FC fragments that bind to FcRn and method of use

Abstract

This specification describes Fc fragment variants that bind to the neonatal Fc receptor (FcRn) and methods of use thereof, which effectively block the binding of IgG to FcRn. In certain embodiments, this specification describes methods for inhibiting the biological activity of FcRn. In certain embodiments, this specification describes pharmaceutical compositions comprising Fc fragments. In certain embodiments, the Fc fragments and methods described herein are used for the treatment of antibody-related diseases or disorders (e.g., autoimmune diseases or undesirable side effects of therapeutic antibodies).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 492,170, filed on 24 March 2023, which is incorporated in its entirety by reference herein.

[0002] This application includes a sequence listing XML file, which was filed electronically and is incorporated in its entirety herein by reference. The XML copy was created on 22 March 2024, is named VRD-018WO_SL.xml, and has a size of 29,298 bytes. [Background technology]

[0003] Immunoglobulin gamma (IgG) antibodies play a crucial role in the pathology of many disorders, including autoimmune diseases, inflammatory diseases, and disorders in which the pathology is characterized by overexpression of IgG antibodies (e.g., hypergammaglobulinemia) (see, for example, Junghans, Immunologic Research 16(1):29(1997)).

[0004] The half-life of serum IgG is prolonged compared to the serum half-lives of other plasma proteins (Roopenian et al, J. Immunology 170:3528 (2003), Junghans and Anderson, Proc. Natl. Acad. Sci. USA 93:5512 (1996)). This long half-life is partly due to the binding of the Fc region of IgG to the Fc receptor, FcRn (in this application, unless otherwise specified, the FcRn / b2m complex is generally referred to as its active form). FcRn was originally characterized as a neonatal transport receptor for maternal IgG, but in adults it also functions to protect IgG from degradation. FcRn prevents IgG from being transported to degradable lysosomes by binding to pinocytosed IgG and recycling it back into the extracellular compartment, where it can be released from FcRn and resume its biological function. This recycling is facilitated by pH-dependent binding of IgG to FcRn, where the IgG / FcRn interaction is strong at acidic endosomal pH but weak to absent at extracellular physiological pH. Therefore, at physiological pH, IgG is released from binding to FcRn, the antibody is recycled, and thus the antibody's half-life is extended.

[0005] In certain cases, such as autoimmune or inflammatory diseases, it is desirable to prevent IgG recycling. Preventing IgG recycling has previously been achieved through agents that reduce or block IgG binding to FcRn, or increase IgG binding to FcRn at extracellular physiological pH. An example of such an agent is an antibody against FcRn that blocks IgG binding (see, e.g., WO2002 / 43658). Peptides that bind to and antagonize FcRn function have also been disclosed in the art (see, e.g., US6,212,022 and US8,101,186). In addition, IgG molecules containing mutant Fc receptors with enhanced FcRn binding and reduced pH-dependent release have been identified (see, e.g., U.S. Patent No. 8,163,881). These IgG molecules occupy the FcRn receptor, preventing the FcRn receptor from binding to other IgG antibodies and recycling them. Fc fragments that form homodimers and contain two Fc regions occupying FcRn receptors have also been developed, exhibiting enhanced FcRn binding and reduced pH-dependent release (see, for example, US10,316,073). However, in the art, there is a need for additional and / or improved agents that reduce or block FcRn binding of such intact antibodies to IgG for use in treating antibody-mediated disorders caused by intact antibodies. [Overview of the project]

[0006] This disclosure provides, in particular, Fc fragment mutants that bind to the neonatal Fc receptor (FcRn) and have increased affinity for FcRn at endosomal pH (e.g., pH 6.0) and physiological pH (e.g., pH 7.4) compared to wild-type Fc fragments. As described herein, the present invention is in part based on the identification of a novel set of mutations in Fc that are remarkably effective in inhibiting IgG from binding to FcRn. Specifically, the Fc fragment mutants of this disclosure are characterized by having a low IC50 value (e.g., ≤2.5 nM) and high affinity for FcRn for blocking IgG from binding to FcRn at both endosomal pH and physiological pH (e.g., ≤10 nM and ≤300 nM, respectively). This is important because the Fc fragment mutants of the present invention can be used at lower doses and / or less frequently to achieve therapeutic effects compared to other Fc fragments. The Fc fragment variant of the present invention promises more potent treatment for pathogenic IgG-related diseases, including autoimmune diseases, myasthenia gravis, and thyroid eye disease (TED).

[0007] In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to a neonatal Fc receptor (FcRn) derived from human, cynomolgus monkey, mouse, or rat, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises at least one amino acid substitution selected from M428L, H433R, and N434Y. In some embodiments, the isolated Fc fragment comprises an additional amino acid substitution at one or more of the amino acid positions 252, 254, and 256 of SEQ ID NO: 1. In some embodiments, the additional amino acid substitutions are M252Y, S254T, and / or T256E.

[0008] In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises an amino acid substitution at position 428. In some embodiments, the amino acid substitution is M428L. In some embodiments, the Fc fragment comprises an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 433, and 434 of SEQ ID NO: 1. In some embodiments, the additional amino acid substitutions are M252Y, S254T, T256E, H433K, H433R, N434F, and / or N434Y.

[0009] In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to a neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution H433R. In some embodiments, the additional amino acid substitution is located at one or more of the amino acid positions 252, 254, 256, 428, and 434 of SEQ ID NO: 1. In some embodiments, one or more additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0010] In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution N434Y. In some embodiments, the Fc fragment comprises an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 428, and 433 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions are M252Y, S254T, T256E, M428L, H433K, and / or H433R.

[0011] In certain embodiments, the Fc fragment described herein is an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) derived from a human, cynomolgus monkey, mouse, or rat, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution H433K and an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 428, and 434. In certain embodiments, the additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0012] In some embodiments, the isolated Fc fragment described herein comprises a sequence selected from any one of sequence numbers 3 to 9.

[0013] The amino acid substitutions described herein result in a lower K for FcRn at pH 6.0 compared to IgG containing the wild-type Fc region. D and K for FcRn that can be measured at pH 7.4 D This results in an Fc fragment containing [a specific feature]. The Fc region of wild-type IgG is approximately 8 × 10 at pH 6.0. -7 K for FcRn of M D It possesses a high affinity for FcRn at pH 6.0 and / or its ability to remain bound to FcRn at pH 7.4 increases the occupancy rate of FcRn by the Fc fragment, and therefore reduces the amount of FcRn available to bind to IgG containing the wild-type Fc region. As a result, IgG containing the wild-type Fc region becomes more susceptible to degradation within lysosomes, effectively shortening its half-life.

[0014] In some embodiments, the amino acid substitution results in an extended Fc fragment half-life compared to the Fc fragment containing the wild-type human IgG1 Fc region. In some embodiments, the Fc fragment blocks or reduces the natural recycling of the IgG antibody. In some embodiments, the Fc fragment results in an increased catabolism of pathogenic IgG antibodies. In some embodiments, the pathogenic IgG antibodies are antibodies associated with autoimmune diseases.

[0015] In some embodiments, the Fc fragment has a K of about 1×10 -8 M or less, or about 1×10 -9 M, 2×10 -9 M, 3×10 -9 M, 4×10 -9 M, 5×10 -9 M, 6×10 -9 M, 7×10 -9 M, 8×10 -9 M, or 9×10 -9 M or less and binds to the FcRn sequence set forth in SEQ ID NOs: 10-17. In some embodiments, the Fc fragment has a K of about 1×10 D M or less, or about 1×10 -8 M, 2×10 -9 M, 3×10 -9 M, 4×10 -9 M, 5×10 -9 M , 6×10 -9 M, 7×10 -9 M, 8×10 -9 M, 9×10 -9 M, or 9×10 -9 M or less and binds to the FcRn sequence set forth in SEQ ID NO: 10 or 11. In some embodiments, the Fc fragment has a K of about 1×10 D M, or about 1×10 -8 M, 2×10 -9 M, 3×10 -9 M, 4×10 -9 M, 5×10 -9 M, 6×10 -9 M-9 M, 7×10 -9 M, 8×10 -9 M, or 9x10 -9 K below M D The Fc fragment then binds to the FcRn sequence described in Sequence ID No. 16 or 17. In some embodiments, the Fc fragment, when measured by surface plasmon resonance (SPR), has a value of approximately 1 × 10⁻¹⁶ at approximately pH 6.0. -8 M, 2×10 -8 M, 3×10 -8 M, 4×10 -8 M, 5×10 -8 M, 6×10 -8 M, 7×10 -8 M, 8×10 -8 M, or 9×10 -8 K below M D The Fc fragment then binds to the FcRn sequences described in Sequence IDs 10-17. In some embodiments, the Fc fragment, when measured by surface plasmon resonance (SPR), is approximately 1 × 10⁻¹⁶ at pH 6.0. -8 M, 2×10 -8 M, 3×10 -8 M, 4×10 -8 M, 5×10 -8 M, 6×10 -8 M, 7×10 -8 M, 8×10 -8 M, or 9×10 -8 K below M D The Fc fragment then binds to the FcRn sequence described in Sequence ID No. 12 or 13. In some embodiments, the Fc fragment, when measured by surface plasmon resonance (SPR), has a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 × 10¹⁶ at approximately pH 6.0. -10 K below M D The Fc fragment then binds to the FcRn sequences described in Sequence IDs 10-17. In some embodiments, the Fc fragment, when measured by surface plasmon resonance (SPR), is approximately 1 × 10⁻¹⁶ at pH 6.0. -10 M, 2×10 -10 M, 3×10 -10 M, 4×10 -10 M, 5×10 -10 M, 6×10 -10 M, 7×10 -10 M, 8×10-10 M, or 9×10 -10 K below M D Then, it binds to the FcRn sequence described in sequence number 14 or 15.

[0016] In some embodiments, the Fc fragment exhibits a melting temperature above 55°C when measured by differential scanning fluorescence (DSF).

[0017] In some embodiments, human Fc fragments exhibit aggregation temperatures of approximately 65°C or higher, or approximately 70°C or higher, as measured by static light scattering (SLS).

[0018] In certain embodiments, isolated human Fc fragments disclosed herein are used to treat antibody-related disorders or diseases, such as autoimmune diseases or disorders associated with undesirable side effects from therapeutic antibodies. In some embodiments, isolated human Fc fragments disclosed herein are used to treat diseases or disorders selected from the group consisting of generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuritis, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disorder (MOG antibody disorder), membranous nephropathy, lupus nephritis, thyroid eye disease, warm autoimmune hemolytic anemia, fetal and neonatal hemolytic disease, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, and cutaneous lupus erythematosus.

[0019] In some embodiments, the isolated Fc fragments disclosed herein are used to treat generalized myasthenia gravis (gMG). In some embodiments, the isolated Fc fragments disclosed herein are used to treat immune thrombocytopenia (ITP). In some embodiments, the treatment reduces disease severity in the patient, and disease severity is assessed by the gMG disease severity outcome scale.

[0020] In certain embodiments, what is described herein is an isolated polynucleotide or set of polynucleotides for the expression of an isolated Fc fragment of any of the embodiments disclosed herein. Thus, what is disclosed herein is an isolated polynucleotide or set of polynucleotides encoding an isolated Fc fragment of any of the embodiments disclosed herein, wherein the polynucleotide or set of polynucleotides optionally comprises mRNA or cDNA.

[0021] In certain embodiments, what is described herein is a vector or set of vectors for the expression of an isolated Fc fragment of any of the embodiments disclosed herein, comprising a polynucleotide or set of polynucleotides disclosed herein.

[0022] In certain embodiments, what is described herein is a host cell comprising a polynucleotide or set of polynucleotides, or a vector or set of vectors, as disclosed herein.

[0023] In certain embodiments, the method described herein is a method for producing an Fc fragment, comprising expressing the Fc fragment in a host cell disclosed herein and isolating the expressed Fc fragment.

[0024] In certain embodiments, what is described herein is a pharmaceutical composition comprising an isolated Fc fragment from any one of the embodiments disclosed herein and a pharmaceutically acceptable excipient.

