FcRn antagonists and uses thereof

Abstract

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

Description

[0001] Related application data

[0002] This application claims priority to Australian Patent Application No. 2022903917, filed on December 20, 2022, entitled "FcRn Antagonists and Uses Thereof". The entire content thereof is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application is filed together with a sequence listing in electronic form. The entire content of the sequence listing is hereby incorporated by reference. Technical Field

[0005] The present disclosure relates to antagonists of FcRn that have an extended half-life. Background Art

[0006] Immunoglobulin gamma (IgG) plays an important role in many disorders, such as autoimmune diseases, inflammatory diseases, and disorders that are pathologically characterized by overexpression of IgG (e.g., hypergammaglobulinemia) (see, e.g., Junghans, Immunol Res. 16:29 (1997)).

[0007] The half-life of IgG in serum is extended relative to the serum half-life of other plasma proteins. This longer half-life is partly attributed to the binding of the Fc domain of IgG to the neonatal Fc receptor (FcRn). In adults, FcRn is used to protect IgG from degradation. FcRn binds to endocytosed IgG and protects IgG from delivery to degradative lysosomes by recycling IgG back to the extracellular compartment. This recycling is facilitated by the pH-dependent binding of IgG to FcRn, where the IgG / FcRn interaction is stronger at acidic endosomal pH than at extracellular physiological pH.

[0008] When the serum concentration of IgG reaches above the content of available FcRn molecules, unbound IgG is not protected by the degradation mechanism and will thus have a reduced serum half-life. Thus, inhibiting IgG binding to FcRn reduces the serum half-life of IgG by preventing intracellular recycling of IgG. Therefore, agents for antagonizing the binding of IgG to FcRn are suitable for modulating, treating, or preventing antibody-mediated disorders, such as autoimmune diseases.

[0009] Currently, some autoimmune diseases are treated by intravenous infusion (IVIg) of pooled IgG from human donors. Since many of these autoimmune diseases are chronic, affected subjects may require repeated administration of IVIg and / or other suitable therapies to manage their disease.

[0010] In another approach, blocking antibodies to FcRn have been developed to inhibit the binding of IgG to FcRn. Peptides that bind to FcRn and antagonize FcRn function have also been identified.

[0011] Recently, an FcRn inhibitor has been developed that is a modified form of the human IgG1 Fc domain (efgartigimod). This compound has been approved for the treatment of myasthenia gravis (MG). A drawback of this compound is its relatively short half-life, requiring regular infusions for effective treatment. SUMMARY OF THE INVENTION

[0012] In the studies that led to the present disclosure, the inventors herein recognized that the relatively short half-life of FcRn antagonists, such as efgartigimod, is a limitation of the molecule and a potential problem for patient compliance, and sought to generate FcRn antagonists with an extended half-life. The inventors herein 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 herein found that connecting the C-terminus of the Fc domain to the N-terminus of albumin resulted in a longer half-life and that the resulting FcRn antagonist more potently inhibited FcRn than in the relative orientation (i.e., the N-terminus of Fc connected to the C-terminus of albumin). This finding can be considered counterintuitive because most Fc fusion proteins contain the fusion partner at the N-terminus of the protein, i.e., away from the stable structure formed by the Fc C H 3 domain. An FcRn antagonist comprising an Fc domain with its C-terminus fused to the N-terminus of albumin results in a longer half-life than efgartigimod.

[0013] The inventors herein extended their studies using variants of albumin that have a reduced affinity for FcRn or do not bind to FcRn. The inventors herein hypothesized that using wild-type albumin in the antagonist could affect endogenous albumin recycling via FcRn and that there may be a risk of dyslipidemia or elevated cholesterol known to be associated with weakened albumin stability and hypoalbuminemia. On the other hand, a potential drawback of using albumin with a reduced affinity for FcRn or that does not bind FcRn is that it may not extend the half-life of the antagonist to the same extent. The inventors herein demonstrated that an FcRn antagonist comprising albumin retains its ability to inhibit the binding of IgG to FcRn and retains its ability to extend the half-life, the albumin having a reduced binding to FcRn or not binding to FcRn.

[0014] The inventors of the present case further show that FcRn antagonists can be administered subcutaneously and reduce the circulating IgG levels.

[0015] Based on the foregoing, the present disclosure provides an FcRn antagonist comprising an Fc domain from an immunoglobulin or a fragment thereof and albumin or a fragment thereof.

[0016] For example, the present disclosure provides an FcRn antagonist comprising:

[0017] (i) an immunoglobulin Fc domain or a fragment thereof capable of binding to FcRn; and

[0018] (ii) at least one albumin or a fragment thereof capable of extending the half-life of the FcRn antagonist as compared to the half-life of the immunoglobulin Fc domain or a fragment thereof.

[0019] The FcRn antagonists of the present disclosure inhibit or reduce the binding of immunoglobulins (e.g., IgG) to FcRn. For example, administration of the FcRn antagonists of the present disclosure to a subject inhibits or reduces the binding of immunoglobulins (e.g., IgG) to FcRn. Such inhibition can be determined using standard methods known in the art, for example, by administering an antibody (“tracer antibody”) to a subject and measuring the clearance of the tracer antibody from the circulation, wherein an increase in the clearance and / or a more rapid clearance of the tracer antibody from the circulation as compared to the clearance level or rate in a control subject indicates an FcRn antagonist.

[0020] In the present disclosure, the Fc domain or a fragment thereof of the FcRn antagonist is not an immunoglobulin. For example, the FcRn antagonists of the present disclosure only comprise the Fc domain of an immunoglobulin or comprise the Fc domain and the hinge region, but do not comprise C H 1, C L and variable domains.

[0021] In one example, the present disclosure provides an FcRn antagonist comprising:

[0022] (i) a region of an immunoglobulin consisting of an Fc domain or a fragment thereof capable of binding to FcRn; and

[0023] (ii) at least one albumin or a fragment thereof capable of extending the half-life of the FcRn antagonist as compared to the half-life of the immunoglobulin Fc or a fragment thereof.

[0024] In one example, the present disclosure provides an FcRn antagonist consisting of:

[0025] (i) an immunoglobulin Fc domain or a fragment thereof capable of binding to FcRn;

[0026] (ii) at least one albumin or a fragment thereof capable of extending the half-life of the FcRn antagonist as compared to the half-life of the immunoglobulin Fc domain or a fragment thereof; and

[0027] (iii) optionally, a linker disposed between (i) and (ii).

[0028] In one example, the FcRn antagonist has a longer serum half-life as compared to the serum half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the half-life of the FcRn antagonist is at least about two-fold or three-fold or four-fold or five-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the half-life of the FcRn antagonist is at least about two-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the half-life of the FcRn antagonist is at least about three-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the half-life of the FcRn antagonist is at least about four-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone. For example, the half-life of the FcRn antagonist is at least about five-fold longer than the half-life of the immunoglobulin Fc domain or a fragment thereof alone.

[0029] In one instance, the half-life of the FcRn antagonist 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 instance, the half-life of the FcRn antagonist is at least 25 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 30 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 35 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 40 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 45 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 50 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 55 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 60 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 65 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 70 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 75 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone. In one instance, the half-life of the FcRn antagonist is at least 80 hours longer than the half-life of the immunoglobulin Fc domain or fragment thereof alone.

[0030] In one instance, the antagonist binds to human FcRn with an affinity constant (K D ) of at least 700 nM at neutral pH and / or with a K D of at least 100 nM at acidic pH, wherein the antagonist is immobilized on a solid support and the binding of soluble human FcRn to the antagonist is determined using surface plasmon resonance (SPR). In one instance, the antagonist binds to human FcRn with a K D of at least 650 nM or 600 nM at neutral pH and / or with a K DBind to human FcRn, wherein the antagonist is immobilized on a solid support and the binding of soluble human FcRn to the antagonist is determined using SPR. In one example, the antagonist has a K of at least 600 nM at neutral pH D Bind to human FcRn and / or has a K of at least 65 nM at acidic pH D Bind to human FcRn, wherein the antagonist is immobilized on a solid support and the binding of soluble human FcRn to the antagonist is determined using SPR.

[0031] For purposes of clarification, and based on the description herein, it will be apparent to those skilled in the art that reference to "an affinity of at least about" or "at least" will be understood to mean that the affinity is equal to or greater than the value (i.e., lower as an affinity than the value), i.e., an affinity of 2 nM is greater than an affinity of 3 nM. In other words, this term can be "an affinity of X or lower" or "an affinity of not more than X", where X is the value recited herein.

[0032] In one example, the antagonist has a K of at least 500 nM at neutral pH D Bind to cynomolgus macaque FcRn and / or has a K of at least 100 nM at acidic pH D Bind to cynomolgus macaque FcRn, wherein the antagonist is immobilized on a solid support and the binding of soluble cynomolgus macaque FcRn to the antagonist is determined using surface plasmon resonance (SPR).

[0033] In one example, the antagonist has a K of at least 20 nM at neutral pH D Bind to mouse FcRn and / or has a K of at least 1 nM at acidic pH D Bind to mouse FcRn, wherein the antagonist is immobilized on a solid support and the binding of soluble mouse FcRn to the antagonist is determined using surface plasmon resonance (SPR).

[0034] In one example, the antagonist has a K of at least 160 nM at neutral pH D Bind to rat FcRn and / or has a K of at least 2 nM at acidic pH D Bind to rat FcRn, wherein the antagonist is immobilized on a solid support and the binding of soluble rat FcRn to the antagonist is determined using surface plasmon resonance (SPR).

[0035] In one example, the antagonist has a K of at least 10 nM at neutral pH DBinding to human FcRn, where the FcRn is immobilized on a solid support and the binding of the FcRn antagonist to FcRn is determined using surface plasmon resonance (SPR). In one example, the antagonist has a K of at least 5 nM or 4 nM or 3 nM at neutral pH D Binding to human FcRn, where the FcRn is immobilized on a solid support and the binding of the FcRn antagonist to FcRn is determined using SPR. In one example, the antagonist has a K of at least 2.5 nM at neutral pH D Binding to human FcRn, where the FcRn is immobilized on a solid support and the binding of the FcRn antagonist to FcRn is determined using SPR.

[0036] In one example, "neutral pH" is about pH 7.3. In one example, "acidic pH" is about pH 6.

[0037] In one example, the binding of the antagonist to FcRn or the K D is conferred by an immunoglobulin Fc domain or a fragment thereof.

[0038] In one example of the present disclosure, the albumin or a fragment thereof is a human albumin variant or a fragment thereof. In one example, the human albumin variant or a fragment thereof is a naturally occurring albumin variant. Those skilled in the art will recognize that in the case of an albumin variant, references to "the position corresponding to the recited amino acid residue" may need to be adjusted according to any insertions or deletions of amino acid residues.

[0039] For example, the albumin variant or a fragment thereof binds to FcRn with a reduced affinity compared to the albumin set forth in SEQ ID NO:1. For example, the binding affinity is measured at neutral and / or acidic pH.

[0040] In one example, the albumin variant or a 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 binding to human FcRn. In one example, the albumin variant or a fragment thereof has a K of greater than 10 μM at neutral pH or pH 6.0 D binding to human FcRn.

[0041] In one example, the albumin variant or a fragment thereof undetectably binds to FcRn on the surface of HEK-293 cells expressing FcRn.

[0042] In one example, the albumin variant or a fragment thereof contains an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1.

[0043] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 391 of SEQ ID NO:1.

[0044] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 402 of SEQ ID NO:1.

[0045] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 418 of SEQ ID NO:1.

[0046] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 420 of SEQ ID NO:1.

[0047] [[ID=1...]]In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 424 of SEQ ID NO:1.

[0048] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 429 of SEQ ID NO:1.

[0049] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 462 of SEQ ID NO:1.

[0050] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 492 of SEQ ID NO:1.

[0051] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 501 of SEQ ID NO:1.

[0052] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 545 of SEQ ID NO:1.

[0053] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 547 of SEQ ID NO:1.

[0054] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 422 of SEQ ID NO:1.

[0055] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 510 of SEQ ID NO:1.

[0056] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 535 of SEQ ID NO:1.

[0057] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 463 of SEQ ID NO:1.

[0058] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 467 of SEQ ID NO:1.

[0059] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 505 of SEQ ID NO:1.

[0060] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 509 of SEQ ID NO:1.

[0061] In one instance, the albumin comprises an amino acid substitution at the position corresponding to amino acid 519 of SEQ ID NO:1.

[0062] Exemplary albumin variants or fragments thereof comprise one or more amino acid substitutions selected from the group consisting of:

[0063] (i) Aspartic acid substitutes asparagine at the position corresponding to amino acid 391 of SEQ ID NO:1;

[0064] (ii) Glutamic acid substitutes lysine at the position corresponding to amino acid 402 of SEQ ID NO:1;

[0065] (iii) Methionine substitutes valine at the position corresponding to amino acid 418 of SEQ ID NO:1;

[0066] (iv) Alanine substitutes threonine at the position corresponding to amino acid 420 of SEQ ID NO:1;

[0067] (v) Isoleucine substitutes isoleucine at the position corresponding to amino acid 424 of SEQ ID NO:1;

[0068] (vi) Aspartic acid substitutes asparagine at the position corresponding to amino acid 429 of SEQ ID NO:1;

[0069] (vii) Methionine substitutes valine at the position corresponding to amino acid 462 of SEQ ID NO:1;

[0070] (viii) Glycine substitutes glutamic acid at the position corresponding to amino acid 492 of SEQ ID NO:1;

[0071] (ix) Valine substitutes glutamic acid at the position corresponding to amino acid 501 of SEQ ID NO:1;

[0072] (x) Glutamic acid replaces lysine at the position corresponding to amino acid 545 of SEQ ID NO:1;

[0073] (xi) Alanine replaces valine at the position corresponding to amino acid 547 of SEQ ID NO:1;

[0074] (xii) Glutamine replaces histidine at the position corresponding to amino acid 464 of SEQ ID NO:1;

[0075] (xiii) Tryptophan replaces threonine at the position corresponding to amino acid 422 of SEQ ID NO:1;

[0076] (xiv) Methionine replaces threonine at the position corresponding to amino acid 422 of SEQ ID NO:1;

[0077] (xv) Glutamine replaces histidine at the position corresponding to amino acid 510 of SEQ ID NO:1;

[0078] (xvi) Arginine replaces histidine at the position corresponding to amino acid 510 of SEQ ID NO:1;

[0079] (xvii) Phenylalanine replaces histidine at the position corresponding to amino acid 535 of SEQ ID NO:1;

[0080] (xviii) Glutamine replaces histidine at the position corresponding to amino acid 535 of SEQ ID NO:1;

[0081] (xix) Tryptophan replaces leucine at the position corresponding to amino acid 463 of SEQ ID NO:1;

[0082] (xx) Methionine replaces threonine at the position corresponding to amino acid 467 of SEQ ID NO:1;

[0083] (xxi) Lysine replaces glutamic acid at the position corresponding to amino acid 505 of SEQ ID NO:1;

[0084] (xxii) Glycine replaces glutamic acid at the position corresponding to amino acid 505 of SEQ ID NO:1;

[0085] (xxiii) Arginine replaces glutamic acid at the position corresponding to amino acid 505 of SEQ ID NO:1;

[0086] (xxiv) Tryptophan replaces phenylalanine at the position corresponding to amino acid 509 of SEQ ID NO:1;

[0087] (xxv) a glutamate substitution for lysine at the position corresponding to amino acid 519 of SEQ ID NO:1; and

[0088] (xxvi) combinations thereof.

[0089] In one instance, the albumin variant or fragment thereof comprises:

[0090] (i) an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0091] (ii) an amino acid substitution at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0092] (iii) an amino acid substitution at the position corresponding to amino acid 422 of SEQ ID NO:1 and an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0093] (iv) an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0094] (v) an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at the position corresponding to amino acid 509 of SEQ ID NO:1; or

[0095] (vi) an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at the position corresponding to amino acid 519 of SEQ ID NO:1; or

[0096] (vii) an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at the position corresponding to amino acid 510 of SEQ ID NO:1; or

[0097] (viii) an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at the position corresponding to amino acid 505 of SEQ ID NO:1; or

[0098] (ix) an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1 and an amino acid substitution at the position corresponding to amino acid 467 of SEQ ID NO:1;

[0099] (x) An amino acid substitution at the position corresponding to amino acid 418 of SEQ ID NO:1, an amino acid substitution at the position corresponding to amino acid 420 of SEQ ID NO:1, an amino acid substitution at the position corresponding to amino acid 505 of SEQ ID NO:1, and an amino acid substitution at the position corresponding to amino acid 547 of SEQ ID NO:1; or

[0100] (xi) An amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1; and an amino acid substitution at the position corresponding to amino acid 463 of SEQ ID NO:1.

