Improved stability of anti-FcRn antibody or antigen-binding fragment

Abstract

The present disclosure relates to an anti-FcRn antibody or an antigen-binding fragment thereof with improved stability and its use. The anti-FcRn antibody or the antigen-binding fragment thereof binds to FcRn non-competitively with IgG or the like with respect to the parent antibody HL161AN, thereby having improved stability. For example, the production rate of aggregates is decreased, and at the same time, the biological activity of significantly reducing the amount of pathogenic autoantibodies in the blood is maintained. Therefore, it can be more efficiently used for the treatment of autoimmune diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the merits of Korean Patent Application No. 10-2022-0066176 filed on 30 May 2022, U.S. Provisional Patent Application No. 63 / 352,948 filed on 16 June 2022, U.S. Provisional Patent Application No. 63 / 370,772 filed on 8 August 2022, U.S. Provisional Patent Application No. 63 / 377,283 filed on 27 September 2022, and U.S. Provisional Patent Application No. 63 / 499,116 filed on 28 April 2023, each of which is incorporated herein by reference in its entirety.

[0002] Inclusion by referencing the sequence list The contents of a text file named MUNO-008_001WO_SeqList.xml, created on May 25, 2023, and measuring 61,442 bytes, are incorporated herein by reference in their entirety.

[0003] This invention relates to an anti-FcRn antibody with improved stability or an antigen-binding fragment thereof, and its use. [Background technology]

[0004] The causes of autoimmune diseases have long been studied from genetic, environmental, and immunological perspectives, but the precise causes of these diseases remain unclear. Numerous recent studies have revealed that some autoimmune diseases are caused by IgG-type autoantibodies. Indeed, research into the diagnosis and treatment of autoimmune diseases has extensively investigated the relationship between the presence, absence, or reduction of disease-specific autoantibodies and their therapeutic effects.

[0005] For the treatment of such autoimmune diseases, the first-line treatment is systemic high-dose steroid injection. If symptoms are severe or difficult to control with steroids, high-dose IVIG (intravenous immunoglobulin) administration or plasmapheresis is applied. Repeated use of high-dose steroids can lead to weaker effects or serious side effects. In the case of IVIG and plasmapheresis, the treatment costs are high, and there are various side effects and risks of infection, so the development of treatments in this area is urgently needed.

[0006] On the other hand, in recent years, therapeutic drugs for autoimmune diseases using FcRn antibodies have been studied (Korean Patent Registration No. 10-1815265). This drug has a novel mechanism in which the antibody blocks FcRn (neonatal Fc receptor) involved in the recirculation of IgG, thereby increasing the catabolism of IgG in the body, and thus reducing autoantibody levels to treat the disease. This anti-FcRn antibody is expected to be a product that can solve the problems of existing therapeutic drugs.

[0007] However, in order to apply this antibody to severe autoimmune diseases caused by the production of autoantibodies against autoantigens in the body, such as pemphigus or neuromyelitis, it is necessary to develop an antibody that is stable while maintaining the affinity and biological activity of the existing antibody. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Korean Registered Patent No. 10-1815265 [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, the inventors have completed the present invention by generating a variant of the anti-FcRn antibody with improved stability, which significantly reduces the rate of aggregate production while maintaining the affinity and biological activity of the previously developed anti-FcRn antibody HL161AN. [Means for solving the problem]

[0010] In aspects of the present invention, an anti-FcRn antibody or antigen-binding fragment is provided, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 1, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, wherein the amino acid at N3 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, or the amino acid at C2 in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid. In some embodiments, the amino acid at N3 in the amino acid sequence of SEQ ID NO: 1 is substituted with Ser(S) or Gln(Q). In some embodiments, the amino acid at C2 in the amino acid sequence of SEQ ID NO: 5 is substituted with Ser(S) or Tyr(Y).

[0011] Another aspect of the present invention provides an anti-FcRn antibody or antigen-binding fragment comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, wherein the amino acid at N25 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid, and the amino acid at C32 or Q82 in the amino acid sequence of SEQ ID NO: 8 is substituted with another amino acid. In some embodiments, the amino acid at N25 in the amino acid sequence of SEQ ID NO: 4 is substituted with Ser(S) or Gln(Q). In some embodiments, the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 is substituted with Ser(S) or Tyr(Y). In some embodiments, the amino acid at Q82 in the amino acid sequence of SEQ ID NO: 8 may be substituted with Glu(E), but is not limited thereto.

[0012] In some embodiments, the anti-FcRn antibody or the antigen-binding fragment provided herein includes a heavy chain variable region comprising a framework consisting of framework region 1 (FR1) of the amino acid sequence of SEQ ID NO: 15, FR2 of the amino acid sequence of SEQ ID NO: 16, FR3 of the amino acid sequence of SEQ ID NO: 17, and FR4 of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the amino acid Q16 in the amino acid sequence of SEQ ID NO: 17 is substituted with another amino acid. In some embodiments, the amino acid Q16 in the amino acid sequence of SEQ ID NO: 17 is substituted with Glu(E).

[0013] In some embodiments, an anti-FcRn antibody or an antigen-binding fragment thereof is provided, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7.

[0014] In some embodiments, the anti-FcRn antibody comprises an Fc region, and this Fc region is an IgG1 Fc region or an IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region comprising the amino acid substitutions Leu234Ala and Leu235Ala. In some embodiments, the Fc region is an IgG4 Fc region comprising the S228P mutation.

[0015] In another aspect of the invention, there is provided a polynucleotide encoding an anti-FcRn antibody or an antigen-binding fragment thereof.

[0016] In another aspect of the invention, there is provided a recombinant expression vector comprising this polynucleotide.

[0017] In another aspect of the invention, there is provided a host cell transformed with this recombinant expression vector.

[0018] In another aspect of the invention, there is provided a method for producing an anti-FcRn antibody or an antigen-binding fragment thereof, comprising culturing a host cell to produce an antibody, and isolating and purifying this produced antibody to recover an antibody that specifically binds to FcRn.

[0019] In another aspect of the invention, there is provided a pharmaceutical composition for treating an autoimmune disease, comprising an anti-FcRn antibody or an antigen-binding fragment thereof. In some embodiments, the autoimmune disease is an autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaac's syndrome, vitiligo syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid gestationis, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture's syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, IgA nephropathy (Berger's disease), dermatomyositis, necrotizing autoimmune myopathy, and membranous nephropathy, which is an autoimmune disease selected from the group consisting of.

[0020] Another aspect of the present invention provides a method for treating an autoimmune disease in a subject requiring such treatment, comprising administering an anti-FcRn antibody or an antigen-binding fragment or pharmaceutical composition to the subject. In some embodiments, the subject is a human. In some embodiments, the autoimmune disease is selected from the group consisting of autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus deciduousis, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Graves' disease, Goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), lupus nephritis, and membranous nephropathy.

[0021] In some embodiments, administration is parenteral. In some embodiments, administration is subcutaneous or intravenous. In some embodiments, administration is at doses of 300 mg to 2400 mg. In some embodiments, administration is once a week, once every two weeks, once every three weeks, or once a month. In some embodiments, administration is subcutaneous once a week at doses of 300 mg to 900 mg. In some embodiments, administration is subcutaneous once every two weeks at doses of 300 mg to 1800 mg. In some embodiments, administration does not result in a decrease in blood albumin levels in subjects of more than 5% or more than 10% compared to blood albumin levels before administration of anti-FcRn antibody or antigen-binding fragment. In some embodiments, administration does not result in an increase in blood total cholesterol or low-density lipoprotein (LDL) levels in subjects of more than 5% or more than 10% compared to blood total cholesterol or low-density lipoprotein (LDL) levels before administration of anti-FcRn antibody or antigen-binding fragment. [Effects of the Invention]

[0022] The FcRn-specific antibody according to the present invention exhibits improved stability compared to the parent antibody HL161AN, for example, with a reduced rate of aggregate production, while simultaneously possessing excellent productivity and biological activity. The antibody of the present invention not only has high affinity for FcRn, but is also expected to be highly specific and non-immunogenic or low-immunogenic, binding to FcRn and significantly reducing the amount of pathogenic autoantibodies in the blood. Therefore, it can be used in the treatment of autoimmune diseases. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 is a flowchart of the in silico design of the HL161AN mutant. [Figure 2] Figure 2 shows the predicted post-translational modification (PTM) sites of the HL161AN light chain. [Figure 3] Figure 3 shows the predicted post-translational modification (PTM) sites of the HL161AN heavy chain. [Figure 4] Figure 4 shows the 100% sequence inclusion confirmed by treating an in-house standard sample of HL161AN obtained from an HL161AN-producing cell line (#8G7) with two enzymes, trypsin + LysC mixed enzyme and chymotrypsin, respectively. [Figure 5] Figure 5 shows the results of in silico design of HL161AN mutants (top) and the results of paratope analysis of HL161AN against human FcRn (bottom). Specifically, it shows the results of designing mutants by modifying three amino acid sites (L: Asn25, H: Cys32, H: Gln82) that may affect the stability of the HL161AN antibody. Here, the oval indicates the substitution site of the selected PTM site. In addition, the paratope of HL161AN is shown in bold, and the Kabat-type CDR region of IgBlast is shown underlined. * indicates the substitution site of the selected amino acid. [Figure 6] Figure 6 shows the SDS-PAGE results for the HL161AN mutant. [Figure 7]Figure 7 shows the SPR results for the HL161AN mutant. [Figure 8] Figure 8 shows the results obtained by confirming the binding of the HL161AN mutant and hFcRn using FACS. [Figure 9] Figure 9 shows the results obtained by confirming the effect of FACS on blocking hFcRn in the HL161AN mutant. [Figure 10] Figure 10 shows the degree of increase in aggregates of the HL161AN mutant (1 mg / mL) in a stability test conducted under accelerated conditions at 40°C. [Figure 11] Figure 11 shows the degree of fragment increase of the HL161AN mutant (1 mg / mL) in stability tests performed under accelerated conditions at 40°C. [Figure 12] Figure 12 shows the results obtained by confirming the binding of hFcRn to eight high-concentration HL161AN variants selected after the first in vitro screening using FACS. [Figure 13] Figure 13 shows the results obtained by confirming the hFcRn-blocking effect of eight high-concentration HL161AN variants selected after the first in vitro screening using FACS. [Figure 14] Figure 14 shows the SDS-PAGE results for eight high-concentration HL161AN mutant samples. [Figure 15] Figure 15 shows the SEC-HPLC results for eight high-concentration HL161AN mutant samples. [Figure 16] Figure 16 is a graph showing the results obtained by comparing aggregation formation among HL161AN mutant samples. [Figure 17]Figure 17 shows the results of biotin-hIgG catabolism in transgenic mice (Tg) with the HL161AN mutant (20 mg / kg). Blood samples were collected at 24, 48, 72, 96, 120, and 168 hours after tracer administration, and the biotin-hIgG concentration was measured by ELISA. [Figure 18] Figure 18 shows the decrease in IgG in monkeys after administration of HL161ANS (IMVT-1402). [Figure 19] Figures 19A-19C show albumin levels, LDL levels, and cholesterol levels, respectively, in monkeys after administration of HL161ANS (IMVT-1402). [Figure 20] Figure 20 is a schematic diagram showing the design of the clinical trial described in Example 13. Abbreviations: AUC = Area under the blood concentration-time curve; Cmax = Maximum observed blood concentration; IV = Intravenous administration; MAD = Multiple dose escalations; PK = Pharmacokinetics; QW = Once a week; SAD = Single dose escalation; SC = Subcutaneous administration. [Modes for carrying out the invention]

[0024] Anti-FcRn antibody variant or this antigen-binding fragment In aspects of the present invention, an improved stability of an anti-FcRn antibody or antigen-binding fragment thereof is provided, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 1, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7.

[0025] Next, the amino acid at position N3 in the amino acid sequence of SEQ ID NO: 1 may be substituted with another amino acid, or the amino acid at position C2 in the amino acid sequence of SEQ ID NO: 5 may be substituted with another amino acid. "The amino acid at position N3 in the amino acid sequence of SEQ ID NO: 1" refers to the third amino acid of SEQ ID NO: 1 (asparagine), and "the amino acid at position C2 in the amino acid sequence of SEQ ID NO: 5" refers to the second amino acid of SEQ ID NO: 5 (cysteine).

[0026] As used herein, the terms "FcRn" or "neonatal Fc receptor" refer to an MHC class I-related protein expressed in vascular endothelial cells that binds to IgG and albumin. A key characteristic is that the binding between IgG and FcRn is strong at weakly acidic pH, while no binding force exists at neutral pH. Therefore, IgG that has entered cells via pinocytosis or endocytosis can evade the degrading lysosomal pathway by strongly binding to FcRn, a type of Fc gamma receptor (FcγR), within endosomes under pH 6.0 conditions. During reverse recirculation to the cell membrane, IgG rapidly dissociates from FcRn in the bloodstream at pH 7.4. This receptor-mediated recirculation mechanism has been shown to extend the half-life of IgG by efficiently blocking the degradation of IgG within lysosomes. In other words, FcRn plays a crucial role in maintaining serum antibody levels by acting as a receptor that binds to and recovers antibodies of IgG isotypes.

[0027] On the other hand, it has been suggested that autoimmune diseases caused by autoantibodies can be treated by shortening the serum half-life of IgG by inhibiting the binding of IgG to FcRn (Li et al., J. Clin. Invest., 115:3440, 2005).

[0028] As used herein, the term "anti-FcRn antibody" is interchangeable with "anti-FcRn antibody variant" and refers to an antibody specific to FcRn. This antibody includes not only the intact antibody type but also the antigen-binding fragment of the antibody.

[0029] Generally, antibody molecules obtained from humans are associated with one of the immunoglobulin classes (IgG, IgM, IgA, IgE, and IgD) that are distinct from one another due to the properties of the heavy chain present in the molecule. Certain classes also have subclasses such as IgG1, IgG2, IgG3, or IgG4, among others. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain. Therefore, in one embodiment, the antibody disclosed herein is a human IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the anti-FcRn antibody described herein is a synthetic antibody based on a protein scaffold that has the ability to bind to FcRn.

[0030] In some embodiments, the anti-FcRn antibody is a monoclonal antibody. The terms “monoclonal antibody” (MAb or mAb) or “monoclonal antibody composition” as used herein refer to a group of antibody molecules that contain a single molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.

[0031] As used herein, the term “antigen-binding fragment” refers to one or more fragments of an intact antibody that possess the ability to specifically bind to a given antigen, i.e., FcRn. The antibody fragments according to the present invention include, but are not limited to, single-chain antibodies, bispecific antibodies, trispecific antibodies, polyspecific antibodies, e.g., diabodies, triabodies, and tetrabodies, Fab fragments, F(ab')2 fragments, Fd, scFv, domain antibodies, dual-specific antibodies, minibodies, scap (sterol-regulated binding protein cleavage-activating protein), chelated recombinant antibodies, tribodies, vibodies, intrabodies, nanobodies, SMIP (small module immunotherapy), binding domain immunoglobulin fusion proteins, camel-shaped antibodies, VHH-containing antibodies, and antibody constant region derivatives.

[0032] The anti-FcRn antibody or antigen-binding fragment provided herein may be a humanized antibody, a human antibody, a chimeric antibody, or a mouse antibody.

[0033] It will be apparent to those skilled in the art that, as long as the binding function to FcRn is maintained, any type of fragment of the antibody according to this disclosure will exhibit the same properties as the antibody according to this disclosure.

[0034] Examples of CDR sequences for anti-FcRn antibodies disclosed herein are listed in Table 1. These CDRs are defined according to the Kabat scheme.

[0035] [Table 1]

[0036] In one embodiment, the anti-FcRn antibody or the antigen-binding fragment comprises a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 1, LCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, wherein the amino acid at N3 in the amino acid sequence of SEQ ID NO: 1 may be substituted with another amino acid, or the amino acid at C2 in the amino acid sequence of SEQ ID NO: 5 may be substituted with another amino acid.

