Fc fragments that bind fcRn and methods of use

Abstract

This article describes Fc fragment variants that effectively block IgG binding to the neonatal Fc receptor (FcRn) and methods of using them. In some aspects, this article describes methods for inhibiting the biological activity of FcRn. In some aspects, this article describes pharmaceutical compositions comprising said Fc fragments. In some aspects, the Fc fragments and methods described herein are intended for the treatment of antibody-related diseases or conditions (e.g., autoimmune diseases or undesirable side effects of therapeutic antibodies).

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202480021679.4, filed on March 25, 2024, entitled "Fc fragment in combination with FcRn and method of use". The original application was the national phase application with international application No. PCT / US2024 / 021300.

[0002] This international application claims priority to U.S. Provisional Patent Application No. 63 / 492,170, filed on March 24, 2023, which is hereby incorporated by reference in its entirety.

[0003] This application contains a sequence listing XML, which was submitted electronically and is hereby incorporated in its entirety by reference. The copy of the XML was created on March 22, 2024, named VRD-018WO_SL.xml, and is 29,298 bytes in size. Background Technology

[0004] Immunoglobulin γ (IgG) antibodies play an important role in the pathology of many diseases, such as autoimmune diseases, inflammatory diseases, and diseases whose pathology is characterized by IgG antibody overexpression (e.g., hypergammaglobulinemia) (see, for example, Junghans, Immunologic Research 16 (1):29 (1997)).

[0005] IgG has a longer serum half-life compared to other plasma proteins (Roopenian et al., J. Immunology 170:3528 (2003); Junghans and Anderson, Proc. Natl. Acad. Sci. USA93:5512 (1996)). This long half-life is partly due to the binding of the Fc region of IgG to the Fc receptor FcRn (unless otherwise specified, FcRn generally refers to the FcRn / b2m complex as its active form in this application). Although FcRn was initially characterized as a neonatal transport receptor for maternal IgG, it also functions in adults to protect IgG from degradation. FcRn binds to pinocytic IgG and protects IgG from transport to degrading lysosomes by recycling IgG back to the extracellular compartment, where IgG is released from FcRn and its biological function is restored. The pH-dependent binding of IgG to FcRn promotes this recycling process, with the IgG / FcRn interaction being stronger at acidic endosome pH but weaker, or even nonexistent, at extracellular physiological pH. Therefore, at physiological pH, IgG is released from its binding to FcRn, and the antibody is recycled, thus prolonging its half-life.

[0006] In certain situations, it is necessary to prevent IgG recycling, such as in autoimmune or inflammatory diseases. IgG recycling has previously been prevented by agents that reduce or block the binding of IgG to FcRn or increase the binding of IgG to FcRn at extracellular physiological pH. An example of such agents is an FcRn antibody that blocks IgG binding (see, for example, WO2002 / 43658). Peptides that bind to FcRn and antagonize FcRn function are also disclosed in the art (see, for example, US6,212,022 and US8,101,186). Additionally, IgG molecules comprising variant Fc receptors have been identified that exhibit enhanced binding to FcRn and reduced pH-dependent release (see, for example, US Patent No. 8,163,881). These IgG molecules occupy FcRn receptors, preventing them from being used to bind and recycle other IgG antibodies. Fc fragments comprising two Fc regions that form homodimers and occupy FcRn receptors have also been developed, exhibiting enhanced FcRn binding and reduced pH-dependent release (see, for example, US10,316,073). However, there is a need in the art for additional and / or modified agents that reduce or block FcRn binding to intact antibody IgG for the treatment of antibody-mediated conditions caused by such intact antibodies. Summary of the Invention

[0007] This disclosure provides, in particular, an Fc fragment variant that binds to the neonatal Fc receptor (FcRn) and exhibits increased affinity for FcRn at endosomal pH (e.g., pH 6.0) and physiological pH (e.g., pH 7.4) compared to the wild-type Fc fragment. As described herein, the invention is based in part on the identification of novel mutants in Fc that significantly and effectively inhibit IgG binding to FcRn. Specifically, the Fc fragment variant of this disclosure is characterized by a low IC50 value (e.g., ≤2.5 nM) for blocking IgG binding to FcRn and high affinity for FcRn at both endosomal and physiological pH (e.g., ≤10 nM and ≤300 nM, respectively). This is important because the Fc fragment variant of the present invention can achieve therapeutic effects with lower doses and / or lower administration frequencies compared to other Fc fragments. The inventive Fc fragment variant of the present invention holds promise for more potent treatment of diseases associated with pathogenic IgG, including autoimmune diseases, myasthenia gravis, and thyroid ophthalmopathy (TED).

[0008] In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant contains at least one amino acid substitution selected from M428L, H433R, and N434Y. In some embodiments, the isolated Fc fragment contains an additional amino acid substitution at one or more of amino acid positions 252, 254, and 256 of SEQ ID NO: 1. In some embodiments, said additional amino acid substitution is M252Y, S254T, and / or T256E.

[0009] In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant comprises an amino acid substitution at position 428. In some embodiments, said amino acid substitution is M428L. In some embodiments, said Fc fragment comprises additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 433, and 434 of SEQ ID NO: 1. In some embodiments, said additional amino acid substitution is M252Y, S254T, T256E, H433K, H433R, N434F, and / or N434Y.

[0010] In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant comprises the amino acid substitution H433R. In some embodiments, additional amino acid substitutions are made at one or more of amino acid positions 252, 254, 256, 428, and 434 of SEQ ID NO: 1. In some embodiments, said one or more additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0011] In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant comprises the amino acid substitution N434Y. In some embodiments, said Fc fragment comprises additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 428, and 433 of SEQ ID NO: 1. In some embodiments, said amino acid substitutions are M252Y, S254T, T256E, M428L, H433K, and / or H433R.

[0012] In some aspects, this document describes an isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) of humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: a variant having the amino acid sequence shown in SEQ ID NO: 1, said variant comprising the amino acid substitution H433K and additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 428, and 434. In some embodiments, said additional amino acid substitution is M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0013] In some implementations, the isolated Fc fragment described herein comprises a sequence selected from any of SEQ ID NO: 3-9.

[0014] Compared to IgG containing the wild-type Fc region, the anticipated amino acid substitutions described herein result in the Fc fragment exhibiting K+ kinase activity against FcRn at pH 6.0. D Lower and at pH 7.4, the K of FcRn D Measurable. The Fc region of wild-type IgG exhibits K+ ionization of FcRn at pH 6.0. D Approximately 8 × 10 -7 Furthermore, no binding to FcRn was detected at pH 7.4. The higher affinity for FcRn at pH 6.0 and / or the ability to maintain binding to FcRn at pH 7.4 result in a higher occupancy of FcRn by the Fc fragment of this invention, thus reducing the amount of FcRn available for binding IgG containing the wild-type Fc region. Consequently, IgG containing the wild-type Fc region is more readily degraded in lysosomes, effectively shortening the half-life of IgG.

[0015] In some embodiments, the amino acid substitutions result in a prolonged half-life of the Fc fragment compared to an Fc fragment containing the Fc region of wild-type human IgG1. In some embodiments, the Fc fragment blocks or reduces the recycling of naturally occurring IgG antibodies. In some embodiments, the Fc fragment leads to increased catabolism of pathogenic IgG antibodies. In some embodiments, the pathogenic IgG antibody is an antibody associated with an autoimmune disease.

[0016] In some embodiments, the Fc fragment is at pH 6.0 at a concentration of less than or equal to about 1 × 10⁻⁶. -8 M is less than or equal to approximately 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 of K D The Fc fragments are bound to the FcRn sequences shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragments are at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -8 M is less than or equal to approximately 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 of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 10 or 11, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -8 M is less than or equal to approximately 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 of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 16 or 17, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at about pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -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 M of K D The Fc fragments are bound to the FcRn sequences shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragments are at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -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 M of K D This is combined with the FcRn sequence shown in SEQ ID NO: 12 or 13, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at about pH 6.0 with a concentration less than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, or 9 × 10⁻⁶. -10 M of K D The Fc fragments are bound to the FcRn sequences shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragments are at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -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 M of K DCombined with the FcRn sequence shown in SEQ ID NO: 14 or 15, as measured by surface plasmon resonance (SPR).

[0017] In some embodiments, the Fc fragment exhibits a melting temperature greater than 55°C, as measured by differential scanning fluorometry (DSF).

[0018] In some implementations, human Fc fragments exhibit aggregation temperatures equal to or greater than about 65°C or greater than about 70°C, as measured by static light scattering (SLS).

[0019] In some respects, the isolated human Fc fragments disclosed herein are used to treat antibody-related conditions or diseases, such as autoimmune diseases or conditions associated with unwanted side effects from therapeutic antibodies. In some embodiments, the isolated human Fc fragments disclosed herein are used to treat diseases or conditions selected from the group consisting of: generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuropathy, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disease (MOG antibody disease), membranous nephropathy, lupus nephritis, thyroid ophthalmopathy, warm antibody-type autoimmune hemolytic anemia, hemolytic diseases of the fetus and newborn, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, foliaceous pemphigoid, pemphigus vulgaris, and cutaneous lupus erythematosus.

[0020] 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 thrombocytopenic purpura (ITP). In some embodiments, the treatment reduces the severity of the patient's disease, and the disease severity is assessed using a gMG disease severity outcome measure.

[0021] In some respects, this document describes a separated polynucleotide or polynucleotide set for expressing a separated Fc fragment of any of the embodiments disclosed herein. Therefore, this document discloses a separated polynucleotide or polynucleotide set encoding a separated Fc fragment of any of the embodiments disclosed herein, and optionally said polynucleotide or polynucleotide set comprises mRNA or cDNA.

[0022] In some respects, this document describes a vector or set of vectors for expressing any of the embodiments disclosed herein, comprising the polynucleotides or sets of polynucleotides disclosed herein.

[0023] In some respects, this document describes a host cell that contains the polynucleotides or polynucleotide groups or vectors or vector groups disclosed herein.

[0024] In some respects, this document describes a method for generating an Fc fragment, the method comprising expressing the Fc fragment in a host cell disclosed herein and isolating the expressed Fc fragment.

[0025] In some respects, this document describes a pharmaceutical composition comprising a separated Fc fragment of any of the embodiments disclosed herein and a pharmaceutically acceptable excipient.

[0026] In some respects, this document describes a pillbox containing a separated Fc fragment of any of the embodiments disclosed herein or a pharmaceutical composition disclosed herein, along with instructions for use.

[0027] In some aspects, this document describes a method for treating or preventing antibody-related conditions or diseases (e.g., autoimmune diseases) or conditions associated with unwanted side effects of therapeutic antibodies in a mammalian subject of need, the method comprising administering to the mammalian subject a therapeutically effective amount of any of the isolated Fc fragments of the embodiments disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the disease or condition is selected from the group consisting of: generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuropathy, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disease (MOG antibody disease), membranous nephropathy, lupus nephritis, thyroid ophthalmopathy, warm antibody-type autoimmune hemolytic anemia, hemolytic diseases of the fetus and newborn, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, foliaceous pemphigoid, pemphigus vulgaris, and cutaneous lupus erythematosus. In some embodiments, the inflammatory condition or disease is an autoimmune disease. In some embodiments, the inflammatory condition or disease is gMG. In some embodiments, the method reduces the severity of the patient's disease, and the severity of the disease is assessed using a gMG disease severity outcome measure.