[0101] Exemplary albumin variants or fragments thereof comprise one or more amino acid substitutions selected from the group consisting of:

[0102] (i) Glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1;

[0103] (ii) Tryptophan substitution for threonine at the position corresponding to amino acid 422 of SEQ ID NO:1;

[0104] (iii) Glutamine substitution for histidine at the position corresponding to amino acid 510 of SEQ ID NO:1;

[0105] (iv) Phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; and

[0106] (v) Combinations thereof.

[0107] In one instance, the exemplary albumin variant or fragment thereof comprises glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1.

[0108] In one instance, the albumin variant or fragment thereof comprises:

[0109] (i) An amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0110] (ii) An amino acid substitution at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0111] (iii) An amino acid substitution at the position corresponding to amino acid 422 of SEQ ID NO:1; and an amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0112] (iv) An amino acid substitution at the position corresponding to amino acid 464 of SEQ ID NO:1; and an amino acid substitution at the position corresponding to amino acid 535 of SEQ ID NO:1.

[0113] In one example, the albumin variant or its fragment comprises:

[0114] (i) A glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0115] (ii) A phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0116] (iii) A glutamine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0117] (iv) A tryptophan substitution for threonine at the position corresponding to amino acid 422 of SEQ ID NO:1, and a glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0118] (v) A glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1, and a phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0119] (vi) A methionine substitution for threonine at the position corresponding to amino acid 422 of SEQ ID NO:1, and a glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0120] (vii) A tyrosine substitution for phenylalanine at the position corresponding to amino acid 509 of SEQ ID NO:1, and a glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0121] (viii) A glutamate substitution for lysine at the position corresponding to amino acid 519 of SEQ ID NO:1, and a glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0122] (ix) An arginine substitution for histidine at the position corresponding to amino acid 510 of SEQ ID NO:1, and a glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0123] (x) Arginine substitution for histidine at the position corresponding to amino acid 510 of SEQ ID NO:1, and phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0124] (xi) Lysine substitution for glutamate at the position corresponding to amino acid 505 of SEQ ID NO:1, and glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0125] (xii) Methionine substitution for threonine at the position corresponding to amino acid 467 of SEQ ID NO:1, and glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0126] (xiii) Tyrosine substitution for leucine at the position corresponding to amino acid 519 of SEQ ID NO:1, and glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1.

[0127] In one example, the albumin variant or its fragment comprises:

[0128] (i) Glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0129] (ii) Phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0130] (iii) Glutamine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or

[0131] (iv) Tryptophan substitution for threonine at the position corresponding to amino acid 422 of SEQ ID NO:1, and glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or

[0132] (v) Glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1, and phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1.

[0133] In one example of the present disclosure, the immunoglobulin Fc domain or its fragment is the Fc domain or its fragment from IgG.

[0134] In one example of the present disclosure, the immunoglobulin Fc domain or its fragment is the Fc domain or its fragment from IgG1.

[0135] In one example of the present disclosure, the immunoglobulin Fc domain or fragment thereof is the Fc domain or fragment thereof from IgG4. In one example of the present disclosure, the immunoglobulin Fc domain or fragment thereof does not come from IgG4 or its fragment.

[0136] In one example of the present disclosure, the immunoglobulin Fc domain or fragment thereof is an Fc domain variant or fragment thereof. For example, the Fc domain variant or fragment thereof is an IgG1 Fc domain variant or fragment thereof or an IgG4 Fc domain variant or fragment thereof.

[0137] In one example, the IgG1 Fc domain variant or fragment thereof or the IgG1 Fc domain variant or fragment thereof binds to human FcRn with increased affinity compared to the IgG1 Fc domain set forth in SEQ ID NO:2.

[0138] For example, the binding affinity is measured at neutral and / or acidic pH. Exemplary K of the Fc domain variant or fragment thereof D Regarding the K of the antagonist of the present disclosure against FcRn above D described and adapted mutatis mutandis to the examples of the present disclosure.

[0139] In one example, the Fc domain variant or fragment thereof comprises one or more amino acid substitutions selected from the group consisting of:

[0140] (i) a tyrosine substitution for methionine at the position corresponding to amino acid 252 according to the EU numbering system;

[0141] (ii) a threonine substitution for serine at the position corresponding to amino acid 254 according to the EU numbering system;

[0142] (iii) a glutamate substitution for threonine at the position corresponding to amino acid 256 according to the EU numbering system;

[0143] (iv) a glutamate substitution for asparagine at the position corresponding to amino acid 286 according to the EU numbering system;

[0144] (v) a proline substitution for valine at the position corresponding to amino acid 308 according to the EU numbering system;

[0145] (vi) a lysine substitution for histidine at the position corresponding to amino acid 433 according to the EU numbering system;

[0146] (vii) a tyrosine substitution for asparagine at the position corresponding to amino acid 434 according to the EU numbering system;

[0147] (viii) Substitution of asparagine with phenylalanine at the position corresponding to amino acid 434 according to the EU numbering system;

[0148] (ix) Substitution of tyrosine with histidine at the position corresponding to amino acid 436 according to the EU numbering system; and

[0149] (x) Combinations thereof.

[0150] For example, the Fc domain variant or a fragment thereof comprises:

[0151] (i) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system; substitution of valine with proline at the position corresponding to amino acid 308 according to the EU numbering system and substitution of asparagine with tyrosine at the position corresponding to amino acid 434 according to the EU numbering system; or

[0152] (ii) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system; substitution of serine with threonine at the position corresponding to amino acid 254 according to the EU numbering system and substitution of threonine with glutamate at the position corresponding to amino acid 256 according to the EU numbering system; or

[0153] (iii) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system; substitution of asparagine with glutamate at the position corresponding to amino acid 286 according to the EU numbering system and substitution of asparagine with tyrosine at the position corresponding to amino acid 434 according to the EU numbering system; or

[0154] (iv) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system; substitution of serine with threonine at the position corresponding to amino acid 254 according to the EU numbering system, substitution of threonine with glutamate at the position corresponding to amino acid 256 according to the EU numbering system, substitution of histidine with lysine at the position corresponding to amino acid 433 according to the EU numbering system and substitution of asparagine with phenylalanine at the position corresponding to amino acid 434 according to the EU numbering system.

[0155] In one instance, the FcRn antagonist of the present disclosure comprises:

[0156] (i) The Fc domain comprises substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system; substitution of valine with proline at the position corresponding to amino acid 308 according to the EU numbering system and substitution of asparagine with tyrosine at the position corresponding to amino acid 434 according to the EU numbering system; and

[0157] (ii) The albumin comprises substitution of histidine with glutamine at the position corresponding to amino acid 464.

[0158] In one instance, the Fc domain or a fragment thereof comprises a tyrosine substitution for methionine at the position corresponding to amino acid 252 according to the EU numbering system; a threonine substitution for serine at the position corresponding to amino acid 254 according to the EU numbering system, a glutamate substitution for threonine at the position corresponding to amino acid 256 according to the EU numbering system, a lysine substitution for histidine at the position corresponding to amino acid 433 according to the EU numbering system, and a phenylalanine substitution for asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0159] In one instance, the Fc domain or a fragment thereof comprises a tyrosine substitution for methionine at the position corresponding to amino acid 252 according to the EU numbering system; a proline substitution for valine at the position corresponding to amino acid 308 according to the EU numbering system, and a tyrosine substitution for asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0160] In one instance, the Fc domain or a fragment thereof is linked to albumin. In some instances, the Fc domain can form a dimer of two Fc domains, and the antagonist of the present disclosure can comprise an Fc domain dimer, wherein each Fc domain is linked to albumin, i.e., the antagonist comprises two albumins.

[0161] In another instance, one Fc domain 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.

[0162] In one instance, each Fc domain of the FcRn antagonist is linked to two or more albumins or fragments thereof.

[0163] In one instance, each Fc domain is linked to albumin or a fragment thereof.

[0164] In one instance, the Fc domain or a fragment thereof is indirectly linked to the albumin or a fragment thereof, for example via a linker. For example, the antagonist comprises a linker disposed between the Fc domain or a fragment thereof and the albumin or a fragment thereof. In one instance, the linker is a peptide or polypeptide. For example, the linker is a peptide linker comprising between 2 and 31 amino acids in length. For example, the linker comprises glycine or glycine and serine. For example, the linker comprises one or more repeat sequences of Gly4Ser.

[0165] In another instance, the Fc domain or a fragment thereof is directly linked to the albumin or a fragment thereof, for example in the absence of an intervening linker.

[0166] In an exemplary form of the present disclosure, 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.

[0167] In another form of the present disclosure, the C-terminus of albumin or a fragment thereof is indirectly or directly linked to the N-terminus of an Fc domain or a fragment thereof.

[0168] In another form of the present disclosure, the C-terminus of an Fc domain or a fragment thereof is indirectly or directly linked to the C-terminus of albumin or a fragment thereof.

[0169] In another form of the present disclosure, the N-terminus of an Fc domain or a fragment thereof is indirectly or directly linked to the N-terminus of albumin or a fragment thereof.

[0170] In one example, the Fc domain or a fragment thereof is a monomeric Fc domain or a fragment thereof. In another example, albumin or a fragment thereof is a monomeric albumin or a fragment thereof. For example, a monomeric Fc domain is fused to a monomeric albumin.

[0171] In another example, the FcRn antagonist comprises a single albumin or a fragment thereof. In these examples, the FcRn antagonist is a heterodimer. For example, a heterodimeric FcRn antagonist comprises a single albumin or a fragment thereof fused to a dimeric Fc domain or a fragment thereof.

[0172] In one example, the FcRn antagonist comprises two albumins or fragments thereof. For example, the FcRn antagonist is a homodimer.

[0173] The present disclosure further provides an FcRn antagonist comprising:

[0174] (i) an immunoglobulin Fc domain or a fragment thereof comprising a tyrosine substitution for methionine at the position corresponding to amino acid 252 according to the EU numbering system; a threonine substitution for serine at the position corresponding to amino acid 254 according to the EU numbering system; a glutamate substitution for threonine at the position corresponding to amino acid 256 according to the EU numbering system; a lysine substitution for histidine at the position corresponding to amino acid 433 according to the EU numbering system; and a phenylalanine substitution for asparagine at the position corresponding to amino acid 434 according to the EU numbering system; and

[0175] (ii) albumin or a fragment thereof comprising a glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1,

[0176] 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.

[0177] The present disclosure further provides an FcRn antagonist comprising the sequence set forth in SEQ ID NO:8.

[0178] The present disclosure further provides an FcRn antagonist comprising:

[0179] (i) The immunoglobulin Fc domain or a fragment thereof comprises a tyrosine substitution for methionine at the position corresponding to amino acid 252 according to the EU numbering system; a proline substitution for valine at the position corresponding to amino acid 308 according to the EU numbering system; and a tyrosine substitution for asparagine at the position corresponding to amino acid 434 according to the EU numbering system; and

[0180] (ii) Albumin or a fragment thereof comprises a glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1,

[0181] wherein the C-terminus of the Fc domain or its fragment is indirectly or directly linked to the N-terminus of the albumin or its fragment.

[0182] The present disclosure further provides an FcRn antagonist comprising the sequence set forth in SEQ ID NO:11.

[0183] The FcRn antagonist of the present disclosure comprises the sequence set forth in any one of SEQ ID NOs: 3-12. For example, the FcRn antagonist of the present disclosure comprises the sequence set forth in any one of SEQ ID NO: 3, 5, 8, 9 or 11.

[0184] 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.

[0185] The present disclosure further provides a composition comprising the FcRn antagonist of the present disclosure and a pharmaceutically acceptable carrier.

[0186] The present disclosure further provides a nucleic acid encoding the FcRn antagonist of the present disclosure. In one example, the nucleic acid is DNA. In another example, the nucleic acid is RNA, including its modified forms. In one example, the nucleic acid is linked to the nucleic acid required for expressing the FcRn antagonist. In some examples, the nucleic acid is contained within a liposome or particle, such as a lipid nanoparticle (LNP). In some examples, the nucleic acid, liposome or particle is within a pharmaceutical formulation.

[0187] The present disclosure further provides an FcRn antagonist or nucleic acid or composition as disclosed herein for inhibiting an immunoglobulin of a subject from binding to FcRn and / or reducing the circulating amount of the immunoglobulin. The present disclosure further provides the use of an FcRn antagonist or nucleic acid or composition as disclosed herein for manufacturing an agent for inhibiting an immunoglobulin of a subject from binding to FcRn and / or reducing the circulating amount of the immunoglobulin of the subject. The present disclosure further provides a method for inhibiting an immunoglobulin of a subject from binding to FcRn and / or reducing the circulating amount of the immunoglobulin, the method comprising administering an FcRn antagonist or nucleic acid or composition as disclosed herein.

[0188] The present disclosure also provides an FcRn antagonist or nucleic acid or composition of the present disclosure for reducing the circulating Fc-containing protein and / or antibody of a subject, such as a subject suffering from an autoimmune disease and having pathogenic autoantibodies or a subject who has developed and / or is at risk of developing anti-drug antibodies. In one example, the time taken for the antibody content of the subject to decrease is longer than the time taken for the antibody content of the subject to decrease if only the Fc domain or a fragment thereof from the FcRn antagonist is administered (i.e., in the absence of albumin or a fragment thereof).

[0189] The present disclosure also provides an FcRn antagonist or nucleic acid or composition of the present disclosure for treating or preventing the progression of an antibody-mediated disorder in a subject in need thereof.

[0190] The present disclosure further provides a method for reducing the circulating autoantibodies of a subject, the method comprising administering an FcRn antagonist or nucleic acid or composition of the present disclosure to the subject. In one example, the time taken for the autoantibody content of the subject to decrease is longer than the time taken for the autoantibody content of the subject to decrease if only the Fc domain or a fragment thereof from the FcRn antagonist is administered (i.e., in the absence of albumin or a fragment thereof).

[0191] The present disclosure further provides a method for reducing the circulating anti-drug antibodies of a subject, the method comprising administering an FcRn antagonist or nucleic acid or composition of the present disclosure to the subject. In one example, the time taken for the anti-drug antibody content of the subject to decrease is longer than the time taken for the anti-drug antibody content of the subject to decrease if only the Fc domain or a fragment thereof from the FcRn antagonist is administered (i.e., in the absence of albumin or a fragment thereof).

[0192] The present disclosure also provides a method for treating or preventing the progression of an antibody-mediated disorder in a subject in need thereof, the method comprising administering an FcRn antagonist or nucleic acid or composition of the present disclosure to the subject.

[0193] The present disclosure also provides the 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.

[0194] The present disclosure also provides the 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.

[0195] The present disclosure also provides the 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 disorder in a subject.

[0196] In one example, the subject has an autoimmune disease, has produced unwanted antibodies, 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.

[0197] In one example, the subject has produced or is at risk of producing anti-drug antibodies. For example, the subject is undergoing a therapeutic treatment with a protein therapy, such as an antibody, immunoadhesin, or coagulation factor, and has produced or is at risk of producing antibodies against the therapy.

[0198] In one example, the FcRn antagonist is administered in an amount effective to:

[0199] (i) reduce the endogenous IgG content by at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold (compared to the case where no FcRn antagonist is administered); and / or

[0200] (ii) reduce the endogenous albumin content by no more than 20%, 15%, 10%, 5%, or 1% (compared to the case where no FcRn antagonist is administered).

[0201] In one instance, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, 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 administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 10% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 20% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 30% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, 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 administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, 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 administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 60% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, the FcRn antagonist is administered in an amount effective to reduce endogenous IgG levels by at least about 70% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone.In one instance, the FcRn antagonist is administered in an amount that is effective to reduce endogenous IgG levels by at least about 80% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, the FcRn antagonist is administered in an amount that is effective to reduce endogenous IgG levels by at least about 90% compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin and / or administering the Fc domain or fragment thereof from the FcRn antagonist alone.

[0202] In one instance, the FcRn antagonist antagonizes IgG recycling without significantly, substantially, or detectably reducing endogenous albumin levels.

[0203] In one instance, the FcRn antagonist antagonizes IgG recycling without substantially antagonizing albumin recycling. As discussed above, the inventors of the present case contemplate this advantage of an FcRn antagonist comprising albumin or a fragment thereof that does not substantially bind to FcRn as it does not interfere with endogenous albumin recycling and / or homeostasis.

[0204] In one instance, 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 administering an FcRn antagonist comprising wild-type human albumin. For example, administration of the FcRn antagonist does not induce dyslipidemia. In another instance, administration of the FcRn antagonist induces a smaller increase in serum cholesterol levels compared to the levels observed after administering an FcRn antagonist comprising wild-type human albumin.

