0088] The FcRn antagonist of the present disclosure comprises a sequence set forth in any one of SEQ ID NOs: 3 to 12. For example, the FcRn antagonist of the present disclosure comprises a sequence set forth in any one of SEQ ID NOs: 3, 5, 8, 9, or 11.

[0089] In one example, the FcRn antagonist comprises the sequence set forth in SEQ ID NO: 3. In one example, the FcRn antagonist comprises the sequence set forth in SEQ ID NO: 5. In one example, the FcRn antagonist comprises the sequence set forth in SEQ ID NO: 8. In one example, the FcRn antagonist comprises the sequence set forth in SEQ ID NO: 9. In one example, the FcRn antagonist comprises the sequence set forth in SEQ ID NO: 11.

[0090] The present disclosure further provides compositions comprising an FcRn antagonist of the present disclosure and a pharmaceutically acceptable carrier.

[0091] The present disclosure further provides nucleic acids encoding the FcRn antagonists of the present disclosure. In one embodiment, the nucleic acid is DNA. In another embodiment, the nucleic acid is RNA, including modified forms thereof. In one embodiment, the nucleic acid is linked to nucleic acid necessary for expression of the FcRn antagonist. In some embodiments, the nucleic acid is contained within a liposome or particle, such as a lipid nanoparticle (LNP). In some embodiments, the nucleic acid, liposome, or particle is present in a pharmaceutical formulation.

[0092] The present disclosure further provides an FcRn antagonist, nucleic acid, or composition disclosed herein for use in inhibiting immunoglobulin binding to FcRn and / or reducing circulating immunoglobulin levels in a subject. The present disclosure further provides use of an FcRn antagonist, nucleic acid, or composition disclosed herein in the manufacture of a medicament for inhibiting immunoglobulin binding to FcRn and / or reducing circulating immunoglobulin levels in a subject. The present disclosure further provides a method for inhibiting immunoglobulin binding to FcRn and / or reducing circulating immunoglobulin levels in a subject, the method comprising administering an FcRn antagonist, nucleic acid, or composition disclosed herein.

[0093] The present disclosure also provides an FcRn antagonist, nucleic acid, or composition of the present disclosure for use in reducing circulating Fc-containing proteins and / or antibodies in a subject, e.g., a subject suffering from an autoimmune disease and having pathogenic autoantibodies, or a subject that has developed and / or is at risk of developing anti-drug antibodies. In one example, the time period during which antibody levels are reduced in the subject is longer than the time period during which antibody levels are reduced in the subject when the FcRn antagonist-derived Fc domain or a fragment thereof is administered alone, i.e., in the absence of albumin or a fragment thereof.

[0094] The present disclosure also provides an FcRn antagonist, nucleic acid, or composition of the present disclosure for use in a subject in need of treatment or prevention of progression of an antibody-mediated disease.

[0095] The present disclosure further provides a method for reducing circulating autoantibodies in a subject, the method comprising administering to the subject an FcRn antagonist, nucleic acid, or composition of the present disclosure. In one example, the time period during which the autoantibody level is reduced in the subject is longer than the time period during which the autoantibody level is reduced in the subject when the Fc domain derived from the FcRn antagonist or a fragment thereof is administered alone, i.e., in the absence of albumin or a fragment thereof.

[0096] The present disclosure further provides a method for reducing circulating anti-drug antibodies in a subject, the method comprising administering to the subject an FcRn antagonist, nucleic acid, or composition of the present disclosure. In one example, the time period during which the anti-drug antibody level is reduced in the subject is longer than the time period during which the anti-drug antibody level is reduced in the subject when the Fc domain derived from the FcRn antagonist or a fragment thereof is administered alone, i.e., in the absence of albumin or a fragment thereof.

[0097] The present disclosure also provides a method for treating or preventing the progression of an antibody-mediated disease in a subject in need thereof, the method comprising administering to the subject an FcRn antagonist, nucleic acid, or composition of the present disclosure.

[0098] The present disclosure also provides use of an FcRn antagonist, nucleic acid, or composition of the present disclosure in the manufacture of a medicament for reducing circulating autoantibodies in a subject.

[0099] The present disclosure also provides use of an FcRn antagonist, nucleic acid, or composition of the present disclosure in the manufacture of a medicament for reducing circulating anti-drug antibodies in a subject.

[0100] The present disclosure also provides use of an FcRn antagonist, nucleic acid, or composition of the present disclosure in the manufacture of a medicament for treating or preventing the progression of an antibody-mediated disease in a subject.

[0101] In one example, the subject has an autoimmune disease and develops and / or is at risk of developing unwanted antibodies. For example, the unwanted antibodies are autoantibodies. In another example, the unwanted antibodies are anti-drug antibodies.

[0102] In one example, the subject has developed or is at risk of developing anti-drug antibodies, e.g., the subject has received therapeutic therapy with a protein therapy, e.g., an antibody, immunoadhesin, or clotting factor, and has developed or is at risk of developing antibodies to the therapy.

[0103] In one embodiment, the FcRn antagonist is (i) reducing endogenous IgG levels by at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold (compared to not administering the FcRn antagonist); and / or (ii) administered in an amount effective to reduce endogenous albumin levels by no more than 20%, 15%, 10%, 5%, or 1% (compared to when the FcRn antagonist is not administered).

[0104] In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. For example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 40% to about 80% compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 10% compared to the level observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 20% compared to the level observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 30% compared to the level observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 40% compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 50% compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone.In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 60% compared to the level observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 70% compared to the level observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 80% compared to the level observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone. In one example, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 90% compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from the FcRn antagonist or a fragment thereof alone.

[0105] In one example, the FcRn antagonist antagonizes IgG recycling but does not significantly, substantially, or detectably reduce endogenous albumin levels.

[0106] In one embodiment, the FcRn antagonist antagonizes IgG recycling but does not substantially antagonize albumin recycling. As noted above, the inventors consider this to be an advantage because an FcRn antagonist comprising albumin or a fragment thereof that does not substantially bind to FcRn will not interfere with endogenous albumin recycling and / or homeostasis.

[0107] In one example, administration of the FcRn antagonist does not induce dyslipidemia and / or induces a smaller increase in serum cholesterol levels compared to levels observed after administration of an FcRn antagonist comprising wild-type human albumin. For example, administration of the FcRn antagonist does not induce dyslipidemia. In another example, administration of the FcRn antagonist induces a smaller increase in serum cholesterol levels compared to levels observed after administration of an FcRn antagonist comprising wild-type human albumin.

[0108] In one example, administration of an FcRn antagonist does not change endogenous albumin, cholesterol, low-density lipoprotein (LDL) cholesterol, and / or triglyceride levels and / or induces smaller changes compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of an FcRn antagonist does not change endogenous albumin levels and / or induces smaller changes compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of an FcRn antagonist does not change endogenous cholesterol levels and / or induces smaller changes compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of an FcRn antagonist does not change endogenous low-density lipoprotein (LDL) cholesterol levels and / or induces a smaller change compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of an FcRn antagonist does not change endogenous triglyceride levels and / or induces a smaller change compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin.

[0109] In one example, the FcRn antagonist reduces endogenous IgG levels within at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of administration. For example, the FcRn antagonist reduces endogenous IgG levels within 2 to 9 days of administration. In one example, the FcRn antagonist reduces endogenous IgG levels within at least 2 days of administration. In one example, the FcRn antagonist reduces endogenous IgG levels within at least 3 days of administration. In one example, the FcRn antagonist reduces endogenous IgG levels within at least 4 days of administration. In one example, the FcRn antagonist reduces endogenous IgG levels within at least 5 days of administration. In one example, the FcRn antagonist reduces endogenous IgG levels within at least 6 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 7 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 8 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 9 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 10 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 11 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 12 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 13 days of administration. In one embodiment, the FcRn antagonist reduces endogenous IgG levels within at least 14 days of administration.

[0110] In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. For example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 50% to about 80%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 10% to about 90%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 10%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 20%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 30%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 40%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 50%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 60%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 70%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 80%. In one example, administration of an FcRn antagonist reduces endogenous IgG levels by at least about 90%.

[0111] In one example, administration of an FcRn antagonist does not change or does not substantially change endogenous IgA levels.

[0112] In one embodiment, the FcRn antagonist increases endogenous IgM levels within at least 5, 6, 7, 8, or 9 days after administration. In one embodiment, the FcRn antagonist increases endogenous IgM levels within at least 5 days after administration. In one embodiment, the FcRn antagonist increases endogenous IgM levels within at least 6 days after administration. In one embodiment, the FcRn antagonist increases endogenous IgM levels within at least 7 days after administration. In one embodiment, the FcRn antagonist increases endogenous IgM levels within at least 8 days after administration. In one embodiment, the FcRn antagonist increases endogenous IgM levels within at least 9 days after administration.

[0113] In one example, administration of an FcRn antagonist may improve the volume of distribution (V) observed after administration of an FcRn antagonist comprising wild-type human albumin and / or after administration of the Fc domain derived from an FcRn antagonist (e.g., huG1FcYTEKF) or a fragment thereof alone. SS ), compared to V SS In one example, administration of an FcRn antagonist reduces V observed after administration of the Fc domain or fragment thereof derived from the FcRn antagonist alone. SS Compared to V SS For example, administration of an FcRn antagonist reduces the V observed after administration of an Fc domain or fragment thereof derived from the FcRn antagonist alone. SS Compared to V SS In one example, administration of an FcRn antagonist reduces the V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone by at least about 400 mL to 500 mL. SS Compared to V SS In one example, administration of an FcRn antagonist reduces V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone by at least about 100 mL. SS Compared to V SS In one example, administration of an FcRn antagonist reduces V by at least about 200 mL compared to V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone. SS Compared to V SS In one example, administration of an FcRn antagonist reduces V by at least about 250 mL compared to V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone. SS Compared to V SS In one example, administration of an FcRn antagonist reduces V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone by at least about 300 mL. SS Compared to V SSIn one example, administration of an FcRn antagonist reduces V by at least about 2500 mL compared to the V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone. SS Compared to V SS In one example, administration of an FcRn antagonist reduces V by at least about 400 mL compared to V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone. SS Compared to V SS In one example, administration of an FcRn antagonist reduces V observed after administration of an Fc domain or fragment thereof derived from an FcRn antagonist alone by at least about 450 mL. SS Compared to V SS The volume is reduced by at least about 500 mL.

[0114] In one example, administration of an FcRn antagonist may reduce or eliminate the V observed after administration of an FcRn antagonist comprising wild-type human albumin. SS Compared to V SS by at least about 40 mL, 50 mL, 60 mL, 70 mL, or 80 mL. For example, administration of an FcRn antagonist reduces the V observed after administration of an FcRn antagonist comprising wild-type human albumin. SS Compared to V SS In one example, administration of an FcRn antagonist reduces the V observed after administration of an FcRn antagonist comprising wild-type human albumin by at least about 40 mL to 80 mL. SS Compared to V SS In one example, administration of the FcRn antagonist reduces the V observed after administration of an FcRn antagonist comprising wild-type human albumin by at least about 40 mL. SS Compared to V SS In one example, administration of the FcRn antagonist reduces the V observed after administration of an FcRn antagonist comprising wild-type human albumin by at least about 50 mL. SS Compared to V SS In one example, administration of the FcRn antagonist reduces the V observed after administration of an FcRn antagonist comprising wild-type human albumin by at least about 60 mL. SS Compared to V SSIn one example, administration of the FcRn antagonist reduces the V observed after administration of an FcRn antagonist comprising wild-type human albumin by at least about 70 mL. SS Compared to V SS The amount of blood in the blood is reduced by at least about 80 mL.

[0115] In one example, administration of an FcRn antagonist increases the area under the curve (AUC) compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone. For example, administration of an FcRn antagonist increases AUC by at least 250 μg / mL*h / mg, 300 μg / mL*h / mg, 350 μg / mL*h / mg, 400 μg / mL*h / mg, 450 μg / mL*h / mg, or 500 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone. In one example, administration of an FcRn antagonist increases AUC by at least 250 μg / mL*h / mg to 500 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone. In one example, administration of an FcRn antagonist increases AUC by at least 250 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone. In one example, administration of an FcRn antagonist increases AUC by at least 250 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone. In one example, administration of an FcRn antagonist increases AUC by at least 350 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone. In one example, administration of an FcRn antagonist increases AUC by at least 400 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone. In one example, administration of the FcRn antagonist increases the AUC by at least 450 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone, hi one example, administration of the FcRn antagonist increases the AUC by at least 500 μg / mL*h / mg compared to the AUC observed after administration of the FcRn antagonist-derived Fc domain or fragment thereof alone.

[0116] In one example, administration of the FcRn antagonist increases the AUC compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. For example, administration of the FcRn antagonist increases the AUC by at least 100 μg / mL*h / mg, 150 μg / mL*h / mg, 200 μg / mL*h / mg, or 250 μg / mL*h / mg compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of the FcRn antagonist increases the AUC by at least 100 μg / mL*h / mg to 250 μg / mL*h / mg compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of the FcRn antagonist increases AUC by at least 100 μg / mL*h / mg compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of the FcRn antagonist increases AUC by at least 150 μg / mL*h / mg compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of the FcRn antagonist increases AUC by at least 200 μg / mL*h / mg compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one example, administration of the FcRn antagonist increases AUC by at least 250 μg / mL*h / mg compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin.

[0117] In another example, administration of an FcRn antagonist crosslinks cell surface FcRn at a lower level than an FcRn antagonist comprising wild-type human albumin.

[0118] In one example, the subject has received, is receiving, or will receive additional therapy, for example, steroidal immunomodulatory drugs, plasma exchange therapy, and / or IVIg therapy.

[0119] The present disclosure further provides a kit for use in reducing circulating autoantibodies in a subject in need thereof, the kit comprising: (i) at least one FcRn antagonist or composition of the disclosure; (ii) instructions for using the kit in reducing circulating autoantibodies in a subject; and (iii) optionally, at least one additional therapy.