[0037] As used herein, the term "heavy chain (HC)" refers to both a full-length heavy chain containing a variable region domain VH with an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3, as well as this fragment. In addition, as used herein, the term "light chain (LC)" refers to both a full-length light chain containing a variable region domain VL with an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen, and a constant region domain CL, as well as this fragment.

[0038] As used herein, the term “variable” refers to the fact that the sequence of a particular portion of the variable region differs significantly between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody to a particular antigen. Variability is concentrated in three segments called hypervariable regions (HVRs), namely the CDRs, within both the light and heavy chain variable regions. The more highly conserved portions of the variable region are called framework (FR) regions. The heavy and light chain variable regions have the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 structures from the N-terminus to the C-terminus.

[0039] As used herein, the term "CDR (Complementarity-Determining Region)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains. The heavy chains (HCDR1, HCDR2, and HCDR3) and light chains (LCDR1, LCDR2, and LCDR3) each contain three CDRs. The CDRs provide key contact residues for the binding of the antibody to the antigen or epitope.

[0040] As used herein, the term “substitution with another amino acid” refers to a substitution with another amino acid residue having properties similar to the original amino acid sequence. This is not particularly limited, as long as the properties of the antibody according to this disclosure are maintained even if an amino acid substitution occurs. The “amino acid” introduced by the above substitution may be any one selected from the group consisting of lysine (K), alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). The substitution may also be described, for example, as “the amino acid at N3 in the amino acid sequence of SEQ ID NO: 1,” which refers to the third N in SEQ ID NO: 1.

[0041] In one embodiment, the amino acid N3 in the amino acid sequence of SEQ ID NO: 1 may be substituted with Ser(S) or Gln(Q), but is not limited thereto.

[0042] In one embodiment, the C2 amino acid in the amino acid sequence of SEQ ID NO: 5 may be substituted with Ser(S) or Tyr(Y), but is not limited thereto.

[0043] In one embodiment, variant number 1 may include a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 1 may be represented as HL161ANS, HL161ANS(IgG1null), or HL161ANS(IgG4).

[0044] In one embodiment, variant number 12 may include a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 13, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 12 may be represented as HL161ANQ, HL161ANQ(IgG1null), or HL161ANQ(IgG4).

[0045] In one embodiment, variant number 2 may include a light chain variable region comprising LCDR1 containing an amino acid sequence in which the N3 amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with Ser(S), LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing an amino acid sequence in which the C2 amino acid in the amino acid sequence of SEQ ID NO: 5 is substituted with Tyr(Y), HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 2 may be represented as HL161ANSY, HL161ANSY(IgG1null), or HL161ANSY(IgG4).

[0046] In one embodiment, variant number 13 may include a light chain variable region comprising LCDR1 containing an amino acid sequence in which the N3 amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with Gln(Q), LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing an amino acid sequence in which the C2 amino acid in the amino acid sequence of SEQ ID NO: 5 is substituted with Tyr(Y), HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 13 may be represented as HL161ANQY, HL161ANQY(IgG1null), or HL161ANQY(IgG4).

[0047] In one embodiment, variant number 23 may include a light chain variable region comprising LCDR1 containing an amino acid sequence in which the N3 amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with Ser(S), LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing an amino acid sequence in which the C2 amino acid in the amino acid sequence of SEQ ID NO: 5 is substituted with Tyr(Y), HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 23 may be represented as HL161ANSY, HL161ANSY(IgG1null), or HL161ANSY(IgG4).

[0048] In some embodiments, the anti-FcRn antibody or this antigen-binding fragment comprises a VL CDR1 containing the sequence GGX1NIGSTSV (SEQ ID NO: 38), where X1 is N, S, or Q. In some embodiments, the anti-FcRn antibody or this antigen-binding fragment comprises a VH CDR1 containing the sequence SX2VMT (SEQ ID NO: 39), where X2 is C, S, or Y.

[0049] In one embodiment, the heavy chain variable region of the anti-FcRn antibody or this antigen-binding fragment may include a framework consisting of FR1 of the amino acid sequence of SEQ ID NO: 15, FR2 of the amino acid sequence of SEQ ID NO: 16, FR3 of the amino acid sequence of SEQ ID NO: 17, and FR4 of the amino acid sequence of SEQ ID NO: 18.

[0050] Within the FR3 framework on the heavy chain variable region, certain amino acids may be substituted with other amino acids, but are not limited to this. Specifically, the amino acid at Q16 in the amino acid sequence of SEQ ID NO: 17 may be substituted with another amino acid.

[0051] As stated above, the "substitution with another amino acid" described above is not particularly limited, as long as the properties of the antibody according to this disclosure are maintained even if an amino acid substitution occurs.

[0052] In one embodiment, the amino acid Q16 in the amino acid sequence of SEQ ID NO: 17 may be substituted with Glu(E), but is not limited thereto.

[0053] In one embodiment, variant number 8 includes a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, which may include FR3 of an amino acid sequence in which the amino acid Q16 in the amino acid sequence of SEQ ID NO: 17 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 8 may be represented as HL161ANSE, HL161ANSE(IgG1null), or HL161ANSE(IgG4).

[0054] In one embodiment, variant number 19 includes a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 13, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, which may include FR3 of an amino acid sequence in which the amino acid Q16 in the amino acid sequence of SEQ ID NO: 17 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 19 may be represented as HL161ANQE, HL161ANQE(IgG1null), or HL161ANQE(IgG4).

[0055] In one embodiment, variant number 11 includes a light chain variable region comprising LCDR1 containing an amino acid sequence in which the N3 amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with Ser(S), LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising HCDR1 containing an amino acid sequence in which the C2 amino acid in the amino acid sequence of SEQ ID NO: 5 is substituted with Tyr(Y), HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region which may include FR3 of an amino acid sequence in which the Q16 amino acid in the amino acid sequence of SEQ ID NO: 17 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 11 may be represented as HL161ANSYE, HL161ANSYE(IgG1null), or HL161ANSYE(IgG4).

[0056] In one embodiment, variant number 22 includes a light chain variable region comprising LCDR1 containing an amino acid sequence in which the N3 amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with Gln(Q), LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising HCDR1 containing an amino acid sequence in which the C2 amino acid in the amino acid sequence of SEQ ID NO: 5 is substituted with Tyr(Y), HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region which may include FR3 of an amino acid sequence in which the Q16 amino acid in the amino acid sequence of SEQ ID NO: 17 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof. Variant number 22 may be represented as HL161ANQYE, HL161ANQYE(IgG1null), or HL161ANQYE(IgG4).

[0057] Another aspect of this disclosure provides an anti-FcRn antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0058] In this regard, in the anti-FcRn antibody or this antigen-binding fragment, the amino acid at N25 in the amino acid sequence of SEQ ID NO: 4 may be substituted with another amino acid, and the amino acid at C32 or Q82 in the amino acid sequence of SEQ ID NO: 8 may be substituted with another amino acid.

[0059] The "anti-FcRn antibody," "antigen-binding fragment," and "substitution with another amino acid" are as described above.

[0060] In one embodiment, the amino acid at N25 in the amino acid sequence of SEQ ID NO: 4 may be substituted with Ser(S) or Gln(Q), but is not limited thereto.

[0061] In one embodiment, the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 may be substituted with Ser(S) or Tyr(Y), but is not limited thereto.

[0062] In one embodiment, the amino acid Q82 in the amino acid sequence of SEQ ID NO: 8 may be substituted with Glu(E), but is not limited thereto.

[0063] In one embodiment, variant number 1 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12. In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof.

[0064] In one embodiment, variant number 12 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 14 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12. In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof.

[0065] In one embodiment, variant number 2 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 10, and a heavy chain variable region containing an amino acid sequence in which the C32 amino acid in the amino acid sequence of SEQ ID NO: 8 is substituted with Tyr(Y). In this regard, the Fc region of the antibody may be IgG1 or its variant, IgG4 or its variant.

[0066] In one embodiment, variant number 13 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a heavy chain variable region containing an amino acid sequence in which the C32 amino acid in the amino acid sequence of SEQ ID NO: 8 is substituted with Tyr(Y). In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof.

[0067] In one embodiment, variant number 8 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 10, and a heavy chain variable region containing an amino acid sequence in which the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 is substituted with Ser(S) and the amino acid at Q82 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or a variant thereof, IgG4 or a variant thereof.

[0068] In one embodiment, variant number 19 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a heavy chain variable region containing an amino acid sequence in which the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 is substituted with Ser(S) and the amino acid at Q82 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or its variant, IgG4 or its variant.

[0069] In one embodiment, variant number 11 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 10, and a heavy chain variable region containing an amino acid sequence in which the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 is substituted with Tyr(Y) and the amino acid at Q82 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or its variant, IgG4 or its variant.

[0070] In one embodiment, variant number 22 may include a light chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a heavy chain variable region containing an amino acid sequence in which the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 is substituted with Tyr(Y) and the amino acid at Q82 is substituted with Glu(E). In this regard, the Fc region of the antibody may be IgG1 or its variant, IgG4 or its variant.

[0071] In some embodiments, the anti-FcRn antibody includes a light chain variable region containing a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in SEQ ID NO: 4. In some of these embodiments, the sequence of the CDR in the light chain variable region is 100% identical to that of SEQ ID NO: 4. In some embodiments, the anti-FcRn antibody includes a light chain variable region containing a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in SEQ ID NO: 10. In some of these embodiments, the sequence of the CDR in the light chain variable region is 100% identical to that of SEQ ID NO: 10. In some embodiments, the anti-FcRn antibody includes a light chain variable region containing a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in SEQ ID NO: 14. In some of these embodiments, the CDR sequence of the light chain variable region is 100% identical to that of SEQ ID NO: 14.

[0072] In some embodiments, the anti-FcRn antibody includes a heavy chain variable region containing a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in SEQ ID NO: 8. In some of these embodiments, the sequence of the CDR in the heavy chain variable region is 100% identical to that of SEQ ID NO: 8. In some embodiments, the anti-FcRn antibody includes a heavy chain variable region containing a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in SEQ ID NO: 12. In some of these embodiments, the sequence of the CDR in the heavy chain variable region is 100% identical to that of SEQ ID NO: 12.

[0073] In some embodiments, the anti-FcRn antibody comprises one of the CDRs, light chain variable regions, and / or heavy chain variable regions described herein, and further comprises an IgG1 Fc region. In some embodiments, the anti-FcRn antibody comprises one of the CDRs, light chain variable regions, and / or heavy chain variable regions described herein, and further comprises an IgG1 Fc region ("IgG1-LALA") containing the Leu234Ala / Leu235Ala ("LALA") amino acid substitution.

[0074] In some embodiments, the anti-FcRn antibody comprises one of the CDRs, light chain variable regions, and / or heavy chain variable regions described herein, and further comprises an IgG4 Fc region. In some embodiments, the anti-FcRn antibody comprises one of the CDRs, light chain variable regions, and / or heavy chain variable regions described herein, and further comprises an IgG4 Fc region containing the S228P amino acid substitution ("IgG4 S228P").

[0075] In some embodiments, the anti-FcRn antibody is an IgG1 antibody and includes a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-FcRn antibody is an IgG4 antibody and includes a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7.

[0076] In some embodiments, the anti-FcRn antibody is an IgG1-LALA antibody and includes a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-FcRn antibody is an IgG4 S228P antibody and includes a light chain variable region comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, as well as a heavy chain variable region comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7.

[0077] In one embodiment, HL161ANS(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 21 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 22.

[0078] In one embodiment, HL161ANS(IgG4) may include a light chain region containing the amino acid sequence of SEQ ID NO: 21 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 23.

[0079] In one embodiment, HL161ANQ(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 22.

[0080] In one embodiment, HL161ANQ(IgG4) may include a light chain region containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 23.

[0081] In one embodiment, HL161ANSY(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 21 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 25.

[0082] In one embodiment, HL161ANSE(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 21 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 26.

[0083] In one embodiment, HL161ANSYE(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 21 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 27.

[0084] In one embodiment, HL161ANQY(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 25.

[0085] In one embodiment, HL161ANQE(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 26.

[0086] In one embodiment, HL161ANQYE(IgG1null) may include a light chain region containing the amino acid sequence of SEQ ID NO: 24 and a heavy chain region containing the amino acid sequence of SEQ ID NO: 27.

[0087] In one embodiment, the anti-FcRn antibody was an anti-FcRn antibody variant prepared by substituting amino acid residues at specific sites of the parent antibody HL161AN. It was confirmed that the aggregation rate was reduced while maintaining high affinity and specificity for FcRn, similar to the parent antibody. In one embodiment, the anti-FcRn antibody was an anti-FcRn antibody variant prepared by substituting amino acid residues at specific sites of the parent antibody HL161AN. It was confirmed that the aggregation rate was reduced while maintaining high affinity for FcRn, similar to the parent antibody. In detail, the anti-FcRn antibody variant according to this disclosure has improved stability, for example, the aggregation rate is reduced to less than half by up to half, and at the same time, it has excellent biological activity, similar to the parent antibody HL161AN developed to date (Korean Patent No. 10-1954906). For example, it was confirmed that it has productivity, acts as an inhibitor of IgG and FcRn binding, and IgG catabolism, i.e., promotes IgG clearance.

[0088] Therefore, the anti-FcRn antibody variant according to this disclosure, which exhibits significantly improved stability while simultaneously possessing excellent biological activity similar to the parent antibody HL161AN developed to date, can be usefully used in the treatment of autoimmune diseases.

[0089] In some embodiments, administration of the anti-FcRn antibody described herein to a subject results in a decrease of 30%, 40%, 50%, 60%, 65%, or 80% or more in the subject's blood immunoglobulin level (e.g., IgG level) compared to the subject's blood immunoglobulin level (e.g., IgG level) before administration of the anti-FcRn antibody.

[0090] In some embodiments, upon administration to a subject, the anti-FcRn antibody described herein lowers the subject's blood immunoglobulin level (e.g., IgG level) more effectively than another anti-FcRn antibody. In some embodiments, the anti-FcRn antibody described herein lowers the subject's blood immunoglobulin level (e.g., IgG level) more effectively than batoclimab. In some embodiments, upon administration to a subject, the anti-FcRn antibody described herein lowers the subject's blood immunoglobulin level (e.g., IgG level) to approximately the same level as when an equivalent dose of batoclimab is administered. Batoclimab is known in the art and is described, for example, in International Patent Application Publication No. WO2015 / 167293, which is incorporated herein by reference in its entirety, and is referred to as "HL161BKN" in WO2015 / 167293.

[0091] In some embodiments, administration of an effective dose of the anti-FcRn antibody described herein to a subject (e.g., a human) results in a decrease in blood albumin levels of less than 5%, less than 10%, less than 20%, or less than 30% compared to the blood albumin level before administration. In some embodiments, administration of an effective dose of the anti-FcRn antibody described herein to a subject (e.g., a human) results in an increase in blood cholesterol levels of less than 5%, less than 10%, less than 20%, or less than 30% compared to the blood cholesterol level before administration.

[0092] In some embodiments, when an effective dose of the anti-FcRn antibody described herein is administered to a subject (e.g., a human), the total blood cholesterol level increases by less than 5%, less than 10%, less than 20%, or less than 30% compared to the total blood cholesterol level before administration.

[0093] In some embodiments, when an effective dose of the anti-FcRn antibody described herein is administered to a subject (e.g., a human), the blood LDL level increases by less than 5%, less than 10%, less than 20%, or less than 30% compared to the blood LDL level before administration.

[0094] In some embodiments, administration of an effective dose of the anti-FcRn antibody described herein to a subject (e.g., a human) results in a decrease of at least about 40%, 50%, 65%, or 80% in blood immunoglobulin levels compared to pre-administration levels, but with (i) no or minimal effect on blood albumin levels (e.g., a decrease of less than 20%, 10%, 5%, 2%, or 1% in blood albumin), and / or (ii) no or minimal effect on blood total cholesterol levels and / or blood LDL levels (e.g., an increase of less than 20%, 10%, 5%, 2%, or 1% in cholesterol).