[0028] In some respects, this document describes a method for treating a pathology associated with elevated IgG levels in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment of any of the embodiments disclosed herein or a pharmaceutical composition disclosed herein.

[0029] In some aspects, this document describes a method for reducing IgG bioactivity in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment of any of the embodiments disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the disease is an autoimmune disease.

[0030] In some respects, this document describes a method for preventing a condition in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of the isolated Fc fragment or pharmaceutical composition described herein, wherein the condition is an unwanted side effect of a therapeutic antibody.

[0031] In one aspect, embodiments of the invention particularly provide an Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions M428L and N434F compared to the amino acid sequence shown in SEQ ID NO: 1.

[0032] In some embodiments, the Fc fragment variant further comprises an amino acid substitution at position 433 compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the Fc fragment comprises an amino acid substitution of H433K or H433R compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the Fc fragment comprises an amino acid substitution of N434F compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the Fc fragment comprises amino acid substitutions of both H433K and N434F compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, it comprises amino acid substitutions of H433R and N434F compared to the amino acid sequence shown in SEQ ID NO: 1.

[0033] In one aspect, the present invention particularly provides an Fc fragment variant that binds to a neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions (i) N434Y and (ii) H433R or H433K compared to the amino acid sequence shown in SEQ ID NO: 1.

[0034] In some embodiments, the Fc fragment contains amino acid substitutions H433K and N434Y compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the Fc fragment contains amino acid substitutions H433R and N434Y compared to the amino acid sequence shown in SEQ ID NO: 1.

[0035] In one aspect, the present invention particularly provides an Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions M428L and N434Y compared to the amino acid sequence shown in SEQ ID NO: 1.

[0036] In some implementations, the Fc fragment also contains an amino acid substitution for H433K.

[0037] In one aspect, the present invention particularly provides an Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions M428L and H433R compared to the amino acid sequence shown in SEQ ID NO: 1.

[0038] In some embodiments, the Fc fragment further comprises an amino acid substitution N434Y.

[0039] In one aspect, the present invention particularly provides an Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions H433R and H434F compared to the amino acid sequence shown in SEQ ID NO: 1.

[0040] In some implementations, the Fc fragment also contains amino acid substitutions for M252Y, S254T, and T256E.

[0041] In some embodiments, the Fc fragment comprises amino acid substitutions for M252Y, S254T, T256E, M428L, H433K, and N434F. In some embodiments, the Fc fragment comprises amino acid substitutions for M252Y, S254T, T256E, M428L, H433K, and N434Y. In some embodiments, the Fc fragment comprises amino acid substitutions for M252Y, S254T, T256E, H433K, and N434Y. In some embodiments, the Fc fragment comprises amino acid substitutions for M252Y, S254T, T256E, M428L, and N434F. In some embodiments, the Fc fragment comprises amino acid substitutions for M252Y, S254T, T256E, H433R, and N434F. In some embodiments, the Fc fragment comprises amino acid substitutions of M252Y, S254T, T256E, M428L, H433R, and N434F.

[0042] In some embodiments, the Fc fragment variant does not contain amino-substituted L309D.

[0043] In some embodiments, the Fc fragment comprises an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 3.

[0044] In some embodiments, the Fc fragment comprises an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 4.

[0045] In some embodiments, the Fc fragment comprises an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 5.

[0046] In some embodiments, the Fc fragment comprises an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 6.

[0047] In some embodiments, the Fc fragment comprises an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 7.

[0048] In some embodiments, the Fc fragment comprises an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 8.

[0049] In some embodiments, the Fc fragment comprises an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 9.

[0050] In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 2.6 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 2.5 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 2.3 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 2.2 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 2.0 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.9 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.8 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.7 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.6 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.5 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.4 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.3 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.2 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.1 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 1.0 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 0.9 nM. In some implementations, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 0.8 nM.

[0051] In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.5-2.2 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-2.0 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.8 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.6 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.5 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.4 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.3 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.2 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.2 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-1.0 nM.

[0052] In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 2.0 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.9 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.8 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.7 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.5 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.4 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.3 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.2 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.1 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 1.0 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 0.9 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 0.8 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 0.7 nM. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of 0.6 nM.

[0053] In some embodiments, the Fc fragment variant effectively inhibits IgG binding to human FcRn at a pH of approximately 5.8–7.5. In some embodiments, the Fc fragment variant effectively inhibits IgG binding to human FcRn at a pH of approximately 6.0–7.4.

[0054] In some embodiments, the Fc fragment variant effectively inhibits IgG binding to human FcRn at pH 6.0. In some embodiments, the Fc fragment variant effectively inhibits IgG binding to human FcRn at pH 7.0. In some embodiments, the Fc fragment variant effectively inhibits IgG binding to human FcRn at pH 7.4.

[0055] In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-2.2 nM at pH 6.0. In some embodiments, the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of about 0.7-2.2 nM at pH 7.4.

[0056] 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 is a variable neoantigen receptor (VNAR).

[0057] 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 HSA-binding domain.

[0058] In one aspect, the present invention particularly provides a method for inhibiting IgG binding to FcRn by applying an Fc fragment variant described herein. Detailed Implementation

[0059] definition Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art. In some cases, terms having their commonly understood meaning are defined herein for clarity and / or convenience of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a difference from the commonly understood meaning in the art. Those skilled in the art generally have a good understanding of the techniques and procedures described or mentioned herein and typically employ conventional methods, such as the widely used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, unless otherwise indicated, procedures involving the use of commercially available kits and reagents are generally performed according to the protocols and conditions defined by the manufacturer.

[0060] Unless otherwise indicated, as used herein, the singular forms “a,” “an,” and “described” include a plural number of references.

[0061] It should be understood that the aspects and embodiments of the invention described herein include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "substantially consisting of" aspects and embodiments.

[0062] For all compositions described herein and all methods using the compositions described herein, the compositions may comprise the listed components or steps, or may be “substantially composed of the listed components or steps.” When a composition is described as “substantially composed of the listed components,” the composition contains the listed components and may contain other components that do not substantially affect the disease being treated, but does not contain any other components besides those explicitly listed that substantially affect the disease being treated; or, if the composition contains additional components besides those listed that substantially affect the disease being treated, the composition does not contain a sufficient concentration or amount of the additional components to substantially affect the disease being treated. When a method is described as “substantially composed of the listed steps,” the method contains the listed steps and may contain other steps that do not substantially affect the disease being treated, but the method does not contain any other steps besides those explicitly listed that substantially affect the disease being treated. As a non-limiting specific example, when a composition is described as "consisting substantially of components", the composition may additionally contain any amount of pharmaceutically acceptable carriers, mediators or diluents and other such components that do not substantially affect the disease being treated.

[0063] As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. This term includes vectors that exhibit a self-replicating nucleic acid structure as well as vectors incorporated into the genome of a host cell to which they have been introduced. Some vectors are capable of guiding the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0064] 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 their progeny. Host cells include “transformers” (or “transformed cells”) and “transfectants” (or “transfected cells”), each comprising primary transformed or transfected cells and their progeny. The nucleic acid content of such progeny may not be exactly the same as that of the parent cells, and they 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 uptake, transduction, f-coordination, or other methods known in the art for producing recombinant host cells).

[0065] As used in this article, the term "eukaryote" refers to organisms belonging to the phylogenetic domain eukaryotes, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidiids, protozoa, etc.

[0066] As used in this article, the term "prokaryote" refers to a prokaryotic organism. For example, non-eukaryotic organisms may belong to the lineage 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 thermoautotrophicum, Halobacterium such as Halofera volcanii, and Halobacterium species NRC-1, Archaeoeglobus fulgidus, Pyrococcus furiosus, Pyrococcus huskae). Phylogenetic domains such as horikoshii and Aeropyrum pernix.

[0067] 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, either as a single dose or as part of a series of doses, which, alone or in combination with another mode of treatment, is effective in producing or promoting the desired therapeutic effect. Examples of desired therapeutic effects include a reduction in IgG levels and improvement of one or more symptoms. An effective dose may be given in one or more doses.

[0068] The term "treating" (and its variations such as "treat" or "treatment") refers to a clinical intervention that attempts to alter the natural course of a disease or ailment in a subject with a need. Treatment may be administered during the clinical pathological course of the disease. Desired effects of treatment include preventing disease recurrence, relieving symptoms, mitigating any direct or indirect pathological consequences of the disease, preventing cancer metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.

[0069] The term "sufficient amount" refers to an amount sufficient to produce the desired effect, such as an amount sufficient to modulate the immune response of a subject.

[0070] As used herein, the term "subject" or "individual" refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, rabbits, and sheep. In some embodiments, the subject is a human.

[0071] The term "in vitro" refers to a process that occurs in living cells grown separately from living organisms, such as in tissue cultures.

[0072] The term "in vivo" refers to processes that occur within a living organism.

[0073] The term "instructions for use" refers to the instructions for use that are typically included in the commercial packaging of therapeutic or diagnostic products (such as medicine boxes), which contain information about indications, usage, dosage, administration, combination therapy, contraindications and / or warnings related to the use of such therapeutic or diagnostic products.

[0074] The term "pharmaceutical composition" refers to a formulation which is in a form that is effective in treating a subject by means of the biological activity of the active ingredient contained therein and which does not contain any additional components that would have unacceptable toxicity to the subject in the amount provided in the pharmaceutical composition.

[0075] The terms "co-administration," "co-administer," and "combination" include the simultaneous, concurrent, or sequential administration of two or more therapeutic agents within a non-specific timeframe. In one embodiment, the agents are simultaneously present in cells or within the subject's body, or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In some embodiments, the first agent may be administered prior to the administration of the second therapeutic agent.

[0076] The terms "modulate" and "modulation" refer to reducing or inhibiting, or activating or increasing, the described variable.

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

[0078] The terms “reduction” and “suppression” refer to a reduction of the described variable by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more.

[0079] The term "about" indicates and encompasses a specified value and a range above and below that value. In some embodiments, the term "about" indicates a specified value ± 10%, ± 5%, or ± 1%. In some embodiments, where applicable, the term "about" indicates a specified value ± one standard deviation of that value.

[0080] For any of the structural and functional features described herein, the methods for determining these features are known in the art.

[0081] The term “optionally” when used sequentially means to include one to all of the listed combinations and to cover all sub-combinations.

[0082] The term "amino acid" refers to twenty common, naturally occurring amino acids. These 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).

[0083] The term "affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an Fc fragment) and its binding partner (e.g., FcRn). Unless otherwise indicated, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., Fc fragment and FcRn). Affinity is related to K... D Proportional.

[0084] When referring to an amino acid sequence, the term "variant" as used herein means an amino acid sequence that contains one or more changes (including one or more substitutions) compared to a reference amino acid sequence.

[0085] As used herein, the term "kd" (sec⁻¹) refers to the dissociation rate constant of a specific antibody-antigen or protein-protein interaction. This value is also known as the koff value.

[0086] As used herein, the term "ka" (M⁻¹×sec⁻¹) refers to the association rate constant of a specific antibody-antigen or protein-protein interaction. This value is also known as the kon value.

[0087] As used in this article, the term "KD" or "K" refers to... D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen or protein-protein interaction. KD = kd / ka. In some embodiments, the affinity of a protein is described as the KD of the interaction between the protein and its binding chaperone. For clarity, as is known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction."

[0088] As used in this article, the terms "measurable KD" or "measurable KD" are used in this context. D This means less than 1 M, less than 0.1 M, less than 0.01 M, less than 0.001 M, and less than 1 × 10⁻⁶. -4 M, less than 1×10 -5 M or less than 1×10 -6 The KD value of M.