[0205] In one instance, administration of an FcRn antagonist does not change the levels and / or induces smaller changes in endogenous albumin, cholesterol, low density lipoprotein (LDL) cholesterol, and / or triglycerides as compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of an FcRn antagonist does not change the level of endogenous albumin and / or induces smaller changes as compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of an FcRn antagonist does not change the level of endogenous cholesterol and / or induces smaller changes as compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of an FcRn antagonist does not change the level of endogenous low density lipoprotein (LDL) cholesterol and / or induces smaller changes as compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of an FcRn antagonist does not change the level of endogenous triglycerides and / or induces smaller changes as compared to the levels observed after administration of an FcRn antagonist comprising wild-type human albumin.

[0206] In one instance, the FcRn antagonist reduces the level of endogenous IgG 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 the level of endogenous IgG within 2 to 9 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 2 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 3 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 4 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 5 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 6 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 7 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 8 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 9 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 10 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 11 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 12 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 13 days of administration. In one instance, the FcRn antagonist reduces the level of endogenous IgG within at least 14 days of administration.

[0207] In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%. For example, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 50% to about 80%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 10% to about 90%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 10%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 20%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 30%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 40%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 50%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 60%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 70%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 80%. In one instance, administration of an FcRn antagonist reduces the endogenous IgG content by at least about 90%.

[0208] In one instance, administration of an FcRn antagonist does not change or substantially changes the endogenous IgA content.

[0209] In one instance, the FcRn antagonist increases the endogenous IgM content within at least 5, 6, 7, 8, or 9 days of administration. In one instance, the FcRn antagonist increases the endogenous IgM content within at least 5 days of administration. In one instance, the FcRn antagonist increases the endogenous IgM content within at least 6 days of administration. In one instance, the FcRn antagonist increases the endogenous IgM content within at least 7 days of administration. In one instance, the FcRn antagonist increases the endogenous IgM content within at least 8 days of administration. In one instance, the FcRn antagonist increases the endogenous IgM content within at least 9 days of administration.

[0210] In one instance, compared to that observed after administration of an FcRn antagonist comprising wild-type human albumin and / or administration of the Fc domain or a fragment thereof from the FcRn antagonist alone (e.g., huG1FcYTEKF), administration of the FcRn antagonist reduces the volume of distribution (V SS )). In one instance, compared to V SS observed after administration of the Fc domain or a fragment thereof from the FcRn antagonist alone, administration of the FcRn antagonist reduces V SS . In one instance, compared to V SSAt least about 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL. In one instance, compared to V observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 400 mL to 500 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 100 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 200 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 250 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS [[ID=-- --]]by at least about 300 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 2500 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 400 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 450 mL. In one instance, compared to V SS observed after administering the Fc domain of an FcRn antagonist or a fragment thereof alone, administration of the FcRn antagonist reduces V SS by at least about 500 mL. SS At least about 500 mL.

[0211] In one instance, compared to V SS observed after administering an FcRn antagonist comprising wild-type human albumin, administration of the FcRn antagonist reduces V SSAt least about 40 mL, 50 mL, 60 mL, 70 mL, or 80 mL. For example, compared to V observed after administration of an FcRn antagonist comprising wild-type human albumin SS administration of the FcRn antagonist reduces V SS by at least about 40 mL to 80 mL. In one instance, compared to V observed after administration of an FcRn antagonist comprising wild-type human albumin SS administration of the FcRn antagonist reduces V SS by at least about 40 mL. In one instance, compared to V observed after administration of an FcRn antagonist comprising wild-type human albumin SS administration of the FcRn antagonist reduces V SS by at least about 50 mL. In one instance, compared to V observed after administration of an FcRn antagonist comprising wild-type human albumin SS administration of the FcRn antagonist reduces V SS by at least about 60 mL. In one instance, compared to V observed after administration of an FcRn antagonist comprising wild-type human albumin SS administration of the FcRn antagonist reduces V SS by at least about 70 mL. In one instance, compared to V observed after administration of an FcRn antagonist comprising wild-type human albumin SS administration of the FcRn antagonist reduces V SS by at least about 80 mL.

[0212] In one instance, administration of an FcRn antagonist increases the area under the curve (AUC) compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the 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 Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the AUC by at least 250 μg / mL*h / mg to 500 μg / mL*h / mg compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the AUC by at least 250 μg / mL*h / mg compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the AUC by at least 300 μg / mL*h / mg compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the AUC by at least 350 μg / mL*h / mg compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the AUC by at least 400 μg / mL*h / mg compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the AUC by at least 450 μg / mL*h / mg compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone. In one instance, administration of an FcRn antagonist increases the AUC by at least 500 μg / mL*h / mg compared to the AUC observed after administration of the Fc domain or fragment thereof from the FcRn antagonist alone.

[0213] In one instance, administration of the FcRn antagonist increases the AUC as compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, 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 as compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of the FcRn antagonist increases the AUC by at least 100 μg / mL*h / mg to 250 μg / mL*h / mg as compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of the FcRn antagonist increases the AUC by at least 100 μg / mL*h / mg as compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of the FcRn antagonist increases the AUC by at least 150 μg / mL*h / mg as compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of the FcRn antagonist increases the AUC by at least 200 μg / mL*h / mg as compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin. In one instance, administration of the FcRn antagonist increases the AUC by at least 250 μg / mL*h / mg as compared to the AUC observed after administration of an FcRn antagonist comprising wild-type human albumin.

[0214] In another instance, the FcRn antagonist is administered at a lower content as compared to an FcRn antagonist comprising wild-type human albumin to crosslink cell surface FcRn.

[0215] In one instance, the subject has received, is receiving, or will receive additional therapy. By way of example, the additional therapy is a steroid immunomodulator, plasmapheresis, and / or IVIg therapy.

[0216] The present disclosure further provides a kit for reducing circulating autoantibodies in a subject in need thereof, the kit comprising:

[0217] (i) at least one FcRn antagonist or composition of the present disclosure;

[0218] (ii) instructions for using the kit to reduce circulating autoantibodies in the subject; and

[0219] (iii) optionally, at least one additional therapy.

[0220] The present disclosure also provides a kit for treating or preventing the progression of an antibody-mediated disorder in a subject in need thereof, the kit comprising:

[0221] (i) At least one FcRn antagonist or composition of the present disclosure;

[0222] (ii) Instructions for using the kit to treat or prevent the progression of an antibody-mediated disorder in a subject; and

[0223] (iii) Optionally, at least one additional therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0224] Figure 1A A graphical representation showing the levels of IgG “tracer” antibodies in human FcRn (huFcRn) transgenic mice dosed with various FcRn antagonists of the present disclosure.

[0225] Figure 1B A graphical representation showing the levels of IgG “tracer” antibodies in human FcRn (huFcRn) transgenic mice dosed with various FcRn antagonists of the present disclosure.

[0226] Figure 1C A graphical representation showing the in vivo half-lives of various FcRn antagonists of the present disclosure after administration to huFcRn transgenic mice.

[0227] Figure 2A And Panel B is a graphical representation showing the levels of tracer antibody (CSL360) in huFcRn transgenic mice dosed with various FcRn antagonists of the present disclosure.

[0228] Figure 2C A graphical representation showing the pharmacokinetics of the FcRn antagonists described herein when administered intravenously or subcutaneously. The antagonists are as indicated and described herein. As shown, the antagonists exhibit similar pharmacokinetics whether administered intravenously or subcutaneously.

[0229] Figure 2D A graphical representation showing the dose-dependent reduction in the levels of tracer antibody (CSL360) in huFcRn transgenic mice dosed with different doses of huG1FcYPY-HSA. Controls are as indicated.

[0230] Figure 2E A graphical representation showing the dose-dependent reduction in the levels of tracer antibody (CSL360) in huFcRn transgenic mice dosed with different doses of huG1FcYPY-HSA (H464Q). Controls are as indicated.

[0231] Figure 3A And Panel B is a graphical representation showing the effect on endogenous murine IgG levels in mice after a single administration of huG1FcYPY-HSA, huG1FcYPY-HSA (H464Q), or huG1FcYTEKF.

[0232] Figure 3C And D are graphical representations showing the in vivo half-lives of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF in mice.

[0233] Figure 4A Are graphical representations showing the effects on the content of fluorescently labeled IgG in bone marrow-derived macrophages at specified concentrations and time points in the absence or presence of the FcRn antagonists huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF and in the presence or absence of a protease inhibitor (PI).

[0234] Figure 4B Are graphical representations showing the effects on the content of fluorescently labeled albumin in bone marrow-derived macrophages at specified concentrations and time points in the absence or presence of the FcRn antagonists huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF and in the presence or absence of a protease inhibitor (PI).

[0235] Figure 5 Is a graphical representation depicting how to use fluorescence to evaluate the degradation and recycling of molecules in cells.

[0236] Figure 6 Is a schematic diagram depicting the putative protein complex of FcRn / β2m and huG1FcYPY-HSA(H464Q).

[0237] Figure 7 Is a schematic diagram depicting the FcRn-binding pocket of HSA.

[0238] Figure 8A Are graphical representations showing the effects of the FcRn antagonist on the content of endogenous cynomolgus macaque IgG up to 2016 hours (84 days) after a single intravenous administration of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF.

[0239] Figure 8B Are graphical representations showing the effects of the FcRn antagonist on the content of endogenous cynomolgus macaque IgG up to 420 hours (17.5 days) after a single intravenous administration of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF.

[0240] Figure 8CGraphical representations of the in vivo PK of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), or huG1FcYTEKF in cynomolgus monkeys up to 420 hours (17.5 days) after a single intravenous administration.

[0241] Figure 9A -C is a graphical representation of the endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of the huG1FcYPY-HSA molecule.

[0242] Figure 9D -F is a graphical representation of the endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of the huG1FcYPY-HSA(H464Q) molecule.

[0243] Figure 9G -I is a graphical representation of the endogenous IgG and IgM levels in individual cynomolgus monkeys after a single intravenous administration of the huG1FcYTEKF molecule.

[0244] Figure 10A Graphical representations of the effect of an FcRn antagonist on endogenous cynomolgus monkey IgG levels up to 2016 hours (84 days) after a single subcutaneous administration of huG1FcYPY-HSA(H464Q) or huG1FcYTEKF.

[0245] Figure 10B Graphical representations of the effect of an FcRn antagonist on endogenous cynomolgus monkey IgG levels up to 420 hours (17.5 days) after a single subcutaneous administration of huG1FcYPY-HSA(H464Q) or huG1FcYTEKF.

[0246] Figure 10C Graphical representations of the in vivo half-life of huG1FcYPY-HSA(H464Q) or huG1FcYTEKF in cynomolgus monkeys up to 420 hours (17.5 days) after a single subcutaneous administration.

[0247] Figure 11A -C is a graphical representation of the endogenous IgG and IgM levels in individual cynomolgus monkeys after a single subcutaneous administration of the huG1FcYPY-HSA(H464Q) molecule.

[0248] Figure 11D -F is a graphical representation of the endogenous IgG and IgM levels in individual cynomolgus monkeys after a single subcutaneous administration of the huG1FcYTEKF molecule.

[0249] Keywords of the Sequence Listing

[0250] SEQ ID NO:1 Human Serum Albumin (HSA) SEQ ID NO:2 Human IgG1 Fc (huG1Fc) SEQ ID NO:3 huG1Fc-HSA SEQ ID NO:4 HSA-huG1Fc SEQ ID NO:5 huG1FcYTEKF-HSA SEQ ID NO:6 HSA-huG1FcYTEKF SEQ ID NO:7 HSA(H464Q)-huG1FcYTEKF SEQ ID NO:8 huG1FcYTEKF-HSA(H464Q) SEQ ID NO:9 huG1FcYPY-HSA SEQ ID NO:10 HSA-huG1FcYPY SEQ ID NO:11 huG1FcYPY-HSA(H464Q) SEQ ID NO:12 HSA(H464Q)-huG1FcYPY SEQ ID NO:13 huG1FcYTEKF Detailed implementation manners

[0251] General

[0252] Throughout this specification, unless specifically stated otherwise or the context otherwise requires, reference to a single step, a composition of substances, a group of steps, or a group of compositions of substances will be considered to encompass one and more than one (i.e., one or more) of those steps, compositions of substances, groups of steps, or groups of compositions of substances.

[0253] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0254] The scope of the present disclosure is not limited by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0255] Unless specifically stated otherwise, any example of the present disclosure herein shall be applicable to any other example of the present disclosure with necessary modifications in details. In other words, any specific example of the present disclosure can be combined with any other specific example of the present disclosure (except in cases of mutual exclusion).

[0256] Any example of the present disclosure that discloses a specific feature or group of features or a method or method step will be considered to provide explicit support for the waiver of the specific feature or group of features or the method or method step.

[0257] Unless otherwise specifically noted, all technical and scientific terms used herein shall be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0258] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those of skill in the art. Such techniques are described and explained throughout the literature in sources such as: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and J. E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0259] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof in this text can be further clarified by the discussions in the following references: 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.

[0260] The term "and / or", e.g., "X and / or Y", should be understood to mean "X and Y" or "X or Y", and should be taken as providing explicit support for both meanings or either meaning.

[0261] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" should be understood to imply the inclusion of the stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0262] As used herein, the term "derived from..." should be understood to indicate that a specified integer can be obtained from a particular source, although not necessarily directly from that source.

[0263] Definition of selection

[0264] "Albumin", "albumin", or "blood albumin" is the most abundant blood protein and serves as a carrier protein for steroids, fatty acids, and thyroid hormones in the blood, and plays a major role in stabilizing extracellular fluid volume. For nomenclature purposes only and without limitation, exemplary sequences of human albumin are set forth in NCBI GenBank accession numbers ID: AEE60908 and SEQ ID NO: 1. It is understood that reference to "albumin" or "albumin" includes prealbumin, which comprises an N-terminal peptide, prealbumin, and secreted albumin. The position of amino acids is referred to herein with reference to the secreted albumin protein consisting of 585 amino acids (e.g., as set forth in SEQ ID NO: 1). Albumin contains three homologous domains, where each domain is the product of two subdomains with a common structural motif. Domains I, II, and III can be defined with reference to human albumin (as set forth in SEQ ID NO: 1). For example, domain I contains amino acids 1 (±1 to 15 amino acids) to 194 (±1 to 15 amino acids) of SEQ ID NO: 1, domain II contains amino acids 192 (±1 to 15 amino acids) to 387 (±1 to 15 amino acids) of SEQ ID NO: 1, and domain III contains 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 residue can deviate 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 amino acids to the C-terminal and / or N-terminal of the stated 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).

[0265] Additional sequences of albumin from other species (e.g., primate albumin (e.g., chimpanzee albumin, gorilla albumin), rodent albumin (e.g., hamster albumin, guinea pig albumin, mouse albumin, and rat albumin), bovine albumin, equine albumin, donkey albumin, rabbit albumin, goat albumin, sheep albumin, canine albumin, chicken albumin, and porcine albumin) can be determined using the sequences provided herein and / or publicly available databases and / or using standard techniques (e.g., as described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0266] The term “Fc” herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native Fc and Fc domain variants. In some instances, the human IgG heavy chain Fc domain region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the Fc produced by a host cell can undergo one or more (specifically, one or two) post-translational cleavages of amino acids from the C-terminus of the heavy chain. Thus, the Fc domain can include cleavage variants of the full-length heavy chain. This can be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the EU index numbering). Thus, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc domain region may or may not be present. When 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. U.S. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0267] For nomenclature purposes only and without limitation, an exemplary sequence of the human IgG1 Fc domain is set forth in SEQ ID NO:2 or Uniprot accession number P01857.

[0268] In the context of the FcRn antagonists of the present disclosure, an Fc domain or a fragment thereof is capable of dimerizing with another Fc domain or a fragment thereof. In one instance, the FcRn antagonist of the present disclosure is a homodimer.

[0269] As used herein, the phrase "corresponding to" when referring to the position of an amino acid in a SEQ ID NO: should be understood as referring to an amino acid residue or position within a polypeptide or protein (e.g., albumin or Fc), and does not necessarily refer to a sequence that includes the recited SEQ ID NO. For example, referring to "the position corresponding to amino acid 522 of SEQ ID NO:1 in an albumin sequence with a 10 amino acid N-terminal truncation" will necessarily refer to the amino acid at position 512.

[0270] When discussing the localization of a mutation within albumin in a fusion protein, for example, the numbering of the position is relative to albumin and not relative to the entire fusion protein. Thus, when discussing a mutation in albumin, if an Fc domain is fused to the N-terminus of albumin, the first residue is the first residue of albumin.

[0271] The "EU numbering system" of the term "Kabat or EU numbering system" should be understood to mean that the numbering of immunoglobulin heavy chains is according to the EU index taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of human IgG1 EU.

[0272] As discussed herein, a reference to an albumin "fragment" should be understood as a reference to an albumin fragment that has the ability to extend the half-life of a molecule to which it binds and does not require the fragment to bind to FcRn. The fragment can comprise or consist of one or more domains of albumin, fragments of said domains, or combinations thereof.