[0095] In some embodiments, administration of an effective dose of the anti-FcRn antibody described herein to a subject (e.g., a human) results in a decrease of at least about 40%, 50%, 65%, or 80% in blood immunoglobulin levels compared to pre-administration levels, but with no or minimal effect on blood albumin levels (e.g., a decrease of less than 10%, less than 5%, less than 2%, or less than 1%) and no or minimal effect on blood cholesterol (e.g., an increase of less than 10%, less than 5%, less than 2%, or less than 1%).

[0096] In some embodiments, subcutaneous administration of an effective dose of the anti-FcRn antibody described herein to human subjects (e.g., long-term subcutaneous administration to humans) results in a decrease of at least about 40%, 50%, 65%, or 80% in blood immunoglobulin levels compared to pre-administration levels, but with no or minimal effect on blood albumin levels (e.g., a decrease of less than 10%, less than 5%, less than 2%, or less than 1%) and no or minimal effect on blood cholesterol (e.g., an increase of less than 10%, less than 5%, less than 2%, or less than 1%).

[0097] In some embodiments, administration of an effective dose of the anti-FcRn antibody described herein to a subject (e.g., a human) results in a decrease of at least about 40%, 50%, 65%, or 80% in serum immunoglobulin levels compared to pre-administration levels, but with no or minimal effect on serum albumin levels (e.g., a decrease of less than 10%, less than 5%, less than 2%, or less than 1%) and no or minimal effect on serum LDL levels (e.g., an increase of less than 10%, less than 5%, less than 2%, or less than 1%).

[0098] In some embodiments, subcutaneous administration of an effective dose of the anti-FcRn antibody described herein to human subjects (e.g., long-term subcutaneous administration to humans) results in a decrease of at least about 40%, 50%, 65%, or 80% in serum immunoglobulin levels compared to pre-administration levels, but with no or minimal effect on serum albumin levels (e.g., a decrease of less than 10%, less than 5%, less than 2%, or less than 1%) and no or minimal effect on serum LDL levels (e.g., an increase of less than 10%, less than 5%, less than 2%, or less than 1%).

[0099] In some embodiments, long-term subcutaneous administration of an effective dose of the anti-FcRn antibody described herein to humans for 12 weeks or more results in a decrease of at least 40%, 50%, 65%, or 80% in blood immunoglobulin levels compared to pre-administration levels, but with no or minimal effect on blood albumin levels (e.g., a decrease of less than 10%, less than 5%, less than 2%, or less than 1%) and no or minimal effect on blood LDL levels and / or blood cholesterol levels (e.g., an increase of less than 10%, less than 5%, less than 2%, or less than 1%).

[0100] This disclosure also encompasses functional variants of the antibodies or antigen-binding fragments described herein. As used herein, the term "functional variant" includes modifications or chemical equivalents of the amino acid and nucleic acid sequences disclosed herein that perform substantially the same function as In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan share similar characteristics in that they have uncharged polar side chains, while alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine share similar characteristics in that they have nonpolar side chains. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine share similar characteristics in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that substitution of amino acid residues in groups exhibiting the similar characteristics described above does not result in any specific change in the characteristics. Furthermore, polypeptide variants include additions and deletions to the polypeptide sequences disclosed herein. In addition, the nucleotide sequences of the variants include analogs and derivatives thereof. The binding protein variants disclosed herein include proteins that bind to the same antigen or epitope as the binding protein.

[0101] As will be apparent to those skilled in the art, the C-terminal lysine of immunoglobulin heavy chains can be cleaved during production. Therefore, in some embodiments, the antibodies described herein contain two C-terminal lysine residues (i.e., one C-terminal lysine residue on each heavy chain). In some embodiments, the antibodies described herein contain one C-terminal lysine residue (i.e., one heavy chain contains a C-terminal lysine residue, while the other heavy chain does not). In some embodiments, the antibodies described herein do not contain a C-terminal lysine residue on either heavy chain.

[0102] The effector functions of the antibodies described herein may be desirable to be modified, for example, to enhance the efficacy of the antibodies in the treatment of related diseases and disorders. For example, by introducing cysteine ​​residues (or more) into the Fc region, interchain disulfide bonds can be formed within this region. Homodimerated antibodies produced in this manner may have improved internal distribution and / or increased complement-mediated cytotoxicity and antibody-dependent cytotoxicity (ADCC) (see Caron et al., J Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies may be modified to have enhanced complement-lytic and ADCC activity by possessing a dual Fc region (see Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)). Mutations affecting the effector function of antibodies are known in the art, and for examples of Fc modifications that can be introduced into the antibodies described herein, see, for example, Saunders et al., 2019; Front. Immunol. 10:1296 and Wang et al., 2018, Protein Cell 9(1): 63-73. Each of these is incorporated herein by reference in its entirety. Unless otherwise indicated, Fc mutations in this section are described in accordance with Kabat's numbering system for immunoglobulins.Explanatory mutations and mutation combinations that can be introduced into Fc in the anti-FcRn antibodies provided herein include: Lys326Trp / Glu333Ser, Ser267Glu / His268Phe / Ser324Thr, Lys326Trp / Glu333Ser, Lys326Ala / Glu333Ala, Lys326Met / Glu333Ser, Cys221Asp / Asp222Cys, Ser267Glu, His268Phe, Ser324Thr, Glu345Arg, S239D / I332E, S239D / I332E / A330L, Ar Examples include, but are not limited to, g435His, Met252Tyr / Ser254Thr / Thr256Glu ("YTE"), Met428Leu / Asn434Ser, Thr252Leu / Thr253Ser / Thr254Phe, Leu235Glu, Leu234Ala / Leu235Ala ("LALA"), Ser228Pro / Leu235Glu, Leu234Ala / Leu235Ala / Pro329Gly, Pro331Ser / Leu234Glu / Leu235Phe, Asp265Ala, and Ala330Leu.

[0103] In some embodiments, the anti-FcRn antibody is an IgG1 antibody containing the Leu234Ala / Leu235Ala ("LALA") mutation (i.e., an "IgG1-LALA" antibody). In some embodiments, the anti-FcRn antibody is an IgG4 antibody containing the S228P mutation (i.e., an "IgG4 S228P" antibody).

[0104] Polynucleotides encoding anti-FcRn antibody variants Another aspect of this disclosure provides an anti-FcRn antibody or a polynucleotide encoding the antigen-binding fragment thereof.

[0105] More specifically, the polynucleotide encoding the heavy chain may include the sequences of SEQ ID NOs. 31, 33, 35, or 37. In addition, the polynucleotide encoding the light chain may include the sequences of SEQ ID NOs. 30, 32, 34, or 36.

[0106] This polynucleotide may contain nucleic acid sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NOs.

[0107] In some embodiments, the polynucleotide includes the sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide includes the sequence of SEQ ID NO: 31. In some embodiments, the polynucleotide includes the sequences of SEQ ID NO: 30 and 31. In some embodiments, the polynucleotide includes the sequence of SEQ ID NO: 32. In some embodiments, the polynucleotide includes the sequence of SEQ ID NO: 33. In some embodiments, the polynucleotide includes the sequences of SEQ ID NO: 32 and 33.

[0108] Polynucleotides can be mutated by substitution, deletion, insertion, or combination thereof of one or more bases. When nucleotide sequences are prepared by chemical synthesis, synthetic methods known in the art, such as those described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), may be used, including methods using triesters, phosphates, phosphoramidites, and H-phosphates, PCR and other automated primer methods, and methods for synthesizing oligonucleotides on solid supports.

[0109] A vector filled with polynucleotides encoding an anti-FcRn antibody variant. Another aspect of this disclosure provides a recombinant expression vector comprising this polynucleotide.

[0110] In addition, polynucleotides can be packed with heavy and light chains in a single vector, or each in two separate vectors.

[0111] More specifically, when packed into a single vector, the polynucleotides may include the polynucleotide of SEQ ID NO: 30 and the polynucleotide of SEQ ID NO: 31, the polynucleotide of SEQ ID NO: 32 and the polynucleotide of SEQ ID NO: 33, the polynucleotide of SEQ ID NO: 34 and the polynucleotide of SEQ ID NO: 35, or the polynucleotide of SEQ ID NO: 36 and the polynucleotide of SEQ ID NO: 37.

[0112] The vectors provided herein may include polynucleotides encoding an anti-FcRn antibody or the light chain of the antigen-binding fragment and / or the heavy chain of the anti-FcRn antibody or the antigen-binding fragment.

[0113] In some embodiments, the vector comprises a polynucleotide containing the sequence of SEQ ID NO: 30. In some embodiments, the vector comprises a polynucleotide containing the sequence of SEQ ID NO: 31. In some embodiments, the vector comprises a polynucleotide containing the sequence of SEQ ID NO: 30 (encoding the light chain) and the sequence of SEQ ID NO: 31 (encoding the heavy chain).

[0114] In some embodiments, the vector comprises a polynucleotide containing the sequence of SEQ ID NO: 32. In some embodiments, the vector comprises a polynucleotide containing the sequence of SEQ ID NO: 33. In some embodiments, the vector comprises a polynucleotide containing the sequence of SEQ ID NO: 32 (encoding the light chain) and the sequence of SEQ ID NO: 33 (encoding the heavy chain).

[0115] Vectors can be introduced into host cells, recombined, and inserted into the host cell genome. Alternatively, vectors are understood as nucleic acid-based means containing polynucleotide sequences that can spontaneously replicate as episomes. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0116] In detail, vectors can be plasmid DNA, phage DNA, etc. In addition, commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis plasmids (pUB110, pTP5, etc.), yeast plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retroviruses, adenoviruses, vaccinia viruses, etc.), and insect virus vectors (baculoviruses, etc.). Since vectors exhibit various protein expression levels and modifications depending on the host cell, it is preferable to select and use the host cell that is most suitable for the purpose.

[0117] The vectors disclosed herein can be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of sequences that can be fused include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6×His (hexahistidine; Quiagen, USA).

[0118] In addition, since the protein expressed by the vector of this disclosure is an antibody, the expressed antibody can be easily purified using a protein A column or the like without adding any further sequences for purification.

[0119] Transformed cells expressing anti-FcRn antibody variants Another aspect of this disclosure provides host cells transformed with a recombinant expression vector.

[0120] The host cells of transformed cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, mammalian cells, plant cells, insect cells, fungal cells, or cells of cell origin. Escherichia coli can be used as an example of a prokaryotic cell. In addition, yeast can be used as an example of a eukaryotic cell. In addition, as mammalian cells, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, HEK293T cells, etc., can be used, but are not limited to these, and any cells known to those skilled in the art that can be used as mammalian host cells are available.

[0121] In this disclosure, “transformation” or “transfection” by host cells includes any method for introducing nucleic acids into an organism, cell, tissue, or organ, and as known in the art, a standard technique suitable for the host cell may be selected and implemented. For example, the CaCl2 precipitation method, the Hanahan method which improves efficiency by using DMSO (dimethyl sulfoxide) as a reducing agent in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, protoplast fusion, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation, transformation methods using PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation may be used.

[0122] Method for generating anti-FcRn antibody variants or this antigen-binding fragments Another aspect of this disclosure provides a method for producing an anti-FcRn antibody or an antigen-binding fragment thereof, comprising culturing host cells to generate an antibody, and isolating and purifying the generated antibody to recover an antibody that specifically binds to FcRn.

[0123] The FcRn-specific antibodies according to this disclosure may be produced in large quantities by culturing transformants expressing a recombinant vector in a nutrient medium, and the medium and culture conditions that are tolerant to the host cell can be appropriately selected and used. During cultivation, conditions such as temperature, medium pH, and culture period can be appropriately adjusted to suit cell proliferation and large-scale protein production. The antibodies or antibody fragments produced by recombinant as described above may be recovered from the medium or cell lysates and isolated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)). Examples include, but are not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoadsorption chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various modified methods and combinations thereof. In particular, isolation and purification using protein A are preferred.

[0124] In particular, the anti-FcRn antibody or the antigen-binding fragment thereof is preferably prepared by expression and purification using a recombination method. More specifically, it is preferable to prepare the variable region encoding the antibody according to this disclosure that specifically binds to FcRn by co-expression in a single host cell.

[0125] Use of anti-FcRn antibody variants or this antigen-binding fragment Another aspect of this disclosure provides a pharmaceutical composition for treating an autoimmune disease, comprising an anti-FcRn antibody or an antigen-binding fragment thereof. Furthermore, this specification provides a method for treating an autoimmune disorder, comprising administering an effective amount of the anti-FcRn antibody or antigen-binding fragment or pharmaceutical composition described herein to a subject in need.

[0126] The "anti-FcRn antibody" and "antigen-binding fragment" are as described above.

[0127] As used herein, the term “autoimmune disease” is a general term for diseases that result from an immune system abnormality of unknown cause in which the immune system attacks normal tissues, organs, or other body components. These are systemic diseases that can occur in almost any part of the body, including the nervous system, gastrointestinal tract, endocrine system, skin, skeletal system, vascular tissue, etc.

[0128] The pharmaceutical composition may be applied to all autoimmune diseases mediated by IgG and FcRn. Representative autoimmune diseases include autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus deciduousis, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Graves' disease, Goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), lupus nephritis, membranous nephropathy, islet graft rejection, alopecia areata, ankylosing spondylitis, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune adrenal disorders, and warm autoimmune disorders. Epidemic hemolytic anemia (WAIHA), autoimmune hepatitis, autoimmune myocarditis, autoimmune oophoritis and autoimmune orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behçet's disease, cardiomyopathy, Castleman syndrome, celiac plue dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, pemphigoid scarring, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, dilated cardiomyopathy, discoid lupus, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia / fibromyositis, glomerulonephritis, thyroid eye disease (TED, also known as Graves' ophthalmopathy);Graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic membranous nephropathy, idiopathic pulmonary fibrosis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease, lichen planus, lichen sclerosing, lupus erythematosus, Meniere's disease, mixed connective tissue disease, type 1 diabetes, multifocal motor neuropathy (MMN), paraneoplastic bullous pemphigoid, pemphigus foliaceus It may be an autoimmune disease selected from the group consisting of foliaceus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Raynaud's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, Sjögren's syndrome, parenchymal organ transplant rejection, Stiff Man syndrome, systemic lupus erythematosus, Takayasu's arteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), temporal arteritis, giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, herpetiform dermatitis, vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegener's granulomatosis. In some embodiments, the autoimmune disorder is an autoimmune channel disease, which includes autoimmune limbic encephalitis, neuromyelitis optica, Lambert-Eaton myasthenic syndrome, myasthenia gravis, anti-N-methyl-D-aspartate (NMDA) receptor encephalitis, anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor encephalitis, Morvan syndrome, neurogenic myotonia, streptococcal infection-associated childhood autoimmune neuropsychiatric disorders (PANDAS), and glycine The following conditions are appropriately selected, but are not limited to, those comprising receptor antibody-associated disorders, myositis, myelin oligodendrocyte glycoprotein antibody disorders (MOG antibody disorders), fetal and neonatal hemolytic disorders, cutaneous lupus erythematosus, refractory rheumatoid arthritis, immune thrombocytopenia, anti-GBM disease, primary membranous nephropathy, necrotizing autoimmune myopathy, anti-synthesis syndrome, ANCA vasculitis, hidradenitis suppurativa, alveolar proteinosis (PAP), and systemic lupus erythematosus (SLE).

[0129] In some embodiments, the disease treated according to the method herein is characterized by or exhibiting elevated total IgG levels in the blood in the subject being treated. In some embodiments, the disease treated according to the method herein is any one of the autoimmune diseases referred to herein. In some embodiments, the disease is autoimmune Graves' disease. In some embodiments, the disease is thyroid eye disease (TED, also known as Graves' eye disease). In some embodiments, the disease is warm autoimmune hemolytic anemia (WAIHA). In some embodiments, the disease is myasthenia gravis. In some embodiments, the disease is chronic inflammatory demyelinating polyneuropathy (CIDP). In some embodiments, the disease is pemphigus vulgaris, pemphigus foliaceus, pemphigus deciduousus, bullous pemphigoid, bullous pemphigoid of pregnancy, or mucosal pemphigoid.