[0089] As used herein, the term "KA" (M-1) refers to the association equilibrium constant of a specific antibody-antigen or protein-protein interaction. KA = ka / kd.

[0090] The term "antibody" is used in its broadest sense here and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibody-specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0091] The terms “full-length antibody,” “intact antibody,” and “complete antibody” are used interchangeably herein to refer to an antibody that has a structure substantially similar to that of naturally occurring antibodies and has 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 consists of two heavy chains and two light chains.

[0092] The terms “Fc domain”, “Fc region”, or “Fc fragment” used in this document are used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. These terms include native sequence Fc regions and variant Fc regions.

[0093] As used herein, "Fc fragment" refers to a fragment that specifically binds to the Fc domain of the target protein FcRn. In some embodiments, the Fc fragment is a variant of SEQ ID NO: 1.

[0094] The term "human Fc fragment" refers to an Fc fragment having an amino acid sequence corresponding to the amino acid sequence of the Fc region of an antibody produced by humans or human cells or derived from a non-human source using a human antibody library or a sequence encoding a human antibody (e.g., derived from a human source or redesigned).

[0095] The term “substantially purified” means that the constructs described herein or variants thereof are substantially or substantially free of components typically found in or interacting with said proteins in their native environment (i.e., native cells) or, in the case of proteins produced in a recombinant manner, in host cells. In some embodiments, they are substantially free of cellular material and include protein formulations having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (on a dry weight basis) of contaminating proteins.

[0096] The ranges listed in this document should be understood as abbreviations of all values ​​within the range, including the stated endpoints. For example, the range 1 to 50 should be understood as including any numerical value, combination of numerical values, or subrange from the following groups: 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.

[0097] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used in this specification and the appended claims include a plurality of indicators.

[0098] Fc fragment Fc fragment structure The Fc fragment may include the Fc fragment described herein, such as the amino acid sequence shown in the table. In some embodiments, the Fc fragment is an IgG subclass IgG1, IgG2, or IgG4.

[0099] In some implementations, the Fc fragment is generated by recombinant cells engineered to express the desired constant domain.

[0100] Sequence of Fc fragments combined with FcRn In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant contains at least one amino acid substitution selected from M428L, H433R, and N434Y. In some embodiments, the isolated Fc fragment contains an additional amino acid substitution at one or more of amino acid positions 252, 254, and 256 of SEQ ID NO: 1. In some embodiments, the additional amino acid substitution is M252Y, S254T, and / or T256E.

[0101] In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant comprises an amino acid substitution at position 428. In some embodiments, said amino acid substitution is M428L. In some embodiments, said Fc fragment comprises additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 433, and 434 of SEQ ID NO: 1. In some embodiments, said additional amino acid substitution is M252Y, S254T, T256E, H433K, H433R, N434F, and / or N434Y.

[0102] In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant comprises the amino acid substitution H433R. In some embodiments, additional amino acid substitutions are made at one or more of 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 made as M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0103] In some aspects, this document describes an isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising a variant having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant comprises the amino acid substitution N434Y. In some embodiments, said Fc fragment comprises additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 428, and 433 of SEQ ID NO: 1. In some embodiments, said amino acid substitutions are M252Y, S254T, T256E, M428L, H433K, and / or H433R.

[0104] In some aspects, this document describes an isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) of humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: a variant having the amino acid sequence shown in SEQ ID NO: 1, said variant comprising the amino acid substitution H433K and additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 428, and 434. In some embodiments, said additional amino acid substitution is M252Y, S254T, T256E, M428L, N434F, and / or N434Y.

[0105] In some embodiments, the Fc fragment comprises a sequence selected from the sequences shown in SEQ ID NO: 3-9.

[0106] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 3, provided that the fragment contains mutations in M252Y, S254T, T256E, M428L, H433K, and N434F.

[0107] In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 91% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 3. In some embodiments, the Fc fragment variant comprises an amino acid sequence having 100% identity with SEQ ID NO: 3. In some embodiments, for each of the aforementioned variants having a percentage identity with SEQ ID NO: 3, the variant comprises mutations in M252Y, S254T, T256E, M428L, H433K, and N434F.

[0108] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 4, provided that the fragment contains mutations in M252Y, S254T, T256E, M428L, H433K, and N434Y.

[0109] In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 91% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 4. In some embodiments, the Fc fragment variant comprises an amino acid sequence having 100% identity with SEQ ID NO: 4. In some embodiments, for each of the aforementioned variants having a percentage identity with SEQ ID NO: 4, the variant comprises mutations in M252Y, S254T, T256E, M428L, H433K, and N434Y.

[0110] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 5, provided that the fragment contains mutations in M252Y, S254T, T256E, H433K, and N434Y.

[0111] In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 91% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 5. In some embodiments, the Fc fragment variant comprises an amino acid sequence having 100% identity with SEQ ID NO: 5. In some embodiments, for each of the aforementioned variants having a percentage identity with SEQ ID NO: 5, the variant comprises mutations in M252Y, S254T, T256E, H433K, and N434Y.

[0112] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 6, provided that the fragment contains mutations in M252Y, S254T, T256E, M428L, and N434F.

[0113] In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 36. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 91% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 6. In some embodiments, the Fc fragment variant comprises an amino acid sequence having 100% identity with SEQ ID NO: 6. In some embodiments, for each of the aforementioned variants having a percentage identity with SEQ ID NO: 6, the variant comprises mutations in M252Y, S254T, T256E, M428L, and N434F.

[0114] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 7, provided that the fragment contains mutations in M252Y, S254T, T256E, H433R, and N434Y.

[0115] In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 91% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 7. In some embodiments, the Fc fragment variant comprises an amino acid sequence having 100% identity with SEQ ID NO: 7. In some embodiments, for each of the aforementioned variants having a percentage identity with SEQ ID NO: 7, the variant comprises mutations in M252Y, S254T, T256E, H433R, and N434Y.

[0116] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 8, provided that the fragment contains mutations in M252Y, S254T, T256E, H433R, and N434F.

[0117] In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 91% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 8. In some embodiments, the Fc fragment variant comprises an amino acid sequence having 100% identity with SEQ ID NO: 8. In some embodiments, for each of the aforementioned variants having a percentage identity with SEQ ID NO: 8, the variant comprises mutations in M252Y, S254T, T256E, H433R, and N434F.

[0118] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 9, provided that the fragment contains mutations in M252Y, S254T, T256E, M428L, H433R, and N434F.

[0119] In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 91% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 9. In some embodiments, the Fc fragment variant comprises an amino acid sequence having 100% identity with SEQ ID NO: 9. In some embodiments, for each of the aforementioned variants having a percentage identity with SEQ ID NO: 9, the variant comprises mutations in M252Y, S254T, T256E, M428L, H433R, and N434F.

[0120] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 18, provided that the fragment contains mutations in M252Y, S254T, T256E, L309D, H433K, and N434F.

[0121] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 19, provided that the fragment contains mutations in M252Y, S254T, T256E, L309D, H433K, and N434Y.

[0122] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 20, provided that the fragment contains mutations in M252Y, S254T, T256E, L309D, M428L, H433K, and N434F.

[0123] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 21, provided that the fragment contains mutations in M252Y, S254T, T256E, L309D, M428L, H433K, and N434Y.

[0124] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 22, provided that the fragment contains mutations in M252Y, S254T, T256E, L309D, Q311K, H433K, and N434F.

[0125] In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence comprising SEQ ID NO: 23, provided that the fragment contains mutations in M252Y, S254T, T256E, L309D, Q311K, H433K, and N434Y.

[0126] For sequence comparison, typically one sequence serves as a reference sequence, and a second sequence is compared to the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and coordinates and algorithm parameters can be specified if necessary. Any suitable algorithm can be used, including (but not limited to) the Smith-Waterman alignment algorithm, Viterbi, Bayesians, Hidden Markov, etc. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm can then be used to calculate the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters. Any suitable algorithm can be used to calculate the percentage of identity. For example, some programs calculate the percentage of identity by dividing the number of aligned residue positions by the total number of aligned positions.

[0127] The sequence alignment methods used for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by: local homology algorithms, Smith and Waterman, Adv. Appl. Math. 2:482 (1981); homology alignment algorithms, Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); similarity retrieval methods, Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); manual alignment and visual inspection (see, for example, Current Protocols, Molecular Biology (Ausubel et al., ed., Supplement 1995)), each of which is hereby incorporated in its entirety by reference. Exemplary computer software for determining identity between two sequences includes (but is not limited to): the GCG package, Devereux, 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 which is hereby incorporated in its entirety by reference.

[0128] In some embodiments, the Fc fragment includes additional amino acid residues 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 1, 2, 3, 4, or 5 additional amino acid residues at the N-terminus and / or C-terminus.

[0129] Fc area Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, an “Fc polypeptide” of a dimer Fc refers to one of two polypeptides forming the dimer Fc domain, namely a polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain, which is capable of stable self-association. For example, the Fc polypeptide of a dimer IgG Fc contains the IgG CH2 and IgG CH3 constant domain sequences. In some respects, the Fc fragments disclosed herein contain the C-terminal 226 amino acids of the complete human IgG Fc region. Fc may belong to the IgG class and may be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4. In some respects, the Fc fragments described herein belong to the IgG1 subclass. In some respects, the Fc fragment described herein is a variant of the human IgG1 Fc region shown in SEQ ID NO: 1. In some respects, the Fc fragment described herein belongs to the IgG2 subclass. In some respects, the Fc fragment described herein belongs to the IgG4 subclass.

[0130] 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 native human FcR sequence. Typically, an FcR is an FcR that binds to IgG antibodies (γ receptors) and includes FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternative splice forms of these receptors). FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), both of which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Other isotypes of immunoglobulins may also be bound by certain FcRs (see, for example, Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). The terminology also includes neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)). The Fc fragment described herein selectively binds to FcRn. In some respects, the Fc fragment selectively binds to mammalian FcRn, including cynomolgus monkey, rat, and / or mouse FcRn.

[0131] The activating receptor FcγRIIA contains an activating motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The repressive receptor FcγRIIB contains an repressive motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain (reviewed in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed 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). The term “FcR” as used herein encompasses other FcRs, including those to be identified in the future.

[0132] Modifications in the CH2 domain can affect the binding of FcR to Fc. Several amino acid modifications in the Fc region are known in the art to selectively alter the affinity of Fc for different Fcγ receptors.

[0133] 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. 28 Feb 2011; 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. 15 Sep 2007; 67(18):8882-90; Nordstrom JL, Gorlatov S, Zhang W et al., Breast Cancer Res. 30 Nov 2011; 13(6):R123); F243L (Stewart R, Thom G, Levens M et al., Protein Eng Des Sel. Sep 2011;24(9):671-8.), S298A / E333A / K334A (Shields RL, Namenuk AK, Hong K et al., JBiol Chem. Mar 2 2001;276(9):6591-604); S239D / I332E / A330L, S239D / I332E (Lazar GA, Dang W, Karki S et al., ProcNatl Acad Sci US A. March 14, 2006; 103(11):4005-10); S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S et al., Mol Immunol. Sep 2008;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 mutations listed in WO2011 / 120134 and WO2011 / 120135, which are incorporated herein by reference. Therapeutic Antibody Engineering (William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine, Vol. 11, ISBN 1 907568 37 9, October 2012) Mutations are listed on page 283.