[0273] As used herein, "amino acid substitution" means that an amino acid at a specific position in a polypeptide sequence is replaced by another amino acid.

[0274] As used herein, the term "FcRn" refers to the neonatal Fc receptor, also known as the Brambell receptor, and is a heterodimer of the truncated heavy chain of the major histocompatibility complex class I Fc receptor (FCGRT) and β-2-microglobulin.

[0275] As used herein, the term "variant" refers to a protein having one or more amino acid substitutions made by well-known techniques induced by site-directed mutagenesis or any other conventional method.

[0276] As used herein, the term "binding", when referring to the interaction of an Fc domain or a fragment thereof with FcRn, means that said interaction is dependent on the presence of specific structures on the Fc domain and FcRn.

[0277] For purposes of clarification and based on the exemplary subject matter herein being apparent to those skilled in the art, references in this specification to "affinity" refer to the interaction, binding or association of albumin or a fragment thereof or an Fc domain or a fragment thereof with FcRn.

[0278] For purposes of clarification and based on the description herein, it will be apparent to those skilled in the art that reference to "an affinity of at least about" will be understood to mean an affinity equal to or higher than the value (i.e., lower as an affinity the value), i.e., an affinity of 2 nM is greater than an affinity of 3 nM. In other words, this term can be "an affinity of X or lower", where X is the value recited herein.

[0279] As used herein, the term "does not bind in a detectable manner" should be understood to mean that an albumin variant binds to FcRn at a level less than 20%, or 10%, or 6%, or 5% above background. The background can be the level of binding signal detected in the absence of the albumin variant or FcRn comprising the albumin variant and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the binding level detected in the presence of a negative control antigen. For example, the binding level is detected using ELISA, where the antigen is immobilized and contacted with the albumin variant.

[0280] As used herein, in the context of the present disclosure, the terms "half-life", "serum half-life" or "plasma half-life" refer to the time period required for the concentration or amount of an Fc domain or albumin or an FcRn antagonist in the body to decrease by 50% (i.e., one half), for example due to degradation and / or clearance or chelation 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., normal state, body size / weight). For example, in healthy human subjects, the serum half-life of albumin is 19 to 20 days and the serum half-life of IgG1 is about 21 days. Methods for determining the serum half-life of a protein are known in the art and include, for example, pharmacokinetic analysis.

[0281] The term "recombinant" should be understood to mean the product of artificial gene recombination. Recombinant proteins also encompass proteins expressed by artificial recombination when the protein is within, for example, the cell, tissue or subject in which it is expressed.

[0282] The term "protein" should be used to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., polypeptide complex). For example, a series of polypeptide chains can be covalently linked using suitable chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0283] The term "polypeptide" or "polypeptide chain" will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0284] As used herein, the terms "treating", "treat" or "treatment" include administering an FcRn antagonist described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition or slow the progression of the disease or condition.

[0285] As used herein, the terms "preventing", "prevent" or "prevention" include providing prophylaxis against the occurrence or recurrence of a bleeding disorder or symptoms of a bleeding disorder in a subject. The subject may be predisposed to having the disorder or a recurrence of the disorder or at risk of developing the disorder or a recurrence of the disorder, but has not been diagnosed with the disorder or recurrence.

[0286] "Effective amount" means at least an effective amount that can effectively achieve the desired result at the desired dose and time period. For example, the desired result can be a therapeutic or prophylactic result. The effective amount can be provided in one or multiple administrations. In some examples of the present disclosure, the term "effective amount" means the amount necessary to achieve the treatment of the disease or condition described above. In some examples of the present disclosure, the term "effective amount" means the amount necessary to achieve a change in a factor associated with the disease or condition described above. The effective amount can vary depending on the disease or condition to be treated or the factor to be changed and also depending on body weight, age, ethnic background, gender, health and / or physical condition, and other factors associated with the mammal to be treated. Generally, the effective amount will fall within a relatively wide range (e.g., a "dose" range), which can be determined by a practitioner via routine tests and experiments. Therefore, this term should not be construed as limiting the present disclosure to a specific quantity. The effective amount can be administered as a single dose or as doses repeated one or several times over a treatment period.

[0287] "Therapeutically effective amount" is at least the minimum concentration required to effect a measurable improvement in a particular disease or condition. The therapeutically effective amount herein can vary according to, for example, the disease condition, age, sex and weight of the patient, and the ability of the albumin conjugate to elicit the desired response in the subject. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the FcRn antagonist are outweighed by a therapeutically beneficial effect. In one instance, a therapeutically effective amount is employed to mean the amount of albumin conjugate sufficient to reduce or inhibit one or more symptoms of a bleeding disorder or its complications.

[0288] As used herein, the term "prophylactically effective amount" shall be deemed to mean the amount of albumin conjugate sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a condition.

[0289] As used herein, the term "subject" shall be understood to mean any animal, including a human, such as a mammal. Exemplary subjects include (but are not limited to) humans and non-human primates. By way of example, the subject is a human.

[0290] Fc domain and fragments thereof

[0291] As described herein, the FcRn antagonists of the present disclosure comprise an Fc domain or an FcRn-binding fragment thereof.

[0292] In one instance, the Fc domain is from IgG.

[0293] By way of example, the Fc domain is from human IgG.

[0294] By way of example, the Fc domain is from IgG1.

[0295] By way of example, the Fc domain is from human IgG1.

[0296] By way of example, the Fc domain is from IgG4.

[0297] By way of example, the Fc domain is from human IgG4.

[0298] By way of example, the Fc domain is not from IgG4.

[0299] By way of example, the Fc domain is not from human IgG4.

[0300] In one instance, the FcRn-binding fragment of the Fc domain refers to a portion of an immunoglobulin heavy chain, such as an IgG1 heavy chain, that extends generally from EU position 243 to EU position 261, and from EU position 275 to EU position 293, and from EU position 302 to EU position 319, and from EU position 336 to EU position 348, and from EU position 367 to EU positions 393 and 408, and from EU position 424 to EU position 440.

[0301] In certain instances, the Fc domain or fragment is not a full-length immunoglobulin. For example, the Fc domain or fragment or FcRn antagonist does not contain a variable domain. In some instances, the Fc domain or fragment or FcRn antagonist does not contain a variable domain or C H 1 domain. However, in certain instances, the FcRn antagonist can contain an Fc domain or a fragment thereof that is linked to one or more other binding domains or moieties, including variable domains.

[0302] In one instance, the Fc domain is an Fc domain variant or an FcRn-binding fragment thereof that binds to FcRn with increased affinity specificity compared to the Fc domain from wild-type human IgG1. In one instance, the variant Fc domain or fragment thereof has a reduced pH-dependence of binding to FcRn relative to the native Fc domain region. The exemplary Fc domain or fragment thereof inhibits or reduces the binding of immunoglobulins and / or other Fc-containing proteins (e.g., immunoadhesins and antibody-drug conjugates) to FcRn in vivo, which causes an increased rate of degradation of the immunoglobulin or Fc-containing protein and concomitantly causes a decrease in the serum levels of these immunoglobulins or Fc-containing proteins.

[0303] Any Fc domain region can be modified to produce the Fc domain variants of the present disclosure. As discussed herein, generally the Fc domain is from a human immunoglobulin. However, the Fc domain can 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 primate (e.g., chimpanzee, macaque) species. Additionally, the Fc domain can 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 instances, the Fc domain is an IgG Fc domain (e.g., a human IgG region). In certain instances, the Fc domain is an IgG1 Fc domain (e.g., human IgG1). In certain instances, the Fc domain is a chimeric Fc domain that comprises portions of several different Fc domains. Suitable examples of chimeric Fc domains are described in US20110243966. It should be understood that the scope of the present disclosure encompasses alleles, variants, and mutations of the Fc domain.

[0304] The Fc domain can be further truncated or include deletions to produce its minimal FcRn-binding fragment. The ability of the Fc domain fragment to bind FcRn can be determined using any binding assay recognized in the art (e.g., ELISA).

[0305] To enhance the manufacturability of the FcRn antagonists disclosed herein, it is desirable that the component Fc domain does not contain any non-disulfide-bonded cysteine residues. Thus, in certain instances, the Fc domain does not contain free cysteine residues.

[0306] In one instance, the Fc domain variant comprises an amino acid modification that enhances the binding of the Fc domain to FcRn, such as a substitution. In one instance, the amino acid modification enhances the binding of the Fc domain to FcRn at neutral pH and / or acidic pH. In one instance, the amino acid modification enhances the binding of the Fc domain to FcRn at neutral pH and acidic pH.

[0307] In some instances, the Fc domain variant comprises substitutions at one or more positions selected from the group consisting of, according to the EU numbering system: 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. 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.

[0308] In some instances, the Fc domain variant comprises at least one substitution selected from the group consisting of, according to the EU numbering system: 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 269G, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 296G, 296W, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 334N, 352S, 378V, 397M and 434Y.

[0309] Other known Fc domain variants that can be used in the FcRn antagonists disclosed herein include, but are not limited to, those disclosed in the following: 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., 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., 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.

[0310] 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 the 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 the 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 additionally taught that the following combinations of residues are suitable for enhancing the binding of the Fc domain to: FcRn M252W or M252Y or M252Y / T256Q or M252F / T256D or V203T / L309P / Q311S or H433K / N434F / Y436H or H433R / N434Y / Y436H or M252Y / S254T / T256E / H433K / N434F / Y436H or M252Y / S254T / T256E / G385R / Q386T / P387R / N389P (according to the EU numbering system). Additional substitutions that enhance the binding of the Fc domain to FcRn include T307A / E380A / N434A or M428L / N434S.

[0311] Mackness et al., (MABS 11:1276 - 1288, 2019) showed the following substitutions that bind to FcRn and have a substantially reduced dissociation rate compared to wild - type Fc: M252Y, T256D / E, K288D / N, T307A / E / F / M / Q / W, E380C, N434F / P / Y, and Y436H / N / W. The authors showed that the substituted M252Y / T256D, T256D / T307Q, and T256D / T307W combinations showed enhanced binding to FcRn while maintaining other activities such as thermal stability and FcγRIIIa and rheumatoid factor binding.

[0312] Monnet et al., (Front. Immunol, 12:728322, 2021) showed that the following substitutions exhibit improved binding to FcRn: Y296W / K334N / P352S / A378V / V397M / N434Y.

[0313] Additional substitutions to the Fc domain are described in WO2010 / 106180; WO2016177984; WO2018007453; WO2018078138; WO2017006052; and WO2019115773.

[0314] WO2015 / 100299 describes substitutions at residues 252, 254, 256, 433, 434, and 436 (according to the EU numbering system) that increase the binding of the Fc domain to FcRn. For example, the Fc domain is identical to efgartigimod.

[0315] Other exemplary substitutions are described, for example, in WO2002 / 060919, which describes substitutions at residues 251, 253, 255, 285 - 290, 308 - 314, 385 - 389, and 428 - 435 (according to the EU numbering system) that increase the binding of the Fc domain to FcRn.

[0316] In one instance, the Fc domain variant or fragment thereof comprises one or more amino acid substitutions selected from the group consisting of:

[0317] (i) Substitution of methionine for the amino acid at the position corresponding to amino acid 252 according to the EU numbering system;

[0318] (ii) Substitution of serine for the amino acid at the position corresponding to amino acid 254 according to the EU numbering system;

[0319] (iii) Substitution of threonine for the amino acid at the position corresponding to amino acid 256 according to the EU numbering system;

[0320] (iv) Substitution of asparagine for the amino acid at the position corresponding to amino acid 286 according to the EU numbering system;

[0321] (v) Substitution of tyrosine for the amino acid at the position corresponding to amino acid 296 according to the EU numbering system;

[0322] (vi) Substitution of valine for the amino acid at the position corresponding to amino acid 308 according to the EU numbering system;

[0323] (vii) Substitution of lysine for the amino acid at the position corresponding to amino acid 334 according to the EU numbering system;

[0324] (viii) Substitution of proline for the amino acid at the position corresponding to amino acid 352 according to the EU numbering system;

[0325] (ix) Substitution of alanine for the amino acid at the position corresponding to amino acid 378 according to the EU numbering system;

[0326] (x) Amino acid substitution of valine at the position corresponding to amino acid 397 according to the EU numbering system;

[0327] (xi) Amino acid substitution of histidine at the position corresponding to amino acid 433 according to the EU numbering system;

[0328] (xii) Amino acid substitution of asparagine at the position corresponding to amino acid 434 according to the EU numbering system; and

[0329] (xiii) Combinations thereof.

[0330] In one example, the Fc domain variant or a fragment thereof comprises one or more amino acid substitutions selected from the group consisting of:

[0331] (i) Amino acid substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system;

[0332] (ii) Amino acid substitution of serine with threonine at the position corresponding to amino acid 254 according to the EU numbering system;

[0333] (iii) Amino acid substitution of threonine with glutamic acid at the position corresponding to amino acid 256 according to the EU numbering system;

[0334] (iv) Amino acid substitution of asparagine with glutamic acid at the position corresponding to amino acid 286 according to the EU numbering system;

[0335] (v) Amino acid substitution of tyrosine with tryptophan at the position corresponding to amino acid 296 according to the EU numbering system;

[0336] (vi) Amino acid substitution of valine with proline at the position corresponding to amino acid 308 according to the EU numbering system;

[0337] (vii) Amino acid substitution of lysine with asparagine at the position corresponding to amino acid 334 according to the EU numbering system;

[0338] (viii) Amino acid substitution of proline with serine at the position corresponding to amino acid 352 according to the EU numbering system;

[0339] (ix) Amino acid substitution of alanine with valine at the position corresponding to amino acid 378 according to the EU numbering system;

[0340] (x) Amino acid substitution of valine with methionine at the position corresponding to amino acid 397 according to the EU numbering system;

[0341] (xi) Amino acid substitution of histidine with lysine at the position corresponding to amino acid 433 according to the EU numbering system;

[0342] (xii) Amino acid substitution of asparagine with tyrosine at the position corresponding to amino acid 434 according to the EU numbering system;

[0343] (xiii) Substitution of asparagine with phenylalanine at the position corresponding to amino acid 434 according to the EU numbering system; and

[0344] (xiv) Combinations thereof.

[0345] In one example, the Fc domain variant or a fragment thereof comprises substitutions at each of the following positions 252, 308, and 434.

[0346] For example, the Fc domain variant or a fragment thereof comprises:

[0347] (i) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system;

[0348] (ii) Substitution of valine with proline at the position corresponding to amino acid 308 according to the EU numbering system; and

[0349] (iii) Substitution of asparagine with tyrosine or phenylalanine at the position corresponding to amino acid 434 according to the EU numbering system;

[0350] In one example, the Fc domain variant or a fragment thereof comprises substitutions at each of the following positions 252, 254, and 256.

[0351] For example, the Fc domain variant or a fragment thereof comprises:

[0352] (i) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system;

[0353] (ii) Substitution of serine with threonine at the position corresponding to amino acid 254 according to the EU numbering system; and

[0354] (iii) Substitution of threonine with glutamic acid at the position corresponding to amino acid 256 according to the EU numbering system.

[0355] In one example, the Fc domain variant or a fragment thereof comprises substitutions at each of the following positions 252, 286, and 434.

[0356] For example, the Fc domain variant or a fragment thereof comprises:

[0357] (i) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system;

[0358] (ii) Substitution of asparagine with glutamic acid at the position corresponding to amino acid 286 according to the EU numbering system; and

[0359] (iii) Substitution of asparagine with tyrosine or phenylalanine at the position corresponding to amino acid 434 according to the EU numbering system.

[0360] In one example, the Fc domain variant or a fragment thereof comprises substitutions at each of the following positions 252, 254, 256, 433, and 434.

[0361] For example, the Fc domain variant or a fragment thereof comprises:

[0362] (i) Substitution of methionine with tyrosine at the position corresponding to amino acid 252 according to the EU numbering system;

[0363] (ii) Substitution of serine with threonine at the position corresponding to amino acid 254 according to the EU numbering system;

[0364] (iii) Substitution of threonine with glutamic acid at the position corresponding to amino acid 256 according to the EU numbering system;

[0365] (iv) Substitution of histidine with lysine at the position corresponding to amino acid 433 according to the EU numbering system; and

[0366] (v) Substitution of asparagine with tyrosine or phenylalanine at the position corresponding to amino acid 434 according to the EU numbering system.

[0367] In one example, the Fc domain variant or a fragment thereof comprises substitutions at each of the following positions 296, 334, 352, 378, 397, and 434.

[0368] For example, the Fc domain variant or a fragment thereof comprises:

[0369] (i) Substitution of tyrosine with tryptophan at the position corresponding to amino acid 296 according to the EU numbering system;

[0370] (ii) Substitution of lysine with asparagine at the position corresponding to amino acid 334 according to the EU numbering system;

[0371] (iii) Substitution of proline with serine at the position corresponding to amino acid 352 according to the EU numbering system;

[0372] (iv) Substitution of alanine with valine at the position corresponding to amino acid 378 according to the EU numbering system;

[0373] (v) Substitution of valine with methionine at the position corresponding to amino acid 397 according to the EU numbering system; and

[0374] (vi) Substitution of asparagine with tyrosine at the position corresponding to amino acid 434 according to the EU numbering system.