[0120] The present disclosure also provides a kit for treating or preventing the progression of an antibody-mediated disease in a subject in need thereof, the kit comprising: (i) at least one FcRn antagonist or composition of the disclosure; (ii) instructions for using the kit in treating or preventing the progression of an antibody-mediated disease in a subject; and (iii) optionally, at least one additional therapy. [Brief explanation of the drawings]

[0121] [Figure 1A] 1 is a graphical representation showing the levels of IgG "tracer" antibodies in human FcRn (huFcRn) transgenic mice administered various FcRn antagonists of the present disclosure. [Figure 1B] 1 is a graphical representation showing the levels of IgG "tracer" antibodies in human FcRn (huFcRn) transgenic mice administered various FcRn antagonists of the present disclosure. [Figure 1C] 1 is a graphical representation showing the in vivo half-life of various FcRn antagonists of the present disclosure after administration to huFcRn transgenic mice. [Figure 2A] 1 is a graphical representation showing tracer antibody (CSL360) levels in huFcRn transgenic mice administered various FcRn antagonists of the present disclosure. [Figure 2B] 1 is a graphical representation showing tracer antibody (CSL360) levels in huFcRn transgenic mice administered various FcRn antagonists of the present disclosure. [Figure 2C]1 is a graphical representation showing the pharmacokinetics of an FcRn antagonist described herein when administered intravenously or subcutaneously. The antagonist is as indicated and described herein. As shown, the antagonist exhibits comparable pharmacokinetics when administered intravenously or subcutaneously. [Figure 2D] 1 is a graphical representation showing a dose-dependent decrease in tracer antibody (CSL360) levels in huFcRn transgenic mice administered various doses of huG1FcYPY-HSA, with controls as indicated. [Figure 2E] 1 is a graphical representation showing a dose-dependent decrease in tracer antibody (CSL360) levels in huFcRn transgenic mice administered various doses of huG1FcYPY-HSA(H464Q). Controls are as indicated. [Figure 3A] 1 is a graphic representation showing the effect on endogenous mouse IgG levels in mice after a single administration of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF. [Figure 3B] 1 is a graphic representation showing the effect on endogenous mouse IgG levels in mice after a single administration of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF. [Figure 3C] 1 is a graphic representation showing the in vivo half-life of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF in mice. [Figure 3D] 1 is a graphic representation showing the in vivo half-life of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF in mice. [Figure 4A]FIG. 1 is a graphical representation showing the effect on fluorescently labeled IgG levels in bone marrow-derived macrophages in the absence or presence of FcRn antagonists huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF at the indicated concentrations and time points, and in the presence or absence of protease inhibitors (PIs). [Figure 4B] FIG. 1 is a graphical representation showing the effect on fluorescently labeled albumin levels in bone marrow-derived macrophages in the absence or presence of FcRn antagonists huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF at the indicated concentrations and time points, and in the presence or absence of protease inhibitors (PIs). [Figure 5] 1 is a graphical representation illustrating how fluorescence can be used to assess the degradation and recycling of molecules within cells. [Figure 6] Schematic diagram depicting the predicted protein complex of FcRn / β2m and huG1FcYPY-HSA(H464Q). [Figure 7] Schematic depicting the FcRn-binding pocket of HSA. [Figure 8A] 1 is a graphic representation showing the effect of FcRn antagonists on endogenous cynomolgus monkey IgG levels up to 2016 hours (84 days) following a single intravenous administration of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF. [Figure 8B] 1 is a graphic representation showing the effect of FcRn antagonists on endogenous cynomolgus monkey IgG levels up to 420 hours (17.5 days) following a single intravenous administration of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF. [Figure 8C] 1 is a graphic representation showing the in vivo PK of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF in cynomolgus monkeys up to 420 hours (17.5 days) following a single intravenous dose. [Figure 9A]1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of huG1FcYPY-HSA molecules. [Figure 9B] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of huG1FcYPY-HSA molecules. [Figure 9C] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of huG1FcYPY-HSA molecules. [Figure 9D] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single intravenous administration of the huG1FcYPY-HSA(H464Q) molecule. [Figure 9E] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single intravenous administration of the huG1FcYPY-HSA(H464Q) molecule. [Figure 9F] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single intravenous administration of the huG1FcYPY-HSA(H464Q) molecule. [Figure 9G] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of the huG1FcYTEKF molecule. [Figure 9H] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of the huG1FcYTEKF molecule. [Figure 9I] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of the huG1FcYTEKF molecule. [Figure 10A] 1 is a graphical representation showing the effect of FcRn antagonists on endogenous cynomolgus monkey IgG levels up to 2016 hours (84 days) following a single subcutaneous administration of either huG1FcYPY-HSA(H464Q) or huG1FcYTEKF. [Figure 10B]1 is a graphical representation showing the effect of FcRn antagonists on endogenous cynomolgus monkey IgG levels up to 420 hours (17.5 days) following a single subcutaneous administration of either huG1FcYPY-HSA(H464Q) or huG1FcYTEKF. [Figure 10C] 1 is a graphical representation showing the in vivo half-life of huG1FcYPY-HSA(H464Q) or huG1FcYTEKF in cynomolgus monkeys up to 420 hours (17.5 days) following a single subcutaneous dose. [Figure 11A] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single subcutaneous administration of the huG1FcYPY-HSA(H464Q) molecule. [Figure 11B] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single subcutaneous administration of the huG1FcYPY-HSA(H464Q) molecule. [Figure 11C] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single subcutaneous administration of the huG1FcYPY-HSA(H464Q) molecule. [Figure 11D] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single subcutaneous administration of the huG1FcYTEKF molecule. [Figure 11E] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single subcutaneous administration of the huG1FcYTEKF molecule. [Figure 11F] 1 is a graphical representation of endogenous IgG and IgM levels in individual cynomolgus monkeys following a single subcutaneous administration of the huG1FcYTEKF molecule. DETAILED DESCRIPTION OF THE INVENTION

[0122] [Table A] general Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps or composition of matter should be considered to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps or composition of matter.

[0123] Those skilled in the art will understand that the present disclosure may be susceptible to variations and modifications other than those specifically described. The present disclosure should be understood to include all such variations and modifications. The present disclosure includes all steps, features, compositions, and compounds referred to or shown in the specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0124] The present disclosure is not to be limited in scope by the specific examples set forth herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0125] Any embodiment of the present disclosure described herein shall be deemed to apply mutatis mutandis to any other embodiment of the present disclosure, unless specifically stated otherwise. In other words, any embodiment of the present disclosure may be combined with any other embodiment of the present disclosure (except where mutually exclusive).

[0126] Any embodiment of the present disclosure disclosing a particular feature or group of features, or a method or method step, is also deemed to provide explicit support for disclaiming that particular feature or group of features, or method or method step.

[0127] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0128] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. al. (editors), Current Protocols in Immunology, John Wiley & Sons, and other sources.

[0129] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by consideration of Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991; Bork et al., J. Mol. Biol. 242, 309-320, 1994; Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273, 927-948, 1997.

[0130] The term "and / or" (e.g., "X and / or Y") shall be understood to mean either "X and Y" or "X or Y" and shall be deemed to explicitly endorse both meanings or either meaning.

[0131] Throughout this specification, the word "comprises" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated component, element, or step, or group of components, elements, or steps, but not the exclusion of any other component, element, or step, or group of components, elements, or steps.

[0132] As used herein, the term "derived from" shall be taken to indicate that a specified element may be obtained from a particular source, but not necessarily directly from that source.

[0133] Selected Definitions "Albumin," "albumin," or "blood albumin" is the most abundant protein in the blood. It functions as a carrier protein for steroids, fatty acids, and thyroid hormones in the blood, as well as playing an important role in stabilizing extracellular fluid volume. For purposes of nomenclature only and not by way of limitation, an exemplary sequence of human albumin is set forth in NCBI GenBank Accession No. AEE60908 and SEQ ID NO: 1. References to "albumin" or "albumin" should be understood to include preproalbumin, including the N-terminal peptide, proalbumin, and secreted albumin. As used herein, amino acid positions are referenced with reference to the 585-amino acid secreted albumin protein (e.g., as set forth in SEQ ID NO: 1). Albumin contains three homologous domains, each of which is the product of two subdomains with a common structural motif. Domains I, II, and III may be defined with reference to human albumin (set forth in SEQ ID NO: 1). For example, domain I comprises amino acids 1 (±1 to 15 amino acids) to 194 (±1 to 15 amino acids) of SEQ ID NO: 1, domain II comprises amino acids 192 (±1 to 15 amino acids) to 387 (±1 to 15 amino acids) of SEQ ID NO: 1, and domain III comprises amino acid residues 381 (±1 to 15 amino acids) to 585 (±1 to 15 amino acids) of SEQ ID NO: 1. The phrase "±1 to 15 amino acids" means that the amino acid residues can deviate by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids relative to the C-terminus and / or N-terminus of the recited amino acid position. Exemplary domains I, II, and III are described by Dockal et al. (The Journal of Biological Chemistry, 1999, Vol. 274(41):29303-29310) and Kjeldsen et al. (Protein Expression and Purification, 1998, Vol 13:163-169).

[0134] Additional sequences for albumins from other species (e.g., primate albumins (such as chimpanzee albumin, gorilla albumin), rodent albumins (such as hamster albumin, guinea pig albumin, mouse albumin, and rat albumin), bovine albumin, horse albumin, donkey albumin, rabbit albumin, goat albumin, sheep albumin, dog albumin, chicken albumin, and porcine albumin) can be determined using the sequences provided herein and / or in publicly available databases and / or can be determined using standard techniques (e.g., as described in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to the present) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0135] As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native Fc and Fc domain variants. In some embodiments, the human IgG heavy chain Fc domain region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, Fc produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, the Fc domain may include truncated variants of a full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU index numbering). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc domain region may or may not be present. As specified herein, the numbering of amino acid residues in the Fc domain region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0136] For nomenclature purposes only, and not by way of limitation, an exemplary sequence of a human IgG1 Fc domain is set forth in SEQ ID NO: 2 or Uniprot Accession No. P01857.

[0137] In the context of the FcRn antagonists of the present disclosure, an Fc domain or a fragment thereof can dimerize with another Fc domain or a fragment thereof. In one example, the FcRn antagonists of the present disclosure are homodimers.

[0138] As used herein, the phrase "corresponding to" with respect to an amino acid position of a SEQ ID NO: should be understood as a reference to an amino acid residue or position within a polypeptide or protein (e.g., albumin or Fc), and not necessarily a reference to the sequence that comprises the recited SEQ ID NO. For example, a reference to "a position corresponding to amino acid 522 of SEQ ID NO: 1" in an albumin sequence that includes a 10 amino acid N-terminal truncation necessarily refers to the amino acid at position 512.

[0139] For example, when discussing the placement of mutations in albumin within a fusion protein, the numbering of positions is relative to albumin, not relative to the fusion protein as a whole. Thus, if an Fc domain is fused to the N-terminus of albumin, then when discussing mutations in albumin, the first residue will be the first residue of albumin.

[0140] The term "Kabat EU numbering system" or "EU numbering system" will be understood to mean that the numbering of immunoglobulin heavy chains is according to the EU index as taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of human IgG1 EU.

[0141] As discussed herein, reference to a "fragment" of albumin should be understood as a reference to a fragment of albumin that retains the ability to extend the half-life of the molecule to which it is bound, and that does not require that the fragment bind to FcRn. A fragment may comprise or consist of one or more domains of albumin, fragments of such domains, or combinations thereof.

[0142] As used herein, "amino acid substitution(s)" refers to the substitution of an amino acid at a particular position in a polypeptide sequence with another amino acid.

[0143] As used herein, the term "FcRn" refers to the neonatal Fc receptor, also known as the Brambell receptor, which is a heterodimer of a truncated heavy chain of the major histocompatibility complex class I-like Fc receptor (FCGRT) and beta2-microglobulin.

[0144] As used herein, the term "mutant" refers to a protein that has undergone one or more amino acid substitutions using well-known techniques for site-directed mutagenesis or any other conventional method.

[0145] As used herein, the term "binds" with respect to the interaction between an Fc domain or a fragment thereof and FcRn means that the interaction is dependent on the presence of specific structures on the Fc domain and FcRn.

[0146] For purposes of clarity, and as will be apparent to those skilled in the art based on the exemplified subject matter herein, references herein to "affinity" are references to the interaction, binding, or association of albumin or a fragment thereof, or an Fc domain or a fragment thereof, with FcRn.

[0147] For purposes of clarity, and as will be apparent to one of skill in the art based on the description herein, reference to an "affinity of at least about" is understood to mean that the affinity is equal to or greater than the stated value (i.e., the stated affinity is lower), i.e., an affinity of 2 nM is stronger than an affinity of 3 nM. In other words, the term can refer to an "affinity of less than or equal to X," where X is a value described herein.

[0148] As used herein, the term "does not detectably bind" is understood to mean that the albumin variant binds to FcRn at a level that is less than 20%, less than 10%, less than 6%, or less than 5% of background. Background may be the level of binding signal detected in the absence of the albumin variant or FcRn comprising it, and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or in the presence of a negative control antigen. Binding levels are detected, for example, using ELISA, in which an antigen is immobilized and contacted with the albumin variant.

[0149] As used herein, the terms "half-life," "serum half-life," or "plasma half-life" in the context of this disclosure refer to the period of time required for the concentration or amount of an Fc domain, albumin, or FcRn antagonist in the body to decrease by 50% (i.e., half) due to, for example, degradation and / or clearance or sequestration by natural mechanisms. Those skilled in the art will recognize that the serum half-life of a protein in a subject depends on various physiological conditions (e.g., health status, body size / weight). For example, in healthy human subjects, the serum half-life of albumin is 19-20 days, and the serum half-life of IgG1 is approximately 21 days. Methods for determining the serum half-life of a protein are known in the art and include, for example, pharmacokinetic analysis.

[0150] The term "recombinant" shall be understood to mean the product of artificial genetic recombination. Recombinant protein also encompasses proteins expressed by artificial recombinant means, for example, when present in a cell, tissue, or subject in which it is expressed.