[0134] In some 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), wherein said one or more substitutions result in measurable K at pH 7.4. D The Fc fragment of SEQ ID NO: 1 did not show any binding to FcRn at pH 7.4 without one or more substitutions.

[0135] In some implementations, one or more amino acid substitutions extend the half-life of the Fc fragment at pH 6.0 compared to an Fc fragment containing a wild-type Fc region.

[0136] Combination The affinity of molecule X for its partner Y can be determined by the dissociation equilibrium constant (K). D The following describes in more detail the dynamic components that constitute the dissociation equilibrium constant. 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®).

[0137] Regarding the binding of Fc fragments to target molecules, the terms "binding," "specific binding," "specifically binding to," "specifically binding to," "selectively binding," and "selectively binding to" when used with a specific target (e.g., a peptide target such as FcRn) refer to binding that is significantly different from non-specific or non-selective interactions (e.g., 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 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 for non-target molecules is about 50% less than its affinity for FcRn (i.e., K0 for FcRn). D Compare non-target K D (Approximately twice as low). In some embodiments, the Fc fragment has an affinity for non-target molecules that is about 40% less than that for FcRn. In some embodiments, the Fc fragment has an affinity for non-target molecules that is about 30% less than that for FcRn. In some embodiments, the Fc fragment has an affinity for non-target molecules that is about 20% less than that for FcRn. In some embodiments, the Fc fragment has an affinity for non-target molecules that is about 10% less than that for FcRn. In some embodiments, the Fc fragment has an affinity for non-target molecules that is about 1% less than that for FcRn. In some embodiments, the Fc fragment has an affinity for non-target molecules that is about 0.1% less than that for FcRn.

[0138] When used herein for a first Fc fragment and a second Fc fragment or wild-type IgG (“second molecule”), the terms “competing with” or “cross-competing 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 and 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 and contacted with the FcRn, and then the second molecule is added. If in either assay the presence of a first Fc fragment that reduces the binding of the second molecule, then the Fc fragment competes with the second molecule. The term “competing with” also includes combinations where the first Fc fragment reduces the binding of the second molecule, but where no competition is observed when the first Fc fragment and the second molecule are added in reverse order. However, in some embodiments, the first Fc fragment and the second molecule inhibit each other’s binding to the FcRn, regardless of the order in which they are added. In some embodiments, a competitive binding assay measures that the first Fc fragment 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 use any suitable assay to determine whether Fc fragments compete with each other. Suitable assays are described in, for example, Cox et al., “Immunoassay Methods”, 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 which is incorporated in its entirety by reference.

[0139] If, in a competitive binding assay, an excess of the test Fc fragment (e.g., at least 2, 5, 10, 20, or 100 times) is measured to inhibit or block the binding of the second molecule by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, the test (or first) Fc fragment competes with the second molecule (e.g., the reference Fc fragment). For example, a second competitive Fc fragment can be identified by its ability to compete with the first Fc fragment described herein for binding to the FcRn. In some cases, a competitive binding assay may measure that the second molecule blocks or inhibits the binding of the first Fc fragment by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some cases, the second molecule may replace more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the first Fc fragment.

[0140] In some embodiments, the Fc fragment is combined with the FcRn sequence shown in SEQ ID NO: 10-17.

[0141] In some embodiments, the Fc fragment is at pH 6.0 at a concentration of less than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, 9 × 10⁻⁶. -8 M of K D The Fc fragments are bound to the FcRn sequences shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragments are at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -8 M of K D Incorporation into human FcRn, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 6.0 with a concentration less than or equal to about 1 × 10⁻⁶. -9 M of K D Combined with the FcRn sequence shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR).

[0142] In some implementations, compared to Fc fragments containing wild-type Fc regions at pH 6.0, the Fc fragments exhibit a lower Kc at the same pH. D Combine with FcRn.

[0143] In some embodiments, the Fc fragment provided by the present invention is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -8 1.1×10 -8 1.2×10 -8 1.3×10 -8 1.4×10 -8、1.5×10 -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 、3×10 -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 、1×10 -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 3×10 -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 M of K D Combined with FcRn, as measured by ELISA or any other suitable method known in the art.

[0144] In some embodiments, the Kc of the Fc fragment provided by this invention binds to FcRn at pH 6.0. D Approximately 1.0-1.1 × 10 -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, 1-2×10 -8 M, 1-5×10 -8 M, 2-7×10 -8 M, 3-8×10 -8 M, 3-5×10 -8 M, 4-6×10 -8 M, 5-7×10 -8 M, 6-8×10 -8 M, 7-9×10 -8 M, 7-9.9×10 -8 M, 5-9.9×10 -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 -9M, 1.7-1.8×10 -9 M, 1.8-1.9×10 -9 M, 1.9-2×10 -9 M, 1-2×10 -9 M, 1-5×10 -9 M, 2-7×10 -9 M, 3-8×10 -9 M, 3-5×10 -9 M, 4-6×10 -9 M, 5-7×10 -9 M, 6-8×10 -9 M, 7-9×10 -9 M, 7-9.9×10 -9 M or 5-9.9×10 -9 M, as measured by ELISA or any other suitable method known in the art. In some embodiments, the Fc fragment provided by the present invention is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -8 or less than or equal to approximately 1 × 10 -9 K D Combined with FcRn, as measured by ELISA or any other suitable method known in the art.

[0145] In some embodiments, the Fc fragment provided by the present invention is at a pH of 7.4 with a concentration greater than or equal to about 1 × 10⁻⁶. -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 3×10 -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 1×10 -6 1.1×10 -61.2×10 -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 3×10 -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 1×10 -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 3×10 -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 K D Combined with FcRn, as measured by ELISA or any other suitable method known in the art.

[0146] In some embodiments, the Kc of the Fc fragment provided by this invention binds to FcRn at pH 6.0. D Approximately 1.0-1.1 × 10 -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、1-2×10 -5 M、1-5×10 -5 M、2-7×10 -5 M、3-8×10 -5 M、3-5×10 -5 M、4-6×10 -5 M、5-7×10 -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 -7M, 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 -7 M, 7-9×10 -7 M, 7-9.9×10 -7 M, 5-9.9×10 -7 M, as measured by ELISA or any other suitable method known in the art. In some embodiments, the Fc fragment provided by the present invention is at pH 7.4 with a concentration greater than or equal to about 1 × 10⁻⁶. -5 or greater than or equal to approximately 1 × 10 -6 K D Combined with FcRn, as measured by ELISA or any other suitable method known in the art.

[0147] In some embodiments, the Fc fragment is at pH 6.0 at a concentration of less than or equal to about 1 × 10⁻⁶. -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 -9M or 9×10 -9 M of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 10 or 11, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -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 of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 12 or 13, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -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 M of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 14 or 15, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -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 of K D Combined with the FcRn sequence shown in SEQ ID NO: 16 or 17, as measured by surface plasmon resonance (SPR).

[0148] In some embodiments, the Fc fragment is at pH 7.4 at a concentration greater than or equal to about 1 × 10⁻⁶. -7 M, 2×10 -7M, 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 of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 10 or 11, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 7.4 with a concentration greater than or equal to about 1 × 10⁻⁶. -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, 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 M of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 12 or 13, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 7.4 with a concentration greater than or equal to about 1 × 10⁻⁶. -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 of K D The Fc fragment is bound to the FcRn sequence shown in SEQ ID NO: 14 or 15, as measured by surface plasmon resonance (SPR). In some embodiments, the Fc fragment is at pH 7.4 with a concentration greater than or equal to about 1 × 10⁻⁶. -8 M, 2×10 -8M, 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 M of K D Combined with the FcRn sequence shown in SEQ ID NO: 16 or 17, as measured by surface plasmon resonance (SPR).

[0149] Fc fragment variant fusion protein In some embodiments, the Fc fragment variants of the present invention are fused or compounded with various protein, peptide, and antibody fragments.

[0150] In some embodiments, the Fc fragment variant is fused or compounded with Fab. In some embodiments, the Fc fragment variant is fused or compounded with scFv. In some embodiments, the Fc fragment variant is fused or compounded with sdAb. In some embodiments, the Fc fragment variant is fused or compounded with VHH. In some embodiments, the Fc fragment variant is fused or compounded with VNAR.

[0151] 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 domain. In some embodiments, the Fc fragment variant is fused or complexed with an albumin-binding scFv domain. In some embodiments, the Fc fragment variant is fused or complexed with an albumin-binding sdAb domain. In some embodiments, the Fc fragment variant is fused or complexed with an albumin-binding VHH domain. In some embodiments, the Fc fragment variant is fused or complexed with an albumin-binding VNAR domain.

[0152] In some implementations, the Fc fragment variant is fused or compounded with albumin or a variant thereof.

[0153] In some embodiments, the Fc fragment variant is fused or compounded with an HSA-binding domain. In some embodiments, the Fc fragment variant is fused or compounded with an HSA-binding Fab. In some embodiments, the Fc fragment variant is fused or compounded with an HSA-binding scFv. In some embodiments, the Fc fragment variant is fused or compounded with an HSA-binding sdAb. In some embodiments, the Fc fragment variant is fused or compounded with an HSA-binding VHH. In some embodiments, the Fc fragment variant is fused or compounded with an HSA-binding VNAR.

[0154] Pharmaceutical Composition This application provides compositions comprising Fc fragments, said compositions comprising any and more of the Fc fragments described herein and one or more pharmaceutically acceptable excipients. In some embodiments, the compositions are sterile. The pharmaceutical compositions typically contain an effective amount of the Fc fragment.

[0155] In addition to one or more Fc fragments disclosed herein, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials may depend on the route of administration, such as oral, intravenous, skin or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0156] Pharmaceutical compositions intended for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may include solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions typically include liquid carriers such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. They may include physiological saline solutions, dextran or other sugar solutions, or glycols (e.g., ethylene glycol, propylene glycol, or polyethylene glycol).

[0157] For intravenous, subcutaneous, or subcutaneous injection, or injection at the site of pain, the active ingredient may be in a parenteral acceptable aqueous solution that is pyrogenic and has suitable pH, isotonicity, and stability. Those skilled in the art can readily prepare suitable solutions using isotonic media such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included, as needed.

[0158] The intended Fc fragment to be administered to an individual may be given in a “therapeutic effective dose” or a “prophylactic effective dose” (depending on the specific circumstances, although prophylaxis can also be considered a therapy), which is sufficient to demonstrate benefit to the individual. The actual dose administered, as well as the rate and timing of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Treatment prescriptions, such as the determination of dosage, are within the purview of general practitioners and other physicians, and generally take into account the condition to be treated, the individual patient’s condition, the site of delivery, the method of administration, and other factors known to the professional. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

[0159] In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous injection.

[0160] Depending on the condition to be treated, the composition may be administered alone or in combination with other treatments, simultaneously or sequentially.

[0161] method Preparation method The Fc fragments described herein can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the Fc fragment described herein is provided. This nucleic acid encodes an amino acid sequence comprising an Fc fragment that binds to the FcRn described herein. In another embodiment, one or more vectors (e.g., expression vectors) comprising this nucleic acid are provided. In one embodiment, the nucleic acid is provided in a polycistronic vector. In another embodiment, a host cell comprising this nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., having been transformed with): a vector containing a nucleic acid encoding an amino acid sequence comprising the Fc fragment disclosed herein. In one embodiment, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells or lymphocytes (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method of manufacturing an Fc fragment is provided, wherein the method comprises culturing the host cell provided above containing the nucleic acid encoding the Fc fragment under conditions suitable for expression of the Fc fragment and optionally recovering the Fc fragment from the host cell (or host cell culture medium).