[0375] In one instance, the Fc domain variant or fragment thereof comprises substitutions at each of positions 252, 254, 256, 433, and 434.

[0376] For example, the Fc domain variant or fragment thereof comprises:

[0377] (i) a tyrosine substitution for methionine at the position corresponding to amino acid 252 according to the EU numbering system;

[0378] (ii) a threonine substitution for serine at the position corresponding to amino acid 254 according to the EU numbering system;

[0379] (iii) a glutamate substitution for threonine at the position corresponding to amino acid 256 according to the EU numbering system;

[0380] (iv) a lysine substitution for histidine at the position corresponding to amino acid 433 according to the EU numbering system; and

[0381] (v) a phenylalanine substitution for asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0382] In an exemplary form of the present disclosure, the Fc domain variant or fragment thereof comprises substitutions at each of positions 252, 254, 256, 433, 434, and 436.

[0383] For example, the Fc domain variant or fragment thereof comprises:

[0384] (i) a tyrosine substitution for methionine at the position corresponding to amino acid 252 according to the EU numbering system;

[0385] (ii) a threonine substitution for serine at the position corresponding to amino acid 254 according to the EU numbering system;

[0386] (iii) a glutamate substitution for threonine at the position corresponding to amino acid 256 according to the EU numbering system;

[0387] (iv) a lysine substitution for histidine at the position corresponding to amino acid 433 according to the EU numbering system;

[0388] (v) a tyrosine or phenylalanine substitution for asparagine at the position corresponding to amino acid 434 according to the EU numbering system;

[0389] (vi) a histidine substitution for tyrosine at the position corresponding to amino acid 436 according to the EU numbering system; and

[0390] (vii) combinations thereof.

[0391] In one instance, the Fc domain variant or fragment thereof comprises:

[0392] (i) Tyrosine replaces methionine at the position corresponding to amino acid 252 according to the EU numbering system; proline replaces valine at the position corresponding to amino acid 308 according to the EU numbering system; and tyrosine replaces asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or

[0393] (ii) Tyrosine replaces methionine at the position corresponding to amino acid 252 according to the EU numbering system; threonine replaces serine at the position corresponding to amino acid 254 according to the EU numbering system; and glutamate replaces threonine at the position corresponding to amino acid 256 according to the EU numbering system; or

[0394] (iii) Tyrosine replaces methionine at the position corresponding to amino acid 252 according to the EU numbering system; glutamate replaces asparagine at the position corresponding to amino acid 286 according to the EU numbering system; and tyrosine replaces asparagine at the position corresponding to amino acid 434 according to the EU numbering system; or

[0395] (iv) Tyrosine replaces methionine at the position corresponding to amino acid 252 according to the EU numbering system; threonine replaces serine at the position corresponding to amino acid 254 according to the EU numbering system, glutamate replaces threonine at the position corresponding to amino acid 256 according to the EU numbering system, lysine replaces histidine at the position corresponding to amino acid 433 according to the EU numbering system, and phenylalanine replaces asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0396] In one example, the Fc domain variant or a fragment thereof comprises tyrosine replacing methionine at the position corresponding to amino acid 252 according to the EU numbering system; threonine replacing serine at the position corresponding to amino acid 254 according to the EU numbering system, glutamate replacing threonine at the position corresponding to amino acid 256 according to the EU numbering system, lysine replacing histidine at the position corresponding to amino acid 433 according to the EU numbering system, and phenylalanine replacing asparagine at the position corresponding to amino acid 434 according to the EU numbering system.

[0397] In some examples, the Fc domain variant has an altered (e.g., increased or decreased) binding affinity for additional Fc receptors. The Fc domain variant can have an altered (e.g., increased or decreased) binding affinity for one or more of FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). Any manner recognized in any field for altering the affinity for another Fc receptor can be employed. In certain examples, the amino acid sequence of the variant Fc domain is altered.

[0398] In some instances, the Fc domain or a fragment thereof forms a homodimer.

[0399] In other instances, the Fc domain or a fragment thereof forms a heterodimer. Methods for generating Fc domain heterodimers are known in the art (see, for example, US8216805). In one instance, Fc domain heterodimers are generated using “knob and hole” or “key and hole” technology. For example, one Fc domain in the heterodimer contains at least one amino acid substitution that exposes at least one side chain and forms a “knob,” and the other Fc domain in the heterodimer contains at least one amino acid substitution to form a cavity that can accept the side chain (i.e., the hole). For example, the Fc domain contains a T366W mutation (or knob), and the other Fc domain contains T366S, L368A, and Y407V mutations (or hole). In another instance, the first Fc domain contains T350V, T366L, K392L, and T394W mutations (knob), and the second Fc domain contains T350V, L351Y, F405A, and Y407V mutations (hole). Using such techniques, FcRn antagonists can be formed with a single albumin or one albumin and another protein. For example, one Fc domain in 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.

[0400] In some instances, the Fc domain variant is a single-chain Fc, where the constitutive Fc domain regions are linked together by a linker. Methods for generating single-chain Fc domain regions are known in the art (see, for example, US20090252729 and US20110081345).

[0401] As discussed herein, pathogenic IgG observed in autoimmune diseases is a pathogenic trigger event of these diseases, or causes disease progression and mediates the disease through inappropriate activation of cellular Fc receptors. Aggregated autoantibodies and / or autoantibodies complexed with autoantigens (immune complexes) bind to activated Fc receptors, causing many autoimmune diseases (which occur in part due to immune-mediated inflammation against self-tissues). Thus, for the treatment of antibody-mediated disorders (e.g., autoimmune diseases), removal of harmful autoantibodies and blockade of the interaction of these antibodies with immune complexes of activated Fc receptors (e.g., Fcγ receptors, such as CD16a) would be advantageous.

[0402] In some instances, compared to the corresponding wild-type Fc, Fc domain variants of FcRn antagonists exhibit increased binding to CD16a (e.g., human CD16a). In certain instances, the FcRn antagonist comprises a variant Fc region that contains an N-linked glycan (e.g., at EU position 297). In such cases, by altering the glycan structure, it is possible to increase the binding affinity of the FcRn antagonist for CD16a. Alterations of the N-linked glycans of the Fc domain are known in the art. For example, afucosylated N-linked glycans or N-glycans with a bisecting GlcNac structure have been shown to exhibit increased affinity for CD16a. Thus, in certain instances, the N-linked glycans are afucosylated. Afucosylation can be achieved using any method recognized in the art. For example, the FcRn antagonist can be expressed in cells lacking fucosyltransferase such that fucose is not added to the N-linked glycans at EU position 297 of the variant Fc domain (see, e.g., US 8,067,232, the content of which is incorporated herein by reference in its entirety). In certain instances, the N-linked glycan has a bisecting GlcNac structure. Bisecting GlcNac can be achieved using any method recognized in the art.

[0403] In other instances, Fc domain variants of FcRn antagonists exhibit decreased binding to CD16a (e.g., human CD16a).

[0404] In other instances, compared to the corresponding wild-type Fc, Fc domain variants of FcRn antagonists exhibit similar binding to CD16a (e.g., human CD16a).

[0405] In some instances, the FcRn antagonists described herein are monomers. 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.

[0406] Albumin and its fragments

[0407] The FcRn antagonists of the present disclosure include albumin or its fragments that are capable of extending the half-life of the antagonist. In one instance, the albumin comprises the sequence set forth in SEQ ID NO:1. In one instance, the albumin of the present disclosure comprises a sequence that is at least about 85% or 90% or 95% or 97% or 98% or 99% identical to the sequences disclosed herein.

[0408] In one instance, the albumin is human serum albumin (HSA).

[0409] In one instance, the human albumin variant or fragment thereof is a naturally occurring albumin variant. Exemplary substitutions of 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).

[0410] Peach and Brenan demonstrated various substitutions of naturally occurring albumin variants. Exemplary substitutions include at position 494 (e.g., D494N).

[0411] Iwao et al. demonstrated various substitutions of naturally occurring albumin variants. Exemplary substitutions include at position 541 (e.g., K541E) or at position 560 (e.g., K560E) or at position 501 (e.g., E501K) or at position 570 (e.g., E570K) or at position 573 (e.g., K573E).

[0412] Galliano et al. demonstrated various substitutions of naturally occurring albumin variants. Exemplary substitutions include at position 505 (e.g., E505K).

[0413] In one instance, the fragment of albumin is capable of prolonging the half - life of an FcRn antagonist without necessarily binding to FcRn. In one instance, the albumin fragment binds to FcRn with a reduced affinity compared to wild - type albumin. In another instance, the fragment of albumin binds undetectably to FcRn.

[0414] In some instances, the albumin is an albumin variant. In some instances, the albumin variant binds to FcRn with a reduced affinity compared to wild - type albumin. In some instances, the albumin variant binds undetectably to FcRn. In some instances, the binding of albumin to FcRn is determined by immobilizing soluble FcRn on a soluble surface and detecting the binding of albumin using SPR. In some instances, albumin is immobilized and the binding of soluble FcRn is determined using SPR.

[0415] In one instance, the albumin variant contains an amino acid modification, such as 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.

[0416] Exemplary substitutions of albumin are described herein and include substitutions at position 464 (e.g., H464Q), or position 510 (e.g., H510Q or H510R), or position 535 (e.g., H535Q or H535F) relative to SEQ ID NO:1.

[0417] WO2011 / 051489 shows that various substitutions of albumin reduce FcRn affinity. Exemplary substitutions include those at position 494 (e.g., D494Q, D494N or D494A), or position 495 (e.g., E495Q or E495A), or position 496 (e.g., T496A), or position 417 (e.g., Q417A), or position 499 (e.g., P499A), or position 536 (e.g., K536A), or position 538 (e.g., K538A), or position 550 (e.g., D550N). Additionally, combinations of substitutions reduce the affinity of FcRn, such as at positions 494 and 496 (e.g., D494N and T496A), or positions 494 and 417 (e.g., D494E and Q417H).

[0418] WO2012 / 150319 also shows that various substitutions of albumin reduce FcRn affinity. Exemplary substitutions include those at position 500 (K500A), or position 417 (e.g., Q417A), or position 536 (e.g., K536A), or position 537 (e.g., P537A), or position 538 (e.g., K538A), or position 573 (e.g., K573P), or position 580 (Q580A), or position 111 (e.g., N111H; N111K; N111E), or position 512 (e.g., D512E), or position 527 (e.g., T527A), or position 569 (e.g., A569S), or position 108 (e.g., D108A).

[0419] US10696732 also shows that various substitutions of albumin reduce FcRn affinity. Exemplary substitutions include those at position 494 (e.g., D494N; D494A; D494Q), or position 496 (e.g., T496A), or position 417 (e.g., Q417A), or position 499 (e.g., P499A), or position 500 (e.g., K500A), or position 536 (e.g., K536A), or position 537 (e.g., K537A), or position 538 (e.g., K538A), or position 501 (e.g., E501A or 501Q).

[0420] Schmidt et al. (Structure 21, 1966 - 1978, 2013) also show that various substitutions of albumin reduce FcRn affinity.

[0421] In one instance, the albumin variant or fragment thereof comprises one or more amino acid substitutions selected from the group consisting of:

[0422] (i) substitution of histidine with an amino acid at the position corresponding to amino acid 464 of SEQ ID NO:1;

[0423] (ii) substitution of threonine with an amino acid at the position corresponding to amino acid 422 of SEQ ID NO:1;

[0424] (iii) substitution of histidine with an amino acid at the position corresponding to amino acid 510 of SEQ ID NO:1;

[0425] (iv) substitution of histidine with an amino acid at the position corresponding to amino acid 535 of SEQ ID NO:1; and

[0426] (v) combinations thereof.

[0427] In one instance, the albumin variant or fragment thereof comprises substitution of histidine with glutamine at the position corresponding to amino acid 464 of SEQ ID NO:1.

[0428] In one instance, the albumin variant or fragment thereof comprises substitution of threonine with tryptophan at the position corresponding to amino acid 422 of SEQ ID NO:1.

[0429] In one instance, the albumin variant or fragment thereof comprises substitution of histidine with glutamine at the position corresponding to amino acid 510 of SEQ ID NO:1.

[0430] In one instance, the albumin variant or fragment thereof comprises substitution of histidine with arginine at the position corresponding to amino acid 510 of SEQ ID NO:1.

[0431] In one instance, the albumin variant or fragment thereof comprises substitution of histidine with phenylalanine at the position corresponding to amino acid 535 of SEQ ID NO:1.

[0432] In one instance, the albumin variant or fragment thereof comprises substitution of histidine with glutamine at the position corresponding to amino acid 535 of SEQ ID NO:1.

[0433] In one instance, the albumin variant or fragment comprises an amino acid substitution at the position corresponding to amino acid 463 of SEQ ID NO:1.

[0434] In one instance, the albumin variant or fragment comprises an amino acid substitution at the position corresponding to amino acid 467 of SEQ ID NO:1.

[0435] In one instance, the albumin variant or fragment comprises an amino acid substitution at a position corresponding to amino acid 509 of SEQ ID NO:1.

[0436] In one instance, the albumin variant or fragment comprises an amino acid substitution at a position corresponding to amino acid 519 of SEQ ID NO:1.

[0437] In one instance, the albumin variant or fragment comprises a glutamine substitution for histidine at a position corresponding to amino acid 464 of SEQ ID NO:1.

[0438] In one instance, the albumin variant or fragment comprises a tryptophan substitution for threonine at a position corresponding to amino acid 422 of SEQ ID NO:1, and a glutamine substitution for histidine at a position corresponding to amino acid 464 of SEQ ID NO:1.

[0439] In one instance, the albumin variant or fragment comprises a glutamine substitution for histidine at a position corresponding to amino acid 464 of SEQ ID NO:1, and a phenylalanine substitution for histidine at a position corresponding to amino acid 535 of SEQ ID NO:1.

[0440] In one instance, the albumin variant or its fragment comprises an arginine substitution for histidine at a position corresponding to amino acid 510 of SEQ ID NO:1 and a phenylalanine substitution for histidine at a position corresponding to amino acid ५३५ of SEQ ID NO:1.

[0441] Exemplary methods for generating variant forms of albumin are described herein or are known in the art and include:

[0442] ]● 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);

[0443] ● Introduction of the nucleic acid encoding the polypeptide into mutant cells such as XL-1Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);

[0444] ● DNA shuffling, such as disclosed in Stemmer, Nature 370:389-91, 1994; and

[0445] ● Induced by site-directed mutagenesis, for example as described in Dieffenbach (ed.) and Dveksler (ed.) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0446] Exemplary methods for determining the biological activity of the albumin variants or fragments thereof of the present disclosure will be apparent to those skilled in the art and / or are described herein, such as FcRn affinity. For example, methods for determining the affinity of an albumin variant or fragment thereof include the affinity, binding, dissociation, and therapeutic efficacy described herein.

[0447] In some instances, the FcRn antagonist comprises more than one albumin. In some instances, more than one albumin is linked to the Fc domain. For example, within the FcRn antagonist, a single Fc domain is linked to more than one albumin or fragment thereof. In some instances, the albumin is linked in series, such as to the C-terminus of the Fc domain. In other instances, 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 instances, the Fc domain can dimerize, thus further increasing the number of albumins in the FcRn antagonist.

[0448] In one instance, each Fc domain in the FcRn antagonist is linked to a single albumin. As exemplified herein, linking an albumin or fragment thereof to the C-terminus of the Fc domain provides an extended half-life and increased FcRn antagonism.

[0449] Linker

[0450] In some instances, the components of the FcRn antagonist of the present disclosure are indirectly linked, such as via a linker. In some instances, the linker is a polypeptide linker.

[0451] In some instances, the polypeptide linker comprises or consists of: a gly-ser linker. As used herein, the term "gly-ser linker" refers to a peptide composed of glycine and serine residues. Exemplary gly / ser linkers comprise the amino acid sequence of formula (Gly4Ser) n where n is a positive integer (e.g., 1, 2, 3, 4, or 5). In certain instances, the gly / ser linker is (Gly4Ser)1. In some instances, the gly / ser linker is (Gly4Ser)2. In some instances, the gly / ser linker is (Gly4Ser)3 or (Gly4Ser)4.

[0452] Other linkers suitable for the FcRn antagonists of the present disclosure are known in the art, such as the serine-rich linker disclosed in US 5525491, 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.

[0453] Other exemplary linkers include GS linker (i.e., (GS)n), GGSG linker (i.e., (GGSG)n), GSAT linker, SEG linker, and GGS linker (i.e., (GGSGGS)n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5).