[0130] In some embodiments, the disease treated according to the method herein is a disease that responds to plasma exchange therapy. Subjects treated according to the method herein may have previously received plasma exchange therapy. In some embodiments, the disease treated according to the method herein is a disease that responds to treatment that lowers total IgG levels. Subjects treated according to the method herein may have previously received treatment that lowers total IgG levels. In some embodiments, the disease treated according to the method herein is a disease that responds to rituximab. Subjects treated according to the method herein may have previously received treatment with rituximab.

[0131] In some embodiments, the anti-FcRn antibody described herein or this antigen-specific fragment reduces the level of any antigen-specific immunoglobulin or autoantibody, for example, any autoantibody described herein or known in the art. In some embodiments, the anti-FcRn antibody described herein or this antigen-specific fragment reduces the level of any autoimmune disease-specific autoantibody or autoantibody associated with or known to be associated with an autoimmune disease, for example, any autoantibody described herein or known in the art. In some embodiments, the autoimmune disease is any autoimmune disease disclosed herein.

[0132] In some embodiments, administration (e.g., subcutaneously) of an effective amount of the anti-FcRn antibody described herein to a subject (e.g., a human) results in a reduction of at least about 40%, 50%, 65%, or 80% of the levels of antigen-specific immunoglobulins or autoantibodies (e.g., levels of one or more of the autoantibodies described herein) in the blood. In some embodiments, such administration has no or minimal effect on blood albumin levels compared to pre-administration levels (e.g., a reduction of less than 10%, less than 5%, less than 2%, or less than 1% in blood albumin) and / or has no or minimal effect on blood LDL levels (e.g., an increase of less than 10%, less than 5%, less than 2%, or less than 1% in LDL). In some embodiments, the antigen-specific immunoglobulins or autoantibodies are associated with diseases such as autoimmune diseases.

[0133] In some embodiments, long-term subcutaneous administration of an effective amount of the anti-FcRn antibody described herein to humans for 12 weeks or more results in a decrease of at least 40%, 50%, 65%, or 80% in blood antigen-specific immunoglobulin levels or autoantibody levels (e.g., levels of one or more of the autoantibodies described herein) compared to pre-administration levels, but with no or minimal effect on blood albumin levels (e.g., a decrease of less than 10%, less than 5%, less than 2%, or less than 1%) and no or minimal effect on blood LDL and / or cholesterol levels (e.g., an increase of less than 10%, less than 5%, less than 2%, or less than 1%). In some embodiments, the antigen-specific immunoglobulin or autoantibody is associated with a disease such as an autoimmune disease.

[0134] Explanatory examples of autoantibodies associated with certain autoimmune diseases include: - Anti-desmoglein 3 (anti-Dsg3) antibody (associated with pemphigus vulgaris), - Anti-desmoglein 1 (anti-Dsg1) antibody (associated with pemphigus vulgaris and pemphigus foliaceus), - Anti-bullous pemphigoid 180 (anti-BP180 or anti-collagen XVII) antibody and anti-bullous pemphigoid 230 (anti-BP230) antibody (both associated with bullous pemphigoid), - Antibodies against non-collagenous domain 1 of type IV collagen 3 chain (anti-α3NC1 antibody) and antibodies against non-collagenous domain 1 of type IV collagen 5 chain (anti-α5NC1 antibody) (both associated with anti-GBM disease), - Anti-phospholipase A2 receptor (anti-PLA2R) antibody (associated with idiopathic membranous nephropathy), - Anti-proteinase 3 (anti-PR3) antibody and anti-myeloperoxidase (anti-MPO) antibody (both associated with ANCA-associated vasculitis), - Anti-double-stranded (ds)DNA antibodies, antibodies against anti-Sjögren's syndrome-related antigen A (anti-SSA / Ro antibodies) and antibodies against anti-Sjögren's syndrome-related antigen B (anti-SSB / La antibodies), anti-Smith antibodies, anti-ribonucleoprotein (anti-RNP) antibodies, anti-complement C1q antibodies, antiproliferative cell nuclear antigen (PCNA) antibodies, anti-cardiolipin antibodies, anti-beta-2 glycoprotein antibodies, anti-granzyme B antibodies, and anti-nucleosome antibodies (all associated with systemic lupus erythematosus), - Antinuclear antibodies (associated with systemic lupus erythematosus, systemic sclerosis, and autoimmune hepatitis), - Rheumatoid factor, anti-citrullinated peptide antibody (ACPA), anti-CarP antibody, and anti-acetylated peptide antibody (AAPA) (all associated with rheumatoid arthritis), - Anti-centromere antibodies (ACA), anti-Scl-70 antibodies, anti-endothelial cell antibodies (AECA), angiotensin II type 1 receptor (anti-AT1R) antibodies, and anti-endothelin 1A receptor (anti-ETAR) antibodies (all associated with systemic sclerosis), - Anti-signal recognition particle (anti-SRP) antibody (associated with immune-mediated necrotizing myopathy and polymyositis), - Anti-HMG-CoA reductase (anti-HMGCR) antibody (associated with immune-mediated necrotizing myopathy), - Anti-Jo-1 antibody, anti-PL-7 antibody, anti-PL-12 antibody, anti-EJ antibody, anti-OJ antibody, anti-Mi-2 antibody, anti-U3 ribonucleoprotein (anti-U3 RNP or anti-fibrillarin) antibody, and anti-U2 ribonucleoprotein (anti-U2 RNP) antibody (all associated with polymyositis), - Anti-Ku antibodies (associated with systemic lupus erythematosus and polymyositis), - Antinuclear matrix protein 2 (anti-NXP-2) antibody, anti-Mi2 antibody, anti-melanoma differentiation-associated protein 5 (anti-MDA5) antibody, and anti-transcription mediator 1-gamma (anti-TIF1γ) antibody (all associated with dermatomyositis), - Anti-acetylcholine receptor (anti-AChR) antibodies, anti-muscle-specific kinase (anti-MuSK) antibodies, and anti-lipoprotein-related protein 4 (anti-LRP4) antibodies (all associated with myasthenia gravis), - Anti-neurofascin-155 (anti-Nfasc155) antibody, anti-neurofascin-140 / 186 (anti-Nfasc140 / 186) antibody, anti-contactin 1 (anti-CNTN1) antibody, and anti-contactin-associated protein-like 1 (anti-Caspr1) antibody (all associated with chronic inflammatory demyelinating polyneuropathy (CIDP)). - Anti-ganglioside GM1 (anti-GM1) antibody, anti-ganglioside GT1a (anti-GT1a) antibody, and anti-ganglioside GD1a (anti-GD1a) antibody (all associated with Guillain-Barré syndrome), - Anti-ganglioside GQ1b (anti-GQ1b) antibody (associated with Guillain-Barré syndrome and Miller-Fischer syndrome), - Anti-aquaporin 4 (anti-AQP4) antibody (associated with spectral impairment of neuromyelitis optica), - Anti-NMDAR antibodies (associated with systemic lupus erythematosus and autoantibody-positive autoimmune encephalitis), - Anti-contactin-associated protein-like 2 (anti-Caspr2) antibody, anti-gamma-aminobutyric acid receptor type B (anti-GABAbR) antibody, anti-leucine-rich glioma inactivation 1 (anti-LGI1) antibody, and anti-Kelch-like protein 11 (anti-Klh11) antibody (all associated with autoantibody-positive autoimmune encephalitis), - Anti-β2 glycoprotein I (anti-β2GPI) antibodies and antiphospholipid antibodies (both associated with primary antiphospholipid syndrome), - Anti-FcγRIIIb antibody and anti-CD177 antibody (both associated with primary autoimmune neutropenia), - Anti-smooth muscle actin (anti-SMA) antibodies and anti-hepatorheological microsome (anti-LKM) antibodies (both associated with autoimmune hepatitis), - Anti-ADAMTS13 antibody (associated with idiopathic thrombotic thrombocytopenic purpura), - Antiplatelet GP IIb / IIIa antibodies and antiplatelet GP Ib / IX antibodies (both associated with idiopathic immune thrombocytopenic purpura), - Thyroid-stimulating hormone (TSH) binding inhibitory immunoglobulin (TBII), thyroid-stimulating immunoglobulin (TSI), cytotropin binding inhibitor (TBI) antibodies, and anti-insulin-like growth factor 1 receptor (anti-IFG1R) antibodies (all associated with Graves' disease and thyroid eye disease), - Antithyroid peroxidase (anti-TPO) antibody (associated with autoimmune thyroiditis / Hashimoto's thyroiditis), - Anti-thyroglobulin antibodies (associated with Graves' disease), - Pancreatic islet cytoplasmic autoantibodies (ICA), glutamate decarboxylase autoantibodies (GADA), insulinoma-associated autoantibodies (IA-2A), and insulin autoantibodies (IAA) (all associated with type 1 diabetes), - Anti-adenovirus neutralizing antibodies, anti-lentivirus neutralizing antibodies, anti-AAV1 neutralizing antibodies, anti-AAV2 neutralizing antibodies, anti-AAV3 neutralizing antibodies, anti-AAV4 neutralizing antibodies, anti-AAV5 neutralizing antibodies, anti-AAV6 neutralizing antibodies, anti-AAV7 neutralizing antibodies, anti-AAV8 neutralizing antibodies, and / or anti-AAV9 neutralizing antibodies (as associated with previous gene therapies), as well as - Anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF) antibody (associated with acquired alveolar proteinosis (PAP)).

[0135] In some embodiments, when a human subject is administered an effective dose of the anti-FcRn antibody described herein (e.g., subcutaneous or long-term subcutaneous administration to a human), proteinuria in subjects with lupus nephritis is reduced by at least 40%, 50%, 65%, or 80%, as measured by the urinary protein levels of the subjects.

[0136] The preferred dosage of a pharmaceutical composition may vary depending on the patient's symptoms and weight, the severity of the disease, the form of the drug, the route of administration, and the duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical compositions of this disclosure for the treatment or prevention of autoimmune diseases, the active ingredient may be present in any amount (effective amount) depending on the use, formulation, and intended use, as long as it exhibits therapeutic activity or, in particular, therapeutic action against autoimmune diseases. A typical effective amount is determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. As used herein, the term "effective amount" refers to the amount of the active ingredient that has an effect of treating or improving the condition of an autoimmune disease, in particular, the amount of the active ingredient that can induce an effect of treating or improving the condition of an autoimmune disease. Such an effective amount can be determined empirically by those skilled in the art.

[0137] In one embodiment, the term “treatment” includes any form of administration or application for treating a disease in a mammal, including a human. In addition, the term includes inhibiting or delaying a disease or its progression, restoring or repairing an impairment or loss of function, thereby partially or completely alleviating the disease, stimulating an inefficient process, or alleviating a serious disease.

[0138] In another embodiment, this specification provides a method for preventing autoimmune disorders, comprising administering an effective amount of the anti-FcRn antibody or the antigen-binding fragment or pharmaceutical composition described herein to a subject in need.

[0139] Any embodiment of this specification relating to a method for treating an autoimmune disorder, comprising administering an anti-FcRn antibody, this antigen-binding fragment, or a pharmaceutical composition, also encompasses the use of the anti-FcRn antibody, this antigen-binding fragment, or a pharmaceutical composition for the preparation of a medicament for treating an autoimmune disorder. Similarly, any embodiment of this specification relating to a method for preventing an autoimmune disorder, comprising administering an anti-FcRn antibody, this antigen-binding fragment, or a pharmaceutical composition, also encompasses the use of the anti-FcRn antibody, this antigen-binding fragment, or a pharmaceutical composition for the preparation of a medicament for preventing an autoimmune disorder.

[0140] Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also affect efficacy. Therefore, "improved efficacy" (e.g., improvement in efficacy) may be due to improvements in pharmacokinetic parameters and efficacy, and these parameters, such as clearance rate and the treatment or improvement of autoimmune diseases, can be measured by comparing them in experimental animals or human subjects.

[0141] As used herein, the terms “therapeutably effective amount” or “pharmaceutically effective amount” refer to the amount of a compound or composition effective in preventing or treating a target disease, meaning an amount sufficient to treat the disease, having a reasonable benefit-to-risk ratio applicable to the medical treatment, and without causing side effects. The level of the effective amount may be determined according to factors including the patient’s health status, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration, and the rate of excretion, the duration of treatment, concomitant or concurrently used drugs, and other factors well known in the medical field. In one embodiment, the therapeutically effective amount refers to the amount of a drug effective in treating an autoimmune disease.

[0142] In this regard, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to the patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. In addition, a pharmaceutically acceptable adjuvant (buffer, dispersant) may be included in the pharmaceutical composition.

[0143] In detail, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the active ingredient, and can be prepared as a parenteral formulation according to the route of administration by conventional methods known in the art. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not exceed the toxicity tolerable by the target population.

[0144] When preparing a pharmaceutical composition as a parenteral formulation, it can be formulated in the form of an injection, transdermal preparation, nasal inhalation preparation, and suppository, together with a suitable carrier, according to methods known in the art. Formulations of pharmaceutical compositions are known in the art, and for details, refer to literature such as [Remington's Pharmaceutical Sciences (19th ed., 1995)]. This literature is considered to be part of this specification.

[0145] The preferred dosage of the pharmaceutical composition may vary depending on the patient's symptoms, weight, sex, and age, the severity of the disease, the route of administration, etc., but can be appropriately selected by those skilled in the art.

[0146] The target organisms to which the pharmaceutical composition can be applied (formulated) are mammals and humans, and particularly preferably humans.

[0147] Another aspect of this disclosure provides a method for treating an autoimmune disease, comprising administering an effective amount of an antibody or antigen-binding fragment that specifically binds to FcRn to a patient in need of treatment for an autoimmune disease.

[0148] Another aspect of this disclosure provides a method for alleviating autoimmune or alloimmune symptoms, comprising administering an anti-FcRn antibody or an antigen-binding fragment thereof to a subject requiring such treatment. In addition, it provides a concurrent anti-FcRn specific therapy.

[0149] Methods or anti-FcRn therapies according to this disclosure for alleviating autoimmune or alloimmune symptoms can be achieved by administering the pharmaceutical compositions according to this disclosure to a target. The pharmaceutical compositions according to this disclosure may be administered orally or parenterally. For example, they may be administered via routes of administration such as intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. In some embodiments, the pharmaceutical compositions disclosed herein may be administered subcutaneously.

[0150] The preferred dosage of the pharmaceutical composition may vary depending on the patient's symptoms and weight, the severity of the disease, the form of the drug, the route of administration, and the duration of administration, but may be appropriately selected by those skilled in the art, and may be administered once or repeatedly.

[0151] In various embodiments of the therapeutic methods and uses disclosed herein, the antibody or antigen-binding fragment is administered to the patient as a fixed dose. In various embodiments of the therapeutic methods and uses disclosed herein, the antibody or antigen-binding fragment is administered to the patient as a body weight-based dose; that is, the dose depends on the patient's body weight. In various embodiments of the therapeutic methods and uses disclosed herein, the antibody or antigen-binding fragment is administered to the patient as a body surface area-based dose; that is, the dose depends on the patient's body surface area (BSA). In various embodiments, the dose administered to the patient contains a therapeutically effective amount of the antibody or antigen-binding fragment.