[0162] To generate the Fc fragment through recombination, nucleic acids encoding the Fc fragment, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the Fc fragment.

[0163] When host cells generate Fc fragments in a recombinant manner, in some embodiments, the protein is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the cell dry weight. When host cells generate Fc fragments in a recombinant manner, in some embodiments, the protein is present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less. In some embodiments, the purity level of the "substantially purified" Fc fragment produced by the methods described herein is 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%, at least about 70%, and more specifically at least about 75%, 80%, 85%, and more specifically at least about 90%, at least about 95%, at least about 99% or greater, as determined by appropriate methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0164] The host cells suitable for cloning or expressing vectors encoding Fc fragments include prokaryotic or eukaryotic cells as described herein.

[0165] The recombinant host cell, or host cell, is a cell comprising exogenous polynucleotides, regardless of the method used for insertion (e.g., direct uptake, transduction, f-coordination, or other methods known in the art for generating recombinant host cells). The exogenous polynucleotides may remain as non-integrating vectors, such as plasmids, or may be integrated into the host genome. The host cell may include CHO, CHO derivatives, NSO, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0166] For example, Fc fragments can be produced in bacteria, especially where glycosylation and Fc effector functions are not required. For the expression of Fc fragments and peptides 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 (edited by BKC Lo, HumanaPress, Totowa, NJ, 2003), pp. 245-254, which describes the expression of Fc fragments in *E. coli*). After expression, the Fc fragments can be separated from the bacterial cell paste by soluble fractions and can be further purified.

[0167] Besides 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 have been "humanized," thereby producing Fc fragments with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006).

[0168] Suitable host cells for expressing glycosylated Fc fragments are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfecting fall armyworm (Spodoptera frugiperda) cells.

[0169] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES™ technology for generating antibodies in transgenic plants).

[0170] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension are suitable. Other examples of suitable mammalian host cell lines include monkey kidney CV1 cell lines transformed with SV40 (COS-7); human embryonic kidney cell lines (such as 293 or 293 cells as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK); mouse seteloli cells (such as TM4 cells as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumors (MMT060562); as described, for example, in Mather et al., Annals NY Acad. Sci. TRI cells, MRC 5 cells, and FS4 cells, as described in 383:44-68 (1982), are also applicable. Other suitable 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)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines applicable to the production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, HumanaPress, Totowa, NJ), pp. 255-268 (2003).

[0171] In one embodiment, the Fc fragment described herein is generated 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 at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acid is determined in a transient transfection experiment to determine the relative ratio of the input nucleic acid, thereby maximizing the percentage of the Fc fragment in the expression product.

[0172] In some embodiments, a method is provided for generating glycosylated Fc fragments in stable mammalian cells as described herein, the method comprising identifying and purifying desired glycosylated Fc fragments. In some embodiments, mammalian cells are selected and cultured under conditions intended to generate a large percentage of the desired glycosylated Fc fragments. In some embodiments, the identification of the desired glycosylated Fc fragments is performed by one or both of liquid chromatography and mass spectrometry.

[0173] If necessary, the Fc fragment can be purified or isolated after expression. Proteins can be isolated or purified using a variety of methods known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interactions, affinity, size determination, or gel filtration and reverse phase, performed at atmospheric pressure or under high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoresis, immunoassay, precipitation, dialysis, and chromatographic focusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also applicable. As is well known in the art, many natural proteins bind to the Fc region, and these proteins can be used in this invention to purify the Fc fragment. For example, bacterial proteins A and G bind to the Fc region. Purification can often be achieved using specific fusion partners. For example, glutathione resin (when using GST fusions), Ni +2 Purify the antibody using affinity chromatography (when His-tagged) or by immobilizing the anti-flag antibody (when Flag-tagged). For general guidelines on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Edition, Scopes, Springer-Verlag, NY, 1994, which is incorporated herein by reference in its entirety.

[0174] In some embodiments, the Fc fragment is purified using anion exchange chromatography, including (but not limited to) chromatography on Q-agarose gel, DEAE agarose gel, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, and Fractogel Q and DEAE columns.

[0175] In certain embodiments, the proteins described herein are purified using cation exchange chromatography, including but not limited to SP-agarose gel, CM agarose gel, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and analogues.

[0176] Additionally, 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, WHFreeman & Co., NY, and Hunkapiller et al., Nature, 310:105-111 (1984)). For instance, peptides corresponding to the polypeptide fragments can be synthesized using a peptide synthesizer. Furthermore, atypical amino acids or chemical amino acid analogs can be introduced as substitutions or additions to the polypeptide sequence, as needed. Generally, atypical amino acids include (but are not limited to) D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, sulfoalanine, tert-butylglycine, tert-butylalanine, phenylglycine, alanine cyclohexyl ester, alanine, fluoroamino acids, designer amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs. Furthermore, the amino acids may be D-(dextral) or L-(levorotatory).

[0177] How to use In one aspect, this application provides a method for contacting FcRn with an Fc fragment as described herein, such as in vivo or in vitro contact with a desired Fc fragment, which results in the inhibition of IgG binding to the FcRn receptor. In some embodiments, the FcRn is expressed on a cell surface.

[0178] In one aspect, this application provides a method for treating a subject's condition or disease using the isolated Fc fragments described herein. In other aspects, this application describes a method for treating a subject in need with an Fc fragment as described herein, the method comprising administering to a mammalian subject a therapeutically effective amount of the Fc fragment or a pharmaceutical composition comprising the Fc fragments described herein. In some embodiments, this application provides a method for treating a subject with a condition or disease associated with elevated IgG levels by administering the disclosed Fc fragments.

[0179] In some respects, this article describes a method for treating pathologies associated with IgG activity, the method comprising administering to a mammalian subject a therapeutically effective amount of a separated Fc fragment or a pharmaceutical composition comprising the separated Fc fragment described herein.

[0180] In some respects, this article describes a method for treating pathology associated with elevated FcRn levels in mammalian subjects in need, the method comprising administering to the mammalian subject a therapeutically effective amount of the Fc fragment or pharmaceutical composition described herein.

[0181] In some aspects, this document describes a method for treating or preventing antibody-related conditions or diseases (e.g., autoimmune diseases or conditions associated with unwanted side effects of therapeutic antibodies) in a mammalian subject of need, the method comprising administering to the mammalian subject a therapeutically effective amount of any of the Fc fragments of the embodiments disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the inflammatory condition or disease is an autoimmune disease. In some embodiments, the inflammatory condition or disease is myasthenia gravis (gMG). In some embodiments, the inflammatory condition or disease is immune thrombocytopenic purpura (ITP).

[0182] In some respects, this document describes a method for treating a pathology associated with elevated IgG levels in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of an Fc fragment of any of the embodiments disclosed herein or a pharmaceutical composition disclosed herein.

[0183] In some respects, this document describes a method for reducing the bioactivity of IgG in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of either a separated Fc fragment of any of the embodiments disclosed herein or a pharmaceutical composition disclosed herein.

[0184] In some respects, this document describes a method for treating or preventing autoimmune diseases. In some embodiments, the autoimmune disease is caused by autoreactive antibodies.

[0185] In some implementations, the autoimmune disease is selected from the group consisting of: allogeneic islet transplant rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), adrenal autoimmune diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac disease-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), and Churg-Strauss syndrome. syndromes, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, dilated cardiomyopathy, discoid lupus, acquired epidermolysis bullosa, primary mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyalgia, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease. Diseases including lichen planus, lichen sclerosus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, mucosal pemphigoid, multiple sclerosis, type 1 diabetes, multifocal motor neuropathy (MMN), myasthenia gravis, paraneoplastic bullous pemphigoid, gestational pemphigoid, pemphigus vulgaris, foliaceous pemphigoid, pernicious anemia, polyarteritis nodosa, polychondritis, polygonatum syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Raynaud's phenomenon, and Reiter's syndrome.Rheumatoid arthritis, sarcomatoid disease, scleroderma, Sjogren's syndrome, solid organ transplant rejection, stiff-person syndrome, systemic lupus erythematosus, takayasuarteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, herpetic dermatitis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegner's granulomatosis.

[0186] Methods of application and manufacture In some embodiments, the methods provided herein can be used to treat an individual's disease or condition. In one embodiment, the individual is a human and the treatment includes administering the Fc fragment described herein.

[0187] In some implementations, the Fc fragment is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorally, intraorally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some implementations, the Fc fragment is administered intravenously. An effective amount of the Fc fragment can be administered to treat a disease or condition. The appropriate dose of the Fc fragment can be determined based on the following: the type of disease or condition to be treated, the type of Fc fragment, the severity and duration of the disease or condition, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician.

[0188] This application also includes at least the following implementation schemes: Implementation Scheme 1. An isolated Fc fragment that binds to the neonatal Fc receptor (FcRn) of humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant contains at least one amino acid substitution selected from M428L, H433R, H433K and N434Y.

[0189] Implementation Scheme 2. The isolated Fc fragment as described in Implementation Scheme 1, wherein the Fc fragment contains an additional amino acid substitution at one or more of amino acid positions 252, 254 and 256 of SEQ ID NO: 1.

[0190] Implementation Scheme 3. The isolated Fc fragment as described in Implementation Scheme 2, wherein the additional amino acid is replaced with M252Y, S254T and / or T256E.

[0191] Implementation Scheme 4. The isolated Fc fragment as described in Implementation Scheme 1, wherein the variant comprises amino acid substitutions of M252Y, S254T, T256E, M428L, H433K and N434F, or M252Y, S254T, T256E, H433K and N434Y.

[0192] Implementation Scheme 5. The isolated Fc fragment as described in Implementation Scheme 4, wherein the variant comprises amino acid substitutions M252Y, S254T, T256E, M428L, H433K, and N434F.

[0193] Implementation Scheme 6. The isolated Fc fragment as described in Implementation Scheme 5, wherein the variant comprises the amino acid sequence of SEQ ID NO: 3.

[0194] Implementation Scheme 7. The isolated Fc fragment as described in Implementation Scheme 4, wherein the variant comprises amino acid substitutions M252Y, S254T, T256E, H433K, and N434Y.

[0195] Implementation Scheme 8. The isolated Fc fragment as described in Implementation Scheme 7, wherein the variant comprises the amino acid sequence of SEQ ID NO: 5.

[0196] Implementation Scheme 9. An isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant contains an amino acid substitution at position 428.

[0197] Implementation Scheme 10. The isolated Fc fragment as described in Implementation Scheme 9, wherein the amino acid is substituted with M428L.

[0198] Implementation Scheme 11. The isolated Fc fragment as described in Implementation Scheme 9 or 10, wherein the Fc fragment contains 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.

[0199] Implementation Scheme 12. The isolated Fc fragment as described in Implementation Scheme 11, wherein the additional amino acid substitution is M252Y, S254T, T256E, H433K, H433R, N434F and / or N434Y.

[0200] Implementation Scheme 13. An isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant contains an amino acid substitution H433R.

[0201] Implementation Scheme 14. The isolated Fc fragment as described in Implementation Scheme 13, wherein the Fc fragment contains 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.

[0202] Implementation Scheme 15. The isolated Fc fragment as described in Implementation Scheme 14, wherein one or more additional amino acids are substituted with M252Y, S254T, T256E, M428L, N434F and / or N434Y.

[0203] Implementation Scheme 16. An isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant contains an amino acid substitution N434Y.