[0151] The term "protein" will be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked using appropriate chemicals or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0152] The term "polypeptide" or "polypeptide chain" is understood from the preceding paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0153] As used herein, the terms "treating," "treat," or "treatment" include administering an FcRn antagonist described herein to reduce or eliminate at least one symptom of a particular disease or condition, or to slow the progression of the disease or condition.

[0154] As used herein, the terms "preventing," "prevent," or "prevention" include providing protection against the occurrence or recurrence of a bleeding disorder or symptoms of a bleeding disorder in an individual. An individual may be predisposed to or at risk of developing the disease or a recurrence of the disease, but may not yet be diagnosed with the disease or recurrence.

[0155] An "effective amount" refers to at least an amount effective, at a dosage and for a period of time necessary, to achieve a desired result. For example, the desired result can be a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some embodiments of the present disclosure, the term "effective amount" refers to the amount necessary to effectively treat a disease or condition, as described hereinabove. In some embodiments of the present disclosure, the term "effective amount" refers to the amount necessary to effect a change in a factor associated with a disease or condition, as described hereinabove. An effective amount can vary depending on the disease or condition to be treated or the factor to be modified, as well as the weight, age, ethnic background, sex, health and / or physical condition of the mammal being treated, and other relevant factors. Typically, an effective amount falls within a relatively broad range (e.g., a "dosage" range) that can be determined through routine trial and experimentation by a medical professional. Therefore, this term should not be construed as limiting the present disclosure to a specific amount. An effective amount can be administered in a single dose or repeated one or more times during a treatment period.

[0156] A "therapeutically effective amount" is at least the minimum concentration required to affect a measurable improvement in a particular disease or condition. The therapeutically effective amount herein may vary depending on the patient's condition, age, sex, and weight, as well as the ability of the albumin conjugate to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the FcRn antagonist. In one embodiment, a therapeutically effective amount is understood to mean an amount of albumin conjugate sufficient to alleviate or inhibit one or more symptoms of a bleeding disorder or its complications.

[0157] As used herein, the term "prophylactically effective amount" will be understood to mean an amount of albumin conjugate sufficient to prevent, inhibit, or delay the onset of one or more detectable symptoms of a disease state.

[0158] As used herein, the term "subject" shall be understood to mean any animal, e.g., a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0159] Fc domain and its fragments As described herein, the FcRn antagonists of the present disclosure comprise an Fc domain or an FcRn-binding fragment thereof.

[0160] In one example, the Fc domain is from an IgG.

[0161] For example, the Fc domain is derived from human IgG.

[0162] For example, the Fc domain is from IgG1.

[0163] For example, the Fc domain is derived from human IgG1.

[0164] For example, the Fc domain is from IgG4.

[0165] For example, the Fc domain is derived from human IgG4.

[0166] For example, the Fc domain is not derived from IgG4.

[0167] For example, the Fc domain is not derived from human IgG4.

[0168] In one example, an FcRn-binding fragment of an Fc domain refers to a portion of an immunoglobulin heavy chain, e.g., an IgG1 heavy chain, extending from about EU position 243 to EU position 261, from about EU position 275 to EU position 293, from about EU position 302 to EU position 319, from about EU position 336 to EU position 348, from about EU position 367 to EU position 393 and EU position 408, and from about EU position 424 to EU position 440.

[0169] In certain instances, the Fc domain or fragment is not a full-length immunoglobulin. For example, the Fc domain or fragment, or the FcRn antagonist, does not include a variable domain. In some embodiments, the Fc domain or fragment, or the FcRn antagonist, does not include a variable domain or C H However, in certain embodiments, an FcRn antagonist may comprise an Fc domain or a fragment thereof linked to one or more additional binding domains or moieties, including a variable domain.

[0170] In one example, the Fc domain is an Fc domain variant, or an FcRn-binding fragment thereof, that specifically binds to FcRn with increased affinity, for example, compared to an Fc domain derived from wild-type human IgG1. In one example, the variant Fc domain or fragment thereof exhibits reduced pH-dependence of binding to FcRn compared to the native Fc domain region. Exemplary Fc domains or fragments thereof inhibit or reduce binding of immunoglobulins and / or other Fc-containing proteins (e.g., immunoadhesins and antibody-drug conjugates) to FcRn in vivo, resulting in an increased rate of degradation of the immunoglobulins or Fc-containing proteins and a concomitant decrease in serum concentrations of these immunoglobulins or Fc-containing proteins.

[0171] Any Fc domain region can be modified to generate the Fc domain variants of the present disclosure. As discussed herein, Fc domains are typically derived from human immunoglobulins. However, Fc regions may also be derived from immunoglobulins of any other mammalian species, including, for example, camelid species, rodents (e.g., mouse, rat, rabbit, guinea pig), or non-human primates (e.g., chimpanzee, macaque) species. Furthermore, Fc domains may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the Fc domain is an IgG Fc domain (e.g., a human IgG region). In certain embodiments, the Fc domain is an IgG1 Fc domain (e.g., a human IgG1). In certain embodiments, the Fc domain is a chimeric Fc domain comprising portions of multiple different Fc domains. Suitable examples of chimeric Fc domains are described in US20110243966. It will be understood that the scope of the present disclosure encompasses alleles, variants, and mutations of the Fc domain.

[0172] The Fc domain may be further truncated or contain deletions to generate its minimal FcRn-binding fragment. The ability of the Fc region fragment to bind to FcRn can be determined using any art-recognized binding assay, such as ELISA.

[0173] To improve the manufacturability of the FcRn antagonists disclosed herein, the constituent Fc region desirably does not contain any non-disulfide-bonded cysteine ​​residues, and thus, in certain embodiments, the Fc domain does not contain any free cysteine ​​residues.

[0174] In one example, the Fc domain variant comprises an amino acid modification, e.g., a substitution, that enhances binding of the Fc domain to FcRn. In one example, the amino acid modification enhances binding of the Fc domain to FcRn at neutral and / or acidic pH. In one example, the amino acid modification enhances binding of the Fc domain to FcRn at neutral and acidic pH.

[0175] In some embodiments, the Fc domain variants comprise substitutions at one or more positions selected from the group consisting of 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 252, 254, 256, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332, 333, and 334 according to the EU numbering system. Some exemplary substitutions are described in US5624821, US6277375, US6737056, WO01 / 58957, WO02 / 06919, WO2004 / 016750, WO2004 / 029207, WO 2004 / 035752, WO2005 / 040217 and WO2015 / 100299.

[0176] In some embodiments, the Fc domain variants are 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L according to the EU numbering system. 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T , 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651 , 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 269G, 269H, 269Y, 269F, 269R, 296E, 296Q, 296 D, 296N, 296S, 296T, 296L, 2961, 296H, 296G, 296W, 297S, 297D, 297E, 298H, 2981, 298T, 298F, 2991, 299 L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 32 7G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 32 and at least one substitution selected from the group consisting of 9H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 334N, 352S, 378V, 397M, and 434Y.

[0177] Other known Fc domain variants that can be used in the FcRn antagonists disclosed herein include those described in Ghetie et al., 1997, Nat. Biotech. 15:637-40; Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol. 147:2657-2662; Lund et al., 1992, Mol. Immunol. 29:53-59; Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc. Natl. Acad. Sci. USA 92:11980-11984; Jefferis et al., 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J.,9:115-119, Jefferis et al,1996,Immunol Lett.,54:101-104,Lund et al,1996,J.Immunol,157:4963-4969,Armour et al,1999,Eur J Immunol 29:2613-2624,Idusogie et al. al,2000,J.Immunol,164:4178-4184,Reddy et al,2000,J.Immunol,164:1925-1933,Xu et al,2000,Cell Immunol,200:16-26,Idusogie et al. al,2001,J.Immunol,166:2571-2575, Shields et al,2001,J Biol. Chem., 276:6591-6604; Jefferis et al., 2002, Immunol Lett., 82:57-65; Presta et al., 2002, Biochem Soc Trans., 30:487-490.

[0178] Additional exemplary substitutions will be apparent to those skilled in the art. For example, substitutions at residues 250 and 428 (according to the EU numbering system), such as T250Q and M428L, have been shown to increase binding of the Fc domain to FcRn (Hinton, et al., J. Biol. Chem. 279, 6213-6216, 2004). Similarly, substitutions at residues 252, 254, and 256 (according to the EU numbering system), such as M252Y, S254T, and T256E, have been shown to increase binding of the Fc domain to FcRn (Dall'Acqua, et al., J Immunol 169, 5171-5180, 2002 and Dall'Acqua, et al., The Journal of Biological Chemistry 281, 23514-23524, 2006). Dall'Acqua et al., 2002 further teaches that the following residue combinations (according to the EU numbering system) are useful for enhancing binding of the Fc domain to FcRn: M252W, M252Y, M252Y / T256Q, M252F / T256D, V203T / L309P / Q311S, H433K / N434F / Y436H, H433R / N434Y / Y436H, M252Y / S254T / T256E / H433K / N434F / Y436H, or M252Y / S254T / T256E / G385R / Q386T / P387R / N389P. Additional substitutions that enhance binding of the Fc domain to FcRn include T307A / E380A / N434A or M428L / N434S.

[0179] Mackness et al. (MABS 11:1276-1288, 2019) demonstrated that the M252Y, T256D / E, K288D / N, T307A / E / F / M / Q / W, E380C, N434F / P / Y, and Y436H / N / W substitutions bind to FcRn and significantly reduce the off-rate compared to wild-type Fc. The authors showed that the M252Y / T256D, T256D / T307Q, and T256D / T307W substitution combinations enhance binding to FcRn while maintaining other activities such as thermostability, FcγRIIIa, and rheumatoid factor binding.

[0180] Monnet et al., (Front. Immunol, 12:728322, 2021) showed that the Y296W / K334N / P352S / A378V / V397M / N434Y substitutions improved binding to FcRn.

[0181] Additional exemplary substitutions into the Fc domain are described in WO2010 / 106180, WO2016177984, WO2018007453, WO2018078138, WO2017006052, and WO2019115773.

[0182] WO2015 / 100299 describes substitutions at residues 252, 254, 256, 433, 434, and 436 (according to the EU numbering system) that increase binding of the Fc domain to FcRn. For example, the Fc domain is the same as efgartigimod.

[0183] Additional exemplary substitutions are described, for example, in WO2002 / 060919, which describe substitutions at residues 251, 253, 255, 285-290, 308-314, 385-389, and 428-435 (according to the EU numbering system) that increase binding of the Fc domain to FcRn.

[0184] In one embodiment, the Fc domain variant or fragment thereof comprises: (i) an amino acid substituted with methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) an amino acid substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) an amino acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) an amino acid substituted with asparagine at the position corresponding to amino acid 286 according to the EU numbering system; (v) an amino acid substituted with tyrosine at the position corresponding to amino acid 296 according to the EU numbering system; (vi) an amino acid substituted with valine at the position corresponding to amino acid 308 according to the EU numbering system; (vii) an amino acid substituted with lysine at the position corresponding to amino acid 334 according to the EU numbering system; (viii) an amino acid substituted with proline at the position corresponding to amino acid 352 according to the EU numbering system; (ix) an amino acid substituted with alanine at the position corresponding to amino acid 378 according to the EU numbering system; (x) an amino acid substituted with valine at the position corresponding to amino acid 397 according to the EU numbering system; (xi) an amino acid substituted with histidine at the position corresponding to amino acid 433 according to the EU numbering system; (xii) an amino acid substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system, and (xiii) Contains one or more amino acid substitutions selected from the group consisting of:

[0185] In one embodiment, the Fc domain variant or fragment thereof comprises: (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) glutamic acid substituted for asparagine at the position corresponding to amino acid 286 according to the EU numbering system; (v) tryptophan substituted for tyrosine at the position corresponding to amino acid 296 according to the EU numbering system; (vi) proline substituted with valine at the position corresponding to amino acid 308 according to the EU numbering system; (vii) an asparagine substituted with a lysine at a position corresponding to amino acid 334 according to the EU numbering system; (viii) serine substituted with proline at the position corresponding to amino acid 352 according to the EU numbering system; (ix) valine d substituted with alanine at the position corresponding to amino acid 378 according to the EU numbering system; (x) methionine substituted with valine at the position corresponding to amino acid 397 according to the EU numbering system; (xi) a lysine substituted with a histidine at the position corresponding to amino acid 433 according to the EU numbering system; (xii) tyrosine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (xiii) a phenylalanine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system, and (xiv) contains one or more amino acid substitutions selected from the group consisting of:

[0186] In one example, the Fc domain variant or fragment thereof comprises a substitution at each of the following positions: 252, 308, and 434.

[0187] For example, the Fc domain variant or fragment thereof may be (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and (iii) containing a tyrosine or phenylalanine substituted for asparagine at the position corresponding to amino acid 434 according to the EU numbering system;

[0188] In one example, the Fc domain variant or fragment thereof comprises a substitution at each of the following positions: 252, 254, and 256.

[0189] For example, the Fc domain variant or fragment thereof may be (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system, and (iii) contains a glutamic acid substituted for threonine at the position corresponding to amino acid 256 according to the EU numbering system.

[0190] In one example, the Fc domain variant or fragment thereof comprises a substitution at each of the following positions: 252, 286, and 434.

[0191] For example, the Fc domain variant or fragment thereof may be (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) glutamic acid substituted with asparagine at the position corresponding to amino acid 286 according to the EU numbering system, and (iii) contains a tyrosine or phenylalanine substituted for asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0192] In one example, the Fc domain variant or fragment thereof comprises a substitution at each of the following positions: 252, 254, 256, 433 and 434. For example, the Fc domain variant or fragment thereof may be (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) a lysine substituted with a histidine at the position corresponding to amino acid 433 according to the EU numbering system, and (v) Contains a tyrosine or phenylalanine substituted for asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0193] In one example, the Fc domain variant or fragment thereof comprises a substitution at each of the following positions: 296, 334, 352, 378, 397 and 434.