[0152] In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 300 mg to approximately 500 mg. In some embodiments, the antibody or antigen-binding fragment is administered subcutaneously to the patient in a dose of approximately 300 mg to approximately 500 mg, for example, once a week, once a month, once every two weeks, or once every three weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in doses of approximately 300 mg, approximately 310 mg, approximately 320 mg, approximately 330 mg, approximately 340 mg, approximately 350 mg, approximately 360 mg, approximately 370 mg, approximately 380 mg, approximately 390 mg, approximately 400 mg, approximately 410 mg, approximately 420 mg, approximately 430 mg, approximately 440 mg, approximately 450 mg, approximately 460 mg, approximately 470 mg, approximately 480 mg, approximately 490 mg, or approximately 500 mg (for example, once a week, once a month, once every two weeks, or once every three weeks) (for example, subcutaneously). In some embodiments, the antibody or antigen-binding fragment is administered to the patient in doses of approximately 300 mg or more (for example, once a week, once a month, or once every two weeks) (for example, subcutaneously). In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 340 mg. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 340 mg once a week, once a month, or once every two weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 340 mg once a week. In some embodiments, the antibody or antigen-binding fragment is administered to the patient as a single subcutaneous injection once a week in a dose of approximately 340 mg. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 340 mg once a week for at least two weeks (e.g., two, three, four, five, six, seven, eight, ten, twelve weeks, or more). In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 340 mg once a week for at least four weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a week at a dose of approximately 340 mg for at least 7 weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a week at a dose of approximately 340 mg for at least 12 weeks. In some embodiments, administration is subcutaneous.

[0153] In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 500 mg to approximately 700 mg. In some embodiments, the antibody or antigen-binding fragment is administered subcutaneously to the patient in a dose of approximately 500 mg to approximately 700 mg, for example, once a week, once a month, once every two weeks, or once every three weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in doses of approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, approximately 580 mg, approximately 590 mg, approximately 600 mg, approximately 610 mg, approximately 620 mg, approximately 630 mg, approximately 640 mg, approximately 650 mg, approximately 660 mg, approximately 670 mg, approximately 680 mg, approximately 690 mg, or approximately 700 mg (for example, once a week, once a month, once every two weeks, or once every three weeks) (for example, subcutaneously). In some embodiments, the antibody or antigen-binding fragment is administered to the patient in doses of approximately 750 mg or 700 mg or less (for example, once a week, once a month, or once every two weeks) (for example, subcutaneously). In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 680 mg. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 680 mg once a week, once a month, or once every two weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 680 mg once a week. In some embodiments, the antibody or antigen-binding fragment is administered to the patient as a series of subcutaneous injections of approximately 680 mg once a week for two or more weeks (e.g., two times). In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 680 mg once a week for at least two weeks (e.g., two, three, four, five, six, seven, eight, ten, twelve weeks, or more). In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose of approximately 680 mg once a week for at least four weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a week at a dose of approximately 680 mg for at least 7 weeks. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a week at a dose of approximately 680 mg for at least 12 weeks. In some embodiments, administration is subcutaneous.

[0154] In some embodiments, the antibody or antigen-binding fragment is administered to the patient in doses of approximately 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg. In some embodiments, the antibody or antigen-binding fragment is administered intravenously to the patient in doses of approximately 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg. In some embodiments, the antibody or antigen-binding fragment is administered subcutaneously to the patient in doses of approximately 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg.

[0155] In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose range between any of the dose values ​​mentioned herein. In some embodiments, the antibody or antigen-binding fragment is administered to the patient in a dose less than any of the dose values ​​mentioned herein.

[0156] In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a week (e.g., subcutaneously) in doses of approximately 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg.

[0157] In some embodiments, the antibody or antigen-binding fragment is administered to the patient every two weeks (e.g., subcutaneously) in doses of approximately 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg.

[0158] In some embodiments, the antibody or antigen-binding fragment is administered to the patient once every three weeks (e.g., subcutaneously) in doses of approximately 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg.

[0159] In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a month (e.g., subcutaneously) in doses of approximately 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, or 2500 mg.

[0160] In some embodiments, the antibody or antigen-binding fragment is administered to the patient in doses of approximately 300 mg, approximately 600 mg, approximately 900 mg, approximately 1200 mg, approximately 1500 mg, approximately 1800 mg, approximately 2100 mg, or approximately 2400 mg. In some embodiments, the antibody or antigen-binding fragment is administered intravenously to the patient in doses of approximately 300 mg, approximately 600 mg, approximately 900 mg, approximately 1200 mg, approximately 1500 mg, approximately 1800 mg, approximately 2100 mg, or approximately 2400 mg. In some embodiments, the antibody or antigen-binding fragment is administered subcutaneously to the patient in doses of approximately 300 mg, approximately 600 mg, approximately 900 mg, approximately 1200 mg, approximately 1500 mg, approximately 1800 mg, approximately 2100 mg, or approximately 2400 mg. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a week (e.g., subcutaneously) in doses of approximately 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, or 2100 mg. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once every two weeks (e.g., subcutaneously) in doses of approximately 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, or 2400 mg. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once every three weeks (e.g., subcutaneously) in doses of approximately 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, or 2400 mg. In some embodiments, the antibody or antigen-binding fragment is administered to the patient once a month in doses of approximately 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, or 2400 mg (e.g., subcutaneously).

[0161] In some embodiments, the antibody is administered to the patient in doses of approximately 150 mg, 300 mg, 450 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, or 2400 mg, or any dose between any two of these doses (e.g., subcutaneously) (e.g., subcutaneously once a week, subcutaneously once every two weeks, subcutaneously once every three weeks, or subcutaneously once a month). In some embodiments, the doses presented herein are suitable doses for administration to humans.

[0162] In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week or once a month. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least twelve weeks, at least twenty weeks, at least twenty-four weeks, at least thirty weeks, at least forty weeks, at least fifty weeks, at least sixty weeks, at least seventy weeks, at least seventy weeks, at least eighty weeks, or longer.

[0163] In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once every two or three weeks. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once every two weeks (i.e., once every two weeks) for at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least twelve weeks, at least twenty weeks, at least twenty-four weeks, at least thirty weeks, at least forty weeks, at least fifty weeks, at least sixty weeks, at least seventy weeks, at least seventy weeks, at least eighty weeks, or longer.

[0164] In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once every three weeks for at least three weeks, at least six weeks, at least nine weeks, at least twelve weeks, at least eighteen weeks, at least 21 weeks, at least 24 weeks, at least 30 weeks, at least 42 weeks, at least 48 weeks, at least 60 weeks, at least 72 weeks, at least 78 weeks, at least 81 weeks or longer.

[0165] In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a month for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least twelve months, at least twenty months, at least twenty four months, at least thirty months, at least forty months, at least fifty months, at least sixty months, at least seventy months, at least seventy months, at least seventy months, at least eighty months, or longer.

[0166] In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for 6 to 76 weeks or any period in between. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 6 weeks. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for at least 4 weeks. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for at least 7 weeks. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for at least 12 weeks. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for at least 24 weeks. In some embodiments, the antibody, antigen-binding fragment, or pharmaceutical composition is administered to the patient once a week for at least 52 weeks.

[0167] In some embodiments, the antibody or antigen-binding fragment is administered to the patient in one or more doses (e.g., two or more different doses). For example, in some embodiments, the antibody or antigen-binding fragment is administered to the patient in two different doses, e.g., at least one high dose followed by at least one low dose. The high dose (e.g., the higher of the two different doses) may be referred herein to as the “induction” dose, i.e., a dose capable of reducing the level of at least one autoantibody and / or pathogenic antibody (e.g., at least one IgG) in the patient and / or patient-derived samples. The low dose (e.g., the lower of the two different doses) may be referred herein to as the “maintenance” dose, i.e., a dose capable of maintaining the reduction in the level of at least one autoantibody and / or pathogenic antibody (e.g., at least one IgG) in the patient and / or patient-derived samples [e.g., about 20–80% of the pre-induction (pre-dose) value] after at least one induction dose of the antibody or antigen-binding fragment. In some embodiments, the maintenance dose maintains the level of at least one autoantibody and / or pathogenic antibody (e.g., at least one IgG) in the patient and / or patient-derived sample at or below approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the pre-induction dose value.

[0168] In some embodiments, the induction dose and maintenance dose may be modified, for example, by administering the induction dose to the patient for a first period, then the maintenance dose for a first period, then the induction dose for a second period, and then the maintenance dose for a second period as appropriate. In some embodiments, the induction dose-maintenance dose cycle (one cycle consists of a period of administration of the induction dose followed by a period of administration of the maintenance dose) is repeated 1, 2, 3, 4, 5, 6, 7, or 10 times. In some embodiments, the induction dose-maintenance dose cycle is repeated for about 3 months, about 6 months, about 12 months, about 15 months, about 18 months, about 24 months, or longer.

[0169] In some embodiments, at least one high dose and / or induction dose is about 680 mg or more per dose (e.g., about 680 mg, about 700 mg per dose, about 720 mg per dose, about 750 mg per dose or more). In some embodiments, at least one high dose and / or induction dose is about one dose, about two doses, about three doses, about four doses or about five doses of about 680 mg or more per dose (e.g., about 680 mg, about 700 mg per dose, about 720 mg per dose, about 750 mg per dose or more). In some embodiments, at least one high dose and / or induction dose is about three doses of about 680 mg or more per dose (e.g., about 680 mg, about 700 mg per dose, about 720 mg per dose, about 750 mg per dose or more).

[0170] In some embodiments, at least one high dose and / or induction dose is administered to the patient once, once a week, once every two weeks, or once a month. In some embodiments, at least one high dose and / or induction dose is administered intravenously to the patient. In some embodiments, at least one high dose and / or induction dose is administered subcutaneously to the patient. In some embodiments, each high dose is administered to the patient as one or more subcutaneous injections. In some embodiments, each high dose is administered to the patient as two consecutive subcutaneous injections.

[0171] In some embodiments, at least one low dose and / or maintenance dose is about 340 mg or more per dose (e.g., about 340 mg, about 360 mg, about 380 mg, about 400 mg or more per dose). In some embodiments, at least one low dose and / or maintenance dose is about one, two, three, four, or five doses of about 340 mg or more per dose (e.g., about 340 mg, about 360 mg, about 380 mg, about 400 mg or more per dose). In some embodiments, at least one low dose and / or maintenance dose is about three doses of about 340 mg or more per dose (e.g., about 340 mg, about 360 mg, about 380 mg, about 400 mg or more per dose). In some embodiments, at least one low dose and / or maintenance dose is administered to the patient once, once a week, once every two weeks, or once a month. In some embodiments, at least one low dose and / or maintenance dose is administered to the patient subcutaneously. In some embodiments, each low dose is administered to the patient as one or more subcutaneous injections. In some embodiments, each low dose is administered to the patient as a single subcutaneous injection. This disclosure relates, for example, to the following: [Section 1] A light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 1, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and Heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7. An anti-FcRn antibody or an antigen-binding fragment thereof, comprising An anti-FcRn antibody or this antigen-binding fragment in which the amino acid at N3 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, and / or the amino acid at C2 in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid. [Section 2] The anti-FcRn antibody described in item 1, or the antigen-binding fragment thereof, wherein the amino acid at N3 in the amino acid sequence of SEQ ID NO: 1 is substituted with Ser(S) or Gln(Q). [Section 3] An anti-FcRn antibody or antigen-binding fragment as described in item 1 or 2, wherein the amino acid at C2 in the amino acid sequence of SEQ ID NO: 5 is substituted with Ser(S) or Tyr(Y). [Section 4] An anti-FcRn antibody or antigen-binding fragment according to any one of items 1 to 3, wherein the heavy chain variable region comprises a framework consisting of FR1 of the amino acid sequence of SEQ ID NO: 15, FR2 of the amino acid sequence of SEQ ID NO: 16, FR3 of the amino acid sequence of SEQ ID NO: 17, and FR4 of the amino acid sequence of SEQ ID NO: 18. [Section 5] The anti-FcRn antibody described in item 4, or the antigen-binding fragment thereof, wherein the amino acid Q16 in the amino acid sequence of SEQ ID NO: 17 is substituted with another amino acid. [Section 6] The anti-FcRn antibody described in item 5, or the antigen-binding fragment thereof, wherein the amino acid Q16 in the amino acid sequence of SEQ ID NO: 17 is substituted with Glu(E). [Section 7] An anti-FcRn antibody or an antigen-binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 8, An anti-FcRn antibody or this antigen-binding fragment, wherein the amino acid at N25 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid, and the amino acid at C32 or Q82 in the amino acid sequence of SEQ ID NO: 8 is substituted with another amino acid. [Section 8] The anti-FcRn antibody described in item 7, or the antigen-binding fragment thereof, wherein the amino acid at N25 in the amino acid sequence of SEQ ID NO: 4 is substituted with Ser(S) or Gln(Q). [Section 9] An anti-FcRn antibody or antigen-binding fragment as described in item 7 or 8, wherein the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 is substituted with Ser(S) or Tyr(Y). [Section 10] An anti-FcRn antibody or antigen-binding fragment described in any one of items 7-9, wherein the amino acid at Q82 in the amino acid sequence of SEQ ID NO: 8 is substituted with Glu(E). [Section 11] A light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and Heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7 An anti-FcRn antibody or an antigen-binding fragment thereof, including. [Section 12] The anti-FcRn antibody according to any one of claims 1 to 11, wherein the anti-FcRn antibody comprises an Fc region, and the Fc region is either an IgG1 Fc region or an IgG4 Fc region. [Section 13] The anti-FcRn antibody according to item 12, wherein the Fc region is an IgG1 Fc region containing amino acid substitutions of Leu234Ala and Leu235Ala. [Section 14] An anti-FcRn antibody as described in any one of items 1 to 13, or a polynucleotide encoding this antigen-binding fragment. [Section 15] A recombinant expression vector containing the polynucleotide described in item 14. [Section 16] Host cells transformed with the recombinant expression vector described in item 15. [Section 17] The process involves culturing the host cells described in item 16 to produce antibodies, The aforementioned generated antibody is isolated and purified to recover an antibody that specifically binds to FcRn. A method for producing an anti-FcRn antibody or an antigen-binding fragment thereof, including the above. [Section 18] A pharmaceutical composition comprising an anti-FcRn antibody or an antigen-binding fragment described in any one of items 1 to 13, and a pharmaceutically acceptable carrier. [Section 19] A pharmaceutical composition for treating an autoimmune disease, comprising an anti-FcRn antibody or an antigen-binding fragment as described in any one of items 1 to 13. [Section 20] The pharmaceutical composition according to item 19, wherein the autoimmune disease is an autoimmune disease selected from the group consisting of autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus deciduousis, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, and membranous nephropathy. [Section 21] A method for treating an autoimmune disease in a subject requiring such treatment, comprising administering an anti-FcRn antibody or an antigen-binding fragment thereof as described in any one of items 1 to 13, or a pharmaceutical composition as described in any one of items 18 to 20, wherein the subject may be a human. [Section 22] The method according to item 21, wherein the autoimmune disease is selected from the group consisting of autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus deciduousis, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, and membranous nephropathy. [Section 23] The method according to item 21 or 22, wherein the administration is parenteral. [Section 24] The method according to item 23, wherein the administration is subcutaneous or intravenous. [Section 25] The method according to any one of items 21 to 24, wherein the administration is at a dose of 300 mg to 2400 mg. [Section 26] The method according to any one of claims 21 to 24, wherein the administration is once a week, once every two weeks, once every three weeks, or once a month. [Section 27] The method according to any one of claims 21 to 26, wherein the administration is a subcutaneous dose of 300 mg to 900 mg once a week. [Section 28] The method according to any one of items 21 to 26, wherein the administration is a subcutaneous dose of 300 mg to 1800 mg once every two weeks. [Section 29] The method according to any one of claims 21 to 28, wherein administration does not result in a decrease in the serum albumin level in the subject of more than 5% or more than 10% compared to the serum albumin level before administration of the anti-FcRn antibody or antigen-binding fragment. [Section 30] The method according to any one of claims 21 to 29, wherein administration does not result in an increase in total blood cholesterol or LDL levels in the subject by more than 5% or more than 10% compared to the total blood cholesterol or low-density lipoprotein (LDL) levels before administration of the anti-FcRn antibody or antigen-binding fragment. [Examples]

[0172] Hereafter, the Disclosure will be described in further detail by the following embodiments. However, these embodiments are merely illustrative of the Disclosure and the scope of the Disclosure is not limited thereto.