[0454] The polypeptide linker of the present disclosure is at least one amino acid in length and can have different lengths. In some instances, the length of the polypeptide linker of the present disclosure is from about 1 to about 50 amino acids. In another instance, the length of the polypeptide linker of the present disclosure is from about 5 to 10 amino acids. In another instance, the length of the polypeptide linker of the present disclosure is from about 10 to 20 amino acids. In another instance, the length of the polypeptide linker of the present disclosure is from about 15 to about 50 amino acids.

[0455] In some instances, the linker comprises or is a chemical linker. In some embodiments, the linker is one or more ethylene glycol (EG) units, such as 2 or more EG units (i.e., polyethylene glycol (PEG)). In some instances, the linker comprises or consists of a polyethylene glycol (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-(0-CH2-CH2)n-OH, where n is a positive integer (e.g., 1, 10, 20, 50, 100, 200, 300, 400, 500, 600). In some instances, the PEG linker is PEG1000. In some instances, the PEG linker is PEG2000. In some instances, the PEG linker is PEG3000.

[0456] In some instances, the FcRn antagonist comprises an Fc domain or a fragment thereof linked to a PEG, which is in turn linked to albumin or a fragment thereof.

[0457] 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 can be linked to the C-terminus of albumin. Chemical linkers are suitable for such linkages.

[0458] Generation of FcRn antagonists

[0459] The present disclosure provides polynucleotides, vectors, and host cells encoding the FcRn antagonists disclosed herein. Also provided are methods of preparing FcRn antagonists, which comprise expressing these polynucleotides.

[0460] The polynucleotides encoding the FcRn antagonists disclosed herein are typically inserted into an expression vector for introduction into a host cell that can be used to produce the desired amount of the required FcRn antagonist. Thus, in some instances, the present disclosure provides expression vectors comprising the polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.

[0461] For the purposes of this specification and claims, the term "vector" or "expression vector" is used herein to mean a vector that is used as an agent for introducing into a cell and expressing a desired gene according to the present disclosure. As is known to those of skill in the art, such vectors can be readily selected from the group consisting of: plasmids, phages, viruses, and retroviruses.

[0462] A number of expression vector systems can be used to achieve the purposes of the present disclosure. For example, one class of vectors utilizes DNA components derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MoMLV), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome entry sites. Additionally, cells in which the DNA has integrated into its chromosome can be selected by introducing one or more markers that allow selection of transfected host cells. The markers can provide prototrophy to auxotrophic hosts, provide biocide resistance (e.g., antibiotics), or resistance to heavy metals (e.g., copper). The selectable marker gene can be directly ligated to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Optimal synthesis of mRNA may also require other components. These components can include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0463] 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. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be achieved by a variety of techniques well known to those skilled in the art. These techniques include (but are not limited to) transfection (including electroporation and electrotransfer), protoplast fusion, calcium phosphate precipitation, cell fusion with encapsulated DNA, microinjection, and infection with intact virus. See Ridgway, A.A.G. “Mammalian Expression Vectors” Chapter 24.2, pages 470 - 472 in Vectors, Rodriguez and Denhardt eds. (Butterworths, Boston, Mass. 1988). Typically, the plasmid is introduced into the host via electroporation. The transformed cells are grown under conditions suitable for the production of the FcRn antagonist, and the expression of the FcRn antagonist is assayed. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and similar techniques.

[0464] In vitro production allows for scale-up to obtain large quantities of the desired FcRn antagonist. Mammalian cell culture techniques under tissue culture conditions are known in the art and include homogeneous suspension cultures, such as in airlift reactors or continuously stirred reactors, or fixed or entrapped cell cultures, such as in hollow fibers, microcapsules, on agarose microbeads, or ceramic cartridges. Depending on the requirements and / or needs, the polypeptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno-)affinity chromatography.

[0465] The gene encoding the FcRn antagonist of the present disclosure can also be expressed in non-mammalian cells, such as bacteria, yeast, or plant cells. In this regard, it should be understood that various single-celled non-mammalian microorganisms, such as bacteria (i.e., microorganisms capable of growing or fermenting in culture), can also be transformed. Bacteria that are readily transformable include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella. It should be further understood that when expressed in bacteria, the FcRn antagonist can become part of inclusion bodies. The FcRn antagonist must be isolated, purified, and subsequently assembled into a functional molecule. In addition to prokaryotes, eukaryotic microorganisms can also be used. Among eukaryotic microorganisms, Saccharomyces cerevisiae or commonly used baking yeast strains are most commonly used, but many other strains can generally also be used.

[0466] Determining the activity of the FcRn antagonist

[0467] The FcRn antagonists of the present disclosure can be readily screened for bioactivity, as described below for example.

[0468] Determine affinity

[0469] Optionally, determine the dissociation constant (Kd) or association constant (Ka) or affinity constant (K D ) of the FcRn antagonist or its component (e.g., Fc domain or its fragment or albumin or its fragment).

[0470] The affinity measurement results can be determined by standard methods such as immunoassays, surface plasmon resonance (SPR; e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., 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.

[0471] In some instances, the FcRn antagonist has a K D similar to or improved K D (i.e., lower K D value) for FcRn compared to the human IgG1 Fc domain or its variant or fragment.

[0472] Flow cytometry can also be used to non-quantitatively determine the binding affinity of FcRn. For example, CHO cells stably expressing the FcRn antagonist or its fragment are stained with alexa-488-labeled FcRn / β2m (to detect target binding) and anti-myc-alexa 647 (to detect expression) at acidic (pH 5.5) and neutral (pH 7.4) pH and analyzed by flow cytometry. The relative binding to FcRn / β2m is determined, for example, by calculating the mean fluorescence intensity relative to the unmodified human IgG1 Fc domain or its variant or fragment.

[0473] Determine half-life

[0474] The FcRn antagonists encompassed by the present disclosure have an improved half-life, e.g., compared to the human IgG1 Fc domain or its variant or fragment modified to extend its half-life. The methods for determining the half-life of the FcRn antagonist will be apparent to those skilled in the art.

[0475] For purposes of clarification and as will be apparent to one of ordinary skill in the art based on the description herein, reference to "half-life" should be understood to refer to an increase in one or more of the following parameters: terminal half-life, mean residence time, area under the curve, decrease in volume of distribution, and / or clearance rate. By way of example, prolonging the half-life of an FcRn antagonist means improving the plasma exposure of the antagonist or reducing the volume of distribution of the antagonist.

[0476] In one example, the FcRn antagonists of the present disclosure have an improved α half-life, i.e., the rate of decline of plasma concentration attributable to the redistribution process.

[0477] 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 by Kim et al., Eur J of Immunol 24:542, 1994. According to this method, the protein is injected intravenously into mice and its plasma concentration is measured periodically over time, e.g., from 3 minutes to 72 hours after injection. The clearance curve obtained therefrom should be biphasic, i.e., the α phase and the β phase. To determine the in vivo terminal half-life of the protein, the clearance rate of the β-phase is calculated and compared with the clearance rate of the human IgG1 Fc domain or its variants or fragments.

[0478] In vitro cell assay

[0479] A variety of in vitro assays can be used to evaluate the ability of FcRn antagonists to treat the diseases or conditions described herein.

[0480] In one example, the uptake and recycling of FcRn antagonists are tested in in vitro cell assays.

[0481] Methods for assessing cell uptake and recycling are known in the art and / or exemplified herein. By way of example, a fluorescently labeled FcRn antagonist is incubated with cells expressing the human FcRn receptor on the cell surface. After addition of the labeled FcRn antagonist, the process of protein recycling can be followed by methods including flow cytometry and fluorescence microscopy (e.g., confocal fluorescence microscopy) and compared with the human IgG1 Fc domain or its variants or fragments. Changes in the normal recycling pathway of specific albumin variants can be identified and characterized.

[0482] Pharmacokinetic analysis

[0483] In one example, the pharmacokinetic (PK) properties of FcRn antagonists are assessed.

[0484] Methods for assessing PK characteristics are known in the art and / or exemplified herein. For example, an FcRn antagonist is injected into transgenic mice expressing the human FcRn receptor or other suitable mammalian hosts (e.g., rats, cynomolgus monkeys). In one example, the transgenic mouse expressing the human FcRn receptor is the "hFcRn Tg32" homozygous mouse (i.e., B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ; The Jackson Laboratory stock number 014565; or as described in Roopenian et al., J. Immunol 2003; 170:3528-3533). Using ELISA, the plasma levels of the FcRn antagonist are evaluated using commercially available methods.

[0485] Immunoglobulin clearance

[0486] In one example, the ability of an FcRn antagonist to reduce the levels of circulating immunoglobulins is assessed.

[0487] For example, a known tracer antibody, such as an IgG1 antibody, is administered to a subject, such as a mouse. Subsequently, the FcRn antagonist is administered and the levels of the antibody are determined at different time points. The FcRn antagonist-induced reduction in the levels of the tracer antibody is more rapid compared to that observed in the absence of the antagonist.

[0488] In another example, the levels of endogenous immunoglobulins (e.g., IgG) are determined in the presence or absence of the antagonist.

[0489] Pharmaceutical Compositions

[0490] Suitably, in a composition or method for administering the FcRn antagonist of the present disclosure to a subject, the FcRn antagonist of the present disclosure (i.e., the albumin variant or a fragment thereof conjugated to a compound) is combined with a pharmaceutically acceptable carrier, as understood in the art. Accordingly, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising an FcRn antagonist combined with a pharmaceutically acceptable carrier.

[0491] Generally, a "carrier" means a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating agent, or lubricant that can be safely administered to any subject (e.g., a human). Depending on the particular route of administration, a variety of acceptable carriers known in the art can be used, such as those described in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991).

[0492] The FcRn antagonists of the present disclosure are suitable for parenteral, topical, oral or local administration, aerosol administration or transdermal administration for prophylactic or therapeutic treatment. In one example, the FcRn antagonist is administered parenterally, such as subcutaneously or intravenously. By way of example, the FcRn antagonist is administered intravenously.

[0493] The formulation of the FcRn antagonist to be administered will vary depending on the chosen route of administration and formulation (e.g., solution, emulsion, capsule). Suitable pharmaceutical compositions containing the FcRn antagonist to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral formulations can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or non-volatile oils. A variety of suitable aqueous carriers are known to those of skill in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solutions and glycine. Intravenous formulations can include various additives, preservatives or fluids, nutritional or electrolyte supplements (see generally Remington's Pharmaceutical Science, 16th ed., Mack, Ed. 1980). The composition can contain pharmaceutically acceptable adjuvants as needed to approximate physiological conditions, such as pH regulators and buffers; and toxicity regulators, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The FcRn antagonist can be stored in the liquid phase or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0494] Use of FcRn antagonists

[0495] The FcRn antagonist compositions of the present disclosure are particularly suitable for reducing the serum levels of immunoglobulins or other Fc-containing agents (e.g., antibody-drug conjugates and immunoadhesins). Thus, in one example, the present disclosure provides a method of inhibiting FcRn function in a subject, the method generally comprising administering to the subject an effective amount of an FcRn antagonist or pharmaceutical composition of the present disclosure.

[0496] Reducing the serum levels of immunoglobulins or Fc-containing agents is suitable for treating antibody-mediated disorders (e.g., autoimmune diseases). Thus, in one example, the present disclosure provides a method of treating an antibody-mediated disorder (e.g., autoimmune disease) using an FcRn antagonist composition disclosed herein.

[0497] Any antibody-mediated disorder can be treated using an FcRn antagonist composition disclosed herein.

[0498] The FcRn antagonists of the present disclosure are suitable for treating antibody-mediated disorders characterized by overproduction of serum immunoglobulins. Thus, in some embodiments, the FcRn antagonist compositions are used to treat hypergammaglobulinemia.

[0499] The FcRn antagonists can also be used in combination with one or more additional therapeutic agents. In some instances, the other therapeutic agents are anti-inflammatory agents. Any anti-inflammatory agent can be used in combination with the FcRn antagonists disclosed herein. In some instances, the therapeutic agent is rituximab, daclizumab, basiliximab, muronomab-cd3, infliximab, adalimumab, omalizumab, efalizumab, natalizumab, tocilizumab, eculizumab, golimumab, canakinumab, ustekinumab, or belimumab. In an instance, 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 instances, the leukocyte-depleting agent is a B-cell depleting agent. In some instances, 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 the other therapeutic agent can be administered to a subject simultaneously or sequentially via the same or different routes of administration.

[0500] The FcRn antagonists of the present disclosure are also suitable for reducing the serum content of FcR-containing agents in a subject. Such clearance is advantageous when the Fc-containing agent is toxic (e.g., an antibody-drug conjugate or an immunogenic agent), as it reduces the subject's exposure to the drug. Clearance is also advantageous when the Fc-containing agent is a diagnostic agent that requires a lower serum content to achieve better contrast imaging and / or minimize damage to normal tissues when the Fc-containing agent is radiolabeled. Thus, in some instances, an FcRn antagonist is used to reduce the serum content of the Fc-containing agent in a subject to whom an Fc-containing agent has been administered. The serum content of any Fc-containing agent (e.g., a therapeutic or diagnostic agent) can be reduced using the FcRn antagonists disclosed herein. Non-limiting examples of Fc-containing agents include diagnostic agents (e.g., labeled antibodies), antibody-drug conjugates, or immunogenic agents (e.g., non-human immunoglobulins or immunoadhesins). The FcRn antagonist can be administered concomitantly with the Fc-containing agent or sequentially (e.g., before or after the Fc-containing agent).

[0501] The FcRn antagonists disclosed herein can also be used in combination with a therapeutic protein to enhance the benefit of the therapeutic protein by reducing the content of IgG, which is responsible for reducing the bioavailability of the therapeutic protein.

[0502] In some instances, the present disclosure provides a method of reducing or preventing an immune response against a therapeutic compound.

[0503] One of ordinary skill in the art will be able to determine, by routine experimentation, what effective, non-toxic amount of an FcRn antagonist composition will be used for the purpose of treating antibody-mediated disorders. For example, the therapeutically active amount of a polypeptide can vary depending on factors such as the disease stage of the subject (age, gender, medical complications (e.g., immunosuppressive conditions or diseases), and body weight), and the ability of the antibody to elicit the desired response in the subject. The dosage regimen can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. However, in general, an effective dose is expected to be in the range of about 1 to 200 mg per kilogram of body weight.

[0504] The kits and other compositions of the present disclosure

[0505] Another example of the present disclosure provides a kit containing an FcRn antagonist of the present disclosure, which is suitable for reducing circulating autoantibodies in a subject in need thereof, the kit comprising:

[0506] (i) at least one FcRn antagonist or pharmaceutical composition or nucleic acid of the present disclosure;

[0507] (ii) Instructions for using the kit to reduce circulating autoantibodies in the subject; and

[0508] (iii) Optionally, at least one additional therapy.

[0509] The present disclosure also provides a kit for treating or preventing the progression of an antibody-mediated disorder in a subject in need thereof, the kit comprising:

[0510] (i) At least one FcRn antagonist or pharmaceutical composition or nucleic acid of the present disclosure;

[0511] (ii) Instructions for using the kit to treat or prevent the progression of an antibody-mediated disorder in a subject; and

[0512] (iii) Optionally, at least one additional therapy.

[0513] According to this example of the present disclosure, the instructions (or package insert) are on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of various materials, such as glass or plastic. The label or package insert indicates that the FcRn antagonist is for treating eligible subjects, such as subjects having or susceptible to the diseases described herein, with specific guidance regarding the dosage and time interval of the provided FcRn antagonist and any other agents. The kit can further comprise an additional container that contains a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and / or dextrose solution. The kit can further include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0514] The kit optionally further comprises a container that contains a second agent, where the FcRn antagonist is the first agent, and the kit further comprises instructions on the package insert for treating the subject with the second agent in an effective amount. The second agent can be the therapeutic protein described above.

[0515] The present disclosure includes the following non-limiting examples.

[0516] Example 1: Materials and Methods

[0517] Isolate bone marrow-derived macrophages (BMDM)

[0518] Ten-week-old 32HOM huFcRn mice were euthanized by CO2 asphyxiation and BMDM were generated essentially as previously described Tg / Tg and (Lim et al., Biol Open. 2012 Sep 15;1(9):904-14, 2012). Monocytic cells were cryopreserved at 6x10 6 cells / mL for long-term storage.

[0519] Evaluate the effect of FcRn antagonist on IgG and albumin recycling in macrophages after macropinocytosis.