[0194] For example, the Fc domain variant or fragment thereof may be (i) tryptophan substituted for tyrosine at the position corresponding to amino acid 296 according to the EU numbering system; (ii) an asparagine substituted with a lysine at the position corresponding to amino acid 334 according to the EU numbering system; (iii) a serine substituted with a proline at a position corresponding to amino acid 352 according to the EU numbering system; (iv) valine substituted with alanine at the position corresponding to amino acid 378 according to the EU numbering system; (v) a methionine substituted with a valine at the position corresponding to amino acid 397 according to the EU numbering system, and (vi) containing a tyrosine substituted for asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0195] In one example, the Fc domain variant or fragment thereof comprises a substitution at each of the following positions: 252, 254, 256, 433 and 434.

[0196] For example, the Fc domain variant or fragment thereof may be (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) a lysine substituted with a histidine at the position corresponding to amino acid 433 according to the EU numbering system, and (v) containing a phenylalanine substituted for asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0197] In an exemplary embodiment of the disclosure, the Fc domain variant or fragment thereof comprises a substitution at each of the following positions: 252, 254, 256, 433, 434, and 436.

[0198] For example, the Fc domain variant or fragment thereof may be (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) a lysine substituted for histidine at the position corresponding to amino acid 433 according to the EU numbering system; (v) tyrosine or phenylalanine substituted for asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (vi) a histidine substituted for a tyrosine at a position corresponding to amino acid 436 according to the EU numbering system, and (vii) includes any combination thereof.

[0199] In one example, the Fc domain variant or fragment thereof comprises: (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or (ii) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with a serine at the position corresponding to amino acid 254 according to the EU numbering system, and a glutamic acid substituted with a threonine at the position corresponding to amino acid 256 according to the EU numbering system; or (iii) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a glutamic acid substituted with an asparagine at the position corresponding to amino acid 286 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or (iv) a tyrosine substituted with methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system, a glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system, a lysine substituted with histidine at the position corresponding to amino acid 433 according to the EU numbering system, and a phenylalanine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0200] In one example, the Fc domain variant or fragment thereof comprises a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with a serine at the position corresponding to amino acid 254 according to the EU numbering system, a glutamic acid substituted with a threonine at the position corresponding to amino acid 256 according to the EU numbering system, a lysine substituted with a histidine at the position corresponding to amino acid 433 according to the EU numbering system, and a phenylalanine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0201] In some embodiments, the Fc domain variants alter (e.g., increase or decrease) binding affinity to additional Fc receptors. The Fc domain variants can alter (e.g., increase or decrease) binding affinity to one or more of the following Fcy receptors: FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). Any art-recognized means of altering affinity to additional Fc receptors can be employed. In certain embodiments, the amino acid sequence of the variant Fc domain is altered.

[0202] In some embodiments, the Fc domain or fragment thereof forms a homodimer.

[0203] In other examples, Fc domains or fragments thereof form heterodimers. Methods for generating Fc domain heterodimers are known in the art (see, e.g., U.S. Pat. No. 8,216,805). In one example, Fc domain heterodimers are generated using the "knob-and-hole" or "key-and-hole" technique. For example, one Fc domain in the heterodimer contains at least one amino acid substitution that exposes at least one side chain to form a "knob," and the other Fc domain in the heterodimer contains at least one amino acid substitution that forms a cavity, or hole, that can accommodate a side chain. For example, one Fc domain contains a T366W mutation (or knob) and another Fc domain contains T366S, L368A, and Y407V mutations (or hole). In another example, a first Fc domain contains T350V, T366L, K392L, and T394W mutations (knob), and a second Fc domain contains T350V, L351Y, F405A, and Y407V mutations (hole). Using such technology, FcRn antagonists can be formed with a single albumin or with one albumin and another protein. For example, one Fc domain of the heterodimer is linked to albumin, and the other Fc domain is not linked to any additional protein or is linked to a protein other than albumin.

[0204] In some embodiments, the Fc domain variant is a single chain Fc, in which the constituent Fc domain regions are linked by a linker. Methods for generating single chain Fc domain regions are known in the art (see, e.g., US20090252729 and US20110081345).

[0205] As discussed herein, pathogenic IgG observed in autoimmune diseases triggers the pathogenesis of these diseases or contributes to their progression, mediating disease through inappropriate activation of cellular Fc receptors. Aggregated autoantibodies and / or autoantibodies complexed with autoantigens (immune complexes) bind to activating Fc receptors, causing many autoimmune diseases (which are caused in part by immune-mediated inflammation directed against self-tissues). Therefore, to treat antibody-mediated diseases (e.g., autoimmune diseases), it is advantageous to both remove harmful autoantibodies and inhibit the interaction of these antibody immune complexes with activating Fc receptors (e.g., Fcγ receptors such as CD16a).

[0206] In some embodiments, the Fc domain mutant of an FcRn antagonist exhibits increased binding to CD16a (e.g., human CD16a) compared to the corresponding wild-type Fc. In particular embodiments, the FcRn antagonist comprises a mutant Fc region comprising an N-linked glycan (e.g., EU position 297). In this case, modifying the glycan structure can increase the binding affinity of the FcRn antagonist to CD16a. Modification of the N-linked glycan of an Fc domain is known in the art. For example, afucosylated N-linked glycans or N-linked glycans having a bisected GlcNac structure have been shown to have high affinity for CD16a. Thus, in particular embodiments, the N-linked glycans are afucosylated. Afucosylation can be achieved using any art-recognized means. For example, an FcRn antagonist can be expressed in a cell lacking fucosyltransferase, such that fucose is not added to the N-linked glycan at EU position 297 of the mutant Fc domain (see, e.g., U.S. Pat. No. 8,067,232, the contents of which are incorporated herein by reference in their entirety). In certain examples, the N-linked glycan has a bisected GlcNac structure. The bisected GlcNac structure can be achieved using any art-recognized means.

[0207] In other examples, Fc domain variants of FcRn antagonists exhibit reduced binding to CD16a (eg, human CD16a).

[0208] In other examples, Fc domain variants of FcRn antagonists exhibit similar binding to CD16a (eg, human CD16a) compared to the corresponding wild-type Fc.

[0209] In some embodiments, the FcRn antagonists described herein are monomeric. For example, the Fc domain contains one or more mutations that reduce or prevent dimerization. Exemplary mutations are described in WO2011 / 063348, WO2018 / 144784, WO2013 / 138643, WO2014 / 087299, and WO2013 / 166604.

[0210] Albumin and its fragments The FcRn antagonists of the present disclosure comprise albumin or a fragment thereof capable of extending the half-life of the antagonist. In one example, the albumin comprises the sequence set forth in SEQ ID NO: 1. In one example, the albumin of the present disclosure comprises a sequence that is at least about 85%, 90%, 95%, 97%, 98%, or 99% identical to a sequence disclosed herein.

[0211] In one embodiment, the albumin is human serum albumin (HSA).

[0212] In one example, the human albumin variant or fragment thereof is a naturally occurring albumin variant. Exemplary substitutions for naturally occurring albumin variants are described, for example, in Peach & Brenan (BBA 1097:49-54, 1991), Iwao et al. (BBA Proteins & Proteomics 1774:1582-90, 2007), and Galliano et al. (BBA 1225:27-32, 1993).

[0213] Peach & Brenan present various substitutions for naturally occurring albumin variants. Exemplary substitutions include at position 494 (e.g., D494N).

[0214] Iwao et al. present various substitutions for naturally occurring albumin variants. Exemplary substitutions include at position 541 (e.g., K541E), position 560 (e.g., K560E), position 501 (e.g., E501K), position 570 (e.g., E570K), or position 573 (e.g., K573E).

[0215] Galliano et al. present various substitutions for naturally occurring albumin variants. Exemplary substitutions include at position 505 (e.g., E505K).

[0216] In one example, a fragment of albumin can extend the half-life of an FcRn antagonist without necessarily binding to FcRn. In one example, a fragment of albumin binds to FcRn with reduced affinity compared to wild-type albumin. In another example, a fragment of albumin does not detectably bind to FcRn.

[0217] In some embodiments, the albumin is an albumin variant. In some embodiments, the albumin variant binds to FcRn with reduced affinity compared to wild-type albumin. In some embodiments, the albumin variant does not detectably bind to FcRn. In some embodiments, the binding of albumin to FcRn is determined by immobilizing soluble FcRn on a soluble surface and detecting albumin binding using SPR. In some embodiments, albumin is immobilized and soluble FcRn binding is determined using SPR.

[0218] In one example, the albumin variant comprises an amino acid modification, e.g., a substitution, that reduces the binding of albumin to FcRn. Substitutions that reduce the binding of albumin to FcRn are known in the art and are described, for example, in WO2012 / 150319, WO2011 / 051489, US10696732, or US8822417.

[0219] Exemplary substitutions for albumin are described herein and include substitutions at position 464 (e.g., H464Q), position 510 (e.g., H510Q or H510R), and position 535 (e.g., H535Q or H535F) relative to SEQ ID NO:1.

[0220] WO2011 / 051489 demonstrates that various substitutions in albumin reduce affinity for FcRn. Exemplary substitutions include those at position 494 (e.g., D494Q, D494N, or D494A), position 495 (e.g., E495Q or E495A), position 496 (e.g., T496A), position 417 (e.g., Q417A), position 499 (e.g., P499A), position 536 (e.g., K536A), position 538 (e.g., K538A), or position 550 (e.g., D550N). Furthermore, combinations of substitutions, such as those at positions 494 and 496 (e.g., D494N and T496A) or positions 494 and 417 (e.g., D494E and Q417H), reduce affinity for FcRn.

[0221] WO2012 / 150319 also demonstrates that various substitutions in albumin reduce affinity for FcRn. Exemplary substitutions include those at position 500 (K500A), position 417 (e.g., Q417A), position 536 (e.g., K536A), position 537 (e.g., P537A), position 538 (e.g., K538A), position 573 (e.g., K573P), position 580 (Q580A), position 111 (e.g., N111H, N111K, N111E), position 512 (e.g., D512E), position 527 (e.g., T527A), position 569 (e.g., A569S), or position 108 (e.g., D108A).

[0222] US106967322 also shows that various substitutions in albumin reduce affinity for FcRn. Exemplary substitutions include those at position 494 (e.g., D494N, D494A, D494Q), position 496 (e.g., T496A), position 417 (e.g., Q417A), position 499 (e.g., P499A), position 500 (e.g., K500A), position 536 (e.g., K536A), position 537 (e.g., K537A), position 538 (e.g., K538A), or position 501 (e.g., E501A or 501Q).

[0223] Schmidt et al. (Structure 21, 1966-1978, 2013) also showed that various substitutions in albumin reduce its affinity for FcRn.

[0224] In one embodiment, the albumin variant or fragment thereof comprises: (i) an amino acid substituted with histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; (ii) an amino acid substitution of threonine at a position corresponding to amino acid 422 of SEQ ID NO: 1; (iii) an amino acid substituted with histidine at a position corresponding to amino acid 510 of SEQ ID NO: 1; (iv) an amino acid substituted with histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1, and (v) a combination thereof.

[0225] In one example, the albumin variant or fragment thereof comprises a glutamine substituted for a histidine at a position corresponding to amino acid 464 of SEQ ID NO:1.

[0226] In one example, the albumin variant or fragment thereof comprises a tryptophan substituted for threonine at a position corresponding to amino acid 422 of SEQ ID NO:1.

[0227] In one example, the albumin variant or fragment thereof comprises a glutamine substituted for a histidine at a position corresponding to amino acid 510 of SEQ ID NO:1.

[0228] In one example, the albumin variant or fragment thereof comprises an arginine substituted for a histidine at the position corresponding to amino acid 510 of SEQ ID NO:1.

[0229] In one example, the albumin variant or fragment thereof comprises a phenylalanine substituted for a histidine at a position corresponding to amino acid 535 of SEQ ID NO:1.

[0230] In one example, the albumin variant or fragment thereof comprises a glutamine substituted for a histidine at a position corresponding to amino acid 535 of SEQ ID NO:1.

[0231] In one example, the albumin variant or fragment comprises an amino acid substitution at a position corresponding to amino acid 463 of SEQ ID NO:1.

[0232] In one example, the albumin variant or fragment comprises an amino acid substitution at a position corresponding to amino acid 467 of SEQ ID NO:1.

[0233] In one example, the albumin variant or fragment comprises an amino acid substitution at a position corresponding to amino acid 509 of SEQ ID NO:1.

[0234] In one example, the albumin variant or fragment comprises an amino acid substitution at a position corresponding to amino acid 519 of SEQ ID NO:1.

[0235] In one example, the albumin variant or fragment comprises a glutamine substituted for a histidine at the position corresponding to amino acid 464 of SEQ ID NO:1.

[0236] In one example, the albumin variant or fragment comprises a tryptophan substituted for a threonine at the position corresponding to amino acid 422 of SEQ ID NO:1 and a glutamine substituted for a histidine at the position corresponding to amino acid 464 of SEQ ID NO:1.

[0237] In one example, the albumin variant or fragment comprises a glutamine substituted for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1 and a phenylalanine substituted for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1.

[0238] In one example, the albumin variant or fragment thereof comprises an arginine substituted for histidine at a position corresponding to amino acid 510 of SEQ ID NO:1 and a phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO:1.

[0239] Exemplary methods for making variant forms of albumin are described herein or known in the art: mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525:309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7:18, 2007; and WO1999 / 058661), Introducing a nucleic acid encoding the polypeptide into a mutator cell, such as XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene). DNA shuffling, as disclosed, for example, in Stemmer, Nature 370:389-91, 1994, and Site-directed mutagenesis, as described, for example, in Dieffenbach (ed.) and Dveksler (ed.) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0240] Exemplary methods for determining the biological activity, e.g., FcRn affinity, of the disclosed albumin variants or fragments thereof will be apparent to those of skill in the art and / or are described herein, e.g., methods for determining the affinity of an albumin variant or fragment thereof, such as affinity, association, dissociation, and therapeutic effect, are described herein.

[0241] In some embodiments, the FcRn antagonist comprises multiple albumins. In some embodiments, multiple albumins are linked to the Fc domain. For example, within the FcRn antagonist, a single Fc domain is linked to multiple albumins or fragments thereof. In some embodiments, the albumins are linked in tandem, for example, to the C-terminus of the Fc domain. In other embodiments, one or more albumins or fragments thereof are linked to the N-terminus of the Fc domain and one or more albumins or fragments thereof are linked to the C-terminus of the Fc domain. In some embodiments, the Fc domain can dimerize, thus further increasing the number of albumins within the FcRn antagonist.