[0025] In a particular embodiment, what is described herein is a kit comprising an isolated Fc fragment or a pharmaceutical composition disclosed herein, and instructions for use.

[0026] In certain embodiments, the methods described herein are for treating or preventing an antibody-related disorder or disease, such as an autoimmune disease or a disorder related to an undesirable side effect of a therapeutic antibody, in a mammalian subject requiring such treatment or prevention, comprising administering to the mammalian subject a therapeutically effective amount of an isolated Fc fragment or a pharmaceutical composition disclosed herein from any one of the embodiments disclosed herein. In some embodiments, the disease or disorder is selected from the group consisting of generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuritis, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disorder (MOG antibody disorder), membranous nephropathy, lupus nephritis, thyroid eye disease, warm autoimmune hemolytic anemia, fetal and neonatal hemolytic disease, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, and cutaneous lupus erythematosus. In some embodiments, the inflammatory disorder or disease is an autoimmune disease. In some embodiments, the inflammatory disorder or disease is gMG. In some embodiments, the method reduces disease severity in the patient, and disease severity is assessed by the gMG disease severity outcome scale.

[0027] In certain embodiments, the method described herein is for treating a mammalian subject that requires treatment for a pathology associated with elevated IgG levels, the method comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment of any one embodiment disclosed herein or a pharmaceutical composition disclosed herein.

[0028] In certain embodiments, the method described herein is for reducing the biological activity of IgG in a mammalian subject, comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment of any one embodiment disclosed herein or a pharmaceutical composition disclosed herein. In certain embodiments, the disease is an autoimmune disease.

[0029] In certain embodiments, the method described herein is a method for preventing a disorder in a mammalian subject requiring prevention of a disorder, the method comprising administering to the mammalian subject a therapeutically effective amount of an isolated Fc fragment or pharmaceutical composition described herein, wherein the disorder is an undesirable side effect of a therapeutic antibody.

[0030] In one embodiment, this embodiment provides an Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant includes amino acid substitutions M428L and N434F compared to the amino acid sequence described in SEQ ID NO: 1.

[0031] In some embodiments, the Fc fragment variant further includes an amino acid substitution at position 433 compared to the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the Fc fragment includes an amino acid substitution of H433K or H433R compared to the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the Fc fragment includes an amino acid substitution of N434F compared to the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the Fc fragment includes amino acid substitutions of H433K and N434F compared to the amino acid sequence described in SEQ ID NO: 1. In some embodiments, it includes amino acid substitutions of H433R and N434F compared to the amino acid sequence described in SEQ ID NO: 1.

[0032] In one embodiment, the present invention provides an Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant comprises amino acid substitutions (i)N434Y and (ii)H433R or H433K compared to the amino acid sequence described in SEQ ID NO: 1.

[0033] In some embodiments, the Fc fragment includes the amino acid substitutions H433K and N434Y compared to the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the Fc fragment includes the amino acid substitutions H433R and N434Y compared to the amino acid sequence described in SEQ ID NO: 1.

[0034] In one embodiment, the present invention provides an Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant comprises amino acid substitutions M428L and N434Y compared to the amino acid sequence described in SEQ ID NO: 1.

[0035] In some embodiments, the Fc fragment further comprises the H433K amino acid substitution.

[0036] In one embodiment, the present invention provides an Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant comprises amino acid substitutions M428L and H433R compared to the amino acid sequence described in SEQ ID NO: 1.

[0037] In some embodiments, the Fc fragment further comprises the N434Y amino acid substitution.

[0038] In one embodiment, the present invention provides an Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant comprises amino acid substitutions H433R and H434F compared to the amino acid sequence described in SEQ ID NO: 1.

[0039] In some embodiments, the Fc fragment further comprises the amino acid substitutions M252Y, S254T, and T256E.

[0040] In some embodiments, the Fc fragment includes amino acid substitutions M252Y, S254T, T256E, M428L, H433K, and N434F. In some embodiments, the Fc fragment includes amino acid substitutions M252Y, S254T, T256E, M428L, H433K, and N434Y. In some embodiments, the Fc fragment includes amino acid substitutions M252Y, S254T, T256E, H433K, and N434Y. In some embodiments, the Fc fragment includes amino acid substitutions M252Y, S254T, T256E, M428L, and N434F. In some embodiments, the Fc fragment includes amino acid substitutions M252Y, S254T, T256E, H433R, and N434F. In some embodiments, the Fc fragment includes amino acid substitutions M252Y, S254T, T256E, M428L, H433R, and N434F.

[0041] In some embodiments, the Fc fragment variant does not contain the L309D amino substitution.

[0042] In some embodiments, the Fc fragment includes an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment includes the amino acid sequence of SEQ ID NO: 3.

[0043] In some embodiments, the Fc fragment includes an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment includes the amino acid sequence of SEQ ID NO: 4.

[0044] In some embodiments, the Fc fragment includes an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment includes the amino acid sequence of SEQ ID NO: 5.

[0045] In some embodiments, the Fc fragment includes an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment includes the amino acid sequence of SEQ ID NO: 6.

[0046] In some embodiments, the Fc fragment includes an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment includes the amino acid sequence of SEQ ID NO: 7.

[0047] In some embodiments, the Fc fragment includes an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment includes the amino acid sequence of SEQ ID NO: 8.

[0048] In some embodiments, the Fc fragment includes an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment includes the amino acid sequence of SEQ ID NO: 9.

[0049] In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 2.6 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 2.5 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 2.3 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 2.2 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 2.0 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.9 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.8 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.7 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.6 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.5 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.4 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.3 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.2 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.1 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 1.0 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of less than 0.9 nM.In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn, with an IC50 value of less than 0.8 nM.

[0050] In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.5–2.2 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 0.7–2.0 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.8 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.6 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.5 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.4 nM. In some embodiments, the Fc fragment mutant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.3 nM. In some embodiments, the Fc fragment mutant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.2 nM. In some embodiments, the Fc fragment mutant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.2 nM. In some embodiments, the Fc fragment mutant inhibits IgG from binding to human FcRn with an IC50 value of approximately 0.7–1.0 nM.

[0051] In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 2.0 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.9 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.8 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.7 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.5 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.4 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.3 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.2 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.1 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 1.0 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 0.9 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 0.8 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 0.7 nM. In some embodiments, the Fc fragment variant inhibits IgG from binding to human FcRn with an IC50 value of 0.6 nM.

[0052] In some embodiments, the Fc fragment mutant effectively inhibits IgG from binding to human FcRn at a pH of approximately 5.8–7.5. In some embodiments, the Fc fragment mutant effectively inhibits IgG from binding to human FcRn at a pH of approximately 6.0–7.4.

[0053] In some embodiments, the Fc fragment mutant effectively inhibits IgG from binding to human FcRn at pH 6.0. In some embodiments, the Fc fragment mutant effectively inhibits IgG from binding to human FcRn at pH 7.0. In some embodiments, the Fc fragment mutant effectively inhibits IgG from binding to human FcRn at pH 7.4.

[0054] In some embodiments, the Fc fragment mutant inhibits IgG from binding to human FcRn at pH 6.0, with an IC50 value of approximately 0.7–2.2 nM. In some embodiments, the Fc fragment mutant inhibits IgG from binding to human FcRn at pH 7.4, with an IC50 value of approximately 0.7–2.2 nM.

[0055] In some embodiments, the Fc fragment is fused to or complexed with the half-life extension domain. In some embodiments, the half-life extension domain is a protein. In some embodiments, the half-life extension domain is a polypeptide. In some embodiments, the half-life extension domain is a peptide. In some embodiments, the half-life extension domain is an antibody. In some embodiments, the half-life extension domain is an antibody fragment. In some embodiments, the half-life extension domain is scFv. In some embodiments, the half-life extension domain is sdAb. In some embodiments, the half-life extension domain is Fab. In some embodiments, the half-life extension domain is VHH. In some embodiments, the half-life extension domain is or a variable novel antigen receptor (VNAR).

[0056] In some embodiments, the half-life extension domain is albumin. In some embodiments, the half-life extension domain is an albumin-binding domain. In some embodiments, the half-life extension domain is an aa HSA-binding domain.

[0057] In one embodiment, the present invention provides a method for inhibiting IgG from binding to FcRn by administering, among other things, an Fc fragment mutant as described herein. [Modes for carrying out the invention]

[0058] definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings generally understood by those skilled in the art. In some cases, terms having generally understood meanings are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from the generally understood meaning in the art. The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where necessary, unless otherwise specified, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and conditions defined by the manufacturers.

[0059] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0060] It is understood that the aspects and embodiments of the present invention described herein include "including," "consisting of," and "essentially consisting of."

[0061] For all compositions described herein, and for all methods using the compositions described herein, the compositions may include or "be essentially" of the listed components or steps. Where a composition is described as "be essentially" of the listed components, the composition may contain the listed components and other components that do not substantially affect the condition being treated but do not contain other components that substantially affect the condition being treated other than those explicitly listed. Or, where a composition does not contain any extra components other than those listed that substantially affect the condition being treated, the composition does not contain any extra components in a concentration or amount sufficient to substantially affect the condition being treated. Where a method is described as "be essentially" of the listed steps, the method may include the listed steps and other steps that do not substantially affect the condition being treated, but the method may not include any other steps that substantially affect the condition being treated other than those explicitly listed. As a non-limiting example, where a composition is described as "essentially" consisting of a certain component, the composition may further contain any amount of pharmaceutically acceptable carriers, vehicles, or diluents, and other such components, that does not substantially affect the condition being treated.

[0062] As used herein, the term “vector” refers to a nucleic acid molecule capable of replicating another nucleic acid it is linked to. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of operatively inducing the expression of linked nucleic acids. Such vectors are referred to herein as “expression vectors.”

[0063] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and the offspring of such cells. Host cells include “transformed” (or “transformed cell”) and “transfected” (or “transfected cell”), which respectively include primary transformed or transfected cells and their offspring. Such offspring may not have exactly the same nucleic acid content as the parent cells and may contain mutations. “Recombinant host cell” or “host cell” refers to a cell containing exogenous polynucleotides, regardless of the method used for insertion, e.g., direct incorporation, transduction, f-crossing, or any other method known in the art for producing recombinant host cells.

[0064] As used herein, the term “eukaryote” refers to organisms belonging to the phylogenetic region Eucarya, including but not limited to animals (mammals, insects, reptiles, birds, etc.), ciliates, plants (monocotyledons, dicotyledons, algae, etc.), fungi, yeasts, flagellates, microsporids, and protists.

[0065] As used herein, the term “prokaryote” refers to a prokaryote. For example, non-eukaryotes may belong to the phylogenetic region of Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) or Archaea (including, but not limited to, Methanococcus jannaschii, Methanobacterium thermautotrophicum, Halobacterium (e.g., Haloferax volcanii and Halobacterium species NRC-1), Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeropyrum pernix, etc.).

[0066] As used herein, “effective dose” or “therapeutic effective dose” refers to the amount of a therapeutic compound, such as an Fc fragment, administered to an individual as a single dose or as part of a series of doses, either alone or in combination with another therapeutic modality, that is effective in producing or contributing to a desired therapeutic effect. An example of a desired therapeutic effect is a reduction in the improvement of IgG levels for one or more symptoms. An effective dose may be given in one or more dosages.

[0067] The term “to treat” (and its variations such as “to treat” or “to cure”) refers to a clinical intervention that attempts to do so in a subject who requires an alteration of the natural course of a disease or condition. Treatment can be performed during the course of clinicopathology. Desired effects of treatment include prevention of disease relapse, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis.

[0068] The term "sufficient amount" means an amount sufficient to produce the desired effect, for example, an amount sufficient to modulate the immune response in the subject.

[0069] As used herein, the terms “subject” or “individual” mean a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human.

[0070] The term "in vitro" refers to the processes that occur in living cells that are isolated from a living organism and grown, such as living cells grown in tissue culture.