[0204] Implementation Scheme 17. The isolated Fc fragment as described in Implementation Scheme 16, wherein the Fc fragment contains 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.

[0205] Implementation Scheme 18. The isolated Fc fragment as described in Implementation Scheme 17, wherein the amino acid substitutions are M252Y, S254T, T256E, M428L, H433K and / or H433R.

[0206] Implementation Scheme 19. An isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution H433K, and additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 428 and 434.

[0207] Implementation Scheme 20. The isolated Fc fragment as described in Implementation Scheme 19, wherein the additional amino acid is substituted with M252Y, S254T, T256E, M428L, N434F and / or N434Y.

[0208] Implementation Scheme 21. The isolated Fc fragment as described in any of the above embodiments, wherein the Fc fragment comprises a sequence selected from any of SEQ ID NO: 3-9.

[0209] Implementation Scheme 22. The isolated Fc fragment as described in any one of Implementation Schemes 1-16, wherein the Fc fragment blocks or reduces the recycling of naturally occurring antibodies.

[0210] Implementation Scheme 23. The isolated Fc fragment as described in Implementation Scheme 22, wherein the Fc fragment causes an increase in the catabolism of pathogenic antibodies.

[0211] Implementation Scheme 24. The isolated Fc fragment as described in Implementation Scheme 23, wherein the pathogenic antibody is an antibody associated with an autoimmune disease.

[0212] Implementation Scheme 25. The isolated Fc fragment as described in any one of Implementation Schemes 1-24, wherein, compared to containing a wild-type Fc region and measurable K at pH 7.4 D The Fc fragment, which is at a low Kc at pH 6.0. D It binds to FcRn in humans, cynomolgus monkeys, mice, or rats.

[0213] Implementation Scheme 26. The separated Fc fragment as described in any one of Schemes 1-25, wherein the Fc fragment is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -8 M of K D It binds to the FcRn sequence shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR).

[0214] Implementation Scheme 27. The separated Fc fragments as described in Implementation Scheme 26, wherein the Fc fragments are at about pH 6.0 at a concentration of less than or equal to about 1, 2, 3, 4, 5, 6, 7, 8 or 9 × 10⁻⁶. -9 M of K D It binds to the FcRn sequence shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR).

[0215] Implementation Scheme 28. The separated Fc fragment as described in any one of Implementation Schemes 1-27, wherein the Fc fragment exhibits a melting temperature greater than 55°C, as measured by differential scanning fluorometry (DSF).

[0216] Implementation Scheme 29. The separated Fc fragment as described in any one of Implementation Schemes 1-28, wherein the Fc fragment exhibits an aggregation temperature equal to or greater than 70°C, as measured by differential scanning fluorescence (DSF).

[0217] Implementation Scheme 30. The isolated Fc fragment as described in any one of Implementation Schemes 1-29, wherein the Fc fragment is used to treat an antibody-related condition or disease.

[0218] Implementation Scheme 31. The isolated Fc fragment as described in Implementation Scheme 30, wherein the Fc fragment is used to treat autoimmune diseases.

[0219] Implementation Scheme 32. The isolated Fc fragment as described in Implementation Scheme 30 or 31, said Fc fragment being used to treat diseases or conditions selected from the group consisting of: generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuropathy, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disease (MOG antibody disease), membranous nephropathy, lupus nephritis, thyroid ophthalmopathy, warm antibody-type autoimmune hemolytic anemia, hemolytic diseases of the fetus and newborn, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, foliaceous pemphigus, pemphigus vulgaris, and cutaneous lupus erythematosus.

[0220] Implementation Scheme 33. The isolated Fc fragment as described in Implementation Scheme 32, wherein the treatment reduces the severity of the patient’s disease and wherein the severity of the disease is assessed by a gMG disease severity outcome measure.

[0221] Implementation Scheme 34. An isolated polynucleotide or polynucleotide group encoding the isolated Fc fragment described in any one of the above embodiments, and optionally, wherein the polynucleotide or polynucleotide group comprises mRNA or cDNA.

[0222] Implementation Scheme 35. A vector or vector group comprising the polynucleotide or polynucleotide group described in Implementation Scheme 34.

[0223] Implementation Scheme 36. A host cell comprising the polynucleotide or polynucleotide group of embodiment 30 or the vector or vector group of embodiment 35.

[0224] Implementation Scheme 37. A method for generating an Fc fragment, the method comprising expressing the Fc fragment in a host cell as described in Implementation Scheme 36 and isolating the expressed Fc fragment.

[0225] Implementation Scheme 38. A pharmaceutical composition comprising the isolated Fc fragment of any one of Implementation Schemes 1-33 and a pharmaceutically acceptable excipient.

[0226] Implementation Scheme 39. A method for treating a symptom or disease in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of any one of Implementation Schemes 1-33, or the pharmaceutical composition of Implementation Scheme 33.

[0227] Implementation Scheme 40. The method as described in Implementation Scheme 39, wherein the disease or condition is selected from the group consisting of: generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuropathy, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disease (MOG antibody disease), membranous nephropathy, lupus nephritis, thyroid ophthalmopathy, warm antibody-type autoimmune hemolytic anemia, hemolytic diseases of the fetus and newborn, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, foliaceous pemphigoid, pemphigus vulgaris, and cutaneous lupus erythematosus.

[0228] Implementation Scheme 41. The method as described in Implementation Scheme 40, wherein the condition or disease is gMG.

[0229] Implementation Scheme 42. The method of implementation scheme 41, wherein the method reduces the severity of the patient’s disease and wherein the severity of the disease is assessed by a gMG disease severity outcome measure.

[0230] Implementation Scheme 43. A method for treating a pathology associated with elevated IgG levels in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of the isolated Fc fragment of any one of Implementation Schemes 1-33 or the pharmaceutical composition of Implementation Scheme 38.

[0231] Implementation Scheme 44. A method for reducing the bioactivity of IgG in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of the isolated Fc fragment of any one of Implementation Schemes 1-33 or the pharmaceutical composition of Implementation Scheme 38.

[0232] Implementation Scheme 45. The method as described in Implementation Scheme 44, wherein the disease is an autoimmune disease.

[0233] Implementation Scheme 46. A method for preventing a condition in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of the isolated Fc fragment of any one of Implementation Schemes 1-33 or the pharmaceutical composition of Implementation Scheme 38; wherein the condition is an undesirable side effect of the therapeutic antibody.

[0234] Implementation Scheme 47. An Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions M428L and N434F compared to the amino acid sequence shown in SEQ ID NO: 1.

[0235] Implementation Scheme 48. The Fc fragment variant as described in Implementation Scheme 47, wherein the Fc fragment further comprises an amino acid substitution H433K or H433R.

[0236] Implementation Scheme 49. The Fc fragment variant as described in Implementation Scheme 47, wherein the Fc fragment further comprises amino acid substitutions H433K and N434F.

[0237] Implementation Scheme 50. The Fc fragment variant as described in Implementation Scheme 47, wherein the Fc fragment further comprises amino acid substitutions H433R and N434F.

[0238] Implementation Scheme 51. An Fc fragment variant that binds to a neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions (i) N434Y and (ii) H433R or H433K compared to the amino acid sequence shown in SEQ ID NO: 1.

[0239] Implementation Scheme 52. The Fc fragment variant as described in Implementation Scheme 51, wherein the Fc fragment comprises amino acid substitutions for H433K and N434Y.

[0240] Implementation Scheme 53. The Fc fragment variant as described in Implementation Scheme 51, wherein the Fc fragment comprises amino acid substitutions for H433R and N434Y.

[0241] Implementation Scheme 54. An Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions M428L and N434Y compared to the amino acid sequence shown in SEQ ID NO: 1.

[0242] Implementation Scheme 55. The Fc fragment variant as described in Implementation Scheme 54, wherein the Fc fragment further comprises an amino acid substitution H433K.

[0243] Implementation Scheme 56. An Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions M428L and H433R compared to the amino acid sequence shown in SEQ ID NO: 1.

[0244] Implementation Scheme 57. The Fc fragment variant as described in Implementation Scheme 56, wherein the Fc fragment further comprises an amino acid substitution N434Y.

[0245] Implementation Scheme 58. An Fc fragment variant that binds to a neonatal Fc receptor (FcRn), wherein the Fc fragment variant comprises amino acid substitutions H433R and H434F compared to the amino acid sequence shown in SEQ ID NO: 1.

[0246] Implementation Scheme 59. A variant of the Fc fragment as described in any one of Implementation Schemes 47-58, wherein the Fc fragment further comprises amino acid substitutions M252Y, S254T, and T256E.

[0247] Implementation Scheme 60. The Fc fragment variant as described in Implementation Scheme 59, wherein the Fc fragment comprises amino acid substitutions of M252Y, S254T, T256E, M428L, H433K, and N434F.

[0248] Implementation Scheme 61. The Fc fragment variant as described in Implementation Scheme 60, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:3.

[0249] Implementation Scheme 62. The Fc fragment variant as described in Implementation Scheme 59, wherein the Fc fragment comprises amino acid substitutions of M252Y, S254T, T256E, M428L, H433K, and N434Y.

[0250] Implementation Scheme 63. The Fc fragment variant as described in Implementation Scheme 62, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:4.

[0251] Implementation Scheme 64. The Fc fragment variant as described in Implementation Scheme 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, H433K, and N434Y.

[0252] Implementation Scheme 65. A variant of the Fc fragment as described in Implementation Scheme 64, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:5.

[0253] Implementation Scheme 66. The Fc fragment variant as described in Implementation Scheme 59, wherein the Fc fragment comprises amino acid substitutions of M252Y, S254T, T256E, M428L, and N434F.

[0254] Implementation Scheme 67. A variant of the Fc fragment as described in Implementation Scheme 66, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:6.

[0255] Implementation Scheme 68. The Fc fragment variant as described in Implementation Scheme 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, H433R, and N434Y.

[0256] Implementation Scheme 69. A variant of the Fc fragment as described in Implementation Scheme 68, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:7.

[0257] Implementation Scheme 70. The Fc fragment variant as described in Implementation Scheme 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, H433R, and N434F.

[0258] Implementation Scheme 71. A variant of the Fc fragment as described in Implementation Scheme 70, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:8.

[0259] Implementation Scheme 72. The Fc fragment variant as described in Implementation Scheme 59, wherein the Fc fragment comprises amino acid substitutions M252Y, S254T, T256E, M428L, H433R, and N434F.

[0260] Implementation Scheme 73. The Fc fragment variant as described in Implementation Scheme 72, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:9.

[0261] Implementation Scheme 74. The Fc fragment variant as described in any one of Implementation Schemes 47-73, wherein the Fc fragment variant does not contain amino-substituted L309D.

[0262] Implementation Scheme 75. An Fc fragment variant having at least 85%, 90%, 93%, 95%, 97%, or 99% identity with the following amino acid sequences: SEQ ID NO: 3, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, M428L, H433K and N434F; SEQ ID NO: 4, provided that this variant contains amino acid substitutions M252Y, S254T, 256E, M428L, H433K and N434Y; SEQ ID NO: 5, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, H433K and N434Y; SEQ ID NO: 6, provided that this variant contains amino acid substitutions S254T, T256E, M428L and N434F; SEQ ID NO: 7, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, H433R, and N434Y; SEQ ID NO: 8, provided that this variant contains amino acid substitutions for M252Y, S254T, T256E, H433R, and N434F; or SEQ ID NO: 9, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, M428L, H433R and N434F.