[0242] In one example, each Fc domain of an FcRn antagonist is linked to a single albumin. As exemplified herein, linking albumin or a fragment thereof to the C-terminus of the Fc domain results in increased half-life and FcRn antagonism.

[0243] Linker In some embodiments, the components of the FcRn antagonists of the present disclosure are linked indirectly, for example, via a linker. In some embodiments, the linker is a polypeptide linker.

[0244] In some embodiments, the polypeptide linker comprises or consists of a glycine-serine linker. As used herein, the term "gly-ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly / ser linker has the formula (Gly4Ser): n where n is a positive integer (e.g., 1, 2, 3, 4, or 5). In particular embodiments, the gly / ser linker is (Gly4Ser)1. In some embodiments, the gly / ser linker is (Gly4Ser)2. In some embodiments, the gly / ser linker is (Gly4Ser)3 or (Gly4Ser) 4. is.

[0245] Other linkers suitable for use in the FcRn antagonists of the present disclosure are known in the art and include, for example, the serine-rich linker disclosed in US5525491, the helix-forming peptide linker (e.g., A(EAAAK)nA (n=2-5)) disclosed in Arai et al., Protein Eng 2001;14:529-32, or the stable linker disclosed in Chen et al., Mol Pharm 2011;8:457-65.

[0246] Other exemplary linkers include a GS linker (i.e., (GS)n), a GGSG linker (i.e., (GGSG)n), a GSAT linker, a SEG linker, and a GGS linker (i.e., (GGSGGS)n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5).

[0247] Polypeptide linkers of the present disclosure are at least one amino acid long and can vary in length. In some embodiments, polypeptide linkers of the present disclosure are from about 1 amino acid long to about 50 amino acids long. In other embodiments, polypeptide linkers of the present disclosure are from about 5 amino acids long to about 10 amino acids long. In other embodiments, polypeptide linkers of the present disclosure are from about 10 amino acids long to about 20 amino acids long. In other embodiments, polypeptide linkers of the present disclosure are from about 15 amino acids long to about 50 amino acids long.

[0248] In some embodiments, the linker comprises or is a chemical linker. In some embodiments, the linker is one or more ethylene glycol (EG) units, e.g., two or more EG units (i.e., polyethylene glycol (PEG)). In some embodiments, the linker comprises a polyethylene glycol (PEG) linker or consists of a PEG linker. Polyethylene glycol or PEG refers to a compound composed of repeating ethylene glycol units. Exemplary "PEG linkers" include compounds of the formula: H-(O-CH2-CH2)n-OH, where n is a positive integer (e.g., 1, 10, 20, 50, 100, 200, 300, 400, 500, 600). In some embodiments, the PEG linker is PEG1000. In some embodiments, the PEG linker is PEG2000. In some embodiments, the PEG linker is PEG3000.

[0249] In some embodiments, the FcRn antagonist comprises an Fc domain or a fragment thereof linked to PEG, which is further linked to albumin or a fragment thereof.

[0250] As discussed herein, the N-terminus of the Fc domain can be linked to the N-terminus of albumin, or the C-terminus of the Fc domain to the C-terminus of albumin. Chemical linkers are suitable for such linkages.

[0251] Production of FcRn antagonists The present disclosure provides polynucleotides, vectors, and host cells encoding the FcRn antagonists disclosed herein, as well as methods for producing FcRn antagonists, including expressing these polynucleotides.

[0252] Polynucleotides encoding the FcRn antagonists disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce desired amounts of the FcRn antagonist. Thus, in some embodiments, the present disclosure provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0253] The term "vector" or "expression vector" is used herein and in the claims to mean a vector used in accordance with the present disclosure as a vehicle for introducing and expressing a desired gene into a cell. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses, and retroviruses.

[0254] For purposes of this disclosure, numerous expression vector systems may be employed. For example, one class of vector utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others include the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. Markers may provide, for example, prototrophy for an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. Selectable marker genes can either be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0255] More generally, once a vector or DNA sequence encoding an FcRn antagonist has been prepared, the expression vector can be introduced into a suitable host cell; i.e., the host cell can be transformed. Introduction of the plasmid into the host cell can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, in Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass., 1988). Most preferably, introduction of the plasmid into the host is by electroporation. The transformed cells are grown under conditions suitable for production of the FcRn antagonist and assayed for expression of the FcRn antagonist. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0256] In vitro production allows for scale-up to obtain large quantities of the desired FcRn antagonist. Techniques for culturing mammalian cells under tissue culture conditions are known in the art, and include, for example, homogeneous suspension culture in airlift reactors or continuous stirrer reactors, or immobilized or entrapped cell culture in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the polypeptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and / or (immuno)affinity chromatography.

[0257] Genes encoding the FcRn antagonists of the present disclosure can also be expressed in non-mammalian cells, such as bacteria, yeast, or plant cells. In this regard, it will be understood that various unicellular non-mammalian microorganisms, such as bacteria, i.e., microorganisms capable of growth in culture or fermentation, can also be transformed. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella. Furthermore, it will be understood that when expressed in bacteria, the FcRn antagonists may become part of inclusion bodies. The FcRn antagonists must be isolated and purified before assembly into functional molecules. In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although many other strains are commonly available.

[0258] FcRn antagonist activity assay FcRn antagonists of the present disclosure are readily screened for biological activity, for example, as described below.

[0259] Affinity measurement Optionally, the dissociation constant (Kd), association constant (Ka), or affinity constant (K) of the FcRn antagonist or a component thereof (e.g., an Fc domain or fragment thereof, or albumin or a fragment thereof) is determined. D ) is measured.

[0260] Affinity measurements can be measured by standard methodologies, such as immunoassays, surface plasmon resonance (SPR, e.g., BIAcore surface plasmon resonance (BIAcore, Piscataway, NJ) (Rich and Myszka Curr. Opin. Biotechnol 11:54, 2000; Englebienne Analyst. 123:1599, 1998), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art.

[0261] In some embodiments, the FcRn antagonist has a K equivalent to that of a human IgG1 Fc domain or a variant or fragment thereof. Dor improved K D (i.e., lower K D value) for FcRn.

[0262] Binding affinity to FcRn can also be measured non-quantitatively using flow cytometry. For example, CHO cells stably expressing an FcRn antagonist or a fragment thereof are stained with alexa-488-labeled FcRn / β2m (to detect target binding) and anti-myc-alexa647 (to detect expression) at acidic (pH 5.5) and neutral (pH 7.4) pH and analyzed by flow cytometry. Relative binding to FcRn / β2m is determined, for example, by calculating the mean fluorescence intensity relative to the unmodified human IgG1 Fc domain or a variant or fragment thereof.

[0263] Determining half-life The FcRn antagonists encompassed by the present disclosure have improved half-lives, e.g., are modified to have extended half-lives compared to the human IgG1 Fc domain or a variant or fragment thereof. Methods for determining the half-life of an FcRn antagonist will be apparent to those skilled in the art.

[0264] For purposes of clarity, and as will be apparent to those skilled in the art based on the present disclosure, reference to "half-life" is understood to refer to an increase in one or more of the following parameters: terminal half-life, mean residence time, area under the curve, reduction in volume of distribution, and / or clearance rate. For example, increasing the half-life of an FcRn antagonist means improving the plasma exposure of the antagonist or decreasing the volume of distribution of the antagonist.

[0265] In one example, the FcRn antagonists of the present disclosure have an improved alpha half-life, ie, the rate at which plasma concentrations decline due to the redistribution process.

[0266] The half-life of the FcRn antagonists of the present disclosure can also be measured by pharmacokinetic studies, for example, according to the method described in Kim et al., Eur J of Immunol 24:542, 1994. According to this method, the protein is intravenously injected into mice, and its plasma concentration is measured periodically as a function of time, for example, from 3 minutes to 72 hours after injection. The clearance curve thus obtained should be biphasic, i.e., have an alpha phase and a beta phase. To determine the in vivo terminal half-life of the protein, the clearance rate in the beta phase is calculated and compared with the clearance rate of the human IgG1 Fc domain or a variant or fragment thereof.

[0267] In vitro cell assay To assess the ability of an FcRn antagonist to treat a disease or condition described herein, various in vitro assays are available.

[0268] In one example, the uptake and recycling of FcRn antagonists is tested in an in vitro cellular assay.

[0269] Methods for assessing cellular uptake and recycling are known in the art and / or exemplified herein. For example, a fluorescently labeled FcRn antagonist is incubated with cells expressing human FcRn receptors on the cell surface. After adding the labeled FcRn antagonist, the progress of protein recycling can be tracked and compared with that of the human IgG1 Fc domain or its variants or fragments using methods including flow cytometry and fluorescence microscopy (e.g., confocal fluorescence microscopy). The alterations of specific albumin variants to the normal recycling pathway can be identified and characterized.

[0270] Pharmacokinetic analysis In one example, the pharmacokinetic (PK) properties of an FcRn antagonist are evaluated.

[0271] Methods for assessing PK properties are known in the art and / or exemplified herein. For example, an FcRn antagonist is injected into a transgenic mouse expressing a human FcRn receptor or other suitable mammalian host (e.g., rat, cynomolgus monkey). In one example, the transgenic mouse expressing a human FcRn receptor is a "hFcRn Tg32" homozygous mouse (i.e., B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ, Jackson Laboratory stock number 014565, or as described in Roopenian et al., J. Immunol 2003;170:3528-3533). Plasma concentrations of the FcRn antagonist are assessed using ELISA using commercially available methods.

[0272] Immunoglobulin clearance In one example, the ability of an FcRn antagonist to reduce circulating immunoglobulin levels is assessed.

[0273] For example, known tracer antibody, for example, IgG1 antibody, is administered to a subject, for example, a mouse.Then, FcRn antagonist is administered, and the antibody level is measured at various time points.The FcRn antagonist induces a more rapid decrease in tracer antibody level than that observed in the absence of antagonist.

[0274] In another embodiment, levels of endogenous immunoglobulin, eg, IgG, are measured in the presence and absence of an antagonist.

[0275] Pharmaceutical Compositions Preferably, in compositions or methods for administering the FcRn antagonists of the present disclosure to a subject, the FcRn antagonists of the present disclosure (i.e., albumin variants or fragments thereof conjugated to compounds) are combined with a pharmaceutically acceptable carrier, as understood in the art. Accordingly, one embodiment of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising an FcRn antagonist combined with a pharmaceutically acceptable carrier.

[0276] In general terms, a "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating agent, emulsifier, wetting agent, solvent, suspending agent, coating agent, or lubricant that can be safely administered to any subject, for example, a human. Depending on the route of administration, a variety of acceptable carriers known in the art can be used, for example, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0277] The FcRn antagonists of the present disclosure are useful for parenteral, topical, oral, local, aerosol, or transdermal administration for prophylactic or therapeutic treatment. In one embodiment, the FcRn antagonist is administered parenterally, such as subcutaneously or intravenously. For example, the FcRn antagonist is administered intravenously.

[0278] The formulation of the administered FcRn antagonist will vary depending on the selected route of administration and formulation (e.g., solution, emulsion, capsule). Suitable pharmaceutical compositions containing the administered FcRn antagonist can be prepared in a physiologically acceptable carrier. In the case of solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. A variety of suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can contain various additives, preservatives, or fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition may optionally contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The FcRn antagonist may be stored in a liquid state or lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0279] Use of FcRn antagonists The FcRn antagonist compositions of the present disclosure are particularly useful for reducing serum levels of immunoglobulins or other Fc-containing agents (e.g., antibody-drug conjugates and immunoadhesins). Accordingly, in one example, the present disclosure provides a method for inhibiting FcRn function in a subject, which generally comprises administering to the subject an effective amount of an FcRn antagonist or pharmaceutical composition of the present disclosure.

[0280] Reduction of serum levels of immunoglobulins or Fc-containing drugs is applicable to the treatment of antibody-mediated diseases (e.g., autoimmune diseases). Accordingly, in one example, the present disclosure provides methods of treating antibody-mediated diseases (e.g., autoimmune diseases) using the FcRn antagonist compositions disclosed herein.

[0281] The FcRn antagonist compositions disclosed herein can be used to treat any antibody-mediated disease.

[0282] The FcRn antagonists of the present disclosure are suitable for treating antibody-mediated diseases characterized by excessive production of serum immunoglobulins. Thus, in some embodiments, FcRn antagonist compositions are used to treat hypergammaglobulinemia.

[0283] FcRn antagonists can also be used in combination with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an anti-inflammatory agent. Any anti-inflammatory agent can be used in combination with the FcRn antagonists disclosed herein. In one embodiment, the additional therapeutic agent is rituximab, daclizumab, basiliximab, muronomab-cd3, infliximab, adalimumab, omalizumab, efalizumab, natalizumab, tocilizumab, eculizumab, golimumab, canakinumab, ustekinumab, or belimumab. In some embodiments, the additional therapeutic agent is a leukocyte-depleting agent (e.g., a B cell or T cell-depleting agent). Any leukocyte-depleting agent can be used in combination with the FcRn antagonists disclosed herein. In some embodiments, the leukocyte-depleting agent is a B cell-depleting agent. In some embodiments, the leukocyte-depleting agent is an antibody against a cell surface marker. Suitable cell surface markers include, but are not limited to, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD70, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, or CD86. The FcRn antagonist and additional therapeutic agent(s) can be administered to a subject simultaneously or sequentially via the same or different route(s) of administration.