[0071] The term "in vivo" refers to processes that occur within a living organism.

[0072] The term “package insert” is used to refer to the instructions typically included in the market packaging (e.g., a kit) of such therapeutic or diagnostic products, including information relating to indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings for the use of such products.

[0073] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained herein is effective in treating the subject, and which does not contain additional ingredients that are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0074] The terms “co-administration,” “co-administration,” and “in combination with” include administering two or more therapeutic agents simultaneously, concurrently, or sequentially, without specific time constraints. In one embodiment, the agents are simultaneously present in cells or within the body of the subject, or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are the same composition or unit dosage form. In other embodiments, the therapeutic agents are distinct compositions or unit dosage forms. In certain embodiments, the first agent may be administered before the administration of the second therapeutic agent.

[0075] The terms "to regulate" and "to modulate" refer to reducing or inhibiting the described variable, or alternatively activating or increasing it.

[0076] The terms "increase" and "activate" refer to increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or more in the variable described.

[0077] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or more in the variable described.

[0078] The term “approximately” indicates and encompasses the given value and the range above and below that value. In certain embodiments, the term “approximately” indicates a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term “approximately” indicates a specified value(s) ± 1 standard deviation of that value(s).

[0079] For any of the structural and functional properties described herein, methods for determining these properties are known in the art.

[0080] The term "arbitrarily" when used consecutively means to include all of any of the listed combinations, intending all subcombinations.

[0081] The term "amino acid" refers to 20 common naturally occurring amino acids. These naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0082] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an Fc fragment) and its binding partner (e.g., an FcRn). Unless otherwise indicated, "affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an Fc fragment and an FcRn). Affinity is K D It is indirectly proportional to.

[0083] When the term "mutant" is used herein to refer to an amino acid sequence, it refers to an amino acid sequence that has one or more changes in its sequence compared to a reference amino acid sequence, and that has one or more substitutions compared to the reference amino acid sequence.

[0084] The term "kd" (sec-1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen or protein-protein interaction. This value is also referred to as the koff value.

[0085] The term "ka" (M-1 × sec-1), as used herein, refers to the rate constant of a particular antibody-antigen interaction or protein-protein association. This value is also referred to as the kon value.

[0086] When used herein, "KD" or "K D The term (M) refers to the dissociation equilibrium constant of a particular antibody-antigen or protein-protein interaction. KD = kd / ka. In some embodiments, protein affinity is described in terms of KD for the interaction between such protein and its binding partner. For clarity, as is known in the art, a smaller KD value indicates a higher affinity interaction, and a larger KD value indicates a lower affinity interaction.

[0087] The term "measurable KD" or "measurable K" as used in this specification. D The term "less than 1M, less than 0.1M, less than 0.01M, less than 0.001M, 1 × 10 -4 Less than M, 1 x 10 -5 Less than M, or 1 × 10 -6 This refers to a KD value less than M.

[0088] The term "KA" (M-1), as used herein, refers to the association equilibrium constant of a particular antibody-antigen or protein-protein interaction. KA = ka / kd.

[0089] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0090] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein to refer to antibodies that have a structure substantially similar to that of naturally occurring antibodies and that have a heavy chain containing an Fc region. For example, when used to refer to an IgG molecule, a “full-length antibody” is an antibody that contains two heavy chains and two light chains.

[0091] In this specification, the terms “Fc domain,” “Fc region,” or “Fc fragment” are used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. This term includes both the native sequence Fc region and the mutant Fc region.

[0092] "Fc fragment," as provided herein, refers to a fragment of the Fc domain that specifically binds to the target protein FcRn. In some embodiments, the Fc fragment is a variant of SEQ ID NO: 1.

[0093] The term "human Fc fragment" refers to an Fc fragment that corresponds to the amino acid sequence of the Fc region of an antibody produced by a human or human cell, or an Fc fragment derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequence (e.g., obtained from a human source or newly designed).

[0094] The term “substantially purified” means, in certain embodiments, substantially free of cytoplasmic material, recombinant-produced proteins, and in the case of such proteins, substantially or essentially free of proteins, i.e., components that normally accompany or interact with native cells or host cells, as found in their naturally occurring environment, and includes protein preparations having contaminating proteins of less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% (dry weight).

[0095] The ranges described herein are understood to be abbreviations for all values ​​within that range, including the endpoints described. For example, the range 1–50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0096] It should be noted that, as used herein and in the accompanying claims, the singular forms "a," "an," and "the" include plural referents unless otherwise explicitly indicated by the context.

[0097] Fc fragment Fc fragment structure The Fc fragment may include those described herein, such as the amino acid sequences listed in the table. In some embodiments, the Fc fragment is an IgG subclass IgG1, IgG2, or IgG4.

[0098] In a particular embodiment, the Fc fragment is produced by recombinant cells engineered to express a desired constant domain.

[0099] Sequence of FcRn-bound Fc fragments In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to a neonatal Fc receptor (FcRn) derived from human, cynomolgus monkey, mouse, or rat, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises at least one amino acid substitution selected from M428L, H433R, and N434Y. In some embodiments, the isolated Fc fragment comprises an additional amino acid substitution at one or more of the amino acid positions 252, 254, and 256 of SEQ ID NO: 1. In some embodiments, the additional amino acid substitutions are M252Y, S254T, and / or T256E.

[0100] In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises an amino acid substitution at position 428. In some embodiments, the amino acid substitution is M428L. In some embodiments, the Fc fragment comprises an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 433, and 434 of SEQ ID NO: 1. In some embodiments, the additional amino acid substitutions are M252Y, S254T, T256E, H433K, H433R, N434F, and / or N434Y.

[0101] In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to a neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution H433R. In some embodiments, the additional amino acid substitution is located at one or more of the amino acid positions 252, 254, 256, 428, and 434 of SEQ ID NO: 1. In some embodiments, one or more additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0102] In certain embodiments, the Fc fragments described herein are isolated Fc fragments that bind to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution N434Y. In some embodiments, the Fc fragment comprises an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 428, and 433 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions are M252Y, S254T, T256E, M428L, H433K, and / or H433R.

[0103] In certain embodiments, the Fc fragment described herein is an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) derived from a human, cynomolgus monkey, mouse, or rat, comprising a variant of the amino acid sequence described in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution H433K and an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 428, and 434. In certain embodiments, the additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0104] In a particular embodiment, the Fc fragment includes a sequence selected from the sequences described in sequence numbers 3 to 9.

[0105] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 3, provided that such a fragment contains the M252Y, S254T, T256E, M428L, H433K, and N434F mutations.

[0106] In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 85% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 88% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 91% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 92% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 93% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 94% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 97% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 98% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 99% identical to SEQ ID NO: 3. In some embodiments, the Fc fragment variant includes an amino acid sequence that is 100% identical to SEQ ID NO: 3. In some embodiments, for each of the aforementioned variants having percent identity with SEQ ID NO: 3, the variant includes the M252Y, S254T, T256E, M428L, H433K, and N434F mutations.

[0107] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 4, provided that such a fragment contains the M252Y, S254T, T256E, M428L, H433K, and N434Y mutations.

[0108] In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 85% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 88% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 91% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 92% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 93% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 94% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 97% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 98% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 99% identical to SEQ ID NO: 4. In some embodiments, the Fc fragment variant includes an amino acid sequence that is 100% identical to SEQ ID NO: 4. In some embodiments, for each of the aforementioned variants having percent identity with SEQ ID NO: 4, the variant includes the M252Y, S254T, T256E, M428L, H433K, and N434Y mutations.

[0109] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 5, provided that such a fragment contains the M252Y, S254T, T256E, H433K, and N434Y mutations.

[0110] In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 85% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 88% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 53. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 91% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 92% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 93% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 94% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 97% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 98% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 99% identical to SEQ ID NO: 5. In some embodiments, the Fc fragment variant includes an amino acid sequence that is 100% identical to SEQ ID NO: 5. In some embodiments, for each of the aforementioned variants having percent identity with SEQ ID NO: 5, the variant includes the M252Y, S254T, T256E, H433K, and N434Y mutations.

[0111] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 6, provided that such a fragment contains the M252Y, S254T, T256E, M428L, and N434F mutations.

[0112] In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 85% identical to SEQ ID NO: 36. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 88% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 91% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 92% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 93% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 94% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 97% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 98% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 99% identical to SEQ ID NO: 6. In some embodiments, the Fc fragment variant includes an amino acid sequence that is 100% identical to SEQ ID NO: 6. In some embodiments, for each of the aforementioned variants having percent identity with SEQ ID NO: 6, the variant includes the M252Y, S254T, T256E, M428L, and N434F mutations.

[0113] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 7, provided that such a fragment contains the M252Y, S254T, T256E, H433R, and N434Y mutations.

[0114] In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 85% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 88% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 91% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 92% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 93% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 94% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 97% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 98% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 99% identical to SEQ ID NO: 7. In some embodiments, the Fc fragment variant includes an amino acid sequence that is 100% identical to SEQ ID NO: 7. In some embodiments, for each of the aforementioned variants having percent identity with SEQ ID NO: 7, the variant includes the M252Y, S254T, T256E, H433R, and N434Y mutations.

[0115] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 8, provided that such a fragment contains the M252Y, S254T, T256E, H433R, and N434F mutations.

[0116] In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 85% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 88% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 91% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 92% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 93% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 94% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 97% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 98% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 99% identical to SEQ ID NO: 8. In some embodiments, the Fc fragment variant includes an amino acid sequence that is 100% identical to SEQ ID NO: 8. In some embodiments, for each of the aforementioned variants having percent identity with SEQ ID NO: 8, the variant includes the M252Y, S254T, T256E, H433R, and N434F mutations.

[0117] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 9, provided that such a fragment contains the M252Y, S254T, T256E, M428L, H433R, and N434F mutations.

[0118] In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 85% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 88% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 91% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 92% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 93% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 94% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 97% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 98% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is at least 99% identical to SEQ ID NO: 9. In some embodiments, the Fc fragment variant includes an amino acid sequence that is 100% identical to SEQ ID NO: 9. In some embodiments, for each of the aforementioned variants having percent identity with SEQ ID NO: 9, the variant includes the M252Y, S254T, T256E, M428L, H433R, and N434F mutations.

[0119] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 18, provided that such a fragment contains the mutations M252Y, S254T, T256E, L309D, H433K, and N434F.

[0120] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 19, provided that such a fragment contains the mutations M252Y, S254T, T256E, L309D, H433K, and N434Y.

[0121] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 20, provided that such a fragment contains the mutations M252Y, S254T, T256E, L309D, M428L, H433K, and N434F.

[0122] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 21, provided that such a fragment contains the mutations M252Y, S254T, T256E, L309D, M428L, H433K, and N434Y.

[0123] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 22, provided that such a fragment contains the mutations M252Y, S254T, T256E, L309D, Q311K, H433K, and N434F.

[0124] In certain embodiments, the Fc fragment contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing SEQ ID NO: 23, provided that such a fragment contains the mutations M252Y, S254T, T256E, L309D, Q311K, H433K, and N434Y.

[0125] For sequence comparison, generally, one sequence serves as the reference sequence on which the second sequence is compared. When using a sequence comparison algorithm, the test and reference sequences may be entered into the computer, subsequent coordinates may be specified as needed, and sequence algorithm program parameters may be specified. Any suitable algorithm may be used, including but not limited to the Smith-Waterman alignment algorithm, Viterbi, Bayesians, Hidden Markov, etc. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm can then be used to calculate the percentage sequence identity of the test sequence to the reference sequence based on the program parameters. Any suitable algorithm may be used to calculate the identity percentage. For example, some programs calculate the identity percentage as the number of aligned positions obtained by dividing the number of identical residues by the total number of aligned positions.

[0126] Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (e.g., Current Protocols in Molecular Biology (Ausubel et al., Eds. 1995)). This can be carried out by (see supplement) (each of these is incorporated herein in its entirety by reference). Exemplary computer software for determining identity between two sequences includes, but is not limited to, the GCG program package, Develeux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA Altschul, SF et al., J Molec. Biol., 215, 403 (1990) (each of these is incorporated herein in its entirety by reference).