[0263] Implementation Scheme 76. The Fc fragment variant as described in Implementation Scheme 75, wherein the Fc fragment variant inhibits IgG binding to human FcRn with an IC50 value of less than 2.5 nM, 2.0 nM, 1.5 nM or 1.0 nM.

[0264] Implementation Scheme 77. The Fc fragment variant as described in Implementation Scheme 76, wherein the Fc fragment variant inhibits IgG binding to human FcRn at pH 6.0.

[0265] Implementation Scheme 78. A variant of the Fc fragment as described in any one of Implementation Schemes 47-77, wherein the Fc fragment is fused or compounded with a half-life extension domain.

[0266] Implementation Scheme 79. The Fc fragment variant as described in Implementation Scheme 78, wherein the half-life extension domain is an albumin, an albumin-binding domain, or an HSA-binding domain.

[0267] Implementation Scheme 80. A method for inhibiting IgG binding to FcRn, the method comprising administering an Fc fragment variant as described in any one of Implementation Schemes 47-79.

[0268] Implementation Scheme 81. A method for treating a symptom or disease in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of any one of the Fc fragment variants described in Implementation Schemes 47-79.

[0269] Implementation Scheme 82. The method as described in Implementation Scheme 81, wherein the disease or condition is selected from the group consisting of: generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuropathy, myositis, autoimmune encephalitis, myelin oligodendrocyte glycoprotein antibody disease (MOG antibody disease), membranous nephropathy, lupus nephritis, thyroid ophthalmopathy, warm antibody-type autoimmune hemolytic anemia, hemolytic diseases of the fetus and newborn, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, foliaceous pemphigoid, pemphigus vulgaris, and cutaneous lupus erythematosus.

[0270] Implementation Scheme 83. The method as described in Implementation Scheme 82, wherein the condition or disease is gMG.

[0271] Implementation Scheme 84. The method of implementation scheme 83, wherein the method reduces the severity of the patient’s disease and wherein the severity of the disease is assessed by a gMG disease severity outcome measure.

[0272] Implementation Scheme 85. A method for treating a pathology associated with elevated IgG levels in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of any one of the Fc fragment variants described in Implementation Schemes 47-79.

[0273] Implementation Scheme 86. A method for reducing the bioactivity of IgG in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of any one of the Fc fragment variants described in Implementation Schemes 47-79.

[0274] Implementation Scheme 87. The method as described in Implementation Scheme 86, wherein the disease is an autoimmune disease.

[0275] Implementation Scheme 88. A method for preventing a condition in a mammalian subject in need, the method comprising administering to the mammalian subject a therapeutically effective amount of any one of the Fc fragment variants of Implementation Schemes 47-79; wherein the condition is an undesirable side effect of the therapeutic antibody.

[0276] Example The following are embodiments for carrying out specific implementations of the invention. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Every effort has been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental error and deviation should, of course, be allowed.

[0277] Unless otherwise specified, the practice of this invention will be carried out using conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are well described in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods in Enzymology (edited by S. Colowick and N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (Plenum Press) Volume A and Volume B (1992).

[0278] method Gene synthesis and plasmid construction The coding sequence for the Fc fragment was generated through DNA synthesis and PCR, and then subcloned into a pcDNA3.1-based plasmid for protein expression in mammalian cell systems. The gene sequence in the expression vector was confirmed by DNA sequencing.

[0279] Expression of Fc fragments Transient antibody expression was performed using the ExpiCHO™ expression system (ThermoFisher, catalog number A29133). ExpiCHO-S cells were expanded from a working cell bank and passaged in ExpiCHO expression medium according to the manufacturer's instructions. The mixture for transfection was prepared following 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 filter and DE as a filter aid (Sartorius, catalog number SDLV-0150-05E0-2), and then immediately processed. Conditioned medium was collected for protein purification.

[0280] Purification of Fc fragments The generated antibodies were obtained from the clear supernatant using a HiTrap MabSelect PrismA 25 mL column (Cytiva, catalog number 17-5498-54) on an ÄKTA Pure 25 FPLC system. Column equilibration and protein binding were performed using 20 mM sodium phosphate with 150 mM NaCl at pH 7.4, followed by protein elution using 100 mM sodium citrate with 150 mM NaCl at pH 3.5. Immediately after elution, the peak corresponding to affinity-purified antibodies was neutralized with 30% 1 M Tris at pH 8.0. To purify the sample and obtain a high-purity monomeric form with a purity >95%, the protein sample was loaded onto a HiLoad 26 / 600 Superdex 200 pg column (Cytiva, catalog number 28-9893-36) on an ÄKTA Pure 25 FPLC system. Elution fractions corresponding to the monomeric antibody were collected from 96-well plates into 50 ml Falcon tubes and filtered through a 0.22 µm PES membrane (Fisher brand, catalog number 15206869, lot number 2103171806) in a laminar flow chamber. Protein samples were then transferred to a 50 kDa MWCO rotary concentrator (Amicon 50K, catalog number UFC905024; lot number 0000187574) for concentration, with each centrifugation performed at 4000 x g for 10 min, repeated until the desired concentration was achieved.

[0281] SEC-HPLC analysis of the Fc fragment Analytical SEC-HPLC was performed using a Thermo Vanquish Flex UHPLC system (Thermo Fisher) and a TSKgel Super SWmAb 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 and 0.05% sodium azide, pH 6.7, with a flow rate of 0.35 mL / min for 10 min at 25 °C.

[0282] Protein thermal stability test by differential scanning fluorometry (DSF) 20 mM sodium acetate containing the Fc fragment at pH 5.5 was loaded into UNi (Unchained Labs). The samples were subjected to thermal heating from 20 to 95 °C at a rate of 0.3 °C / min. Fluorescence intensity at 330 / 350 nm and the ratio of SLS at 266 nm to temperature were acquired. The inflection point (T0) of the transition curve was measured using UNCLE software. m The fluorescence ratio of the second derivative peak was used to determine Tagg, and the relationship between the SLS signal at 266 nm and temperature was used to determine Tagg.

[0283] HIC-HPLC analysis of Fc fragments HIC-HPLC was performed on a Proteomix HIC Butyl column with a Proteomix HIC Butyl pre-column on a UHPLC Vanquish Flex (ThermoFisher Scientific, MA) with a salt gradient at a flow rate of 0.8 mL / min for 15 min. Detection was performed at a wavelength of 220 nm. Substances eluted before the main peak were designated as less hydrophobic, while those eluted after the main peak were designated as more hydrophobic.

[0284] Dynamic light scattering (DLS) Protein particle size distribution and molecular size were monitored using dynamic light scattering (DLS) with UNCLE (Unchained Labs). Three 9 µL samples were loaded into UNI sample holders, and the intensity of scattered light was measured at 20 °C for 10 s each time.

[0285] Non-reducing and reducing sodium dodecyl sulfate capillary electrophoresis (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 Proteinclear HT chip (catalog number CLS1486695) under both reducing and non-reducing conditions, following the manufacturer's instructions. Reduced samples were treated with β-mercaptoethanol, and non-reduced samples were treated with iodoacetamide (IAM) prior to analysis. For CE-SDS using Maurice, 25 µg of each sample was analyzed using a CE-SDS cartridge (ProteinSimple, catalog number PS-MC02-SP), following the manufacturer's instructions. Reduced samples were treated with β-mercaptoethanol.

[0286] Differential scanning calorimetry (DSC) DSC experiments were performed using a Microcal PEAQ DSC-DSC automated differential scanning calorimeter (Malvern Panalytical). All solutions and samples used for DSC were filtered using a 0.22-µm filter and degassed before loading into the calorimeter. Antibodies used for DSC studies were determined to be >95% monomers 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 exchanges). The buffer from this dialyze was then used as a reference buffer for subsequent DSC experiments. Baseline measurements (buffer to buffer) were obtained before sample measurements and subtracted from the sample measurements. 325 μL of each dialyzed sample (0.5 mg / ml) was added in duplicate to the sample, and DSC measurements were performed at a scan rate of 1.5 °C / min. Data analysis and deconvolution were performed using the manufacturer's DSC software.

[0287] PEG precipitation determination PEG precipitation assays were performed in a manner similar to that described by Gibson et al., J. Pharm. Sci. 100: 1009-1021 (2011). Incremental amounts of PEG 4000 were added to precipitate the antibody. Test buffers containing 40% PEG stock solution were prepared, from which eight aliquots were prepared in 96-well plates with different final PEG percentages (ranging from 20% to 0%), diluted 1:1 with the protein sample to a total volume of 100 µL per well. The 96-well plates were incubated overnight at room temperature, and then read using a microplate reader to measure the optical density at 320 nm.

[0288] Capillary isoelectric focusing (cIEF) According to the manufacturer's instructions, this procedure was performed on a Maurice (ProteinSimple, CA) system using a cIEF filter cartridge (ProteinSimple, catalog number PS-MC02-C). Prior to analysis, the sample was diluted in a master mixture containing a mixture of amphoteric electrolytes, pI label, 1% methylcellulose, 12.5% ​​arginine, and 4M urea.

[0289] Example 1: Measurement of antibody fragment-FcRn binding kinetics using surface plasmon resonance The Biacore 8K SPR system (GE Healthcare) equipped with a CM5 Sensor Chip (Cytiva, catalog 29149603) was used to immobilize anti-human Fc-specific antibodies via amine coupling for determination of binding kinetics and affinity constants at 25°C in electrophoresis buffer 1xHBS-EP+pH 6.0 or pH 7.4 (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20) (Cytiva, catalog BR100669). After stabilization in the electrophoresis buffer, 20 nM of the anti-FcRn mAb construct was captured onto flow cell 2 (active) for 60 seconds at a flow rate of 30 μL / min. Self-produced recombinant human FcRn protein at concentrations of 0, 26.25, 52.5, 125, 250, and 500 nM was prepared and injected into flow tank 1 (reference) and flow tank 2 (active) for 120 seconds at a flow rate of 30 μL / min. Recombinant cynomolgus monkey FcRn protein (Imunitrack, catalog ITF06-400) at concentrations of 0, 25, 50, 100, 200, and 400 nM was also prepared and injected into flow tank 1 (reference) and flow tank 2 (active) for 120 seconds at a flow rate of 30 μL / min. 10 nM glycine at pH 1.5 was injected at a flow rate of 30 μL / min for 30 seconds to regenerate the capture surface. Samples were injected onto freshly captured mAbs in a multi-cycle manner. Data were processed and analyzed using Biacore Insight Evaluation software version 2.0.15.12933 (GE Healthcare). The reaction of flow cell 1 (reference) is subtracted from the reaction of flow cell 2 (active). Then, the reactions of two buffer blank injections are subtracted from the data subtracted from the reference (2-1) to obtain dual-reference data, which are fitted to a 1:1 binding model to determine the apparent association (ka) and dissociation rate constant (kd). The ratio provides the apparent equilibrium dissociation constant or affinity constant (KD = kd / ka).