[0284] The FcRn antagonists of the present disclosure are also suitable for reducing the serum concentration of an Fc-containing drug in a subject. Such clearance is advantageous when the Fc-containing drug is toxic (e.g., an antibody-drug conjugate or an immunogenic drug) because it reduces exposure to the drug in the subject. Clearance is also advantageous when the Fc-containing drug is an imaging agent that requires low serum concentrations of the drug to achieve better contrast imaging and / or to minimize damage to normal tissue when the Fc-containing drug is radiolabeled. Thus, in some embodiments, FcRn antagonists are used to reduce the serum concentration of an Fc-containing drug in a subject administered the Fc-containing drug. The FcRn antagonists disclosed herein can be used to reduce the serum concentration of any Fc-containing drug (e.g., a therapeutic or diagnostic agent). Non-limiting examples of Fc-containing agents include imaging agents (e.g., labeled antibodies), antibody-drug conjugates, or immunogenic agents (e.g., non-human immunoglobulins or immunoadhesins). The FcRn antagonist can also be administered simultaneously with the Fc-containing agent or sequentially (e.g., before or after the Fc-containing agent).

[0285] The FcRn antagonists disclosed herein can also be used in combination with therapeutic proteins to increase the efficacy of the therapeutic proteins by reducing IgG levels, where IgG is responsible for the reduced bioavailability of the therapeutic proteins.

[0286] In some examples, the present disclosure provides methods for reducing or preventing an immune response to a therapeutic compound.

[0287] Those skilled in the art can determine, by routine experimentation, an effective, non-toxic amount of an FcRn antagonist composition for the purpose of treating antibody-mediated diseases. For example, a therapeutically effective amount of a polypeptide may vary depending on factors such as the stage of the disease (age, sex, medical complications (e.g., immunosuppressive conditions or diseases), and weight of the subject, as well as the ability of the antibody to elicit a desired response in the subject. Dosage regimens can be adjusted to provide the optimal therapeutic response; for example, several divided doses may be administered daily, or the dose may be proportionally reduced depending on the exigencies of the therapeutic situation. However, in general, an effective amount is expected to be in the range of about 1 to 200 mg / kg body weight.

[0288] Kits and Other Compositions Another embodiment of the present disclosure provides a kit comprising an FcRn antagonist of the present disclosure useful for reducing circulating autoantibodies in a subject in need thereof, the kit comprising: (i) at least one FcRn antagonist, pharmaceutical composition, or nucleic acid of the present disclosure; (ii) instructions for using the kit in reducing circulating autoantibodies in a subject; and (iii) optionally, at least one additional therapy.

[0289] The present disclosure also provides a kit for treating or preventing the progression of an antibody-mediated disease in a subject in need thereof, the kit comprising: (i) at least one FcRn antagonist, pharmaceutical composition, or nucleic acid of the present disclosure; (ii) instructions for using the kit in treating or preventing the progression of an antibody-mediated disease in a subject; and (iii) optionally, at least one additional therapy.

[0290] According to this embodiment of the disclosure, instructions (or package insert) are on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The label or package insert indicates that the FcRn antagonist is used to treat a subject to be treated, e.g., a subject having or predisposed to developing a condition described herein, and provides specific guidance regarding the dosage and administration interval of the FcRn antagonist and any other provided pharmaceutical agents. The kit may further include an additional container containing a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc.

[0291] The kit optionally further comprises a container containing a second pharmaceutical agent, wherein the FcRn antagonist is the first pharmaceutical agent, and the article further comprises instructions on a package insert for treating a subject with an effective amount of the second pharmaceutical agent, which may be a therapeutic protein as described above.

[0292] The present disclosure includes the following non-limiting examples.

[0293] Example 1: Materials and Methods Isolation of bone marrow-derived macrophages (BMDMs) 10-week-old 32HOM huFcRn Tg / Tg Mice were euthanized by CO2 asphyxiation, and BMDMs were generated essentially as previously described (Lim et al., Biol Open. 2012 Sept. 15;1(9):904-14, 2012). Monocyte cells were cultured at 6 x 10 for long-term storage. 6 cells / mL and stored frozen.

[0294] To evaluate the effect of FcRn antagonists on the recycling of IgG and albumin in macrophages after macropinocytosis. Approximately 6x106 Approximately 1.9x10 BMDMs were plated onto an 8-well removable chamber slide (Ibidi, Germany). 5 Cells were seeded at 1000x1000 cells / well. After differentiation in 200 μL of BMDM medium for 3 days, 200 μL of BMDM medium (RPMI, heat-inactivated FCS (15%), 20% L cell culture medium, 500 μL of Pen / Strep, 2 mM Glutamax) was added to further differentiate the cells into macrophage-like cells. Next, the cells were starved for 16 hours by removing the BMDM medium, washing twice with PBS, and replacing it with 200 μL of C-RPMI (RPMI, heat-inactivated FCS (10%), 500 μL of Pen / Strep, 2 mM Glutamax) per well. The medium was then aspirated from the wells, and the cells were preincubated for 4.5 hours in the presence of DMSO (1:100 dilution) or protease inhibitors (1:100 dilution). Thirty minutes before the end of the preincubation, mouse serum (1 / 100 dilution) was added to block surface Fc gamma receptors and incubated on ice for 30 minutes. Treatment samples containing a combination of FcRn antagonist, HSA-AF488, IgG-AF568, and protease inhibitors were prepared and prewarmed at 37°C. After incubation with mouse serum, cells were washed twice with PBS, and premixed, prewarmed experimental treatment solutions were added and pulsed at 37°C (neutral pH) for 15 minutes. Cells were then washed twice with PBS and either immediately fixed (0-minute chase) or incubated in C-RPMI + / - protease inhibitors for a 15-minute chase period, followed by washing and fixation.

[0295] After the 0 and 15 min time points, cells were fixed with 4% PFA for 10 min at room temperature, washed once with 50 mM NH4Cl in PBS to quench excess PFA, and incubated for an additional 10 min at room temperature. After another PBS wash, the cells were blocked with 5% FCS in PBS for 30 min. Samples were then treated with Hoescht staining to stain nuclei, and slides were mounted with Mowiol mounting medium.

[0296] Imaging was performed using a confocal microscope. The fluorescence intensity of the acquired images was analyzed using the "analyze particle" plugin in Image J. Individual values ​​were entered into GraphPad Prism and graphed.

[0297] Evaluation of the affinity of FcRn antagonists The FcRn antagonists tested in Tables 1, 2, and 3 (below) were captured using surface-bound recombinant albumin-binding protein (ABP) as described by Lejon et al. (2004) Journal of Biological Chemistry, 279(41), pp. 42924-42928. ABP (20 μg / mL in 10 mM sodium acetate, pH 5) was directly immobilized to approximately 2,000 RU on the carboxymethyl dextran surface of a CM5 sensor chip using standard NHS / EDC chemistry at pH 5. Control G1Fc homodimer and full-length IgG1 were directly immobilized to approximately 2,000 RU on the carboxymethyl dextran surface of a CM5 sensor chip using standard NHS / EDC chemistry at pH 4.5.

[0298] FcRn antagonist (2 μg / mL) was prepared in pH 7.3 running buffer and captured up to approximately 350 RU on the active sites of each flow cell. This capture was equally stable under neutral and acidic conditions. No protein was captured on the reference surface used for background subtraction. The immobilized ABP surface was prepared by 10 cycles, including conditioning with recombinant HSA (2 μg / mL) (60 s injection) followed by 30 s regeneration with 6 M guanidine-HCl. Sensorgrams were double-subtracted using the reference surface and blank buffer injection data obtained in each experiment. Data were fit to a 1:1 Langmuir model with a local Rmax and zero refractive index (RI = 0). Experimental running buffers (10 mM HEPES, 150 mM NaCl, pH 7.3 or pH 6.0) were prepared and filtered (0.22 μm) before use.

[0299] Purified soluble recombinant mammalian (i.e., human, cynomolgus monkey, mouse, and / or rat) FcRn / β2m (approximately 42 kDa) receptors were tested at concentrations ranging from 39 nM to 20 μM under neutral conditions (pH 7.3) and from 7.8 nM to 2 μM under acidic conditions (pH 6.0). Analyte samples were prepared by two-fold serial dilution in running buffer. Association and dissociation rate constants for FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcRn / β2m receptors were monitored for 120 and 240 seconds, respectively. Association and dissociation rate constants for FcγRI (CD64) were monitored for 240 and 600 seconds, respectively. The flow rate was set at 30 μL / min. Each ligand was tested in at least three replicates.

[0300] Table 2 (below) shows the results of testing FcRn antagonists under avidity conditions against biotinylated human FcRn / β2m bound to a standard streptavidin or neutravidin sensor chip. Biotinylation of soluble recombinant human FcRn / β2m can be performed using either standard amine chemistry (e.g., biotin-NHS) or enzymatic methods (e.g., BirA biotin ligase) with comparable results. FcRn antagonist samples were prepared at two-fold dilutions ranging from 0.15 nM to 20 nM in running buffer. The FcRn / β2m capture surface was prepared by five cycles, including conditioning (30-second injection) of recombinant IgG (100 nM, 15 μg / mL) followed by a 30-second regeneration in 150 mM Tris, pH 8.

[0301] In Table 3 (below), purified soluble recombinant human FcY receptors (i.e., FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a)), and FcRn / β2m were analyzed against FcRn antagonists under neutral conditions (pH 7.3). G1Fc homodimer and full-length IgG1 were used as baseline controls. Soluble analytes were prepared in two-fold serial dilutions in running buffer. Human FcyRIIA (CD32), FcyRIIB (CD32), and FcyRIIIA (CD16a) receptors were tested at concentrations ranging from 39 nM to 50 μM, and FcyRI (CD64) was tested at concentrations ranging from 0.098 nM to 100 nM.

[0302] Sensorgrams were double-subtracted using the reference surface and blank buffer injection data obtained in each experiment. Data were fit to a 1:1 Langmuir model with local Rmax and zero refractive index (RI = 0). Experimental running buffer (10 mM HEPES, 150 mM NaCl, pH 7.3 or pH 6.0) was prepared and filtered (0.22 μm) before use.

[0303] FcRn antagonists exhibited nanomolar affinity for mammalian FcRn / β2m when the Fc receptor was used as the analyte (Table 1) or surface-bound ligand (Table 2). In Table 3, FcRn antagonists exhibited binding affinities for soluble Fcy receptors comparable to those observed with either G1Fc homodimer or full-length IgG1.

[0304] FcRn antagonist binding FcRn / β2m protein complexes (350-400 kDa, 1.28 mg / ml) were prepared in PBS and diluted to 0.1 μM in acetate buffer (pH 6.0). Carbon-coated Cu grids (GSCU400CC, 400 mesh) were glow-discharged at 15 mA for 30 seconds. 4 μl of the diluted protein sample was applied for 45 seconds and blotted (using Whatman No. 1 filter paper). Then, 4 μl of HO was applied and blotted. Finally, the grids were negatively stained with 2% uranyl acetate for 30 seconds, blotted to remove residual stain, and air-dried. The grids were imaged at room temperature using a TF30 (200 keV) TEM at 39,000x magnification and a pixel size of 2.79 Å. Thirty-two image files were imported into cryoSPARC v3.322 using the following input parameters: pixel size (Å) 2.79, acceleration voltage (kV) 200, and spherical aberration (mm) 2.7. Patch CTF, followed by blob picking (150–250 Å), inspect particle picking, and extract particle picking (96-pixel box size), rendered 59,879 particles for 2D classification. Representative 2D class average selection enabled template-based automated particle picking (250 Å diameter), resulting in 39,478 particles after two additional rounds of 2D classification to further remove junk particles. Images from negative staining experiments allowed the discrimination of a large globular domain (i.e., HSA) bound to a long domain (i.e., Fc) and a soluble domain (i.e., FcRn / β2m) loosely attached to the Fc-like long domain, suggesting the presence of a stable FcRn / β2m / antagonist complex in a 2:1 ratio.

[0305] Example 2: Preparation of a half-life extended FcRn antagonist Fusion proteins containing recombinant human serum albumin (HSA) and either the wild-type IgG1 Fc domain (huG1Fc) or its mutants containing the following mutations: M252Y, S254T, T256E, H433K, and N434F (huG1FcYTEKF) were prepared by linking HSA to the N- or C-terminus of huG1Fc or huG1FcYTEKF.

[0306] huG1FcYTEKF was created as a benchmark molecule, modeled after the FcRn antagonist efgartigimod (Vyvgart), with the only difference being the addition of five amino acids (EPKSC) at the N-terminus (SEQ ID NO: 13). Comparison of the internally generated molecule huG1FcYTEKF with the commercially available Vyvgart product revealed a significant difference in binding affinity (K) for surface-bound huFcRn / b2m. D ) were found to be equivalent (i.e., K D K 2.4 nM, pH 7.3 D Furthermore, the efficacy of the internally generated benchmark huG1FcYTEKF to reduce IgG in non-human primate (NHP) studies was comparable to published data on efgartigimod.

[0307] Homodimeric and heterodimeric variants of the FcRn antagonist were generated. To generate homodimers containing two albumins (e.g., Fc-albumin:Fc-albumin or albumin-Fc:albumin-Fc), cells were transfected with a single expression construct (H464Q).

[0308] For heterodimer production, expression constructs encoding either Fc-albumin or albumin-Fc molecules were cotransfected with an expression construct encoding only the Fc sequence at various ratios (e.g., 1:1, 1:0.5, or 1:0.25) to generate a constant amount of heterodimeric FcRn antagonist (i.e., Fc-albumin:Fc or albumin-Fc:Fc). The heterodimeric products were separated from other expression products by protein A affinity chromatography and size-exclusion chromatography. Heterodimers containing huG1FcYPY and a single albumin molecule were also able to bind to FcRn in vitro.

[0309] In initial SPR experiments, heterodimeric FcRn antagonist variants with a single albumin molecule fused to dimeric huIgG1FcYPY (in both Fc-albumin and albumin-Fc orientations) bound to biotinylated human FcRn / β2m with similar affinity at pH 7.3 compared with the corresponding homodimeric FcRn antagonist variants containing two albumins. The heterodimeric FcRn antagonists tested were huG1FcYPY-HSA:huG1FcYPY, huG1FcYPY-HSA(H464Q):huG1FcYPY, HSA-huG1FcYPY:huG1FcYPY, and HSA(H464Q)-huG1FcYPY:huG1FcYPY.