[0127] In some embodiments, the Fc fragment includes an additional amino acid residue at the N-terminus or C-terminus. In some embodiments, the Fc fragment provided herein includes a C-terminal lysine residue. In some embodiments, the Fc fragment has an additional 1, 2, 3, 4, or 5 amino acid residues at the N-terminus and / or C-terminus.

[0128] Fc area Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the “Fc polypeptide” of dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide containing the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-assembly. For example, the Fc polypeptide of dimeric IgG Fc contains the IgG CH2 and IgG CH3 constant domain sequences. In certain embodiments, the Fc fragment disclosed herein contains the 226 amino acids at the C-terminus of the complete human IgG Fc region. Fc may be of class IgG and may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the Fc fragment described herein is IgG 1、 It is a subclass. In certain embodiments, the Fc fragment described herein is a variant of the human IgG1Fc region described in Sequence ID No. 1. In certain embodiments, the Fc fragment described herein is of the IgG2 subclass. In certain embodiments, the Fc fragment described herein is of the IgG2 subclass. 4、 It belongs to a subclass.

[0129] The terms “Fc receptor” and “FcR” are used to describe receptors that bind to the Fc region of an antibody. For example, an FcR can be a naturally occurring human FcR. Generally, FcRs are those that bind to IgG antibodies (gamma receptors) and include the FcγRI, FcγRII, and FcγRIII subclass receptors, which include allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. Other isotypes of immunoglobulins can also bind to certain FcRs (see, for example, Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). This term also includes FcRn, a neonatal receptor involved in the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976), and Kim et al., J.Immunol. 24:249 (1994)). The Fc fragments described herein selectively bind to FcRn. In certain embodiments, the Fc fragments selectively bind to mammalian FcRn, including cynomolgus monkey, rat, and / or mouse FcRn.

[0130] The activating receptor FcγRIIA contains an immunoreceptor tyrosine activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (as studied in Daeeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs have been studied in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are included in the term "FcR" as used herein.

[0131] Modifications within the CH2 domain can affect the binding of FcR to Fc. Several amino acid modifications within the Fc region are known in the art to selectively alter the affinity of Fc to different Fc gamma receptors.

[0132] The following are exemplary mutations that alter the binding of FcR to Fc: S298A / E333A / K334A, S298A / E333A / K334A / K326A(Lu Y, Vernes JM, Chiang N, et al.J Immunol Methods.2011 Feb 28;365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L(Stavenhagen JB, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep 15;67(18):8882-90, Nordstrom JL, Gorlatov S, Zhang W, et al.Breast Cancer Res.2011 Nov 30;13(6):R123); F243L(Stewart R,Thom G,Levens M,et al.Protein Eng Des Sel.2011 Sep;24(9):671-8.), S298A / E333A / K334A(Shields RL,Namenuk AK,Hong K,et al.J BiolChem.2001 Mar 2;276(9):6591-604); S239D / I332E / A330L, S239D / I332E(Lazar GA,Dang W,Karki S,et al.Proc Natl Acad Sci US A.2006 Mar 14;103(11):4005-10); S239D / S267E,S267E / L328F(Chu SY, Vostiar I, Karki S, et al.Mol Immunol.2008 Sep;45(15):3926-33); S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and other variants listed in WO2011 / 120134 and WO2011 / 120135 (incorporated herein by reference). Therapeutic Antibody Engineering (William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 37 9, Oct 2012) lists the mutations on page 283.

[0133] In certain embodiments, the Fc fragment disclosed herein is a variant of SEQ ID NO: 1 (e.g., containing one or more amino acid substitutions compared to SEQ ID NO: 1), and the one or more substitutions result in a measurable K at pH 7.4 compared to the Fc fragment of SEQ ID NO: 1.D As a result, the Fc fragment of SEQ ID NO: 1 does not bind detectably to FcRn at pH 7.4 unless one or more substitutions are present.

[0134] In certain embodiments, one or more amino acid substitutions result in an extended Fc fragment half-life at pH 6.0 compared to an Fc fragment containing the wild-type Fc region.

[0135] join The affinity of molecule X for its partner Y is given by the dissociation equilibrium constant (K D It can be expressed by ). The kinematic components contributing to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) techniques (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0136] Regarding the binding of Fc fragments to target molecules, the terms "bound to," "specifically bound," "specifically bound to," "specific to," "selectively bound to," and "selectively bound to" all refer to binding that is measurably different from nonspecific or nonselective interactions (e.g., interactions with non-target molecules). Specific binding can be measured, for example, by measuring the binding to a target molecule (e.g., FcRn) and comparing it to the binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the target molecule. In this case, specific binding is indicated if the binding of the Fc fragment to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the Fc fragment to a non-target molecule is less than about 50% of the affinity to FcRn (i.e., K to FcRn). D This is K against non-targets D(Twice as low as) In some embodiments, the affinity of the Fc fragment to non-target molecules is less than about 40% of the affinity to FcRn. In some embodiments, the affinity of the Fc fragment to non-target molecules is less than about 30% of the affinity to FcRn. In some embodiments, the affinity of the Fc fragment to non-target molecules is less than about 20% of the affinity to FcRn. In some embodiments, the affinity of the Fc fragment to non-target molecules is less than about 10% of the affinity to FcRn. In some embodiments, the affinity of the Fc fragment to non-target molecules is less than about 1% of the affinity to FcRn. In some embodiments, the affinity of the Fc fragment to non-target molecules is less than about 0.1% of the affinity to FcRn.

[0137] Where used herein in relation to a first Fc fragment and a second Fc fragment or wild-type IgG ("second molecule"), the terms "compete with" or "cross-compete with" indicate that the first Fc fragment and the second molecule compete for binding to a target (e.g., FcRn). In one exemplary assay, FcRn is coated onto a surface, contacted with the first Fc fragment, and then the second molecule is added. In another exemplary assay, the first Fc fragment is coated onto a surface, contacted with FcRn, and then the second molecule is added. In either assay, if the presence of the first Fc fragment reduces the binding of the second molecule, the Fc fragment competes with the second molecule. The term "compete with" also includes combinations in which the first Fc fragment reduces the binding of the second molecule, but no competition is observed when the first Fc fragment and the second molecule are added in the reverse order. However, in some embodiments, the first Fc fragment and the second molecule inhibit each other's binding to FcRn, regardless of the order in which they are added. In some embodiments, the first Fc fragment, when measured in a competitive binding assay, reduces the binding of the second molecule to its receptor by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. Those skilled in the art can select the concentrations of the first Fc fragment and the second molecule used in the competitive assay based on their affinity for FcRn. The assays described in this definition are illustrative, and those skilled in the art can utilize any suitable assay to determine whether the Fc fragments compete with each other.Suitable assays are described, for example, in Cox et al., “Immunoassay Methods,” in Assay Guidance Manual [Internet], updated December 24, 2014 (ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015), Silman et al., Cytometry, 2001, 44:30-37, and Finco et al., J.Pharm.Biomed.Anal., 2011, 54:351-358 (each of these is incorporated herein by reference in its entirety).

[0138] If an excess of test Fc fragment (e.g., at least 2x, 5x, 10x, 20x, or 100x) is measured in a competitive binding assay, for example, if it inhibits or blocks the binding of the second molecule by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, then the test (or first) Fc fragment competes with the second molecule (e.g., the reference Fc fragment). For example, a second competing Fc fragment can be identified by its ability to compete with the first Fc fragment described herein for binding to FcRn. In certain cases, the second molecule can block or inhibit the binding of the first Fc fragment by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, if measured in a competitive binding assay. In certain cases, the second molecule can super-substitute the first Fc fragment by 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0139] In a particular embodiment, the Fc fragment binds to the FcRn sequence described in sequence numbers 10-17.

[0140] In a particular embodiment, when measured by surface plasmon resonance (SPR), the Fc fragment has a density of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 × 10¹⁶ at pH 6.0. -8 K below M Dand binds to the FcRn sequences set forth in SEQ ID NOs: 10-17. In certain embodiments, the Fc fragment has a K of about 1×10 -8 M or less at about pH 6.0, as measured by surface plasmon resonance (SPR), D and binds to the human FcRn sequence. In certain embodiments, the Fc fragment has a K of about 1×10 -9 M or less at pH 6.0, as measured by surface plasmon resonance (SPR), D and binds to the FcRn sequences set forth in SEQ ID NOs: 10-17. I

[0141] In certain embodiments, the Fc fragment has a lower K at pH 6.0, compared to an Fc fragment comprising a wild-type Fc region at the same pH, D and binds to FcRn.

[0142] In some embodiments, the Fc fragments provided herein have a K of about 1×10 -8 1.1×10 -8 1.2×10 -8 1.3×10 -8 1.4×10 -8 1.5×1,0 -8 1.6×10 -8 1.7×10 -8 1.8×10 -8 1.9×10 -8 1.95×10 -8 2×10 -8 2,5×10 -8 3×10 -8 3.5×10 -8 4×10 -8 4.5×10 -8 5×10 -8 6×10 -8 7×10 -8 8×10 -8 9×10 -8 1×10 -9 1.1×10 -9 1.2×10 -9 1.3×10 -9 1.4×10 -9, 1.5×10 -9 , 1.6×10 -9 , 1.7×10 -9 , 1.8×10 -9 , 1.9×10 -9 , 1.95 × 10 -9 , 2×10 -9 , 2.5×10 -9 , 3 x 10 -9 , 3.5×10 -9 , 4×10 -9 , 4.5×10 -9 , 5×10 -9 , 6×10 -9 , 7×10 -9 , 8×10 -9 , 9×10 -9 , 1 x 10 -10 , 1.1 × 10 -10 , 1.2 × 10 -10 , 1.3 × 10 -10 , 1.4×10 -10 , 1.5×10 -10 , 1.6×10 -10 , 1.7×10 -10 , 1.8×10 -10 , 1.9×10 -10 , 1.95 × 10 -10 , 2×10 -10 , 2.5×10 -10 , 3 x 10 -10 , 3.5×10 -10 , 4×10 -10 , 4.5×10 -10 , 5×10 -10 , 6×10 -10 , 7×10 -10 , 8×10 -10 , or 9×10 -10 K below M D It then binds to FcRn at a pH of 6.0.

[0143] In some embodiments, for the binding of FcRn at pH 6.0, the K of the Fc fragment provided herein D When measured by ELISA or any other suitable method known in the art, it is approximately 1.0 to 1.1 × 10⁻⁶. -8 M, 1.1~1.2×10 -8 M, 1.2~1.3×10-8 M, 1.3~1.4×10 -8 M, 1.4~1.5×10 -8 M, 1.5~1.6×10 -8 M, 1.6~1.7×10 -8 M, 1.7~1.8×10 -8 M, 1.8~1.9×10 -8 M, 1.9~2×10 -8 M, 1-2 x 10 -8 M, 1-5 x 10 -8 M, 2-7 x 10 -8 M, 3-8 x 10 -8 M, 3-5 x 10 -8 M, 4-6 x 10 -8 M, 5-7 x 10 -8 M, 6-8 x 10 -8 M, 7-9 x 10 -8 M, 7-9.9 x 10 -8 M, 5-9.9 x 10 -8 M, 1.0~1.1×10 -9 M, 1.1~1.2×10 -9 M, 1.2~1.3×10 -9 M, 1.3~1.4×10 -9 M, 1.4~1.5×10 -9 M, 1.5~1.6×10 -9 M, 1.6~1.7×10 -9 M, 1.7~1.8×10 -9 M, 1.8~1.9×10 -9 M, 1.9~2×10 -9 M, 1-2 x 10 -9 M, 1-5 x 10 -9 M, 2-7×10 -9 M, 3-8 x 10 -9 M, 3-5 x 10 -9 M, 4-6 x 10 -9 M, 5-7 x 10 -9 M, 6-8 x 10 -9 M, 7-9 x 10 -9 M, 7-9.9 x 10 -9 M, or 5-9.9 × 10 -9 It starts from M. In some embodiments, the Fc fragment provided herein, when measured by ELISA or any other suitable method known in the art, is about 1 × 10⁻¹⁶. -8M or less, or approximately 1 × 10 -9 K below M D It then binds to FcRn at pH 6.0.