[0290] Table 1. Binding kinetics of FcRn and Fc fragment variants from different species at pH 6 and pH 7.4 *The kinetic values ​​are adapted from the reference doi.org / 10.1080 / 19420862.2015.1008353 Example 2: Measurement of the binding of IgG and PAL02-0002 to human FcRn by ELISA Seven constructs were used to measure the ability of IgG and PAL02-0002 (C-terminal lysine-free efgartigimod) to bind to human FcRn by an ELISA assay. A competitive binding ELISA assay was used to screen for molecules that prevent IgG from binding to the self-produced recombinant human FcRn protein at pH 6.0. Candidate therapeutic agents prevent FcRn from binding to IgG Ab in an acidic endosomal environment at pH 6.0. In this assay, functional human FcRn was coated at 2.0 µg / ml in 1×PBS onto a 96-well microtiter plate and then used for an ELISA assay to determine the IC50 concentration of the test subject at pH 6.0, starting with 100 nM and serially diluted three times to 0.002 nM in the presence of 0.568 nM biotinylated human IgG1. Binding to biotinylated human IgG1 was demonstrated at a 1:5000 dilution using ExtrAvidin-HRP (Sigma, catalog number E2886-1ML). IC50 values ​​were calculated using a nonlinear regression (curve fitting) of log(antagonist) versus slope of response variables (four parameters) using GraphPad Prism 7 software. The Fc fragment, as a potential therapeutic candidate, was expected to effectively inhibit the binding of human IgG1 to FcRn. A similar procedure was performed to measure PAL02-0002 blockade by replacing biotinylated human IgG1 with biotinylated PAL02-0002.

[0291] As shown in Table 2, constructs 1-7 were particularly effective in blocking the binding of IgG to FcRn. Notably, the IC50 values ​​of constructs 1-6 were half that of igamaide, and specifically, the IC50 value of construct 3 was >3.5 times smaller than that of igamaide.

[0292] Table 2. Measurement of the binding of blocking IgG and PAL02-0002 to human FcRn by ELISA Example 3. Characterization of Fc fragment variants bound to FcRn SEC-HPLC analysis, HIC-HPLC analysis, and thermal stability measurements of the FcRn construct were performed as described in the Methods section disclosed herein.

[0293] Dynamic light scattering (DLS), non-reducing sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS), and reduced sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) are performed as described in the Methods section disclosed herein.

[0294] Table 3. Characterization of Fc fragment variants that bind FcRn Example 4. Overview of Fc variant developmental studies Perform SEC-HPLC analysis as described in the Methods section disclosed herein.

[0295] Dynamic light scattering (DLS), differential scanning calorimetry (DSC), and PEG precipitation determination were performed as described in the Methods section disclosed herein.

[0296] All samples showed high monomer purity (≥99%) before and after 3 freeze / thaw cycles. DLS analysis showed that all samples had the expected pattern hydrodynamic diameter and low to medium intermediate polydispersity (PDI < 0.2). Only the wild type (PAL02-0001) showed a TM value above 65°C. Most of the remaining samples showed a TM value between 60 and 63°C.

[0297] Table 4. Characterization of FcRn constructs Example 5. Short-term stability of Fc variants SEC-HPLC analysis was performed as described in the Methods section of this document to test the short-term stability of constructs 1-7 at 10 mg / mL in 20 mM sodium acetate at pH 5.5, compared to wild-type Fc and PAL02-0002, at 40 °C. Dynamic light scattering (DLS), non-reducing sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS), reduced sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS), and capillary isoelectric focusing (cIEF) were performed as described in the Methods section of this document.

[0298] Table 5. Short-term stability of Fc variants Informal sequence list

Claims

1. An Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein, a) Compared to the amino acid sequence shown in SEQ ID NO: 1, the Fc fragment variant contains amino acid substitutions M428L and N434F; or b) Compared to the amino acid sequence shown in SEQ ID NO: 1, the Fc fragment variant contains amino acid substitutions M428L and N434Y.

2. The Fc fragment variant of claim 1, wherein the Fc fragment variant further comprises an amino acid substitution of H433K or H433R.

3. An Fc fragment variant that binds to the neonatal Fc receptor (FcRn), wherein, a) Compared to the amino acid sequence shown in SEQ ID NO: 1, the Fc fragment variant contains amino acid substitutions of (i) N434Y and (ii) H433R or H433K; b) Compared to the amino acid sequence shown in SEQ ID NO: 1, the Fc fragment variant contains amino acid substitutions M428L and H433R; or c) Compared to the amino acid sequence shown in SEQ ID NO: 1, the Fc fragment variant contains amino acid substitutions H433R and N434F.

4. The Fc fragment variant according to any one of claims 1-3, wherein the Fc fragment variant further comprises amino acid substitutions M252Y, S254T, and T256E.

5. The Fc fragment variant of any one of claims 1-4, wherein the Fc fragment variant comprises: a) Amino acid substitutions for M252Y, S254T, T256E, M428L, H433K, and N434F; b) Amino acid substitutions for M252Y, S254T, T256E, M428L, H433K, and N434Y; c) Amino acid substitutions for M252Y, S254T, T256E, H433K, and N434Y; d) Amino acid substitutions for M252Y, S254T, T256E, M428L, and N434F; e) Amino acid substitutions for M252Y, S254T, T256E, H433R, and N434Y; f) Amino acid substitutions for M252Y, S254T, T256E, H433R, and N434F; or g) Amino acid substitutions of M252Y, S254T, T256E, M428L, H433R and N434F.

6. The Fc fragment variant of any one of claims 1-5, wherein the Fc fragment variant comprises the amino acid sequence of any one of SEQ ID NO:3-9.

7. The Fc fragment variant of any one of claims 1-6, wherein the Fc fragment variant does not contain amino-substituted L309D.

8. An Fc fragment variant having at least 85%, 90%, 93%, 95%, 97%, or 99% identity with the following amino acid sequences: a) SEQ ID NO: 3, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, M428L, H433K and N434F; b) SEQ ID NO: 4, provided that this variant contains amino acid substitutions M252Y, S254T, 256E, M428L, H433K and N434Y; c) SEQ ID NO: 5, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, H433K and N434Y; d) SEQ ID NO: 6, provided that this variant contains amino acid substitutions S254T, T256E, M428L and N434F; e) SEQ ID NO: 7, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, H433R and N434Y; f) SEQ ID NO: 8, provided that this variant contains amino acid substitutions for M252Y, S254T, T256E, H433R, and N434F; or g) SEQ ID NO: 9, provided that this variant contains amino acid substitutions M252Y, S254T, T256E, M428L, H433R and N434F.

9. An Fc fragment that binds to a neonatal Fc receptor (FcRn), wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

3.

10. An isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant contains at least one amino acid substitution selected from M428L, H433R, H433K and N434Y.

11. An isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: a) A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant comprises an amino acid substitution at position 428, optionally said amino acid substitution being M428L; b) A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant comprises an amino acid substitution for H433R; or c) A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant contains an amino acid substitution N434Y.

12. The separated Fc fragment as described in claim 11, wherein, a) The Fc fragment contains an additional amino acid substitution at one or more of amino acid positions 252, 254, 256, 433 and 434 of SEQ ID NO: 1, and optionally, the additional amino acid substitution is M252Y, S254T, T256E, H433K, H433R, N434F and / or N434Y; b) The Fc fragment contains an additional amino acid substitution at one or more of amino acid positions 252, 254, 256, 428, and 434 of SEQ ID NO: 1, and optionally, the additional amino acid substitution is M252Y, S254T, T256E, M428L, N434F, and / or N434Y; or c) The Fc fragment contains an additional amino acid substitution at one or more of amino acid positions 252, 254, 256, 428 and 433 of SEQ ID NO: 1, and optionally, the additional amino acid substitution is M252Y, S254T, T256E, M428L, H433K and / or H433R.

13. An isolated Fc fragment that binds to a neonatal Fc receptor (FcRn) in humans, cynomolgus monkeys, mice, or rats, said Fc fragment comprising: A variant of the amino acid sequence shown in SEQ ID NO: 1, wherein the variant comprises the amino acid substitution H433K, and additional amino acid substitutions at one or more of amino acid positions 252, 254, 256, 428 and 434, and optionally, the additional amino acid substitutions are M252Y, S254T, T256E, M428L, N434F and / or N434Y.

14. The Fc fragment variant or Fc fragment of any one of claims 1-13, wherein the Fc fragment variant or Fc fragment inhibits IgG binding to human FcRn with an IC50 value of less than 2.5 nM, 2.0 nM, 1.5 nM or 1.0 nM.

15. The Fc fragment variant or Fc fragment of any one of claims 1-14, wherein the Fc fragment variant or Fc fragment inhibits IgG binding to human FcRn at pH 6.

0.

16. The Fc fragment variant or Fc fragment of any one of claims 1-15, wherein the Fc fragment variant or Fc fragment blocks or reduces naturally occurring antibody recycling.

17. The Fc fragment variant or Fc fragment of any one of claims 1-16, wherein the Fc fragment variant or Fc fragment causes an increase in the catabolism of pathogenic antibodies.

18. The Fc fragment variant or Fc fragment of claim 17, wherein the pathogenic antibody is an antibody associated with an autoimmune disease.

19. The Fc fragment variant or Fc fragment as claimed in any one of claims 1-18, wherein, compared to containing a wild-type Fc region and measurable K at pH 7.4 D The Fc fragment, the Fc fragment variant or Fc fragment at pH 6.0 with a lower K D It binds to FcRn in humans, cynomolgus monkeys, mice, or rats.

20. The Fc fragment variant or Fc fragment of any one of claims 1-19, wherein the Fc fragment variant or Fc fragment is at pH 6.0 with a concentration of less than or equal to about 1 × 10⁻⁶. -8 M of K D It binds to the FcRn sequence shown in SEQ ID NO: 10-17, as measured by surface plasmon resonance (SPR).

21. The Fc fragment variant or Fc fragment as claimed in any one of claims 1-20, wherein, a) The Fc fragment variant or Fc fragment exhibits a melting temperature greater than 55°C, as measured by differential scanning fluorometry (DSF), and / or b) The Fc fragment variant or Fc fragment exhibits an aggregation temperature equal to or greater than 70°C, as measured by differential scanning fluorometry (DSF).

22. The Fc fragment variant or Fc fragment of any one of claims 1-21, wherein the Fc fragment variant or Fc fragment is fused or complexed with a half-life extension domain, and optionally, the half-life extension domain is albumin, an albumin-binding domain, or an HSA-binding domain.

23. An isolated polynucleotide or vector encoding an Fc fragment variant or Fc fragment according to any one of claims 1-22, optionally wherein, The polynucleotide or vector contains mRNA or cDNA.

24. A method for generating an Fc fragment variant or Fc fragment, the method comprising expressing the Fc fragment variant or Fc fragment in a host cell and isolating the expressed Fc fragment variant or Fc fragment, the host cell comprising the isolated polynucleotide or vector of claim 23.

25. The Fc fragment variant or Fc fragment of any one of claims 1-22, wherein the Fc fragment variant or Fc fragment is used for a method of inhibiting IgG binding to FcRn in a subject.

26. Use of any Fc fragment variant or Fc fragment as described in any one of claims 1-22 in the preparation of a medicament for treating a condition or disease in a subject in need. Optionally, the disease or condition described herein is selected from the group consisting of: generalized myasthenia gravis (gMG), chronic inflammatory demyelinating polyneuropathy, myositis, autoimmune encephalitis, oligodendrocyte glycoprotein antibody disease (MOG antibody disease), membranous nephropathy, lupus nephritis, thyroid ophthalmopathy, warm antibody-type autoimmune hemolytic anemia, hemolytic diseases of the fetus and newborn, idiopathic thrombocytopenic purpura, primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid, foliaceous pemphigoid, pemphigus vulgaris, and cutaneous lupus erythematosus.

27. Use of any Fc fragment variant or Fc fragment as described in any one of claims 1-22 in the preparation of a medicament for treating pathology associated with elevated IgG levels in mammalian subjects in need.

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