[0310] Homodimeric FcRn antagonists were evaluated for their ability to reduce levels of circulating IgG "tracer" antibodies (CSL360) in transgenic mice expressing human FcRn (huFcRn transgenic mice). Negative controls included mice that received no treatment, and positive controls included mice that received huG1FcYTEKF, a known FcRn antagonist. The half-life of the FcRn antagonists was also assessed.

[0311] Figure 1A shows that administration of C- and N-terminal HSA fusions with (wild-type) huG1Fc did not result in a reduction in circulating tracer antibody levels. Figure 1B shows that huG1FcYTEKF-HSA (i.e., HSA fused to the C-terminus of huG1FcYTEKF, SEQ ID NO: 5) reduced in vivo circulating IgG tracer antibody levels to a similar level to that observed with the huG1FcYTEKF positive control. However, the huG1FcYTEKF-HSA fusion protein exhibited a significantly longer in vivo half-life than huG1FcYTEKF (Figure 1C). When albumin was linked to the N-terminus of huG1FcYTEKF, the same reduction in tracer antibody levels was not observed (Figure 1B).

[0312] Example 3: Preparation of alternative extended half-life FcRn antagonists The M252Y, V308P, and N434Y substitutions were introduced into the huG1 Fc domain (huG1FcYPY) and fused to wild-type HSA and the HSA(H464Q) substitution mutant, which is known to have reduced binding to FcRn.

[0313] Figures 2A and 2B show that huG1FcYPY-HSA and huG1FcYPY-HSA[H464Q) (SEQ ID NO: 11) reduced tracer antibody levels to a similar extent compared to huG1FcYTEKF. This result was observed regardless of whether HSA was wild-type or the H464Q substitution mutant. Fusion of HSA to the N-terminus of huG1FcYPY was able to reduce circulating tracer antibody levels, but not to the same extent as C-terminal fusions (i.e., huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q)).

[0314] Figure 2C shows that similar pharmacokinetics were observed when the FcRn antagonists of the present disclosure were administered intravenously or subcutaneously, indicating high bioavailability of the FcRn antagonists after subcutaneous administration. Each FcRn antagonist was used in equimolar amounts.

[0315] Figure 2D shows that huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) each dose-dependently reduced circulating tracer antibody levels. These data suggest that the reduction in antibody levels is mediated by the Fc domain of the fusion protein and that the observed prolonged half-life conferred by HSA may be due to mechanisms other than FcRn binding and FcRn-mediated recycling. These data were of particular interest to us because binding of the HSA portion of FcRn antagonists to FcRn has been shown to lead to reduced recycling of endogenous albumin, potentially affecting albumin homeostasis, which in turn affects lipid homeostasis (Ward et al. Front Immunol. 13:892-534, 2022).

[0316] Figures 3A and 3B show that a single dose of an FcRn antagonist of the present disclosure reduced endogenous mouse IgG in wild-type mice to levels below those observed with huG1FcYTEKF (comparable to efgartigimod). This effect was observed regardless of whether huG1FcYPY was fused to wild-type HSA or HSA(H464Q).

[0317] As shown in Figures 3C and 3D, the FcRn antagonists of the present disclosure had substantially longer half-lives in wild-type mice compared to huG1FcYTEKF.

[0318] Example 4: Affinity of FcRn antagonists Tables 1 and 2 relate to the binding affinity of the disclosed FcRn antagonists to FcRn, as measured using SPR. The data shown in Table 1 were obtained using soluble human, cynomolgus monkey, mouse, and rat FcRn complexed with β2m and surface-bound FcRn antagonists of the present disclosure at pH 6.0 and pH 7.3. The data in Table 2 relate to the binding of the disclosed soluble FcRn antagonists to surface-captured biotinylated human FcRn complexed with β2m (at pH 7.3). Table 3 relates to the binding of soluble FcY receptors to surface-captured FcRn antagonists. [Table 1] [Table 2] [Table 3] [Table 4]

[0319] Example 5: FcRn antagonists that do not affect albumin recycling The ability of various FcRn antagonist molecules to antagonize IgG and albumin recycling was examined in BMDMs derived from huFcRn transgenic mice (Figure 4A and Figure 4B). Figure 5 is a cartoon illustrating how the assay described in the next section is evaluated. Macropinocytotic uptake of fluorescently labeled IgG (IgG1-AF568) was evident after a 10-minute pulse with a 0-minute chase (Figure 4A). After a 15-minute chase, there was no longer detectable IgG1-AF568, and this signal was not restored by protease inhibitors, suggesting that IgG1-AF568 was recycled by the cells rather than transported to the lysosomal compartment for degradation. Similar results were observed in the presence of a control protein, recombinant huG1Fc (the Fc domain of wild-type human IgG1). After fixation and permeabilization, membrane-bound molecules within large macropinosomes are retained, whereas free proteins within the macropinosome core are lost. Proteins trafficked to late endosomal compartments, such as lysosomes, are more effectively retained and protected within tighter membrane structures after the fixation / permeabilization step (as shown in Figure 5). The dose-dependent decrease in signal at time 0 after pulse in the presence of the FcRn antagonists huG1FcYTEKF, huG1FcYPY-HSA, and huG1FcYPY-HSA(H464Q) suggests a decrease in the interaction of IgG1-AF568 with membrane-associated FcRn within macropinosomes and a loss of free IgG1-AF568 from the macropinosome core after the fixation / permeabilization step. After a 15-minute chase before fixation / permeabilization, IgG1-AF568 was readily detected in the presence of protease inhibitors, indicating lysosomal rescue. A similar dose-dependent FcRn antagonism of IgG1-AF568 recycling (corresponding to lysosomal degradation revealed by rescue in the presence of protease inhibitors during a 15-minute chase) was observed with huG1FcYTEKF, huG1FcYPY-HSA, and huG1FcYPY-HSA(H464Q).

[0320] We also examined the effect of FcRn antagonists on albumin recycling (HSA-AF488) (Figure 4B). In control samples, HSA-AF488 was internalized by macropinocytosis, evident at the 10-minute pulse and 0-minute chase. However, after 15 minutes, no signal was observed, even in the presence of protease inhibitors, suggesting complete albumin recycling. The antagonist huG1FcYTEKF similarly had no effect on albumin recycling. At 30 μM, the molecule huG1FcYPY-HSA reduced the HSA-AF488 signal observed at time 0 after pulse after fixation and permeabilization, suggesting a reduction in the interaction of HSA-AF488 with membrane FcRn and a loss of free HSA-AF488 from the macropinosome core. Furthermore, after a 15-minute chase before fixation / permeabilization, HSA-AF488 could be rescued from lysosomal degradation in the presence of protease inhibitors, suggesting that huG1FcYPY-HSA antagonizes albumin recycling. In contrast, the molecule huG1FcYPY-HSA(H464Q) had minimal effect on albumin recycling, with only slight lysosomal rescue of HSA-AF488 observed at the highest concentration of huG1FcYPY-HSA(H464Q). In summary, these data indicate that the H464Q mutant is unlikely to antagonize albumin recycling in vivo due to its reduced ability to antagonize albumin recycling in vitro.

[0321] Example 6: Binding of FcRn antagonists Negative staining experiments demonstrated the structure of a complex of one huG1FcYPY(H464Q) with two FcRn / β2m molecules, suggesting that the HSA(H464Q) moiety does not bind to FcRn / β2m. Figure 6 shows a schematic diagram of the putative complex.

[0322] Example 7: FcRn antagonists with reduced cross-linking ability Fc-albumin fusion proteins (i.e., FcRn antagonists) may cross-link multiple FcRn receptors under acidic conditions (e.g., in cellular endosomal compartments) by bridging adjacent molecules via their Fc and albumin domains. The HSA(H464Q) mutant exhibits an affinity of approximately 10 μM for human FcRn / β2m at acidic pH 6.0, compared with HSA (i.e., approximately 300 nM). Addition of the HSA(H464Q) mutation to albumin in FcRn antagonists reduces the affinity of albumin for FcRn under acidic conditions (i.e., pH 6.0), limiting the FcRn / β2m interaction with the Fc domains of the FcRn antagonists. This is reflected in the reduced stoichiometry of the HSA(H464Q) molecule to human FcRn / β2m as measured by SPR (Table 5). Briefly, the stoichiometry (Seq) of each ligand is calculated at steady state by multiplying the ratio of the maximum response at saturation (Rmax) to the amount of captured ligand (RL) by the ratio of the molecular weight of the ligand (MwL) to the molecular weight of the analyte (MwA). This is a rearrangement of the equation for the expected theoretical Rmax. The capture level (RL) between cycles is normalized by taking the binding level at equilibrium (Req) and the capture level (RL) for each cycle and performing the calculation according to Equation 1. This results in a table of Seq values ​​for each concentration. These values ​​can be plotted as a steady-state fit to show the binding valency at each concentration and extrapolated to its maximum to determine the ligand stoichiometry. These values ​​are then scaled and fitted to a steady-state one-site model, where "Bmax" represents the stoichiometry of each interaction based on the capture and binding levels of the ligand and analyte, and their molecular weights. The molecular weights of the ligand and analyte are assumed to be 185 kDa for huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) fusions, 67 kDa for HSA, and 42 kDa for FcRn / β2m.

[0323] The role of His464 was demonstrated in the elucidation of the FcRn / β2m / HSA complex (Schmidt et al. (2013) Structure 21, 1966-1978). This model shows that His464, His510, and His535 are linked to two conserved FcRn tryptophan residues (W53). FcRn and W59 FcRn Table 5 explains the role of W53 in stabilizing the binding site of W53 in a pH-dependent manner. FcRn and W59 FcRn Our results demonstrate that mutating HSA residues surrounding the binding pocket (Figure 7) can significantly reduce HSA binding to FcRn / β2m compared to HSA(H464Q). Table 5 shows that incorporating additional albumin mutations into huG1FcYPY-HSA(H464Q) neutralizes the albumin domain of the protein, resulting in FcRn / β2m binding affinity comparable to that observed with the G1FcYPY homodimer and huG1FcYPY-HSA[25-384], in which HSA lacks the entire FcRn-binding domain. [Table 5]

[0324] Example 8: Effects on endogenous cynomolgus monkey IgG, IgA, and IgM after a single intravenous administration of an FcRn antagonist Each compound (i.e., huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), and huG1FcYTEKF) was administered as a single bolus intravenous (IV) injection of the FcRn antagonist administered at equimolar amounts to three male animals weighing approximately 4 kg each. Briefly, the FcRn antagonists were administered at equimolar levels of 75 mg / kg for huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) and 21.15 mg / kg for huG1FcYTEKF. No clinical deterioration, hematological abnormalities, blood biochemistry findings, or changes in cytokine profiles were observed after treatment. Albumin and lipids were also largely unaffected.

[0325] Blood was collected pre-dose, 0.0833 hours, 0.5 hours, 3 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 168 hours, and days 8, 12, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 85 after dosing. The lower limit of quantitation (LLOQ) of the test compounds was reported to be 1000 ng / mL for huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) and 250 ng / mL for huG1FcYTEKF.

[0326] Endogenous Ig analysis was performed using a qualified enzyme-linked immunosorbent assay (ELISA) developed with the Isotyping Panel 1 Human / NHP Kit, Cat. No. K15203D.

[0327] IgG levels in all animals followed a consistent trend of gradual decline until 168 hours (day 7) post-dose (Figures 8A and 8B). HuG1FcYPY-HSA(H464Q) was the most effective at reducing IgG levels by up to 75%, with huG1FcYPY-HSA-treated animals reducing IgG by up to 68% and huG1FcYTEKF-treated animals reducing IgG by up to 59%. Endogenous IgG returned to pre-dose levels in all groups by day 57.

[0328] IgA levels remained generally near pre-dose levels in all animals at all time points (not shown).

[0329] IgM concentrations remained generally constant in all animals up to 168 hours post-dose, after which an increase in IgM was observed in animals treated with huG1FcYPY-HSA(H464Q) and huG1FcYPY-HSA molecules (Figure 9). No clear trend in IgM levels was observed in animals treated with huG1FcYTEKF. The increase in IgM concentrations suggests the generation of anti-drug antibodies (ADAs). ADA responses in NHPs to human proteins are not predictive of ADA in humans, but they may result in an underestimation of drug PK and PD. Indeed, in some animals, a strong IgG rebound preceded or correlated with a rapid increase in IgM, which may be partially caused by ADAs (Figures 8B and 9).

[0330] Example 9. Pharmacokinetics of FcRn antagonists in cynomolgus monkey serum Pharmacokinetic analysis of FcRn antagonists was performed following specific quantification and bioanalytical evaluation of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), and huG1FcYTEKF from cynomolgus monkey serum samples after a single intravenous dose. Quantification was performed using a qualified enzyme-linked immunosorbent assay (ELISA). Calibration standards ranging from 75,000 to 1,000 ng / mL were run in duplicate, and the mean signal was regressed against the nominal concentration using a 1 / y-weighted 4PL curve fit. Calibration standards were prepared by adding appropriate amounts of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), and huG1FcYTEKF stock solutions to blank pooled cynomolgus monkey serum. Calibration standards were prepared fresh before each experiment and discarded after use.

[0331] Briefly, FcRn antagonists were quantified in blood samples collected pre-dose and at 0.0833, 0.5, 3, 8, 24, 48, 72, 168 hours post-dose, and on days 8, 12, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 85. The measured concentrations of at least 75% of the calibration standards were within ±20% of the theoretical concentration (within ±25% of the upper and lower limits of quantitation). Furthermore, the coefficient of variation for duplicate wells of each standard solution was required to be 20% or less.

[0332] Noncompartmental analysis performed to compare the PK profiles demonstrated that huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) exhibited distinct time-concentration profiles compared to huG1FcYTEKF (Figure 8C and Table 6). Compounds huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) exhibited two distribution phases (α and β). The terminal phase of these molecules (after day 8) was dominated by a steep terminal gradient, likely due to ADA, as suggested by the increased IgM activity in some animals and the strong IgG repulsion mentioned above. However, the contribution of target-mediated drug elimination (TMDD) cannot be excluded. Drug product was undetectable at 294 hours for huG1FcYPY-HSA(H464Q) and was detected in only one animal for huG1FcYPY-HSA.