[0520] Approximately 6x10 5 cells / well were seeded into 8-well removable slides (Ibidi, Germany) at approximately 1.9x10 6 BMDM. The cells were differentiated in 200 μL of BMDM medium for 3 days, and then an additional 200 μL of BMDM medium (RPMI, heat-inactivated FCS (15%), 20% L-cell conditioned medium, 500 μL Pen / Strep; 2 mM Glutamax) was added to allow the cells to further differentiate into macrophage-like cells. The cells were then starved for 16 h by the following: removing the BMDM medium, washing twice with PBS and replacing with 200 μL of C-RPMI (RPMI, heat-inactivated FCS (10%), 500 μL Pen / Strep, 2 mM Glutamax) per well. Subsequently, the medium was aspirated from the wells and the cells were pre-incubated for 4.5 h in the presence of DMSO (1:100 dilution) or protease inhibitor (1 / 100 dilution). Thirty minutes before the end of the pre-incubation, mouse serum (at 1 / 100 dilution) was added to block surface Fcγ receptors and the cells were incubated on ice for 30 min. A treatment sample comprising a combination of an FcRn antagonist, HSA-AF488, IgG-AF568 and protease inhibitor was prepared and pre-warmed at 37 °C. After incubation with mouse serum, the cells were washed twice with PBS, the pre-mixed pre-warmed experimental treatment was added and the cells were pulsed at 37 °C (neutral pH) for 15 min. The cells were then washed twice in PBS and immediately fixed (= 0 min chase) or C-RPMI + / - protease inhibitor was added and the cells were incubated for a 15 min chase period, then washed and fixed.

[0521] After the 0 min and 15 min chase time points, the cells were fixed in 4% PFA at room temperature for 10 min, washed once in PBS, 50 mM NH4Cl to quench excess PFA, and incubated for a further 10 min at room temperature. They were then washed again in PBS and blocked in 5% FCS / PBS for 30 min, then the samples were treated with Hoescht to stain the nuclei and the slides were mounted in Mowiol mounting reagent.

[0522] Imaging was performed by confocal microscopy. The fluorescence intensity of the acquired images was analyzed by the "Analyze Particles" plugin in ImageJ. Individual values were entered into GraphPad Prism and a graph was generated.

[0523] Evaluate FcRn antagonist affinity

[0524] 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 onto the carboxymethyl dextran surface of a CM5 sensor chip at pH 5 using standard NHS / EDC chemistry to a level of approximately 2,000 RU. Control G1Fc homodimer and full-length IgG1 were directly immobilized to the carboxymethyl dextran surface of a CM5 sensor chip at pH 4.5 using standard NHS / EDC chemistry to a level of approximately 2,000 RU.

[0525] FcRn antagonists (2 μg / mL) were prepared in the operating buffer at pH 7.3 and captured to approximately 350 RU in the active spots of each flow cell. The capture was equally stable under neutral and acidic conditions. Proteins were not captured on the reference surface used for background subtraction. The immobilized ABP surface was prepared with ten cycles that consisted of conditioning with recombinant HSA (at 2 μg / mL) (60 - second injection), followed by regeneration with 6 M guanidine-HCl for 30 seconds. The sensorgrams were double-subtracted using the reference surface and blank buffer injection data obtained within each experiment. The data were fit to a 1:1 Langmuir model with a local Rmax and zero refractive index (RI = 0). The experimental operating buffer (10 mM HEPES, 150 mM NaCl, at pH 7.3 or pH 6.0) was prepared and filtered (0.22 μm) before use.

[0526] The purified soluble recombinant mammalian (i.e., human, cynomolgus monkey, mouse, and / or rat) FcRn / β2m (≈42 kDa) receptor was tested at concentrations in the range of 39 nM to 20 μM at neutral conditions (pH 7.3) and at concentrations in the range of 7.8 nM to 2 μM at acidic conditions (pH 6.0). Analyte samples were prepared in operating buffer as 2-fold serial dilutions. The association and dissociation rate constants of FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), and the FcRn / β2m receptor were monitored for 120 and 240 seconds, respectively. The association and dissociation rate constants of FcyRI (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 triplicate experiments.

[0527] In Table 2 (below), FcRn antagonists were tested under affinity conditions for biotinylated human FcRn / β2m tethered to a standard streptavidin or neutravidin sensor chip. Biotinylation of the soluble recombinant human FcRn / β2m can be performed using standard amine chemistry methods (e.g., biotin-NHS) or enzymatic methods with similar results (e.g., BirA biotin ligase). FcRn antagonist samples were prepared in operating buffer as 2-fold dilutions in the range of 0.15 nM to 20 nM. The capture FcRn / β2m surface was prepared with 5 cycles consisting of conditioning (30-second injection) of recombinant IgG (100 nM, 15 μg / mL), followed by regeneration with 150 mM Tris, pH 8 for 30 seconds.

[0528] In Table 3 (below), the purified soluble recombinant human Fcy receptors (i.e., FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), and FcRn / β2m) were analyzed for FcRn antagonists under neutral conditions (pH 7.3). G1Fc homodimer and full-length IgG1 were used as baseline controls. Soluble analytes were prepared in operating buffer as 2-fold serial dilutions. The human FcyRIIA (CD32), FcyRIIB (CD32), and FcyRIIIA (CD16a) receptors were tested at concentrations in the range of 39 nM to 50 μM, while FcyRI (CD64) was tested at concentrations in the range of 0.098 to 100 nM.

[0529] The sensorgram was double subtracted using the reference surface and blank buffer injection data obtained within each experiment. The data was fit to a 1:1 Langmuir model with a local Rmax and a refractive index of zero (RI = 0). The experimental operating buffer (10 mM HEPES, 150 mM NaCl at pH 7.3 or pH 6.0) was prepared and filtered (0.22 μm) before use.

[0530] When the Fc receptor was used as the analyte (Table 1) or as the surface-bound ligand (Table 2), the FcRn antagonist exhibited nanomolar affinity for mammalian FcRn / β2m. In Table 3, the FcRn antagonist exhibited binding affinity to the soluble Fcy receptor comparable to that observed for the G1Fc homodimer or full-length IgG1.

[0531] FcRn antagonist binding

[0532] The FcRn / β2m protein complex (350 - 400 kDa, 1.28 mg / ml) was prepared in PBS and diluted to 0.1 μM in acetate buffer (pH 6.0). A carbon-coated Cu grid (GSCU400CC, 400 mesh) was glow-discharged for 30 s at 15 mA. 4 μl of the diluted protein sample was applied for 45 s, blotted (Whatman No. 1 filter paper), then 4 μl of H2O, blotted, and finally stained with 2% uranyl acetate for 30 s, blotted to remove residual dye, and air-dried. The grid was imaged at room temperature using a TF30 (200 KeV) TEM at a magnification of x39,000, where the pixel size was 32 picture files were imported into cryoSPARC v3.322 using the following input parameters: pixel size 2.79; acceleration voltage (kV) 200 and spherical aberration (mm) 2.7. Patch CTF, then blob picking Particle picking for detection and extraction (96 px box size) screened out 59,879 particles for 2D classification. The selection of representative 2D class averages enabled template-based automatic particle picking (diameter ), which yielded 39,478 particles after two additional rounds of 2D classification to further remove junk particles. Images from the negative stain experiment allowed the identification of the large globular domain (i.e., HSA) linked to the long domain (i.e., Fc) and the soluble domain (i.e., FcRn / β2m) loosely linked to the Fc-like long domain, indicating the presence of a stable FcRn / β2m / antagonist complex in a 2:1 ratio.

[0533] Example 2: Generation of an FcRn antagonist with an extended half-life

[0534] Produce a fusion protein comprising recombinant human serum albumin (HSA) and a wild-type IgG1 Fc domain (huG1Fc) or a variant thereof, said variant comprising the mutations M252Y, S254T, T256E, H433K and N434F (huG1FcYTEKF). Produce a fusion protein having HSA linked to the N-terminus or C-terminus of huG1Fc or huG1FcYTEKF.

[0535] huG1FcYTEKF was generated as a reference molecule and modeled on the FcRn antagonist efgartigimod (Vyvgart), with the only difference being five additional amino acids (EPKSC) at the N-terminus (SEQ ID NO:13). The internally generated molecule huG1FcYTEKF was compared to the commercially available Vyvgart product and found to have a similar binding affinity for cell surface-bound huFcRn / b2m (K D )(i.e., huG1FcYTEKF; K D 2.4 nM compared to Vyvgart; K D 2.3 nM at pH 7.3). Additionally, the internally generated reference huG1FcYTEKF was comparable to the published data for efgartigimod in reducing IgG in non-human primate (NHP) studies.

[0536] Generate homodimeric and heterodimeric variants of the FcRn antagonist. To generate a homodimer comprising two albumins (e.g., Fc-albumin:Fc-albumin or albumin-Fc:albumin-Fc), cells were transfected with a single expression construct (H464Q).

[0537] To generate heterodimers, expression constructs encoding Fc-albumin or albumin-Fc molecules were co-transfected with an expression construct encoding an Fc sequence only at various ratios (e.g., 1:1, 1:0.5 or 1:0.25) to produce a certain amount of heterodimeric FcRn antagonists (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 with huG1FcYPY and single albumin molecules were also able to bind FcRn in vitro.

[0538] In an initial SPR experiment, a heterodimeric FcRn antagonist variant having a single albumin molecule fused to dimeric huIgG1FcYPY (in each of the Fc-albumin and albumin-Fc orientations) bound to biotinylated human FcRn / β2m with similar affinity at pH 7.3 compared to the corresponding homodimeric FcRn antagonist variant containing two albumins. The heterodimeric FcRn antagonists tested were: huG1FcYPY-HSA:huG1FcYPY; huG1FcYPY-HSA(H464Q):huG1FcYPY; HSA-huG1FcYPY:huG1FcYPY; and HSA(H464Q)-huG1FcYPY:huG1FcYPY.

[0539] The ability of homodimeric FcRn antagonists to reduce the levels of a circulating IgG "tracer" antibody (CSL360) in transgenic mice expressing human FcRn (huFcRn transgenic mice) was evaluated. The negative control group included untreated mice and the positive control group included mice treated with huG1FcYTEKF, a known FcRn antagonist. The half-life of the FcRn antagonists was also evaluated.

[0540] Figure 1A It was shown that administration of C-terminal and N-terminal HSA fusions to (wild-type) huG1Fc did not result in a decrease in the levels of the circulating tracer antibody. Figure 1B It was demonstrated that huG1FcYTEKF-HSA (i.e., HSA fused to the C-terminus of huG1FcYTEKF; SEQ ID NO:5) reduced the in vivo levels of the IgG tracer antibody to levels similar to those observed with the huG1FcYTEKF positive control. However, the huG1FcYTEKF-HSA fusion protein had a substantially longer in vivo half-life compared to huG1FcYTEKF ( Figure 1C ). When albumin was linked to the N-terminus of huG1FcYTEKF, the same level of reduction of the tracer antibody was not observed ( Figure 1B ).

[0541] Example 3: Generation of alternative extended half-life FcRn antagonists

[0542] The M252Y, V308P, N434Y substitutions were introduced into the huG1 Fc domain (huG1FcYPY) and fused to wild-type HSA and the HSA(H464Q) substitution variant. The HSA(H464Q) substitution variant is known to have reduced binding to FcRn.

[0543] Figure 2AAnd B show that huG1FcYPY-HSA and huG1FcYPY-HSA[H464Q] (SEQ ID NO:11) reduce the tracer antibody content to a level similar to that of huG1FcYTEKF. This result was observed regardless of whether HSA was wild-type or the H464Q substitution variant. Fusion of HSA to the N-terminus of huG1FcYPY was able to reduce the circulating tracer antibody content, but not to the same extent as C-terminal fusion (i.e., huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q)).

[0544] Figure 2C Show that similar pharmacokinetics were observed when the FcRn antagonists of the present disclosure were administered intravenously or subcutaneously, indicating a relatively high bioavailability of the FcRn antagonists after subcutaneous administration. Equimolar amounts of each FcRn antagonist were used.

[0545] Figure 2D Demonstrate that huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) reduce the circulating content of the tracer antibody in a dose-dependent manner, respectively. These data suggest that the Fc domain of the fusion protein confers a reduction in antibody content and that the observed extended half-life conferred by HSA can occur via one or more mechanisms other than binding to FcRn and FcRn-mediated recycling. The inventors of the present case are particularly interested in these data because the binding of the HSA portion of the FcRn antagonist to FcRn may cause a reduction in endogenous albumin recycling, affecting albumin homeostasis, and it has also been found to affect lipid homeostasis (Ward et al. Front Immunol. 13:892534, 2022).

[0546] Figure 3A And B show that a single dose of the FcRn antagonists of the present disclosure reduces endogenous murine IgG in wild-type mice to levels lower than those observed with huG1FcYTEKF (comparable to efgartigimod). This effect was observed regardless of whether huG1FcYPY was fused to wild-type HSA or HSA(H464Q).

[0547] As Figure 3C As shown in and D, the FcRn antagonists of the present disclosure have a substantially longer half-life compared to huG1FcYTEKF in wild-type mice.

[0548] Example 4: Affinity of FcRn antagonists

[0549] Tables 1 and 2 relate to the binding affinity of the FcRn antagonists of the present disclosure to FcRn as determined using SPR. The data shown in Table 1 were obtained using soluble human, cynomolgus macaque, 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 soluble FcRn antagonists of the present disclosure to surface-trapped biotinylated human FcRn complexed with β2m (at pH 7.3). Table 3 relates to the binding of soluble Fcγ receptors to surface-trapped FcRn antagonists.

[0550] Table 1

[0551]

[0552]

[0553] Table 2

[0554]

[0555] Table 3

[0556]

[0557] Table 4

[0558]

[0559]

[0560] Example 5: FcRn Antagonists that Do Not Affect Albumin Recycling

[0561] The ability of different FcRn antagonist molecules to antagonize IgG and albumin recycling was examined in BMDMs from huFcRn transgenic mice ( Figure 4A and Figure 4B ). Figure 5 A sketch depicting how the assays described in the following sections were evaluated is shown. After a 10-minute pulse followed by a 0-minute chase, the uptake of fluorescently labeled IgG (IgG1-AF568) by macropinocytosis was evident ( Figure 4A)。After 15 minutes of chase, IgG1-AF568 was no longer detected and this signal was not rescued by protease inhibitors, indicating that IgG1-AF568 was recycled by the cell rather than transported to lysosomal compartments for degradation. Similar results were observed in the presence of the control protein, recombinant huG1Fc (the Fc domain of wild-type human IgG1). It should be understood that after fixation and permeabilization, membrane-associated molecules within macropinosomes are retained, but free proteins within macropinosomes are lost. After the fixation / permeabilization step, proteins delivered to subsequent endosomal compartments (e.g., lysosomes) are more effectively retained / protected within tighter membranous structures (as Figure 5 shown). In the presence of the FcRn antagonists huG1FcYTEKF, huG1FcYPY-HSA, and huG1FcYPY-HSA(H464Q), the dose-dependent decrease in signal at time 0 post-pulse indicated that after the fixation / permeabilization step, the interaction of IgG1-AF568 with membrane-associated FcRn within macropinosomes was reduced and free IgG1-AF568 was absent from the core of macropinosomes. After 15 minutes of chase prior to fixation / permeabilization, IgG1-AF568 was readily detected in the presence of protease inhibitors, indicating lysosomal rescue. Similar degrees of dose-dependent FcRn antagonism of IgG1-AF568 recycling (corresponding to lysosomal degradation revealed by rescue in the presence of protease inhibitors during 15 minutes of chase) were observed for huG1FcYTEKF, huG1FcYPY-HSA, and huG1FcYPY-HSA(H464Q).

[0562] The effect of FcRn antagonists on albumin recycling (HSA-AF488) was also examined ( Figure 4B)。In the control sample, HSA-AF488 was taken up by macropinocytosis, which was evident with a 10-minute pulse and 0-minute chase. However, at 15 minutes, there was no signal even in the presence of protease inhibitors, indicating complete albumin recycling. The antagonist huG1FcYTEKF similarly had no effect on albumin recycling. The molecule huG1FcYPY-HSA showed a decrease in HSA-AF488 signal at time 0 after fixation and permeabilization at 30 μM (indicating reduced interaction of HSA-AF488 with membrane FcRn and loss of free HSA-AF488 from the core of macropinosomes). Additionally, after a 15-minute chase before fixation / permeabilization, HSA-AF488 could be rescued from lysosomal degradation in the presence of protease inhibitors, indicating that huG1FcYPY-HSA antagonizes albumin recycling. In contrast, the molecule huG1FcYPY-HSA(H464Q) had minimal effect on albumin recycling, with only a small amount of lysosomal rescue of HSA-AF488 observed at the highest concentration of huG1FcYPY-HSA(H464Q). In summary, these data suggest that the H464Q variant has a reduced ability to antagonize albumin recycling in vitro and is therefore unlikely to antagonize albumin recycling in vivo.

[0563] Example 6: FcRn antagonist binding

[0564] Negative staining experiments showed the conformation of a complex of huG1FcYPY(H464Q) and two FcRn / β2m and indicated that the HSA(H464Q) moiety did not bind to FcRn / β2m. Figure 6 A schematic diagram of the putative complex is shown.