[0173] Design of mutants to improve the physical properties of I.HL161AN antibody To improve the stability of HL161AN through molecular engineering, amino acid sites that may affect stability were identified and mutants were designed using Lonza's in silico tools and LC-MS / MS analysis. A schematic diagram of the in silico design of the HL161AN mutant is shown in Figure 1.

[0174] Therapeutic proteins, including antibodies, exist in heterogeneous forms due to post-translational modifications (PTMs) and chemical modifications. These modifications, including glycosylation, deamidation, and oxidation, are known to occur depending on the host cell line, the production process, and storage conditions. These modifications are considered a major challenge in the production process of therapeutic proteins because they can lead to decreased efficacy, limited shelf life, and side effects such as immune responses due to aggregation (De Groot 2006).

[0175] HL161AN is an anti-hFcRn antibody in the form of modified human IgG1, and human FcRn (2 × 10⁻¹⁰ -10 It exhibits high affinity for M) and high PD activity in in vivo studies in cynomolgus monkeys. However, it has the disadvantage of low thermal stability (approximately 58.4°C), aggregation occurs at concentrations above 30 mg / mL, and many charged mutants are identified in CEX-HPLC and cIEF analysis. Therefore, using in silico tools, the PTM sites and major aggregation sites were identified based on the results of amino acid sequence and X-ray crystal structure analysis, and the modification sites were identified by LC-MS / MS analysis of HL161AN samples. Subsequently, by integrating the two results, we attempted to design mutants that reduce the risk of PTM and aggregation. [Example 1]

[0176] In silico assessment of aggregation risk and PTM risk The risks of aggregation and post-translational modification were predicted using in silico analysis.

[0177] Protein aggregation is influenced by environmental factors such as pH, concentration, buffer, excipients, and shear force, as well as intrinsic properties. To predict the risk of aggregation based on amino acid sequence, we used Lonza's Sentinel APART® database. This program is validated based on experimental results for the aggregation of over 500 antibodies. By inputting the sequence of the antibody being tested, the risk of aggregation is predicted. The Sentinel APART algorithm predicted an increased risk of aggregation for HL161AN.

[0178] In addition, post-translational modifications (PTMs) were predicted using an in silico tool developed by Lonza, as described below (Figures 2 and 3). Specifically, the prediction of PTM risks was carried out considering i) asparagine deamidation, ii) aspartate isomerization and fragmentation, iii) C-terminal lysine clipping, iv) Fc ADCC / CDC response, half-life, and protein A purification, v) free cysteine ​​thiol groups, vi) N-glycosylation, O-glycosylation, vii) oxidation, and viiii) pyroglutamate formation.

[0179] As a result of amino acid sequence analysis of HL161AN, the possibility of asparagine deamidation was predicted at 4 sites in the light chain (L) and 12 sites in the heavy chain (H). Most of these were predicted to be low-risk as they were conserved asparagine molecules, and although the 4 sites had the potential for deamidation, they did not correspond to the CDR region, so it was confirmed that protein modification was not necessary. Therefore, it was analyzed that only L:Asn25 in the light chain CDR region required protein modification.

[0180] In addition, free cysteinethiol groups can lead to protein misfolding, aggregation, nonspecific tissue binding, immunogenicity, and potentially low productivity. As a result of confirming whether HL161AN contains free cysteinethiol groups, a free thiol group was found at H:Cys32, and it was determined that protein modification of this amino acid is necessary. [Example 2]

[0181] Analysis of PTM sites by LC-MS / MS analysis Deamidation and oxidation modifications of two lots of HL161AN were analyzed by LC-MS / MS. Peptide mapping using two enzymes, namely trypsin + LysC mixed enzyme and chymotrypsin, confirmed 100% sequence inclusion (Figure 4).

[0182] Modification analysis revealed deamidation and oxidation at 7 and 6 sites, respectively, in the HL161AN in-house standard sample, and at 10 and 8 sites, respectively, in the HL161AN B012 sample.

[0183] The deamidation and oxidation sites of the HL161AN sample, as determined by LC-MS / MS, are summarized in Tables 2 and 3 below, respectively.

[0184] [Table 2]

[0185] [Table 3] [Example 3]

[0186] Selection of the part to modify Based on in silico analysis and LC-MS / MS results, substitution amino acids were selected for three amino acid sites (L:Asn25, H:Cys32, H:Gln82) that were determined to require modification. X-ray crystallography data determined that these three amino acid sites are far from the CDR region that binds to the target antigen, and therefore the substitutions would not affect affinity (Figure 5). In selecting amino acid substitutions, it was determined that the risk of PTM could be reduced by substituting serine (Ser) or glutamine (Gln) for L:Asn25, serine (Ser) or tyrosine (Tyr) for H:Cys32, and serine (Ser), threonine (Tyr), or glutamic acid (Glu) for H:Gln82. Chemically similar side-chain substitution methods were used for each amino acid selection method, for example, asparagine was replaced with glutamine (Table 4). Based on these results, two light chains and eleven heavy chains were designed (Table 5).

[0187] The proposed substitutions of selected PTM sites are summarized in Table 4 below. The designed HL161AN variants are also summarized in Table 5 below.

[0188] [Table 4]

[0189] [Table 5]

[0190] Combining the above results, we get the following:

[0191] To improve the stability of HL161AN through molecular engineering, we used Lonza's in silico tools and LC-MS / MS analysis to identify amino acid sites that may affect stability and designed mutants through mutation.

[0192] As a result, it was determined that three amino acid sites (L:Asn25, H:Cys32, H:Gln82) of the HL161AN antibody required modification. It was determined that substitution of L:Asn25 with serine or glutamine, H:Cys32 with serine or tyrosine, and H:Gln82 with serine, threonine, or glutamic acid could reduce the risk of PTM. A total of 22 HL161AN variants (two light chains and eleven heavy chains of HL161AN variants) were designed by amino acid substitution.

[0193] Subsequently, 22 variants were prepared using site-directed mutagenesis, and then antibodies with improved stability were screened through in vitro and in vivo evaluations.

[0194] II. In vitro screening of HL161AN antibody variants HL161AN has excellent pharmacological efficacy in vitro / in vivo, but due to its insufficient physicochemical properties, it has the disadvantage of insufficient stability during storage and difficulty in preparing a high-concentration SC (subcutaneous) formulation. To improve this, the sites that can affect the stability of the antibody were determined by amino acid sequence analysis and molecular structure analysis of HL161AN as described above, and a total of 22 HL161AN variants were prepared by modifying the derived amino acid sites (Table 6). Subsequently, the in vitro evaluation was carried out by generating the obtained 22 HL161AN variants. Preparation Example 1. Preparation of Test Substances Preparation Example 1.1. HL161AN Reference Standard Sample (Reference Standard, RS): Control 1 - Lot Number: HL161AN / 16E11 / ST01 - Concentration: 13 mg / mL - Formulation Buffer: 100 mM Histidine, 100 mM Arginine-HCl, pH 6.0 - Storage Conditions: ≤ -60°C Preparation Example 1.2. HL161AN Variant Plasmid - Light Chain Variant 2 and Heavy Chain Variant 11 - Storage Conditions: ≤ -60°C

[0195] In the amino acid sequence of HL161AN, two or more amino acids were substituted as shown in Table 6 below.

[0196] [Table 6] [Example 4]

[0197] First In Vitro Evaluation of 22 HL161AN Variants The 22 HL161AN variants were evaluated in comparison with the control antibody HL161AN. [Example 4.1]

[0198] Sample Generation One day before transfection, Expi293F cells were seeded at 1×10 6Seeded into a 500 mL Erlenmeyer flask at [cell count] / mL (100 mL volume), and then incubated with shaking for 24 hours in an incubator under conditions of 8% CO2, 125 rpm, and 37°C. On the day of transfection, the cell concentration was adjusted to 2×10 6 cells / mL, and then the plasmid DNA of each variant was transfected into the cells. The antibody plasmid was diluted to 1 μg / μL in LAL water (Lonza Bioscience). 0.12 mL of the heavy chain plasmid and 0.12 mL of the light chain plasmid were each added to 5 mL of Opti-MEM SFM and suspended. 0.24 mL of FectoPro was added to 5 mL of Opti-MEM SFM and suspended.

[0199] The plasmid DNA and FectoPro were mixed at a 1:1 ratio and incubated at room temperature for 10 minutes, and then added to the flask containing the cells. This was incubated with shaking in an incubator under conditions of 8% CO2, 37°C, and 125 rpm for 3 hours, and then 100 μL of Booster was added to continue the culture. On the 6th day from the start of the culture, the culture solution was allowed to stand in a 250 mL centrifuge tube and centrifuged at 3,000 rpm for 15 minutes, and then only the supernatant was collected and filtered through a 0.2 μm bottle top filter. The antibody expression level in the collected culture medium was quantified using a Cedex bioanalyzer.

[0200] As a result, a total of 22 HL161AN antibody variants were transiently expressed using Expi293F cells. Transfection was performed at a 100 mL culture scale to obtain an expression medium of 0.5 mg or more for each antibody, and the culture medium was collected on the 6th day. The expression levels of the obtained media were 65 - 181 mg / L (Table 7). The production results of the 22 HL161AN variants are summarized and shown in Table 7 below.

[0201] [Table 7] [Example 4.2]

[0202] Purification of Antibody Sample purification was performed by packing 1.5 mL of MabSelect SuRe® LX resin into a Poly-prep® chromatography column. Elution was performed by fractionating 1.5 mL of the sample using 1 × PBS (pH 7.4) as the binding and washing buffer, and 0.1 M glycine (pH 3.0) buffer. All fractionation tubes were neutralized to pH 7.0 by adding 1 M Tris-HCl (pH 9.0) and then pooled. The pooled eluates were purified by buffer exchange with citrate phosphate buffer (pH 8.0) and concentration to 1.0 mg / mL using Millipore centricon (cutoff 30 kDa). In vitro evaluation was also performed using HL161AN RS by buffer exchange with citrate phosphate buffer (pH 8.0), similar to the HL161AN variant. For each sample, the A280 value was measured using Nanodrop, and the concentration was calculated. Based on quantitative values, the state of each antibody sample was confirmed by SDS-PAGE analysis.

[0203] As a result, the culture medium obtained by transiently expressing a total of 22 HL161AN antibody variants using Expi293F cells was purified using a Protein A column and concentrated to a concentration of 1.0 mg / mL. SDS-PAGE and SEC-HPLC analyses confirmed that the generated samples had a purity of 97% or higher (Figure 6 and Table 7). [Example 4.3]

[0204] SPR analysis Surface plasmon resonance (SPR) analysis was performed using a ProteonXPR36 instrument under two conditions: pH 6.0 and pH 7.4. Soluble human FcRn (shFcRn) was immobilized on a GLC chip, and antibody samples were reacted at five concentrations; then, sensorgram results were obtained. Kinetic analysis was performed using a 1:1 Langmuir-coupled model, and the mean KD value was obtained by repeating the analysis six times under each of the pH 6.0 and pH 7.4 conditions.

[0205] Chip activation was performed under EDAC / NHS 0.5×, 30 μL / min, and 300 sec. Subsequently, 250 μL of 2 μg / mL shFcRn was prepared using acetate buffer (pH 5.5) and immobilized while flowing at 30 μL / min. The reaction was stopped when the immobilization level corresponded to 200 RU–300 RU. Then, deactivation was performed using ethanolamine at 30 μL / min for 300 sec. Antibody samples were prepared by serial dilutions of 1 / 2–5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.312 nM based on a concentration of 10 nM. Sample dilution was performed using 1× PBST (pH 7.4) or 1× PBST (pH 6.0) buffer depending on the analytical pH. Under the sample analysis conditions, association was performed by a reaction at 50 μL / min for 200 sec, and dissociation by a reaction at 50 μL / min for 600 sec. Subsequently, regeneration was performed by a reaction at 100 μL / min for 18 sec using glycine buffer (pH 2.0). For kinetic analysis, samples were prepared and reacted once in three cells immobilized with shFcRn. Three kinetic values ​​were then obtained, followed by the measurement of the average KD value. When analyzing the reaction signal, interspot-referencing and double-referencing were performed using signals analyzed only with the buffer.

[0206] As described above, SPR analysis was performed on 22 HL161AN antibody variants at pH 6.0 and pH 7.4. As a result, the HL161AN variants showed similar binding affinities to those of HL161AN for shFcRn [1.16E-10M (pH 6.0) and 2.94E-10M (pH 7.4)] (Figure 7 and Table 8).

[0207] The dynamics results of the HL161AN mutant are summarized in Table 8 below.

[0208] [Table 8] [Example 4.4]

[0209] Analysis of hFcRn binding of antibody variants using FACS hFcRn binding was analyzed using FACS under two conditions: pH 6.0 and pH 7.4. HEK293 cells expressing hFcRn (hFcRn HEK293) were diluted with reaction buffer (0.05% BSA in PBS, pH 6.0 or pH 7.4), and 1 × 10⁶ samples were analyzed for each sample. 5 Cells were prepared in double rows in 96-well plates. In addition, each antibody sample was diluted to 10 nM in reaction buffer, then placed in the 96-well plates, and incubated at 4°C for 90 minutes. The plates were then centrifuged to remove the supernatant, and Alexa488 goat anti-hIgG Ab (1:200) was added, followed by incubation again at 4°C for 90 minutes. After the reaction was complete, the supernatant was removed by centrifugation, and the cell pellet was resuspended with 200 μL of reaction buffer. The MFI value was then measured using a FACS instrument.

[0210] As described above, binding evaluation using FACS was performed on 22 HL161AN antibody variants at pH 6.0 and pH 7.4. As a result, the 22 HL161AN variants showed MFI values ​​similar to those of HL161AN (Figure 8). [Example 4.5]

[0211] Analysis of the hFcRn blocking ability of antibodies using FACS hFcRn HEK293 cells 1 x 10 7 Cells were seeded in a T75 flask and cultured for 24 hours. After culturing, cells were removed and 1 × 10⁶ cells were extracted from each sample. 5 Cells were placed in a double row in a 96-well plate. Then, each antibody sample (0.2 nM, 2 nM, 20 nM, and 200 nM) and 100 nM Alexa488-hIgG1 were added in a 1:1 ratio, and incubated at 4°C for 90 minutes. After the reaction was complete, the supernatant was removed by centrifugation, and the cell pellet was resuspended with 200 μL of reaction buffer. The MFI values ​​were then measured using a FACS instrument. Each MFI value was converted to % blockade and fitted with 4-PL to obtain EC 50The value was obtained.

[0212] As a result, blocking evaluations using FACS were performed at 0.1 nM, 1 nM, 10 nM, and 100 nM. Each antibody blocked in a concentration-dependent manner, and EC 50 values were confirmed to be similar. Similar to the results of hFcRn binding analysis using FACS, the EC 50 values of the 22 HL161AN mutants were confirmed to be similar to those of the control antibody HL161AN. Therefore, it was determined that the amino acid substitutions did not affect the function of the HL161AN mutants (Figure 9). [Example 4.6]

[0213] Evaluation of stability 200 μL of each purified sample was aliquoted into 1.5 mL tubes, and then the samples were stored at 40 °C under accelerated conditions for up to 4 weeks. Samples at Week0, Week1, Week2, and Week4 were analyzed by SEC-HPLC. The analysis of each sample was performed under the SEC-HPLC analysis conditions shown in Table 9 below to analyze the degree of stability under accelerated conditions.

[0214] [Table 9]

[0215] The samples were stored at 40 °C under accelerated conditions for up to 4 weeks, and the stability of the samples was analyzed by SEC-HPLC. As a result, in the case of HL161AN, compared with the sample at Week0, the aggregates in the sample increased by 47.5% and the fragments increased by 4.8% at Week4. On the other hand, the production rates of aggregates of the 22 mutants were 5.6% - 39.6%, which were lower than those of HL161AN. Also, the production rates of fragments were 3.2% - 12.3%, which were increased or similar to the production rate of HL161AN (Figure 10, Figure 11, and Table 10).