[0333] The pharmacokinetics of huG1FcYTEKF were characterized by three phases: a steep initial slope (α-phase), followed by a middle slope (β-phase), and a relatively gentle terminal slope. huG1FcYTEKF showed a significantly steeper initial slope, suggesting a larger total volume of distribution compared with the huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) compounds, which may be more confined to plasma space. The dose-normalized area under the curve (AUC) (as DN AUC last (µg / mL*h / mg)) was significantly greater for huG1FcYPY-HSA(H464Q) compared with huG1FcYPY-HSA and huG1FcYTEKF.

[0334] The reduced volume of distribution for both albumin fusions compared to the benchmark molecules is likely due to a combination of reduced tissue penetration and reduced renal clearance. Interestingly, the volume of distribution for the huG1FcYPY-HSA(H464Q) molecule was smaller than that of the WT fusion protein. In addition to its role in albumin and IgG recycling, FcRn has also been shown to play a role in the tissue distribution of albumin (Feng et al., Mol Pharm. 2019 Jun 3;16(6):2385-2393). One possibility is that, while the albumin fusions collectively reduce the volume of distribution for both molecules, impaired interaction between FcRn and the albumin domain within huG1FcYPY-HSA(H464Q) may reduce FcRn-mediated transcytosis, further limiting the tissue distribution of this molecule. Another possibility is that huG1FcYPY-HSA may interact with FcRn (via two Fc domain-binding sites and two albumin domain-binding sites) in a tetrameric fashion, resulting in stronger sequestration within target cells, and cross-linking of FcRn may promote lysosomal degradation of FcRn / drug complexes, as previously reported for FcRn cross-linking (Weflen et al., 2013). Multivalent immune complexes may exclude FcRn from the recycling sorting duct and divert it to lysosomes (Mol Biol Cell, 2013).

[0335] Because hematopoietic and endothelial cells are responsible for the majority of IgG recycling, increasing the plasma retention of FcRn antagonists by accessing these relevant cell types, as achieved with the huF1FcYPY-HSA(H464Q) molecule, may be beneficial. [Table 6]

[0336] Example 10. Effects of a single subcutaneous administration of an FcRn antagonist on cynomolgus monkey IgG, IgA, and IgM Each compound (i.e., huG1FcYPY-HAS, huG1FcYPY-HSA(H464Q), and huG1FcYTEKF) was administered as a single bolus subcutaneous (SC) injection of FcRn antagonist to three male animals weighing approximately 4 kg each (Figures 10A and 10B). The two FcRn antagonists, huG1FcYPY-HSA(H464Q) and huG1FcYTEKF, were administered at equimolar levels of 75 mg / kg and 21.15 mg / kg, respectively. These molecules were well tolerated at the concentrations tested. No clinical deterioration, hematological abnormalities, blood biochemistry findings, or changes in cytokine profiles were observed.

[0337] Blood samples were collected pre-dose, 0.0833 hours, 0.5 hours, 3 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 168 hours, and on days 8, 12, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 85 after dosing. The lower limits of quantitation (LLOQ) of the test compounds were reported as 1000 ng / mL for huG1FcYPY-HSA (H464Q) and 250 ng / mL for huG1FcYTEKF. Endogenous Ig analysis was performed using a qualified enzyme-linked immunosorbent assay (ELISA) developed with the Isotyping Panel 1 Human / NHP Kit, Cat. No. K15203D.

[0338] Animals treated with huG1FcYPY-HSA(H464Q) and huG1FcYTEKF showed a gradual decline in IgG levels by 168 hours (day 7) (Figures 10A and 10B). Similar to the IV study, huG1FcYPY-HSA(H464Q) was more effective at promoting IgG depletion (up to 80% depletion) than the benchmark huG1FcYTEKF (up to 60% depletion). Endogenous IgG returned to pre-treatment levels by day 18 in both groups.

[0339] IgM levels remained roughly constant in all animals up to 168 hours post-dose, after which an increase in IgM was observed in animals treated with huG1FcYPY-HSA(H464Q) (Figure 11). No clear trend in IgM levels was observed in animals treated with huG1FcYTEKF.

[0340] Following subcutaneous administration of the FcRn antagonist, there was little evidence of a drug-related effect on IgM or IgA levels (not shown). [Table 7] [Table B-1] [Table B-2] [Table B-3] [Table B-4]

Claims

1. 1. A neonatal crystallizable fragment receptor (FcRn) antagonist, comprising: (i) an immunoglobulin Fc domain or a fragment thereof capable of binding to FcRn, and (ii) a neonatal crystallizable fragment receptor (FcRn) antagonist comprising at least one albumin or fragment thereof capable of extending the half-life of the FcRn antagonist compared to the half-life of the immunoglobulin Fc domain or fragment thereof.

2. 2. The FcRn antagonist of claim 1, wherein the antagonist has a longer serum half-life compared to the immunoglobulin Fc domain or fragment thereof alone.

3. The antagonist has an affinity constant (K) of at least 500 nM for human FcRn at neutral pH. Ｄ ) and / or binds to human FcRn at acidic pH with a K of at least 100 nM Ｄ The FcRn antagonist of claim 1 or 2, which binds to FcRn at the following structure:

4. The FcRn antagonist according to claim 1 , wherein the albumin or a fragment thereof is a human albumin variant or a fragment thereof.

5. The FcRn antagonist of claim 4 , wherein the albumin variant or fragment thereof binds to FcRn with lower affinity compared to albumin set forth in SEQ ID NO:

1.

6. The FcRn antagonist of claim 5 , wherein the binding affinity is measured at neutral and / or acidic pH.

7. The albumin variant or fragment thereof has a K of greater than 10 μM for human FcRn at neutral pH or pH 6.

0. Ｄ The FcRn antagonist of any one of claims 4 to 6, wherein the FcRn antagonist binds to the FcRn receptor at the FcRn receptor agonist level.

8. The albumin variant or fragment thereof is (i) a glutamine substituted with a histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1; (ii) tryptophan substituted with threonine at a position corresponding to amino acid 422 of SEQ ID NO: 1; (iii) glutamine substituted with histidine at a position corresponding to amino acid 510 of SEQ ID NO: 1; (iv) a phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; and (v) a combination thereof.

9. The albumin variant or fragment thereof is (i) glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO: 1; or (ii) a phenylalanine substituted for histidine at a position corresponding to amino acid 535 of SEQ ID NO: 1; or (iii) tryptophan substituted with threonine at the position corresponding to amino acid 422 of SEQ ID NO:1 and glutamine substituted with histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or (iv) a glutamine substituted with a histidine at a position corresponding to amino acid 464 of SEQ ID NO:1, and a phenylalanine substituted with a histidine at a position corresponding to amino acid 535 of SEQ ID NO:

1.

10. The FcRn antagonist according to any one of claims 1 to 9, wherein the immunoglobulin Fc domain or a fragment thereof is an Fc domain variant or a fragment thereof.

11. The FcRn antagonist of claim 10 , wherein the Fc domain variant or fragment thereof is an IgG1 Fc domain variant or fragment thereof.

12. The FcRn antagonist of claim 10 or 11, wherein the IgG1 Fc domain variant or fragment thereof binds to human FcRn with higher affinity compared to the IgG1 Fc domain set forth in SEQ ID NO:

2.

13. The FcRn antagonist of claim 12 , wherein the binding affinity is measured at neutral and / or acidic pH.

14. The Fc domain variant or fragment thereof has a K of at least 10 μM for human FcRn at neutral pH. Ｄ The FcRn antagonist of any one of claims 10 to 13, wherein the FcRn antagonist binds at

15. The Fc domain variant or fragment thereof is (i) a tyrosine substituted with methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) glutamic acid substituted for asparagine at the position corresponding to amino acid 286 according to the EU numbering system; (v) proline substituted with valine at the position corresponding to amino acid 308 according to the EU numbering system; (vi) a lysine substituted with a histidine at the position corresponding to amino acid 433 according to the EU numbering system; (vii) a tyrosine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (viii) a phenylalanine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system, and (ix) a combination thereof.

16. The Fc domain variant or fragment thereof is (i) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or (ii) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with a serine at the position corresponding to amino acid 254 according to the EU numbering system, and a glutamic acid substituted with a threonine at the position corresponding to amino acid 256 according to the EU numbering system; or (iii) a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a glutamic acid substituted with an asparagine at the position corresponding to amino acid 286 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or (iv) The FcRn antagonist of any one of claims 10 to 15, comprising a tyrosine substituted with methionine at the position corresponding to amino acid 252 according to the EU numbering system, a threonine substituted with serine at the position corresponding to amino acid 254 according to the EU numbering system, a glutamic acid substituted with threonine at the position corresponding to amino acid 256 according to the EU numbering system, a lysine substituted with histidine at the position corresponding to amino acid 433 according to the EU numbering system, and a phenylalanine substituted with asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

17. (i) the Fc domain comprises a tyrosine substituted with a methionine at a position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at a position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at a position corresponding to amino acid 434 according to the EU numbering system; and (ii) The FcRn antagonist of any one of claims 1 to 16, wherein the albumin comprises a glutamine substituted with a histidine at the position corresponding to amino acid 464 according to the EU numbering system.

18. 18. The FcRn antagonist of claim 1, wherein the Fc domain or a fragment thereof is indirectly linked to the albumin or a fragment thereof via a linker.

19. The FcRn antagonist of claim 18, wherein the linker is a peptide linker of 2 to 31 amino acids in length.

20. 18. The FcRn antagonist of claim 1, wherein the Fc domain or a fragment thereof is directly linked to albumin or a fragment thereof.

21. 21. The FcRn antagonist of claim 1, wherein the C-terminus of the Fc domain or a fragment thereof is indirectly or directly linked to the N-terminus of albumin or a fragment thereof.

22. 22. The FcRn antagonist of claim 1, wherein the FcRn antagonist comprises two or more types of albumin or fragments thereof.

23. (i) an immunoglobulin Fc domain or fragment thereof comprising a tyrosine substituted with a methionine at the position corresponding to amino acid 252 according to the EU numbering system, a proline substituted with a valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substituted with an asparagine at the position corresponding to amino acid 434 according to the EU numbering system; and (ii) an FcRn antagonist comprising albumin or a fragment thereof comprising glutamine substituted with histidine at a position corresponding to amino acid 464 of SEQ ID NO: 1, An FcRn antagonist, wherein the C-terminus of the Fc domain or a fragment thereof is indirectly or directly linked to the N-terminus of the albumin or a fragment thereof.

24. A composition comprising the FcRn antagonist of any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

25. 25. The FcRn antagonist of any one of claims 1 to 23, or the composition of claim 24, for use in reducing circulating Fc-containing proteins and / or antibodies in a subject in need thereof.

26. 25. The FcRn antagonist of any one of claims 1 to 23, or the composition of claim 24, for use in treating or preventing the progression of an antibody-mediated disease in a subject in need thereof.

27. 25. A method for reducing circulating antibodies in a subject in need thereof, comprising administering an FcRn antagonist of any one of claims 1 to 23, or a composition of claim 24.

28. 25. A method for treating or preventing the progression of an antibody-mediated disease in a subject in need thereof, comprising administering an FcRn antagonist of any one of claims 1 to 23, or a composition of claim 24.

29. 25. Use of an FcRn antagonist of any one of claims 1 to 23, or a composition of claim 24, in the manufacture of a medicament for reducing circulating antibodies in a subject in need thereof.

30. Use of an FcRn antagonist of any one of claims 1 to 23, or a composition of claim 24, in the manufacture of a medicament for treating or preventing the progression of an antibody-mediated disease in a subject in need thereof.

31. 31. The FcRn antagonist of claim 25 or 26, the method of claim 27 or 28, or the use of claim 29 or 30, wherein the subject suffers from an autoimmune disease, develops anti-drug antibodies, or is at risk of developing anti-drug antibodies.

32. The FcRn antagonist (i) reducing endogenous IgG levels by at least 1-fold (compared to when the FcRn antagonist is not administered); and / or (ii) the FcRn antagonist of any one of claims 25, 26 or 31, the method of any one of claims 27, 28 or 31, or the use of any one of claims 29 to 31, administered in an amount effective to reduce endogenous albumin levels by 20% or less (compared to when the FcRn antagonist is not administered).

33. 33. The FcRn antagonist of any one of claims 25, 26, 31 or 32, the method of any one of claims 27, 28, 31 or 32, or the use of any one of claims 29 to 32, wherein the FcRn antagonist antagonizes IgG recycling but does not substantially antagonize albumin recycling.

34. 33. The FcRn antagonist of any one of claims 25, 26, 31 or 32, the method of any one of claims 27, 28, 31 or 32, or the use of any one of claims 29 to 32, wherein administration of the FcRn antagonist does not induce dyslipidemia and / or induces a smaller increase in serum cholesterol levels compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin.

35. 33. The FcRn antagonist of any one of claims 25, 26, 31 and 32, the method of any one of claims 27, 28, 31 and 32, or the use of any one of claims 29 to 32, wherein administration of the FcRn antagonist crosslinks cell surface FcRn at a lower level than an FcRn antagonist comprising wild-type human albumin.

36. 36. The FcRn antagonist of any one of claims 25, 26, or 31-35, the method of any one of claims 27, 28, or 31-35, or the use of any one of claims 29-35, wherein the subject has received, is receiving, or will receive additional therapy.

37. 37. The FcRn antagonist, method or use of claim 36, wherein the additional therapy is a steroidal immunomodulator, plasma exchange therapy, and / or IVIg therapy.

38. 1. A kit for use in reducing circulating autoantibodies in a subject in need thereof, comprising: (i) at least one FcRn antagonist according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24; (ii) instructions for using the kit in reducing circulating autoantibodies in the subject; and (iii) optionally, at least one additional therapy.

39. 1. A kit for treating or preventing the progression of an antibody-mediated disease in a subject in need thereof, comprising: (i) at least one FcRn antagonist according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24; (ii) instructions for using the kit in treating or preventing the progression of an antibody-mediated disease in the subject; and (iii) optionally, at least one additional therapy.

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