[0565] Example 7: FcRn antagonists with reduced crosslinking ability

[0566] The Fc-albumin fusion protein (i.e., an FcRn antagonist) has the potential to crosslink multiple FcRn receptors under acidic conditions (e.g., the intracellular compartment of cells) by bridging adjacent molecules via its Fc and albumin domains. Compared to HSA (i.e., ~300 nM), the HSA (H464Q) mutant exhibits a 10 μM affinity for human FcRn / β2m at acidic pH 6.0. Addition of the HSA (H464Q) mutant to the albumin in the FcRn antagonist reduces the albumin's affinity for FcRn under acidic conditions (i.e., pH 6.0) that limit the interaction of FcRn / β2m with the Fc domain of the FcRn antagonist. This is reflected by the stoichiometric reduction of the HSA (H464Q) molecule to human FcRn / β2m (Table 5), as measured by SPR. Briefly, the stoichiometry (Seq) of each ligand at steady state is calculated according to the ratio of the maximum reaction at saturation (Rmax) to the amount of captured ligand (RL) multiplied 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 used to predict the theoretical Rmax. The captured content (RL) between cycles is normalized by taking the binding content at equilibrium (Req) and captured content (RL) for each cycle and calculating according to Equation 1. This yields a table of Seq values for each concentration. These values can be plotted similar to a steady-state fit to show the valency of the binding at each concentration and extrapolated to its maximum to determine the stoichiometry of the ligand. The values are adjusted and fit to a steady-state one-site model, where "Bmax" represents the stoichiometry of each interaction based on the captured and bound content and molecular weights of the ligand and analyte. For the huG1FcYPY-HSA and huG1FcYPY-HSA (H464Q) fusions, the molecular weights of the ligand and analyte are assumed to be 185 kDa, 67 kDa for HSA, and 42 kDa for FcRn / β2m.

[0567] The role of His464 was demonstrated in the elucidation of the FcRn / β2m / HSA complex (Schmidt et al. (2013) Structure 21, 1966 - 1978). The model describes the role of His464, His510, and His510 in stabilizing the binding sites of two conserved FcRn tryptophan residues (W53 FcRn and W59 FcRn ) in a pH-dependent manner. Table 5 shows the HSA residues around the binding pockets of W53 FcRn and W59 FcRn [[ID=eleven]]and Figure 7) can be mutated to substantially reduce the binding of HSA to FcRn / β2m compared to HSA (H464Q). Table 5 depicts the incorporation of additional albumin mutations into the albumin domain of the huG1FcYPY-HSA (H464Q) neutralizing protein, yielding FcRn / β2m binding affinities similar to those observed for the G1FcYPY homodimer and huG1FcYPY-HSA[25-384], where HSA lacks the entire FcRn binding domain.

[0568] Table 5

[0569]

[0570] Example 8: Effects of an FcRn antagonist on endogenous cynomolgus macaque IgG, IgA, and IgM after a single intravenous dose

[0571] Each compound (i.e., huG1FcYPY-HSA, huG1FcYPY-HSA (H464Q), and huG1FcYTEKF) was administered as a single bolus intravenous (I.V.) injection of an FcRn antagonist at an equimolar dose in 3 respective male animals of approximately 4 kg. Briefly, for huG1FcYPY-HSA and huG1FcYPY-HSA (H464Q), the FcRn antagonist was administered at an equimolar content of 75 mg / kg, and for huG1FcYTEKF, at 21.15 mg / kg. No negative clinical signs, abnormal hematology, blood biochemistry, or cytokine profiles were observed post-treatment. Albumin and lipids were also largely unaffected.

[0572] Blood samples were taken before dosing, at 0.0833 hours, 0.5 hours, 3 hours, 8 hours, 24 hours, 48 hours, 72 hours, 168 hours, day 8, day 12, day 15, day 22, day 29, day 36, day 43, day 50, day 57, day 64, day 71, and day 85. The lower limit of quantification (LLOQ) for the compounds tested was reported as 1000 ng / mL for huG1FcYPY-HSA and huG1FcYPY-HSA (H464Q) and 250 ng / mL for huG1FcYTEKF.

[0573] Endogenous Ig analysis was completed using a validated enzyme-linked immunosorbent assay (ELISA) method developed with a Subtype Typing Panel 1 Human / NHP kit, catalog number K15203D.

[0574] The IgG content in all animals followed a consistent trend, gradually decreasing until 168 hours (7 days) post-dosing ( Figure 8A and 8B)。HuG1FcYPY-HSA(H464Q) reduced IgG by up to 75%, animals treated with huG1FcYPY-HSA by up to 68% and animals treated with huG1FcYTEKF by up to 59% IgG. By day 57, endogenous IgG had returned to pre-dose levels in all groups.

[0575] The levels of IgA generally remained at approximately pre-dose levels at all time points in all animals (not shown).

[0576] IgM levels remained constant in all animals until 168 hours post-dose, after which an increase in IgM was observed in animals treated with huG1FcYPY-HSA(H464Q) and the huG1FcYPY-HSA molecule (Figure 9). No clear trend in IgM levels was seen in animals treated with huG1FcYTEKF. The increase in IgM indicates the production of anti-drug antibodies (ADA). An ADA response to a human protein in NHP does not predict ADA in humans but can cause an underestimation of the PK and PD of the drug. Indeed, in some animals, a stronger rebound in IgG before or associated with the spike in IgM may have been partially driven by ADA ( Figure 8B and Figure 9).

[0577] Example 9. Pharmacokinetics of FcRn antagonists in cynomolgus monkey serum

[0578] Pharmacokinetic analysis of FcRn antagonists followed the specific quantification and bioanalytical assessment of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q) and huG1FcYTEKF from cynomolgus monkey serum samples after a single intravenous dose administration. Quantification was performed using a validated enzyme-linked immunosorbent assay (ELISA) method. Calibration standards were run in duplicate wells in the range of 75,000 to 1,000 ng / mL and the mean signal was regressed against the nominal concentration using 4PL curve fitting with 1 / y weighting. Calibration standards were prepared by spiking blank pooled cynomolgus monkey serum with appropriate volumes of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q) and huG1FcYTEKF stock solutions. Calibration standards were freshly prepared before each experiment and discarded after use.

[0579] Briefly, FcRn antagonist was quantified in blood samples taken before dosing and at 0.0833 hours, 0.5 hours, 3 hours, 8 hours, 24 hours, 48 hours, 72 hours, 168 hours, Day 8, Day 12, Day 15, Day 22, Day 29, Day 36, Day 43, Day 50, Day 57, Day 64, Day 71 and Day 85. The measured concentration of at least 75% of the calibration standards was within ±20% of their theoretical concentration (±25% at the upper and lower limit of quantification). Additionally, the coefficient of variation for replicate wells for each standard must be ≤20%.

[0580] Non-compartmental analysis was performed to compare the PK profiles, showing the different time-concentration profiles of huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) compared to huG1FcYTEKF ( Figure 8C and Table 6). The compounds huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q) exhibited two distribution phases (α and β phases). The terminal phase (after Day 8) of these molecules was determined by a steep terminal slope, which may be attributed to ADA as indicated by increased IgM and strong IgG rebound in some animals as described above. However, the role of target-mediated drug disposition (TMDD) cannot be excluded. For huG1FcYPY-HSA(H464Q), the drug was detected in only one animal of huG1FcYPY-HSA at 294 h.

[0581] The pharmacokinetics of huG1FcYTEKF was characterized by three phases: a steep initial slope (α phase), followed by an intermediate slope (β phase) and a rather shallow slope during the terminal phase. huG1FcYTEKF exhibited a significantly steeper slope during the initial phase, indicating a higher volume of distribution overall compared to the compounds huG1FcYPY-HSA and huG1FcYPY-HSA(H464Q), which seemed to be more restricted to the plasma space. The dose-normalized area under the curve (AUC) (as (DN AUC last (ug / mL*h / mg)) was significantly larger for huG1FcYPY-HSA(H464Q) compared to huG1FcYPY-HSA and huG1FcYTEKF.

[0582] The reduced volume of distribution of the albumin fusion compared to the reference molecule can be attributed to a combination of both reduced tissue penetration and reduced renal clearance. The volume of distribution of the huG1FcYPY-HSA(H464Q) molecule of interest is less than that of the WT fusion protein. In addition to its role in the recycling of albumin and IgG, FcRn has been found to have 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 albumin fusion reduces the volume of distribution of both molecules, impaired interaction of FcRn with the albumin domain within huG1FcYPY-HSA(H464Q) can further limit the tissue distribution of this molecule due to reduced FcRn-mediated transcytosis. An alternative possibility is that huG1FcYPY-HSA may be more strongly sequestered within target cells due to possible tetrameric interactions with FcRn (via two Fc and two albumin domain binding sites), and crosslinking of FcRn can in turn cause enhanced lysosomal degradation of the FcRn / drug complex, as previously described under FcRn crosslinking (Weflen et al 2013). Multivalent immune complexes transfer FcRn to lysosomes by exclusion from the recycling sorting tubules (Mol Biol Cell 2013).

[0583] Since hematopoietic and endothelial cells are responsible for most IgG recycling, increased retention of an FcRn antagonist within plasma proximal to these relevant cell types, as achieved for the huF1FcYPY-HSA(H464Q) molecule, may be beneficial.

[0584] Table 6. Key non-compartmental analysis parameters of the pharmacokinetics (PK) of huG1FcYPY-HSA, huG1FcYPY-HSA(H464Q), and huG1FcYTEKF detected in the plasma of non-human primates (cynomolgus macaques) after a single intravenous injection of the antagonist.

[0585]

[0586] Example 10. Effects of an FcRn antagonist on IgG, IgA, and IgM in cynomolgus macaques after a single subcutaneous dose

[0587] Each compound (i.e., huG1FcYPY-HAS, huG1FcYPY-HSA(H464Q), and huG1FcYTEKF) was administered as a single bolus subcutaneous (s.c.) injection of the FcRn antagonist in 3 male animals each weighing approximately 4 kg Figure 10A and 10B)。Two FcRn antagonists, huG1FcYPY-HSA(H464Q) and huG1FcYTEKF, were administered at equimolar contents of 75 mg / kg and 21.15 mg / kg, respectively. These molecules were well tolerated at the tested concentrations. No negative clinical symptoms, abnormal hematology, blood biochemistry, or cytokine profile deviations were observed.

[0588] Blood samples were taken before dosing, at 0.0833 hours, 0.5 hours, 3 hours, 8 hours, 24 hours, 48 hours, 72 hours, 168 hours, day 8, day 12, day 15, day 22, day 29, day 36, day 43, day 50, day 57, day 64, day 71, and day 85. The lower limit of quantification (LLOQ) of the tested compounds was reported as 1000 ng / mL for huG1FcYPY-HSA(H464Q) and 250 ng / mL for huG1FcYTEKF. Endogenous Ig analysis was completed using a validated enzyme-linked immunosorbent assay (ELISA) method developed with a Subtype Typing Panel 1 Human / NHP kit, catalog number K15203D.

[0589] Animals treated with huG1FcYPY-HSA(H464Q) and huG1FcYTEKF showed a progressive decrease in IgG content until 168 h (day 7)( Figure 10A and Figure 10B ). As in the IV study, huG1FcYPY-HSA(H464Q) was more effective in driving IgG depletion (up to 80% IgG depletion) than the reference huG1FcYTEKF (up to 60% depletion). By day 18, endogenous IgG had returned to its pre-dose content in both groups.

[0590] IgM content remained constant in all animals until 168 hours after dosing, after which an increase in IgM was observed in animals treated with huG1FcYPY-HSA(H464Q) (Figure 11). No clear trend in IgM content was seen in animals treated with huG1FcYTEKF.

[0591] There was little evidence of drug-related effects on IgM or IgA content after subcutaneous administration of the FcRn antagonists (not shown).

[0592] Table 7. Key non-compartmental analysis parameters for the pharmacokinetics (PK) of huG1FcYPY-HSA(H464Q) and huG1FcYTEKF detected in the plasma of non-human primates (cynomolgus macaques) after a single subcutaneous injection of the antagonist. (*) The calculated t1 / 2 (h) (terminal) phase value was limited by the non-linear terminal clearance rate of huG1FcYPY-HSA(H464Q).

[0593]

[0594]

[0595] Sequence Listing

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[0599]

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Claims

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) at least one albumin or a fragment thereof capable of extending the half-life of the FcRn antagonist compared to the half-life of the immunoglobulin Fc domain or a fragment thereof.

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

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

4. The FcRn antagonist according to any one of claims 1 to 3, wherein the albumin or a fragment thereof is a human albumin variant or a fragment thereof.

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

1.

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

7. An FcRn antagonist according to any one of claims 4 to 6, wherein the albumin variant or fragment thereof binds to human FcRn with a K at neutral pH or pH 6.0 greater than 10 μM D binding to human FcRn.

8. The FcRn antagonist according to any one of claims 4 to 7, wherein the albumin variant or a fragment thereof comprises one or more amino acid substitutions selected from the group consisting of: (i) glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; (ii) tryptophan substitution for threonine at the position corresponding to amino acid 422 of SEQ ID NO:1; (iii) glutamine substitution for histidine at the position corresponding to amino acid 510 of SEQ ID NO:1; (iv) phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; and (v) combinations thereof.

9. The FcRn antagonist according to any one of claims 4 to 8, wherein the albumin variant or a fragment thereof comprises: (i) glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or (ii) phenylalanine substitution for histidine at the position corresponding to amino acid 535 of SEQ ID NO:1; or (iii) tryptophan substitution for threonine at the position corresponding to amino acid 422 of SEQ ID NO:1, and glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1; or (iv) glutamine substitution for histidine at the position corresponding to amino acid 464 of SEQ ID NO:1, and phenylalanine substitution for histidine at the 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 according to claim 10, wherein the Fc domain variant or a fragment thereof is an IgG1 Fc domain variant or a 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 increased 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 FcRn antagonist according to any one of claims 10 to 13, wherein the Fc domain variant or a fragment thereof binds to human FcRn with a K at neutral pH of at least 10 μM D at neutral pH of at least 10 μM 15. The FcRn antagonist of any one of claims 10 to 13, wherein the Fc domain variant or fragment thereof comprises one or more amino acid substitutions selected from the group consisting of: (i) substitution of tyrosine for methionine at the position corresponding to amino acid 252 according to the EU numbering system; (ii) substitution of threonine for serine at the position corresponding to amino acid 254 according to the EU numbering system; (iii) substitution of glutamic acid for threonine at the position corresponding to amino acid 256 according to the EU numbering system; (iv) substitution of glutamic acid for asparagine at the position corresponding to amino acid 286 according to the EU numbering system; (v) a substitution of proline for valine at the position corresponding to amino acid 308 according to the EU numbering system; (vi) a substitution of lysine for histidine at the position corresponding to amino acid 433 according to the EU numbering system; (vii) substitution of tyrosine for asparagine at the position corresponding to amino acid 434 according to the EU numbering system; (viii) a substitution of phenylalanine for asparagine at the position corresponding to amino acid 434 according to the EU numbering system; and (ix) combinations thereof.

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

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

18. The FcRn antagonist according to any one of claims 1 to 17, wherein the Fc domain or fragment thereof is indirectly linked to the albumin or fragment thereof via a linker.

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

20. The FcRn antagonist of any one of claims 1 to 17, wherein the Fc domain or fragment thereof is directly linked to albumin or a fragment thereof.

21. The FcRn antagonist of any one of claims 1 to 20, wherein the C-terminus of the Fc domain or fragment thereof is indirectly or directly linked to the N-terminus of albumin or a fragment thereof.

22. The FcRn antagonist of any one of claims 1 to 21, wherein the FcRn antagonist comprises two or more albumins or fragments thereof.

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

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

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

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

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

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

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

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

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, has developed anti-drug antibodies, or is at risk of developing anti-drug antibodies.

32. 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, wherein the FcRn antagonist is administered in an amount effective to: (i) reducing endogenous IgG levels by at least 1-fold (compared to the absence of FcRn antagonist administration); and / or (ii) reduces endogenous albumin levels by no more than 20% (compared to the absence of FcRn antagonist administration).

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. 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 achieved after administration of the FcRn antagonist comprising wild-type human albumin.

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

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

37. An FcRn antagonist, method or use according to claim 36, wherein the additional therapy is a steroid immunomodulator, plasmapheresis and / or IVIg therapy.

38. A kit for reducing circulating autoantibodies in a subject in need thereof, the kit 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 to reduce circulating autoantibodies in the subject; and (iii) optionally, at least one additional therapy.

39. A kit for treating or preventing the progression of an antibody-mediated disorder in a subject in need thereof, the kit 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 to treat or prevent the progression of the antibody-mediated disorder in the subject; and (iii) optionally, at least one additional therapy.

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