[0216] [Table 10] [Example 4.7]

[0217] DSC analysis Differential scanning calorimetry (DSC) analysis was performed using a NANO DSC instrument (TA Instrument, PN:602000, SN:K10126), and Tm values ​​were analyzed using NanoAnalyze software with a two-state scale model. The DSC analysis was performed by K Bio Health (Osong Medical Innovation Foundation, New Drug Development Support Center).

[0218] The thermal stability of the HL161AN mutant was analyzed. As shown in Table 11 below, the Tm value of HL161AN was 58.4°C, while the Tm value of the HL161AN mutant was 60°C to 61.5°C. Therefore, it was confirmed that the Tm value of the HL161AN mutant was up to 2°C higher compared to HL161AN.

[0219] The thermal stability of the HL161AN mutant is summarized in Table 11 below.

[0220] [Table 11]

[0221] The results of the first in vitro evaluation of the 22 HL161AN variants listed above are summarized in Table 12 below.

[0222] FcRn binding affinity / blockade and stability were evaluated for a total of 22 HL161AN mutants. The results showed that the binding affinity and blockade effects were similar to those of HL161AN, and all HL161AN mutants exhibited improved stability in terms of aggregate formation compared to HL161AN. Therefore, the top eight mutants that reduced aggregate formation to less than 10% after 4 weeks of storage at 40°C were selected. After preparing high-concentration samples and conducting a second in vitro screening, the leading molecule was ultimately selected.

[0223] [Table 12] [Example 5]

[0224] Second in vitro evaluation of high-concentration samples of eight HL161AN mutants. Eight high-concentration HL161AN variants and the control antibody HL161AN were evaluated. [Example 5.1]

[0225] Sample generation The culture method for producing high-concentration samples is the same as that in Example 4.1. To obtain 120 mg or more of antibody, 1 L or more of each antibody variant was cultured. [Example 5.2]

[0226] Antibody purification The sample purification method was the same as in Example 4.2. The eluate was exchanged with 50 mM histidine buffer (pH 5.0) and concentrated to a concentration of approximately 150 mg / mL to complete the purification. In the case of HL161AN RS, the eluate was exchanged with 50 mM histidine (pH 5.0) and concentrated to a concentration of approximately 150 mg / mL to obtain the sample, similar to the case of the HL161AN variant. For each antibody sample, the concentration was calculated using Nanodrop, and based on this, SDS-PAGE analysis was performed to confirm the state of the sample. [Example 5.3]

[0227] Generation of high-concentration samples of the HL161AN mutant The eight HL161AN mutants selected in the first in vitro evaluation were transiently expressed in Expi293F cells in the same manner as in Examples 5.1 and 5.2 above, and purified to concentrations of 150 mg / mL or higher. HL161AN was used by concentrating the standard product to a high concentration. All samples were analyzed by A280 and SEC-HPLC after preparation (Table 13), and all samples except mutants 2 and 13 were confirmed to have a high purity of 98% or higher. In the case of mutants 2 and 13, precipitates were observed during the concentration process, and the SEC-HPLC purity was confirmed to have decreased to approximately 93% (Table 13).

[0228] [Table 13] [Example 5.4]

[0229] Analysis of antibody hFcRn binding using FACS The procedure was carried out in the same manner as in Example 4.4. For the eight high-concentration HL161AN mutants and HL161AN, hFcRn binding was evaluated using FACS at pH 6.0 and pH 7.4. As a result, the eight HL161AN mutants showed MFI values ​​similar to those of HL161AN (pH 6.0: 2952, pH 7.4: 7155) (Figure 12). [Example 5.5]

[0230] Analysis of hFcRn blockade by antibodies using FACS The procedure was carried out in the same manner as in Example 4.5. hFcRn blockade was evaluated using FACS at 0.1 nM, 1 nM, 10 nM, and 100 nM. Each antibody blocked in a concentration-dependent manner, and EC 50 The values ​​were similar to those of the control antibody HL161AN (Figure 13). [Example 5.6]

[0231] Stability evaluation The procedure was carried out in the same manner as in Example 4.6. To evaluate the stability of the eight high-concentration HL161AN mutants, the change in purity of the samples stored at 40°C for four weeks was confirmed by SDS-PAGE and SEC-HPLC. In the case of HL161AN, the aggregation rate was over 50% after four weeks of storage, which was also confirmed by SDS-PAGE (Figure 14). In the case of the eight HL161AN mutants, the aggregation rate was 8.4% to 25.7%, which was higher than that of HL161AN, and the fragmentation rate was 3.5% to 4.4%, which was similar to the production rate of HL161AN (4.1%) (Figure 15). [Example 5.7]

[0232] Viscosity analysis The VROC tip was placed on the main body of the viscometer (m-VROC system), and then approximately 300 μL each of high-concentration HL161AN RS control sample and HL161AN mutant sample were filled into syringes. The VROC tip was connected to the syringe, stabilized at a set temperature of 25°C, and the viscosity was measured by repeating the process three times.

[0233] The viscosity of the control substance HL161AN and four high-concentration HL161AN variants with sufficient sample volume was analyzed. The viscosity of the buffer solution (50 mM histidine) was measured at 0.93 cP. The average viscosity of the four HL161AN variants was measured at 5.57 cP to 7.65 cP, which was below the viscosity suitable for subcutaneous (sc) administration (<20 cP) (Table 14). The viscosities of the four high-concentration HL161AN variants are summarized in Table 14 below.

[0234] [Table 14]

[0235] The results of the second in vitro evaluation of high-concentration (150-170 mg / mL) samples of the eight HL161AN mutants listed above are summarized in Table 15 below.

[0236] For the eight HL161AN variants selected in the first in vitro evaluation, high-concentration samples of 150 mg / mL to 163 mg / mL were prepared, and FcRn binding / blockage and stability were measured to select the primary molecule. As a result, HL161AN variant number 1 and variant number 12, which had FcRn binding / blockage similar to that of HL161AN and improved stability, were selected as the primary molecules (Table 15).

[0237] [Table 15] [Example 6]

[0238] Conclusions from in vitro screening of HL161AN antibody variants In an in vitro evaluation of 22 HL161AN mutants designed to improve the physical properties of HL161AN, a total of 22 HL161AN mutants were generated, and their FcRn binding affinity / blockade and stability were measured. As a result, all 22 mutants showed similar biological activity to that of HL161AN, and the top 8 mutants with improved stability were selected first. High-concentration samples (150 mg / mL to 163 mg / mL) of the selected 8 mutants were prepared, and their stability was evaluated. As a result, all 8 HL161AN mutants showed similar biological activity to that of HL161AN, and their stability was improved compared to HL161AN.

[0239] The light chain N25S mutation was added to HL161AN mutants #1, #2, #8, and #11, and the N25Q mutation was added to mutants #12, #13, #19, and #22. However, no difference in stability was observed between the two groups. In the case of the heavy chain, C32 was further mutated to S or Y, and Q82 was further mutated to S, T, or E. However, in all mutants, the aggregate production rate decreased by at least half compared to the control group HL161AN. In particular, when C32 was mutated to S, the aggregate production rate (≤10%) decreased by more than one-fifth compared to HL161AN, and stability was improved the most compared to the other mutants (Figure 16). Therefore, HL161AN mutant number 1 (LC:N25S, HC:C32S;HL161ANS) and mutant number 12 (LC:N25Q, HC:C32S;HL161ANQ), which had aggregate production rates of 10% or less, were ultimately selected as the primary molecules.

[0240] III. Evaluation of hIgG catabolism using HL161AN antibody mutants in hFcRn gene-transformed mice The in vivo hIgG catabolism efficacy of three HL161AN mutants modified to improve stability was evaluated using hFcRn gene transgenic (Tg)32 mice.

[0241] The test substances for the hIgG catabolism test of anti-FcRn antibody (HL161AN) mutants using hFcRn gene-transformed mice were prepared as follows. Test substance information is shown in Table 16. Three HL161AN mutants, HL161ANS-IgG1LALA, HL161ANQ-IgG1LALA, and HL161ANQ-IgG4 S228P, were diluted to 2 mg / mL in the buffer shown in Table 16 below. 10 mL was prepared for administration to 5 animals, divided into four doses of 2.5 mL / day, and stored at -60°C or below until administration.

[0242] [Table 16]

[0243] In addition, the comparative substance HL161AN was prepared as shown in Table 17. HL161AN was diluted to 2 mg / mL in 50 mM histidine (pH 5.9) dilution buffer. 10 mL was prepared for administration to 5 animals, divided into four doses of 2.5 mL / day, and stored at -60°C or below until administration.

[0244] [Table 17]

[0245] As a control substance, IV-Globulin SN Inj. (IVIG), a commercially available product from GC Biopharma, was used. Product information is shown in Table 18. Specifically, 0.4 mL of IV-Globulin Inj. at a concentration of 50 mg / mL was diluted to a concentration of 2 mg / mL by adding 9.6 mL of PBS (pH 7.4), divided into four doses of 2.5 mL / day, and stored at 4°C for up to 4 days before administration.

[0246] [Table 18]

[0247] In addition, tracers were prepared by mixing 495 mg / kg of total hIgG and 5 mg / kg of biotin-hIgG. Specifically, total hIgG was prepared at a concentration of 49.5 mg / mL, and 495 mg / kg of IV-Globulin Inj. was administered by adding 0.2 mL of PBS (pH 7.4) to 19.8 mL of 50 mg / mL of IV-Globulin Inj. In addition, biotin-hIgG was prepared by adding 267 μL of 10 mM biotin to 20 mg of IV-Globulin Inj., and then incubated at room temperature for 30 minutes. After that, to remove free biotin, the sample was transferred to a dialysis bag and dialysis was performed with 1 × PBS (pH 7.4). Biotin-labeled hIgG was UV 280nm The absorbance was quantified by measuring the absorbance at IgG E 1% = 14.0 at 280 nm. [Example 7]

[0248] Animal experimentation methods The mFcRn that was being raised - / - hFcRn gene transgenic (Tg) 32 mice (Jackson Laboratory, USA) (male, 5-10 weeks old) were identified by numbering their tails and placing five mice in each cage. After registration, these mice were acclimatized to the climate for 1-2 weeks. In addition, they were divided into groups as shown in Table 19 below and administered the sample substance.

[0249] [Table 19]

[0250] In detail, as tracers, 495 mg / kg of total hIgG and 5 mg / kg of biotin-hIgG were administered intraperitoneally (ip) at a dose of 10 mL / kg, with the administration time set to 0 hours. In addition, the test substances HL161ANS(IgG1-LALA), HL161ANQ(IgG1-LALA), and HL161ANQ(IgG4 S228P), the comparator substance HL161AN, and the control substances, the medium and IVIG, were administered intraperitoneally four times at a dose of 20 mg / kg, 24, 48, 72, and 96 hours after tracer administration.

[0251] Subsequently, the tracer administration time was set to 0 hours, and then blood was collected at 24, 48, 72, 96, 120, and 168 hours. The test substance, comparison substance, and control substance were administered at 24, 48, 72, and 96 hours. In this case, blood was collected first, followed by administration. Mice were anesthetized using a respiratory anesthesia device, and then blood was collected from the orbital venous plexus using a microhematocrit capillary tube.

[0252] Approximately 0.2 mL of whole blood was centrifuged at 3000 rpm for 15 minutes. The resulting serum was then transferred to a 1.5 mL microcentrifuge tube and stored at -60°C or below until analysis. [Example 8]

[0253] Confirmation of the degree of catabolism of biotin-hIgG tracers by ELISA analysis. The degree of catabolism of biotin-hIgG tracers in the body was confirmed using serum obtained by the method described above.

[0254] In detail, the diluent / blocking buffer (assay diluent (AD), 1% BSA in 1×PBS, pH 7.4): 10 g of Probumin® was dissolved in 900 mL of 1×PBS (pH 7.4), adjusted to a final volume of 1 L, filtered through a 0.2 μm filter, and then stored at 4°C. In addition, a standard storage solution (500 ng / mL biotin-hIgG) was prepared by mixing 10 μL of biotin-hIgG (4 mg / mL) with 990 μL of PBS to obtain 40 μg / mL. Subsequently, 750 μL of the diluent was mixed with 250 μL of 40 μg / mL biotin-hIgG to prepare 10 μg / mL. A 500 ng / mL standard storage solution was prepared by mixing 50 μL of 10 μg / mL biotin-hIgG with 950 μL of the diluent, and then divided into 50 μL equal portions. The divided standard storage solutions were stored at -80°C. In addition, neutral avidin was used by diluting 10 μL of 2 mg / mL neutral avidin with 10 mL of 1×PBS to 2 μg / mL.

[0255] Subsequently, the samples from the media group and the IVIG group were all diluted 1:10,000 using a diluent to prepare the analytical samples. Except for the HL161ANS(IgG1-LALA) group, the samples from 24hr, 48hr, and 72hr were diluted 1:10,000, and the samples from 96hr, 120hr, and 168hr were diluted 1:1,000. In the case of the HL161ANS(IgG1-LALA) group, only the sample from 168hr was diluted 1:1,000, while all other time point samples were diluted 1:10,000. The diluted samples were packed into double wells of 100 μL each and incubated at room temperature for 2 hours.

[0256] Blood biotin-hIgG concentrations administered at 0 hours were measured by ELISA at 24, 48, 72, 96, 120, and 168 hours after administration of 20 mg / kg of HL161ANS(IgG1-LALA), HL161ANQ(IgG1-LALA), HL161ANQ(IgG4 S228P), HL161AN, and IVIG. As a result, the other test groups showed a significant decrease in biotin-hIgG over time compared to the vehicle and IVIG administration groups. On the other hand, there were no significant differences among the three HL161AN variants and HL161AN. Among the three variants, HL161ANQ(LC:N25Q,HC:C32S)-IgG1-LALA showed a relatively high effect on biotin-hIgG catabolism. However, since there was no statistical significance, this was determined to be a similar effect (Tables 20, 21, and Figure 17).

[0257] The biotin-hIgG concentrations after administration of 20 mg / kg of the HL161AN mutant are summarized in Table 20 below.

[0258] [Table 20]

[0259] The biotin-hIgG catabolism results (%) of the HL161AN mutant in Tg32 mice (24 hours after tracer administration) are summarized in Table 21 below.

[0260] [Table 21] [Example 9]

[0261] Calculation of the half-life of biotin-IgG tracers using pharmacokinetic (PK) analysis. Using the quantitative analysis results of biotin-hIgG by ELISA, the half-life of each tracer was calculated using BA calc (2007, Korea) after administration of each test substance. Specifically, using the ELISA analysis results, the half-lives from 24 hours to 120 hours after tracer administration were calculated for three HL161AN variants (Table 16), a comparative substance, and a control substance.

[0262] As a result, as shown in Table 22, the half-lives were 22.7±9.0 hours for HL161ANS(IgG1-LALA), 15.9±0.9 hours for HL161ANQ(IgG1-LALA), and 17.8±2.6 hours for HL161ANQ(IgG4 S228P). The half-life of the comparison substance HL161AN was 20.6±3.6 hours, which was confirmed to be equivalent to the half-lives of the three HL161AN mutants. In addition, the half-lives of the medium and IVIG were 108.2±29.0 hours and 98.0±16.8 hours, respectively, and were confirmed to be similar. The results of the half-life analysis of biotin-hIgG after administration of HL161AN mutants in Tg mice are summarized in Table 22 below.

[0263] [Table 22] [Example 10]

[0264] hIgG catabolism of HL161AN antibody mutants in hFcRn gene transgenic mice Three HL161AN mutants, modified to improve the stability of HL161AN, were administered to Tg32 mice at a dose of 20 mg / kg, and their hIgG catabolic activity was examined. As a result, HL161ANS(IgG1-LALA), HL161ANQ(IgG1-LALA), and HL161ANQ(IgG4 S228P) were confirmed to have hIgG catabolic activity equivalent to that of the control substance HL161AN.