[0144] In some embodiments, the Fc fragments provided herein, when measured by ELISA or any other suitable method known in the art, are approximately 1 × 10⁻¹⁶. -5 , 1.1 × 10 -5 , 1.2 × 10 -5 , 1.3 × 10 -5 , 1.4×10 -5 , 1.5×10 -5 , 1.6×10 -5 , 1.7×10 -5 , 1.8×10 -5 , 1.9×10 -5 , 1.95 × 10 -5 , 2×10 -5 , 2.5×10 -5 , 3 x 10 -5 , 3.5×10 -5 , 4×10 -5 , 4.5×10 -5 , 5×10 -5 , 6×10 -5 , 7×10 -5 , 8×10 -5 , 9×10 -5 , 1 x 10 -6 , 1.1 × 10 -6 , 1.2 × 10 -6 , 1.3 × 10 -6 , 1.4×10 -6 , 1.5×10 -6 , 1.6×10 -6 , 1.7×10 -6 , 1.8×10 -6 , 1.9×10 -6 , 1.95 × 10 -6 , 2×10 -6 , 2.5×10 -6 , 3 x 10 -6 , 3.5×10 -6 , 4×10 -6 , 4.5×10 -6 , 5×10 -6 , 6×10 -6 , 7×10 -6 , 8×10 -6,9×10 -6 , 1 x 10 -7 1.1 × 10 -7 , 1.2 × 10 -7 , 1.3 × 10 -7 , 1.4×10 -7 , 1.5×10 -7 , 1.6×10 -7 , 1.7×10 -7 , 1.8×10 -7 , 1.9×10 -7 , 1.95 × 10 -7 , 2×10 -7 , 2.5×10 -7 , 3 x 10 -7 , 3.5×10 -7 , 4×10 -7 , 4.5×10 -7 , 5×10 -7 , 6×10 -7 , 7×10 -7 , 8×10 -7 , 9×10 -7 The above K D Then, it binds to FcRn at a pH of 7.4.

[0145] In some embodiments, for the binding of FcRn at pH 6.0, the K of the Fc fragment provided herein D When measured by ELISA or any other suitable method known in the art, it is approximately 1.0 to 1.1 × 10⁻⁶. -5 M, 1.1~1.2×10 -5 M, 1.2~1.3×10 -5 M, 1.3~1.4×10 -5 M, 1.4~1.5×10 -5 M, 1.5~1.6×10 -5 M, 1.6~1.7×10 -5 M, 1.7~1.8×10 -5 M, 1.8~1.9×10 -5 M, 1.9~2×10 -5 M, 1-2 x 10 -5 M, 1-5 x 10 -5 M, 2-7 x 10 -5 M, 3-8 x 10 -5 M, 3-5 x 10 -5 M, 4-6 x 10 -5 M, 5-7 x 10-5 M、6~8×10 -5 M、7~9×10 -5 M、7~9.9×10 -5 M、5~9.9×10 -5 M、1.0~1.1×10 -6 M、1.1~1.2×10 -6 M、1.2~1.3×10 -6 M、1.3~1.4×10 -6 M、1.4~1.5×10 -6 M、1.5~1.6×10 -6 M、1.6~1.7×10 -6 M、1.7~1.8×10 -6 M、1.8~1.9×10 -6 M、1.9~2×10 -6 M、1~2×10 -6 M、1~5×10 -6 M、2~7×10 -6 M、3~8×10 -6 M、3~5×10 -6 M、4~6×10 -6 M、5~7×10 -6 M、6~8×10 -6 M、7~9×10 -6 M、7~9.9×10 -6 M、5~9.9×10 -6 M、1.0~1.1×10 -7 M、1.1~1.2×10 -7 M、1.2~1.3×10 -7 M、1.3~1.4×10 -7 M、1.4~1.5×10 -7 M、1.5~1.6×10 -7 M、1.6~1.7×10 -7 M、1.7~1.8×10 -7 M、1.8~1.9×10 -7 M、1.9~2×10 -7 M、1~2×10 -7 M、1~5×10 -7 M、2~7×10 -7 M、3~8×10 -7 M、3~5×10 -7 M、4~6×10 -7 M、5~7×10 -7 M、6~8×10 -7M, 7-9 x 10 -7 M, 7-9.9 x 10 -7 M, 5-9.9 x 10 -7 It starts from M. In some embodiments, the Fc fragment provided herein, when measured by ELISA or any other suitable method known in the art, is about 1 × 10⁻¹⁶. -5 M or larger, or approximately 1 x 10 -6 M or higher K D It then binds to FcRn at pH 7.4.

[0146] In some embodiments, when the Fc fragment is measured by surface plasmon resonance (SPR), it is approximately 1 × 10⁻⁶ at pH 6.0. -8 M, 2×10 -8 M, 3×10 -8 M, 4×10 -8 M, 5×10 -8 M, 6×10 -8 M, 7×10 -8 M, 8×10 -8 M, 9×10 -8 M, 1×10 -9 M, 2×10 -9 M, 3×10 -9 M, 4×10 -9 M, 5×10 -9 M, 6×10 -9 M, 7×10 -9 M, 8×10 -9 M, or 9×10 -9 K below M D The Fc fragment then binds to the FcRn sequence described in Sequence ID No. 10 or 11. In some embodiments, the Fc fragment, when measured by surface plasmon resonance (SPR), is approximately 1 × 10⁻¹⁶ at pH 6.0. -8 M, 2×10 -8 M, 3×10 -8 M, 4×10 -8 M, 5×10 -8 M, 6×10 -8 M, 7×10 -8 M, 8×10 -8 M, 9×10 -8 K below M DThe FcRn sequence is then bound to the sequence described in Sequence ID No. 12 or 13. In some embodiments, the Fc fragment is approximately 1 × 10⁻¹⁶ at pH 6.0, as measured by surface plasmon resonance (SPR). -10 M, 2×10 -10 M, 3×10 -10 M, 4×10 -10 M, 5×10 -10 M, 6×10 -10 M, 7×10 -10 M, 8×10 -10 M, or 9×10 -10 K below M D The FcRn sequence is then bound to the sequence described in Sequence ID No. 14 or 15. In some embodiments, the Fc fragment is approximately 1 × 10⁻¹⁶ at pH 6.0, as measured by surface plasmon resonance (SPR). -9 M, 2×10 -9 M, 3×10 -9 M, 4×10 -9 M, 5×10 -9 M, 6×10 -9 M, 7×10 -9 M, 8×10 -9 M, or 9×10 -9 K below M D Then, it binds to the FcRn sequence described in sequence number 16 or 17.

[0147] In some embodiments, when measured by surface plasmon resonance (SPR), the Fc fragment yields approximately 1 × 10⁻¹⁶ fragments at pH 7.4. -7 M, 2×10 -7 M, 3×10 -7 M, 4×10 -7 M, 5×10 -7 M, 6×10 -7 M, 7×10 -7 M, 8×10 -7 M, or 9×10 -7 M or higher K D The FcRn sequence is then bound to the sequence number 10 or 11 described in sequence number 10 or 11. In some embodiments, the Fc fragment is approximately 1 × 10⁻¹⁶ at pH 7.4, as measured by surface plasmon resonance (SPR). -7 M, 2×10 -7 M, 3×10 -7M, 4 × 10 -7 M, 5 × 10 -7 M, 6 × 10 -7 M, 7 × 10 -7 M, 8 × 10 -7 M, 9 × 10 -7 M, 1 × 10 -6 M, 2 × 10 -6 M, 3 × 10 -6 M, 4 × 10 -6 M, 5 × 10 -6 M, 6 × 10 -6 M, 7 × 10 -6 M, 8 × 10 -6 M, or 9 × 10 -6 K of M or more D and binds to the FcRn sequence described in SEQ ID NO: 12 or 13. In some embodiments, when measured by surface plasmon resonance (SPR), the Fc fragment is about 1 × 10 at pH 7.4 -9 M, 2 × 10 -9 M, 3 × 10 -9 M, 4 × 10 -9 M, 5 × 10 -9 M, 6 × 10 -9 M, 7 × 10 -9 M, 8 × 10 -9 M, or 9 × 10 -9 K of M or more D and binds to the FcRn sequence described in SEQ ID NO: 14 or 15. In some embodiments, when measured by surface plasmon resonance (SPR), the Fc fragment is about 1 × 10 at pH 7.4 -8 M, 2 × 10 -8 M, 3 × 10 -8 M, 4 × 10 -8 M, 5 × 10 -8 M, 6 × 10 -8 M, 7 × 10 -8 M, 8 × 10 -8 M, or 9 × 10 -8 K of M or more D and binds to the FcRn sequence described in SEQ ID NO: 16 or 17.

[0148] Fc fragment variant fusion protein In some embodiments, the Fc fragment variant of the present invention is fused or complexed with various proteins, peptides, and antibody fragments.

[0149] In some embodiments, the Fc fragment variant is fused or complexed with a Fab. In some embodiments, the Fc fragment variant is fused or complexed with a scFv. In some embodiments, the Fc fragment variant is fused or complexed with a sdAb. In some embodiments, the Fc fragment variant is fused or complexed with a VHH. In some embodiments, the Fc fragment variant is fused or complexed with a VNAR.

[0150] In some embodiments, the Fc fragment variant is fused or complexed with an albumin binding domain. In some embodiments, the Fc fragment variant is fused or complexed with an albumin binding Fab. In some embodiments, the Fc fragment variant is fused or complexed with an albumin binding scFv. In some embodiments, the Fc fragment variant is fused or complexed with an albumin binding sdAb. In some embodiments, the Fc fragment variant is fused or complexed with an albumin binding VHH. In some embodiments, the Fc fragment variant is fused or complexed with an albumin binding VNAR.

[0151] In some embodiments, the Fc fragment variant is fused or complexed with albumin or a variant thereof.

[0152] In some embodiments, the Fc fragment variant is fused or complexed with an HSA binding domain. In some embodiments, the Fc fragment variant is fused or complexed with an HSA binding Fab. In some embodiments, the Fc fragment variant is fused or complexed with an HSA binding scFv. In some embodiments, the Fc fragment variant is fused or complexed with an HSA binding sdAb. In some embodiments, the Fc fragment variant is fused or complexed with an HSA binding VHH. In some embodiments, the Fc fragment variant is fused or complexed with an HSA binding VNAR.

[0153] Pharmaceutical composition This application provides compositions comprising Fc fragments, comprising a pharmaceutical composition comprising one or more Fc fragments described herein, having one or more pharmaceutically acceptable excipients. In some embodiments, the compositions are sterile. The pharmaceutical compositions generally contain an effective amount of Fc fragments.

[0154] These compositions may include, in addition to one or more of the Fc fragments disclosed herein, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials are non-toxic and must not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other materials may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0155] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain physiological saline, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0156] For intravenous, cutaneous, or subcutaneous injection, or injection at the site of pain, the active ingredient may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has a suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using an isotonic vehicle such as sodium chloride injection, Ringer's injection, or Ringer's lactate injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0157] The Fc fragment intended for administration to an individual may be administered in a “therapeutic effective dose” or a “preventive effective dose” (in some cases, prevention may be considered treatment), as long as it is sufficient to demonstrate the benefit to the individual. The actual amount administered, as well as the rate and time course of administration, depends on the nature and severity of the protein aggregation disorder being treated. The determination of the prescription of treatment, e.g., dosage, is the responsibility of the general practitioner and other physicians, and typically takes into account the disorder being treated, the individual patient’s condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols described above can be found in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0158] In certain embodiments, the pharmaceutical compositions described herein are formulated for intravenous injection.

[0159] Depending on the condition being treated, the compositions may be administered simultaneously or sequentially, alone, or in combination with other treatments.

[0160] method Preparation method The Fc fragments described herein can be prepared using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the Fc fragment described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the Fc fragment that binds to the FcRn described herein. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In one embodiment, the nucleic acid is provided in a multicistron vector. In a further embodiment, a host cell comprising such nucleic acid is provided. In such one embodiment, the host cell comprises a vector (e.g., transformed with the vector) comprising the nucleic acid encoding the amino acid sequence comprising the Fc fragment disclosed herein. In one embodiment, the host cell is a eukaryotic cell, for example, a Chinese hamster ovary (CHO) cell, or a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing an Fc fragment is provided, which includes culturing host cells containing nucleic acids encoding an Fc fragment under conditions suitable for the expression of the Fc fragment, as provided above, and optionally recovering the Fc fragment from the host cells (or host cell culture medium).

[0161] For the recombination of Fc fragments, for example, the nucleic acid encoding the Fc fragment is isolated, as described above, and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to the gene encoding the Fc fragment).

[0162] When the Fc fragment is recombinantly produced by host cells, the protein in a particular embodiment is present in the culture medium at approximately 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or less of the dry weight of the cells. In certain embodiments, the "substantially purified" Fc fragments produced by the methods described herein have purity levels of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, and at least about 70%, specifically 75%, 80%, and 85%, and more specifically, at least about 90%, at least about 95%, at least about 99%, or higher, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0163] Suitable host cells for cloning or expressing vectors encoding Fc fragments include prokaryotic or eukaryotic cells as described herein.

[0164] Recombinant host cells or host cells refer to cells containing exogenous polynucleotides, regardless of the method used for insertion, e.g., direct incorporation, transduction, f-crossing, or other methods known in the art for constructing recombinant host cells. The exogenous polynucleotides may be maintained as non-integrated vectors, e.g., plasmids, or alternatively, integrated into the host genome. Examples of host cells include CHO, derivatives of CHO, NS0, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0165] For example, Fc fragments can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of Fc fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of Fc fragments in E. coli.) After expression, the Fc fragments may be isolated from the bacterial cell paste in the soluble fraction and further purified.

[0166] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding Fc fragments, including fungal and yeast strains whose glycosylation pathways are "humanized," resulting in the production of Fc fragments with partially or completely human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0167] Suitable host cells for the expression of glycosylated Fc fragments also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used, in conjunction with insect cells, particularly for the transfection of armyworm cells.

[0168] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for producing antibodies in transgenic plants).

[0169] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include simian kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), simian kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells described in, for example, Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), as well as myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0170] In certain embodiments, the Fc fragments described herein are produced in stable mammalian cells by a method comprising transfecting at least one stable mammalian cell with a nucleic acid encoding the Fc fragment at a predetermined ratio and expressing the nucleic acid in the at least one mammalian cell. In some embodiments, the predetermined ratio of the nucleic acid is determined in transient transfection experiments to determine the relative ratio of input nucleic acid that results in the highest proportion of Fc fragment in the expression product.

[0171] In some embodiments, a method for producing glycosylated Fc fragments in stable mammalian cells as described herein, the method comprising identifying and purifying a desired glycosylated Fc fragment. In certain embodiments, mammalian cells are selected and cultured under conditions for producing a larger percentage of the desired glycosylated Fc fragment. In some embodiments, the identification of the desired glycosylated Fc fragment is by liquid chromatography and / or mass spectrometry.

[0172] If necessary, Fc fragments can be purified or isolated after expression. Proteins can be isolated or purified by various methods known to those skilled in the art. Standard purification methods include ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and chromatographic techniques, including reversed phase, performed at atmospheric pressure or high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoresis, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques are also useful in conjunction with protein concentration. As is well known in the art, various native proteins bind to Fc, and these proteins can be found to be used in the present invention for the purification of Fc fragments. For example, bacterial proteins A and G bind to the Fc region. Purification can often be made possible by a specific fusion partner. For example, antibodies, glutathione resin if GST fusion is employed, and Ni if His tag is employed. +2 Affinity chromatography may be used, and if flag tags are employed, purification may be performed using immobilized anti-flag antibodies. For general guidance on preferred purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Ed. Scopes, Springer-Verlag, NY, 1994, which is incorporated in its entirety by reference.

[0173] In certain embodiments, the Fc fragment is purified using anion exchange chromatography, including but not limited to chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q and DEAE columns.

[0174] In certain embodiments, the proteins described herein are purified using cation exchange chromatography, including but not limited to SP-sepharose, CM sepharose, poros HS, poros CM, Tyopearl SP, Tyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and equivalents.

[0175] In addition, the Fc fragments described herein can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, polypeptides corresponding to polypeptide fragments can be synthesized using a peptide synthesizer. Furthermore, non-classical amino acids or chemical amino acid analogs can be introduced as substitutions or additions to the polypeptide sequence, if desired. Non-classical amino acids include, but are not limited to, D isomers of common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoroamino acids, methylamino acids, C-methylamino acids, N-methylamino acids, and other designer amino acids and amino acid analogs. Furthermore, amino acids can be D (dextrorotatory) or L (levorotatory).

[0176] How to use In some embodiments, the present application provides a method for contacting FcRn with an Fc fragment described herein, for example, a method for contacting an intended Fc fragment in vivo or ex vivo that results in inhibition of IgG binding to an FcRn receptor. In some embodiments, FcRn is expressed on the cell surface.

[0177] In some embodiments, the application provides a method for using the isolated Fc fragments described herein for the treatment of a disorder or disease in a subject. In certain embodiments, described herein is a method for treating a subject in need of treatment with the Fc fragments described herein, the method comprising administering a therapeutically effective amount of the Fc fragments or pharmaceutical composition containing the Fc fragments described herein to a mammalian subject. In certain embodiments, the application provides a method for treating a disorder or disease associated with elevated IgG levels in a subject by administering the disclosed Fc fragments.

[0178] In certain embodiments, the method described herein is for treating a pathology related to IgG activity, comprising administering to a mammalian subject a therapeutically effective amount of an isolated Fc fragment or a pharmaceutical composition comprising an isolated Fc fragment as described herein.

[0179] In certain embodiments, the method described herein is for treating a mammalian subject that requires treatment for a pathology associated with elevated FcRn levels, the method comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment or pharmaceutical composition described herein.

[0180] In certain embodiments, the methods described herein are for treating or preventing an antibody-related disorder or disease, such as an autoimmune disease or a disorder related to an undesirable side effect of a therapeutic antibody, in a mammalian subject requiring such treatment or prevention, comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment of any one embodiment disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the inflammatory disorder or disease is an autoimmune disease. In some embodiments, the inflammatory disorder or disease is myasthenia gravis (gMG). In some embodiments, the inflammatory disorder or disease is immune thrombocytopenia (ITP).

[0181] In certain embodiments, the method described herein is for treating a mammalian subject that requires treatment for a pathology associated with elevated IgG levels, the method comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment of any one embodiment disclosed herein or a pharmaceutical composition disclosed herein.

[0182] In certain embodiments, the method described herein is a method for reducing the biological activity of IgG in a mammalian subject requiring such reduction, comprising administering to the mammalian subject a therapeutically effective amount of an isolated Fc fragment of any one of the embodiments disclosed herein or a pharmaceutical composition disclosed herein.

[0183] In certain embodiments, methods for treating or preventing autoimmune diseases are described herein. In certain embodiments, the autoimmune disease is caused by autoreactive antibodies.

[0184] In some embodiments, autoimmune diseases include allogeneic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic antibodies (ANCA), adrenal autoimmune diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman syndrome, celiac spruce dermatitis, and chronic fatigue immune dysfunction syndrome. Syndrome), chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, bullous pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, dilated cardiomyopathy, lupus discoid, acquired epidermolysis bullosa, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré disease, Goodpasture syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic membranous neuropathy Neuropathies, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, IgM polyneuritis, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease, lichen planus, lichen sclerosing atrophic, systemic lupus erythematosus, Meniere's disease, mixed connective tissue disease, mucosal pemphigoid, multiple sclerosis, type 1 diabetes, multifocal motor neuropathy (MMN), myasthenia gravis, paraneoplastic bullous pemphigoidPemphigoid, pemphigoid of pregnancy, pemphigus vulgaris, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychondritis, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, solid organ transplant rejection, Stiffman syndrome, systemic lupus erythematosus, Takayasu's arteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, dermatitis herpetiformis The group consists of vasculitis, anti-neutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegner's granulomatosis.

[0185] Methods of administration and manufacture In some embodiments, the methods provided herein are useful for treating diseases or disorders in an individual. In some embodiments, the individual is a human, and the treatment comprises the administration of the Fc fragment described herein.

[0186] In some embodiments, the Fc fragment is administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intravenously, or intranasally. In certain embodiments, the Fc fragment is administered intravenously. An effective dose of the Fc fragment may be administered for the treatment of a disease or disorder. The appropriate dosage of the Fc fragment may be determined based on the type of disease or disorder being treated, the type of Fc fragment, the severity and course of the disease or disorder, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician. [Examples]

[0187] The following are examples of specific embodiments for carrying out the present invention. These embodiments are provided for illustrative purposes only and are not intended in any way to limit the scope of the present invention. Although efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should, of course, be acceptable.

[0188] Unless otherwise indicated, the implementation of this invention will utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the scope of the skills of the art. Such techniques are fully described in the literature. For example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993), ALLehninger, Biochemistry (Worth Publishers, Inc., current addition), Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989), Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.), Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990), Carey and Sundberg Advanced Organic Chemistry 3. rd See Ed. (Plenum Press) Vols. A and B (1992).

[0189] method Gene synthesis and plasmid construction The coding sequence of the Fc fragment was generated by DNA synthesis and PCR, and then subcloned into a pcDNA3.1-based plasmid for protein expression in mammalian cell lines. The gene sequence in the expression vector was confirmed by DNA sequencing.

[0190] Expression of Fc fragments Transient antibody expression was performed using the ExpiCHO® Expression System (ThermoFisher, catalog number A29133). ExpiCHO-S cells were grown from a working cell bank and subcultured in ExpiCHO expression medium according to the manufacturer's instructions. The transfection mixture was prepared according to the protocol described in the ExpiCHO-S system manual (catalog number A29133, publication number MAN0014337, revision D.0). ExpiCHO cells were cultured for 14 days and collected by filtration using a 0.22 μm filtration unit and DE (Sartorius, catalog number SDLV-0150-05E0-2) as a filter aid, and then immediately processed. The condition medium was collected for protein purification.

[0191] Purification of Fc fragments The prepared antibodies were captured from the clear supernatant using a HiTrap MabSelect PrismA 25mL column (Cytiva, catalog no. 17-5498-54) on an AeKTA Pure 25 FPLC system. Column equilibration and protein binding were performed using 20 mM sodium phosphate in 150 mM NaCl pH 7.4, and 100 mM sodium citrate in 150 mM NaCl pH 3.5 was used for protein elution. After elution, the peaks corresponding to the affinity purified antibodies were immediately neutralized with 30% 1M Tris pH 8.0. The samples were polished, and the protein samples were loaded onto HiLoad 26 / 600 Superdex 200pg (Cytiva, catalog no. 28-9893-36) on an AeKTA Pure 25 FPLC system to achieve monomeric form with a purity of over 95%. Fractions corresponding to monomeric antibody morphology were pooled from a 96-deep-well plate into 50 ml Falcon tubes and filtered through a 0.22 μm PES membrane (Fisher brand, catalog no. 15206869, lot no. 2103171806) in a laminar flow chamber. The protein samples were transferred to a 50 kDa MWCO spin concentrator (Amicon 50K, catalog no. UFC 905024, lot no. 0000187574) for concentration, and each round was centrifuged at 4000 × g for 10 minutes, repeating until the desired concentration was reached. SEC-HPLC analysis of Fc fragments was performed.

[0192] Analytical SEC-HPLC was performed using a Thermo Vanquish Flex UHPLC system (Thermo Fisher) with a TSKgel Super SW mAb HTP (4.6 mm × 15.0 cm) column (Tosoh Bioscience, catalog number 00228559). Approximately 10 μg of sample was loaded. The mobile phase was 200 mM sodium phosphate, 0.05% sodium azide, pH 6.7, flow rate 0.35 ml / min, 10 minutes, 25°C.

[0193] Protein thermal stability testing by differential scanning fluorescence (DSF) Fc fragments in 20 mM sodium acetate at pH 5.5 were loaded into UNi (Unchained LabS). The samples were subjected to a thermal lamp at 20-95°C (lamp speed 0.3°C / min), and the fluorescence intensity at 330 / 350 nm and the SLS ratio at 266 nm were collected as a function of temperature. The inflection point (T) of the transition curve was determined. m UNCLE software was used to measure the peak of the second derivative of the fluorescence ratio of ), and the tag was determined using the SLS signal at 266 nm against temperature.

[0194] HIC-HPLC analysis of Fc fragments HIC-HPLC was performed on a UHPLC Vanquish Flex (ThermoFisher Scientific, MA) using a Proteomix HIC Butyl column with a Proteomix HIC Butyl pre-column, at a flow rate of 0.8 mL / min for 15 minutes with a salt gradient. Detection was performed using a wavelength of 220 nm. Species eluted before the main peak were assigned as low hydrophobic species, and species eluted after the main peak were assigned as high hydrophobic species.

[0195] Dynamic light scattering (DLS) Protein size distribution and molecular size were monitored by dynamic light scattering (DLS) using UNCLE (Unchained Labs). 9 μL of each sample was loaded into a UNI sample holder in a triple configuration, and the intensity of scattered light was measured at 20°C, with 10 acquisitions of 10 seconds each.

[0196] Non-reducing and reducing capillary electrophoresis of sodium dodecyl sulfate (nrCE-SDS and rCE-SDS) CE-SDS was performed using either LabChip (Perkin Elmer) or Maurice (ProteinSimple, CA). For CE-SDS using LabChip, approximately 2.5 μL of each sample was analyzed using a Protein Clear HT tip (catalog number CLS1486695) under non-reducing and reducing conditions, according to the manufacturer's instructions. Reduced samples were treated with β-mercaptoethanol, and non-reducing samples were treated with iodoacetamide (IAM) before analysis. For CE-SDS using Maurice, 25 μg of each sample was analyzed according to the manufacturer's instructions for the CE-SDS cartridge (ProteinSimple, catalog number PS-MC02-SP). Reduced samples were treated with β-mercaptoethanol.

[0197] Differential Scanning Calorimetry (DSC) DSC experiments were performed using a Microcal PEAQ DSC-DSC automated differential scanning microcalorimeter (Malvern Panalytical). All solutions and samples used for DSC were filtered using a 0.22-μm filter and degassed before loading into the calorimeter. The antibodies used in the DSC tests were monomeric (over 95%), as determined by analytical gel filtration chromatography. Prior to DSC analysis, all samples were thoroughly dialyzed in 20 mM sodium acetate (pH 5.5) (at least three buffer changes). The buffer from this dialyze was then used as a reference buffer for subsequent DSC experiments. Before sample measurement, baseline measurements (buffer vs. buffer) were obtained and subtracted from the sample measurements. 325 μL (0.5 mg / ml concentration) of each dialyzed sample was added in two batches to the sample well, and DSC measurements were performed at a scan rate of 1.5°C / min. Data analysis and deconvolution were performed using the manufacturer's provided DSC software.

[0198] PEG precipitation assay The PEG precipitation assay was performed as described in Gibson et al., J. Pharm. Sci. 100:1009-1021 (2011). Antibodies were precipitated using increasing amounts of PEG 4000. A 40% PEG stock solution was prepared in test buffer, and from this, eight solutions with varying final PEG percentages (ranging from 20% to 0%) were prepared in a 96-well plate by 1:1 dilutions of the protein sample in a total volume of 100 μL per well. The 96-well plate(s) were incubated overnight at room temperature and then read using a microplate reader that measures optical density at 320 nm.

[0199] Capillary isoelectric focusing (cIEF) This study was conducted on a Maurice (ProteinSimple, CA) system using cIEF cartridge ProteinSimple, catalog number PS-MC02-C), according to the manufacturer's instructions. Prior to analysis, samples were diluted in a master mix solution containing a mixture of amphoteric substances, pI markers, 1% methylcellulose, 12.5% ​​arginine, and 4M urea. Example 1: Measurement of antibody fragment-FcRn binding kinetics using surface plasmon resonance. Binding kinetics and affinity constants were determined at 25°C and in running buffer of 1×HBS-EP+pH6.0 or pH7.4 (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (Cytiva, catalog BR100669) at a Biacore 8K SPR system (GE HealthCare) equipped with a CM5 sensor chip (Cytiva, catalog no. 29149603) immobilized with anti-human Fc-specific antibody by amine coupling. After a stabilization period in running buffer, a 20 nM anti-FcRnMAb construct was captured on flow cell 2 (active) at a flow rate of 30 uL / min for 60 seconds. Recombinant in-house human FcRn protein was prepared at concentrations of 0, 26.25, 52.5, 125, 250, and 500 nM and injected into flow cell 1 (reference) and flow cell 2 (active) at a flow rate of 30 μL / min for 120 seconds. Recombinant cynomolgus monkey FcRn protein ((Immunitrack, catalog ITF06-400)) was prepared at concentrations of 0, 25, 50, 100, 200, and 400 nM and injected into flow cell 1 (reference) and flow cell 2 (active) at a flow rate of 30 μL / min for 120 seconds. The capture surface was regenerated by injecting 10 mM glycine pH 1.5 for 30 seconds at a flow rate of 30 μL / min, thereby injecting the sample into newly captured mAbs using a multi-cycle method. The data was processed and analyzed using Biacore Insight Evaluation Software Version 2.0.15.12933 (GE Healthcare) as follows.The response from flow cell 1 (reference) was subtracted from the response from flow cell 2 (active). Next, the responses from the two buffer blank injections were subtracted from the reference subtraction data (2-1) to obtain dual reference data, which was then fitted to a 1:1 binding model to determine the apparent association (ka) and dissociation rate constant (kd). Their ratio provided the apparent equilibrium dissociation constant or affinity constant (KD = kd / ka).

[0200] [Table 1-1]

[0201] [Table 1-2]

[0202] Example 2: Blocking of IgG and PAL02-0002 binding to human FcRn by ELISA. The ability of seven constructs to block the binding of IgG and PAL02-0002 (an Efgartigimod lacking C-terminal lysine) to human FcRn was measured by ELISA assay. A competitive binding ELISA assay was used to screen for molecules that prevent IgG from binding to recombinant in-house human FcRn protein at pH 6.0. Candidate therapeutic agents prevent FcRn from binding to IgGAbs in the acidic pH 6.0 endosomal environment. In this assay, functional huFcRn was coated with 1×PBS at 2.0 μg / ml in 96-well microtiter plates and then used in the ELISA assay to determine the IC50 concentration of test samples, which were serially diluted 3-fold from 100 nM to 0.002 nM at pH 6.0 in the presence of 0.568 nM biotinylated human IgG1. The binding of biotinylated human IgG1 was elucidated using ExtrAvidin-HRP (Sigma, catalog number E2886-1ML) at a 1:5000 dilution. IC50 values ​​were calculated using GraphPad Prism 7 software, applying nonlinear regression (curve fitting) of logarithmic (antagonist) versus response-variable slop (4 parameters). The Fc fragment, which was a therapeutic candidate, was expected to efficiently inhibit the ability of human IgG1 to bind to FcRn. A similar procedure was performed for PAL02-0002 blockade, replacing biotinylated human IgG1 with biotinylated PAL02-0002.

[0203] As shown in Table 2, constructs 1-7 were particularly effective in blocking the binding of IgG to FcRn. In particular, constructs 1-6 had IC50 values ​​that were half the EC50 value of Efigartigimod, and construct 3, in particular, had an IC50 value that was more than 3.5 times lower than the IC50 value of Efigartigimod.

[0204] [Table 2]

[0205] Example 3. Characterization of Fc fragment mutants that bind to FcRn SEC-HPLC, HIC-HPLC analysis, and thermal stability measurements of the FcRn construct were performed as described in the methods section disclosed herein.

[0206] Dynamic light scattering (DLS), non-reducing capillary electrophoresis of sodium dodecyl sulfate (nrCE-SDS), and reducing capillary electrophoresis of sodium dodecyl sulfate (rCE-SDS) were performed as described in the methods section disclosed herein.

[0207] [Table 3-1]

[0208] [Table 3-2]

[0209] Example 4. Summary of Fc mutant development test SEC-HPLC analysis was performed as described in the Methods section disclosed herein.

[0210] Dynamic light scattering (DLS), differential scanning calorimetry (DSC), and PEG precipitation assays were performed as described in the methods section disclosed herein.

[0211] All samples showed high monomer purity (over 99%) before and after three freeze / thaw cycles. DLS analysis showed the expected modal hydrodynamic diameter and low to intermediate polydispersity (PDI < 0.2) for all samples. Only the wild type (PAL02-0001) showed a TM value above 65°C. The majority of the remaining samples showed a TM between 60 and 63°C.

[0212] [Table 4]

[0213] Example 5. Short-term stability of the Fc variant The short-term stability of constructs 1-7 was tested in comparison with wild-type Fc and PAL02-0002 by SEC-HPLC analysis at a concentration of 10 mg / ml, 40°C, and 20 mM sodium acetate, pH 5.5, as described in the Methods section disclosed herein. Dynamic light scattering (DLS), non-reducing capillary electrophoresis of sodium dodecyl sulfate (nrCE-SDS), reducing capillary electrophoresis of sodium dodecyl sulfate (nrCE-SDS), and capillary isoelectric focusing (cIEF) were performed as described in the Methods section disclosed herein.

[0214] [Table 5-1]

[0215] [Table 5-2]

[0216] [Table 5-3]

[0217] [Table 6-1]

[0218] [Table 6-2]

[0219] [Table 6-3]

[0220] [Table 6-4]

[0221] [Table 6-5]

Claims

1. An isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, The isolated Fc fragment comprises a variant of the amino acid sequence described in Sequence ID No. 1, wherein the variant comprises at least one amino acid substitution selected from M428L, H433R, H433K, and N434Y.

2. The isolated Fc fragment according to claim 1, comprising an additional amino acid substitution at one or more of the amino acid positions 252, 254, and 256 of SEQ ID NO:

1.

3. The isolated Fc fragment according to claim 2, wherein the additional amino acid substitutions are M252Y, S254T, and / or T256E.

4. The isolated Fc fragment according to claim 1, wherein the mutant comprises amino acid substitutions of M252Y, S254T, T256E, M428L, H433K, and N434F or M252Y, S254T, T256E, H433K, and N434Y.

5. The isolated Fc fragment according to claim 4, wherein the mutant comprises the amino acid substitutions M252Y, S254T, T256E, M428L, H433K, and N434F.

6. The isolated Fc fragment according to claim 5, wherein the mutant comprises the amino acid sequence of SEQ ID NO:

3.

7. The isolated Fc fragment according to claim 4, wherein the mutant comprises the amino acid substitutions M252Y, S254T, T256E, H433K, and N434Y.

8. The isolated Fc fragment according to claim 7, wherein the mutant comprises the amino acid sequence of SEQ ID NO:

5.

9. An isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, The isolated Fc fragment comprises a variant of the amino acid sequence described in Sequence ID No. 1, wherein the variant includes an amino acid substitution at position 428.

10. The isolated Fc fragment according to claim 9, wherein the amino acid substitution is M428L.

11. The isolated Fc fragment according to claim 9 or 10, comprising an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 433, and 434 of SEQ ID NO:

1.

12. The isolated Fc fragment according to claim 11, wherein the additional amino acid substitutions are M252Y, S254T, T256E, H433K, H433R, N434F, and / or N434Y.

13. An isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, The isolated Fc fragment comprises a variant of the amino acid sequence described in Sequence ID No. 1, wherein the variant comprises the amino acid substitution H433R.

14. The isolated Fc fragment according to claim 13, comprising an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 428, and 434 of SEQ ID NO:

1.

15. The isolated Fc fragment according to claim 14, wherein the one or more additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

16. An isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, The isolated Fc fragment comprises a variant of the amino acid sequence described in Sequence ID No. 1, wherein the variant includes the amino acid substitution N434Y.

17. The isolated Fc fragment according to claim 16, comprising an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 428, and 433 of SEQ ID NO:

1.

18. The isolated Fc fragment according to claim 17, wherein the amino acid substitutions are M252Y, S254T, T256E, M428L, H433K, and / or H433R.

19. An isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) derived from humans, cynomolgus monkeys, mice, or rats, The isolated Fc fragment comprises a variant of the amino acid sequence described in Sequence ID No. 1, wherein the variant comprises an amino acid substitution H433K and an additional amino acid substitution at one or more of the amino acid positions 252, 254, 256, 428, and 434.

20. The isolated Fc fragment according to claim 19, wherein the additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

21. The isolated Fc fragment according to any one of the prior claims, wherein the Fc fragment comprises a sequence selected from any one of sequence numbers 3 to 9.

22. The isolated Fc fragment according to any one of claims 1 to 16, wherein the Fc fragment blocks or reduces the naturally occurring recycling of the antibody.

23. The isolated Fc fragment according to claim 22, wherein the Fc fragment results in increased catabolism of pathogenic antibodies.

24. The isolated Fc fragment according to claim 23, wherein the pathogenic antibody is an antibody associated with an autoimmune disease.

25. The Fc fragment has a lower K at pH 6.0 compared to the Fc fragment having a wild-type Fc region. Ｄ , and K, which can be measured at pH 7.4 Ｄ The isolated Fc fragment according to any one of claims 1 to 24, which binds to human, cynomolgus monkey, mouse, or rat FcRn.

26. When the Fc fragment is measured by surface plasmon resonance (SPR), it yields approximately 1 × 10⁻¹⁶ at pH 6.

0. －８ K below M Ｄ The isolated Fc fragment according to any one of claims 1 to 25, which is bound to the FcRn sequence described in sequence numbers 10 to 17.

27. When the Fc fragment is measured by surface plasmon resonance (SPR), it is found to be approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 × 10 at approximately pH 6.

0. －９ K below M Ｄ The isolated Fc fragment according to claim 26, which is bound to the FcRn sequence described in sequence numbers 10 to 17.

28. The isolated Fc fragment according to any one of claims 1 to 27, wherein the Fc fragment exhibits a melting temperature of more than 55°C when measured by differential scanning fluorescence (DSF).

29. The isolated Fc fragment according to any one of claims 1 to 28, wherein the Fc fragment exhibits an aggregation temperature of 70°C or higher when measured by differential scanning fluorescence (DSF).

30. An isolated Fc fragment according to any one of claims 1 to 29, for use in the treatment of antibody-related disorders or diseases.

31. The isolated Fc fragment according to claim 30 for use in the treatment of autoimmune diseases.

32. An isolated Fc fragment according to claim 30 or 31 for use in the treatment of a disease or disorder selected from the group consisting of generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuritis, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disorder (MOG antibody disorder), membranous nephropathy, lupus nephritis, thyroid eye disease, warm autoimmune hemolytic anemia, fetal and neonatal hemolytic disease, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, and cutaneous lupus erythematosus.

33. The isolated Fc fragment according to claim 32, wherein the treatment reduces the disease severity in the patient, and the disease severity is assessed by the gMG disease severity outcome scale.

34. An isolated polynucleotide or set of polynucleotides encoding an isolated Fc fragment according to any one of the prior claims, wherein the set of polynucleotides or polynucleotides optionally comprises mRNA or cDNA.

35. A vector or set of vectors comprising a polynucleotide or set of polynucleotides as described in claim 34.

36. A host cell comprising a polynucleotide or set of polynucleotides as described in claim 30, or a vector or set of vectors as described in claim 35.

37. A method for producing an Fc fragment, comprising expressing the Fc fragment in a host cell according to claim 36, and isolating the expressed Fc fragment.

38. A pharmaceutical composition comprising an isolated Fc fragment according to any one of claims 1 to 33 and a pharmaceutically acceptable excipient.

39. A method for treating a disorder or disease in a mammalian subject requiring treatment, comprising administering to the mammalian subject a therapeutically effective amount of an isolated Fc fragment according to any one of claims 1 to 33 or the pharmaceutical composition according to claim 33.

40. The method according to claim 39, wherein the disease or disorder is selected from the group consisting of generalized myasthenia gravis (gMG) chronic inflammatory demyelinating polyneuritis, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disorder (MOG antibody disorder), membranous nephropathy, lupus nephritis, thyroid eye disease, warm autoimmune hemolytic anemia, fetal and neonatal hemolytic disease, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, and cutaneous lupus erythematosus.

41. The method according to claim 40, wherein the disorder or disease is gMG.

42. The method according to claim 41, wherein the method reduces the disease severity in the patient, and the disease severity is evaluated by the gMG disease severity outcome scale.

43. A method for treating a mammalian subject in need of treatment for a pathology associated with elevated IgG levels, comprising administering to the mammalian subject a therapeutically effective amount of an isolated Fc fragment according to any one of claims 1 to 33 or the pharmaceutical composition according to claim 38.

44. A method for reducing the biological activity of IgG in a mammalian subject, comprising administering to the mammalian subject a therapeutically effective amount of an isolated Fc fragment according to any one of claims 1 to 33 or the pharmaceutical composition according to claim 38.

45. The method according to claim 44, wherein the disease is an autoimmune disease.

46. A method for preventing a disorder in a mammalian subject requiring prevention, the method comprising administering to the mammalian subject a therapeutically effective amount of an isolated Fc fragment according to any one of claims 1 to 33 or a pharmaceutical composition according to claim 38, wherein the disorder is an undesirable side effect of a therapeutic antibody.

47. An Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant includes amino acid substitutions M428L and N434F compared to the amino acid sequence described in Sequence ID No.

1.

48. The Fc fragment variant according to claim 47, wherein the Fc fragment further comprises an amino acid substitution of H433K or H433R.

49. The Fc fragment variant according to claim 47, wherein the Fc fragment further comprises the amino acid substitutions H433K and N434F.

50. The Fc fragment variant according to claim 47, wherein the Fc fragment further comprises the amino acid substitutions H433R and N434F.

51. An Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant comprises the amino acid substitutions (i) N434Y and (ii) H433R or H433K compared to the amino acid sequence described in Sequence ID No.

1.

52. The Fc fragment variant according to claim 51, wherein the Fc fragment comprises the amino acid substitutions H433K and N434Y.

53. The Fc fragment variant according to claim 51, wherein the Fc fragment comprises the amino acid substitutions H433R and N434Y.

54. An Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant includes amino acid substitutions M428L and N434Y compared to the amino acid sequence described in Sequence ID No.

1.

55. The Fc fragment variant according to claim 54, wherein the Fc fragment further comprises an H433K amino acid substitution.

56. An Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant includes amino acid substitutions M428L and H433R compared to the amino acid sequence described in Sequence ID No.

1.

57. The Fc fragment variant according to claim 56, wherein the Fc fragment further comprises an N434Y amino acid substitution.

58. An Fc fragment mutant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment mutant includes amino acid substitutions H433R and H434F compared to the amino acid sequence described in Sequence ID No.

1.

59. The Fc fragment variant according to any one of claims 47 to 58, wherein the Fc fragment further comprises the amino acid substitutions M252Y, S254T, and T256E.

60. The Fc fragment variant according to claim 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, M428L, H433K, and N434F.

61. The Fc fragment variant according to claim 60, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

3.

62. The Fc fragment variant according to claim 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, M428L, H433K, and N434Y.

63. The Fc fragment variant according to claim 62, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

4.

64. The Fc fragment variant according to claim 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, H433K, and N434Y.

65. The Fc fragment variant according to claim 64, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

5.

66. The Fc fragment variant according to claim 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, M428L, and N434F.

67. The Fc fragment variant according to claim 66, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

6.

68. The Fc fragment variant according to claim 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, H433R, and N434Y.

69. The Fc fragment variant according to claim 68, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

7.

70. The Fc fragment variant according to claim 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, H433R, and N434F.

71. The Fc fragment variant according to claim 70, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

8.

72. The Fc fragment variant according to claim 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, M428L, H433R, and N434F.

73. The Fc fragment variant according to claim 72, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

9.

74. The Fc fragment mutant according to any one of claims 47 to 73, wherein the Fc fragment mutant does not contain the L309D amino substitution.

75. It is an Fc fragment variant, Sequence ID 3 (provided that such a variant includes the amino acid substitutions M252Y, S254T, T256E, M428L, H433K, and N434F), Sequence ID No. 4 (provided that such a variant includes the amino acid substitutions M252Y, S254T, 256E, M428L, H433K, and N434Y), Sequence ID No. 5 (provided that such a variant includes the amino acid substitutions M252Y, S254T, T256E, H433K, and N434Y), Sequence ID 6 (provided that such a variant includes the amino acid substitutions S254T, T256E, M428L, and N434F), Sequence ID 7 (provided that such a variant includes the amino acid substitutions M252Y, S254T, T256E, H433R, and N434Y), Sequence ID No. 8 (provided that such a variant includes the amino acid substitutions M252Y, S254T, T256E, H433R, and N434F), or The Fc fragment variant is at least 85%, 90%, 93%, 95%, 97%, or 99% identical to Sequence ID No. 9 (provided that such variant includes the amino acid substitutions M252Y, S254T, T256E, M428L, H433R, and N434F).

76. The Fc fragment mutant according to claim 75, wherein the Fc fragment mutant inhibits IgG from binding to human FcRn with an IC50 value of less than 2.5 nM, 2.0 nM, 1.5 nM, or 1.0 nM.

77. The Fc fragment mutant according to claim 76, wherein the Fc fragment mutant inhibits the binding of IgG to human FcRn at pH 6.

0.

78. The Fc fragment variant according to any one of claims 47 to 77, wherein the Fc fragment is fused to or complexed with a half-life extension domain.

79. The Fc fragment variant according to claim 78, wherein the semi-extended domain is albumin, an albumin-binding domain, or an HSA-binding domain.

80. A method for inhibiting IgG from binding to FcRn, comprising administering an Fc fragment variant according to any one of claims 47 to 79.

81. A method for treating a disorder or disease in a mammalian subject requiring treatment, comprising administering to the mammalian subject a therapeutically effective amount of the Fc fragment variant described in any one of claims 47 to 79.

82. The method according to claim 81, wherein the disease or disorder is selected from the group consisting of generalized myasthenia gravis (gMG) chronic inflammatory demyelinating polyneuritis, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disorder (MOG antibody disorder), membranous nephropathy, lupus nephritis, thyroid eye disease, warm autoimmune hemolytic anemia, fetal and neonatal hemolytic disease, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, and cutaneous lupus erythematosus.

83. The method according to claim 82, wherein the disorder or disease is gMG.

84. The method according to claim 83, wherein the method reduces the disease severity in the patient, and the disease severity is evaluated by the gMG disease severity outcome scale.

85. A method for treating a mammalian subject in need of treatment for a pathology associated with elevated IgG levels, the method comprising administering to the mammalian subject a therapeutically effective dose of the Fc fragment variant described in any one of claims 47 to 79.

86. A method for reducing the biological activity of IgG in a mammalian subject, comprising administering to the mammalian subject a therapeutically effective amount of the Fc fragment variant described in any one of claims 47 to 79.

87. The method according to claim 86, wherein the disease is an autoimmune disease.

88. A method for preventing a disorder in a mammalian subject requiring prevention, the method comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment variant according to any one of claims 47 to 79, wherein the disorder is an undesirable side effect of a therapeutic antibody.

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