[0265] In conclusion, HL161ANS (light chain: N32S, heavy chain: C32S) and HL161ANQ (light chain: N32Q, heavy chain: C32S), which are substances in which two amino acids in the variable domain of HL161AN have been substituted to improve stability, were confirmed to have in vivo IgG catabolism similar to that of the original molecule HL161AN. In other words, it was confirmed that the three HL161AN variants modified to improve stability did not affect the in vivo efficacy compared to the existing HL161AN. [Example 11]

[0266] Conclusions regarding hIgG catabolism upon administration of anti-FcRn antibody (HL161AN) mutant to hFcRn gene-transformed mice. To evaluate the effects of HL161AN and HL161AN variants (HL161ANS and HL161ANQ) on biotin-hIgG catabolism in vivo, blood was collected from Tg32 mice up to a point after administration of 20 mg / kg, and the blood concentration of biotin-hIgG was measured.

[0267] As a result, in the case of biotin-hIgG, similar biotin-hIgG catabolism was confirmed for all three administered substances. All tested substances (HL161AN, HL161ANS, and HL161ANQ) reduced biotin-hIgG concentrations by more than 90% within 120 hours after administration compared to the IVIG control group.

[0268] In conclusion, hIgG catabolism tests confirmed that there was no significant difference in the hIgG catabolic activity of HL161ANS and HL161ANQ compared to that of HL161AN. [Example 12]

[0269] Comparison of HL161ANS antibody and batoclimab In this one-to-one study in monkeys, the HL161ANS variant (also known as IMVT-1402) was compared to another anti-FcRn antibody, batoclimab (HL161BKN, see, for example, International Patent Application Publication No. WO2015 / 167293).

[0270] Twenty monkeys in four groups were intravenously administered 50 mg / kg of batoclimab, 5 mg / kg of HL161ANS, 50 mg / kg of HL161ANS, or placebo. IgG, albumin, and low-density lipoprotein (LDL) and cholesterol were measured at time points. Cynomolgus monkeys are known to be a reliable pharmacodynamic surrogate system for anti-FcRn-mediated effects on IgG (see, e.g., Lledo-Garcia, et al., UCB Pharma, 2022).

[0271] At comparative doses, the IgG reduction was nearly identical for batoclimab and HL161ANS (Figure 18). The IgG-lowering effect was dose-dependent. Similar effects were demonstrated on albumin, LDL, and cholesterol with HL161ANS and placebo (Figures 19A-19C, respectively). Therefore, there was no substantial effect of HL161ANS on albumin, LDL, or cholesterol levels. [Example 13]

[0272] A 4-week exploratory intravenous pharmacological study in cynomolgus monkeys followed by a 4-week recovery period. The objective of the study was to determine the pharmacological, toxicological, and toxicological (TK) profiles of the test item HL161ANS after intravenous administration to cynomolgus monkeys once a week for four weeks (Day 1, Day 8, Day 15, and Day 22), and to evaluate the persistence, delayed onset, or reversibility of any changes after a four-week recovery period.

[0273] The test item, positive control item, and control / medium item were administered to groups of monkeys once a week by intravenous injection (over approximately 60 seconds) on Day 1, Day 8, Day 15, and Day 22, as shown in Table 23 below.

[0274] [Table 23]

[0275] After the completion of the final week-long administration cycle, all animals were observed for four weeks, and then animals from groups 1, 3, and 4 were euthanized and subjected to autopsy on Day 57.

[0276] Parameters monitored during this study included mortality, clinical findings, and body weight. In addition, clinicopathological parameters (hematology, coagulation, clinical chemistry, and urinalysis) were evaluated. Blood samples were collected for assessment of toxicology, ApoB, IgG, and ADA. Autopsies were performed, and tissue samples were taken for possible histopathological examination.

[0277] No mortality rate or clinical signs associated with HL161ANS were observed.

[0278] No effects of the test items were observed on body weight, hematology, coagulation, clinical chemistry, or urinalysis.

[0279] Splenic plaque formation was observed in HL161ANS at doses of 5 mg / kg and 50 mg / kg, and a high incidence of small thymus was observed in the 50 mg / kg dose. Since microscopic examination was not performed, it was impossible to determine the toxicological significance of these findings.

[0280] In conclusion, intravenous administration of 5 mg / kg and 50 mg / kg of HL161ANS to cynomolgus monkeys once a week for 4 weeks (Day 1, Day 8, Day 15, and Day 22) was well tolerated. [Example 14]

[0281] Toxicity and toxicology studies using HL161ANS via subcutaneous and / or intravenous injection over 6 weeks in cynomolgus monkeys, followed by a 9-week recovery period. The purpose of this study was to evaluate the toxicity and determine the toxicological effects of HL161ANS when administered to cynomolgus monkeys by subcutaneous and / or intravenous (continuous bolus) injection twice a week for at least 6 weeks (a total of 13 administration intervals), and to assess the reversibility or persistence of any effects after a 9-week recovery period.

[0282] Male and female cynomolgus monkeys were assigned to six groups and administered according to the dosages shown in Table 24. The animals were administered subcutaneously (SC) by injection (groups 1-4) and / or intravenously (IV)-(continuous bolus) by injection (groups 1, 5, and 6) at a volume of 1.3 mL / kg / dose on Day 1, Day 4, Day 8, Day 11, Day 15, Day 18, Day 22, Day 25, Day 29, Day 32, Day 36, Day 39, and Day 43 of the administration period. The control medium / diluent was HL161ANS preparation buffer. In addition, KLH was administered to each animal once by subcutaneous injection at a volume of 1 mL / dose on Day 14 of the administration period and once on Day 28 of the convalescence period.

[0283] [Table 24]

[0284] Toxicity assessment was based on mortality, clinical findings, body weight, quantitative food intake, ophthalmic findings, electrocardiogram (ECG) measurements, skin findings, neurobehavioral findings, respiratory rate assessment, and clinical and anatomical pathology. Blood samples were collected and evaluated using toxicological, ADA, and immunotoxicological methods.

[0285] Toxicogenic parameters were generally similar (within twofold) in male and female monkeys. In SC administration, systemic exposure to HL161ANS was C max Value and AUC 0-72 As the values ​​show, it increased with dose level. Since a terminal death occurred on Day 44, this parameter could not be determined on Day 43, therefore the AUC 0-72The evaluation was limited to animals other than the convalescent animals in the 198.4 mg / kg / dose group. This increase was generally greater than the proportional value between the 50.0 mg / kg / dose and 198.4 mg / kg / dose doses. After repeated SC administration twice per week, based on the mixed sex AR mean values, no serum accumulation was observed in the 50.0 mg / kg / dose group from Day 1 to Day 22, with very low values ​​of less than 1.0 in the two individual female animals in the group. Accumulation did not occur at high doses, and the degree of accumulation increased with dose. max Value and AUC 0-72 The accumulation ratio relative to the value was 1.65–1.86 in the 100.0 mg / kg / dose group (Day 1–Day 22) and 1.83–2.88 in the 198.4 mg / kg / dose group (Day 1–Day 22, and Day 43). In IV administration, the C of HL161ANS max Value and AUC 0-72 The value increased with dose level. AUC 0-72 The increase in values ​​was greater than the proportional value between doses of 50.0 mg / kg / dose and 198.4 mg / kg / dose. C after IV bolus administration max A dose-proportionality assessment showed an increase nearly proportional to the dose on Day 1, but due to the contribution of cumulative effects, the interpretation of this assessment became difficult on Days 22 and 43. After repeated IV administration twice a week, C max Serum accumulation based on the following criteria ranged from mild (50.0 mg / kg / dose) to moderate (198.4 mg / kg / dose), with ARs of 1.11–1.23 and 1.40–1.63, respectively. AUC 0-72 In the 50.0 mg / kg / dose group, the mean mixed-sex exposure decreased by half from Day 1 to Day 22 and remained constant until Day 43, with an AR of approximately 0.460. This indicates a very low AR in 5 out of 6 animals in this group. In the 198.4 mg / kg / dose IV group, the AUC 0-72A moderate increase was observed, with an AR of approximately 1.5. Positive anti-drug antibody (ADA) titers were observed in 35 out of 38 animals (92.1%) treated with HL161ANS (one animal treated with 100 mg / kg / dose via SC and two animals treated with 198.4 mg / kg / dose via IV were negative for ADA titers). Since AR tended to decrease with increasing ADA titers, it is possible that HL161ANS, which induced ADA, influenced the accumulation ratio on Day 22 and Day 43. The 50 mg / kg / dose group was more affected than the other groups.

[0286] On Day 30 of the administration period, one female ox that had received 100 mg / kg / dose of SC was euthanized due to emaciation (0.3 kg weight loss), low body condition score, sunken eyes, decreased skin elasticity, liquid feces, and hypothermia (36.4°C). The animal had been examined prior to this and treated on Day 23 of the administration period for swelling and lacerations of the left leg, but the animal's condition worsened. The clinicopathological changes associated with HL161ANS consisted of mild to moderate decreases in total protein and total albumin concentrations. These changes were similar to those observed in animals that survived until planned euthanasia and were likely to be exacerbated by inflammatory responses and the clinical findings of liquid feces.

[0287] Macroscopic findings included cerebral malformations and compression, which microscopically correlated with a slight bulge of the brainstem and were judged to be incidental. A minimal increase in the cellular solidity of myeloid progenitor cells in the sternal bone marrow may correlate with the inflammatory response associated with the finger laceration. The cause of the mortal state was indeterminate based on microscopic findings, but the finger wound, leading to persistent watery stools and decreased motility and food intake, appeared to contribute to the animal's mortal state. Based on available clinical findings and clinical and anatomical pathological results, this mortality was not associated with HL161ANS. No clinical findings associated with HL161ANS were observed; ophthalmic findings; changes in body weight and food intake; effects on respiratory rate; skin findings; effects on ECG; effects on coagulation, urinalysis, and IgM and IgA titers; effects on CH50 levels; effects on immune and lymphocyte subsets; organ weight differences; or macroscopic findings.

[0288] Prior to administration on Day 22 of the administration period, an increase in IgM titer was observed compared to pre-administration on Day 1 of the administration period in most controls, as well as in males and females, that received HL161ANS at a dose of 50 mg / kg / dose or higher. Subsequently, a decrease in IgM titer was observed prior to administration on Day 43 of the administration period and 1344 hours after administration in females that received HL161ANS at a dose of 50 mg / kg / dose or higher, and the values ​​remained at a level comparable to the control. In males that received HL161ANS, the IgM titer prior to administration on Day 43 of the administration period remained at a level comparable to pre-administration on Day 22 of the administration period, and then decreased 1344 hours after administration on Day 43 of the administration period. Prior to administration on Day 4 of the administration period, a decrease in IgG concentration compared to pre-administration on Day 1 of the administration period was observed in all animals that received HL161ANS at a dose of 50 mg / kg / dose or higher. In the group administered 50 mg / kg / dose or more, the percentage change in mean IgG concentration decreased by approximately 48-76% from baseline by Day 43 of the administration period, consistent with pharmacological predictions. Mean IgG concentrations in each group increased from Day 15 of the recovery period and returned to baseline levels by the end of the recovery period. An increase in IgG concentration compared to pre-administration on Day 1 of the administration period was observed from Day 15 of the administration period in two females administered 198.4 mg / kg / dose via SC, after a decrease was observed on Day 4 of the administration period. In both animals, IgG concentrations remained high until Day 43 of the administration period before decreasing on Day 1 of the recovery period. An increase in IgG concentration compared to pre-administration on Day 1 of the administration period was observed from Day 15 of the administration period in one male administered 198.4 mg / kg / dose via SC, after a decrease was observed on Day 4 of the administration period. IgG concentrations remained high until Day 32 of the administration period before decreasing on Day 36 of the administration period. IgG levels decreased to below baseline by Day 50 of the recovery phase. Elevated IgG levels in a single animal may be associated with vascular inflammation observed in various tissues on microscopic examination. Such group-based variability means that the relationship between IgG levels and HL161ANS and / or ADA titers remains unknown.

[0289] A slight increase in C3a levels compared to control and baseline values ​​was observed on Day 43 of the administration period in animals administered 198.4 mg / kg / dose via SC or IV, and was considered potentially associated with HL161ANS due to its consistency between sexes and the small magnitude of the change. C3a is an effector in the complement system and has various stimulating functions on the immune system. The effects associated with HL161ANS in clinical chemistry results consisted of a minimal to mild decrease in total protein concentration in animals administered up to 198.4 mg / kg / dose via SC or IV on Day 43 of the administration period. A minimal to moderate decrease in albumin concentration was observed on Day 16 and / or Day 43 of the administration period in animals administered more than 50 mg / kg / dose via SC, males administered more than 50 mg / kg / dose IV, and females administered 198.4 mg / kg / dose IV. A minimal decrease in globulin concentration was observed on Day 43 of the administration period in males administered 50 or 100 mg / kg / dose via seroconcentrate or more than 50 mg / kg / dose via intravenous injection, and in females administered 50 mg / kg / dose via seroconcentrate or intravenous injection. Evidence of reversibility of albumin action was shown by Day 43 of the administration period in males administered 50 mg / kg / dose via intravenous injection, and all other effects in clinical chemistry results in animals administered 198.4 mg / kg / dose via seroconcentrate or intravenous injection demonstrated evidence of reversibility by Day 22 or 50 of the recovery period.

[0290] The effects of HL161ANS on hematological test results were minor, consisting of a minimal decrease in absolute neutrophil count in animals administered 198.4 mg / kg / dose intravenously on Day 43 of the administration period. These effects provided evidence of reversibility by Day 22 or 50 of the convalescence period, except for one male administered 198.4 mg / kg / dose intravenously. One female administered 198.4 mg / kg / dose via saccharin showed a minimal increase in absolute monocyte count on Day 43 of the administration period, which correlated with microscopic findings of vasculitis and mononuclear cell infiltration. At sacrificial death in the terminal phase, microscopic findings of vasculitis associated with HL161ANS, potentially related to immune complex-mediated damage, were observed in the lungs, liver, gallbladder, kidneys, stomach, small and large intestines, tongue, heart, uterus, and subcutaneous injection site E of one female administered 198.4 mg / kg / dose via saccharin. Further findings in this animal included minute extramedullary hematopoiesis, mild hypertrophy of hepatic Kupffer cells, and minute hypertrophy of the pulmonary vascular media. No findings associated with HL161ANS were observed at the time of sacrificial death during the recovery phase.

[0291] In conclusion, male and female cynomolgus monkeys were administered HL161ANS at a media control product / dilution or 50, 100, or 198.4 mg / kg / dose via sponge injection and / or intravenous injection twice a week for 6 weeks. Elevated C3A levels were observed on Day 43 of the administration period in animals administered 198.4 mg / kg / dose via sponge injection or intravenous injection. This finding was reversible during the recovery period. Clinicopathological changes included a mild decrease in total protein concentration in both sexes receiving up to 198.4 mg / kg / dose via saccharin (SC) or IV, a minimal to moderate decrease in albumin concentration in both sexes receiving up to 198.4 mg / kg / dose via SC, males receiving ≥ 50 mg / kg / dose IV, and females receiving 198.4 mg / kg / dose IV, a minimal decrease in globulin concentration in males receiving 50 or 100 mg / kg / dose via SC or ≥ 50 mg / kg / dose IV, and females receiving 50 mg / kg / dose via SC or IV, and a minimal decrease in absolute neutrophil count in both sexes receiving 198.4 mg / kg / dose IV. These findings were reversible during the recovery phase.

[0292] Microscopic findings associated with HL161ANS included vasculitis in the lungs, liver, gallbladder, kidneys, stomach, small and large intestines, tongue, heart, uterus, and subcutaneous injection site E of one female animal administered 198.4 mg / kg / dose via SC. No findings associated with HL161ANS were observed at sacrificial death during the recovery phase. Due to the systematic nature of the microscopic findings in animals administered 198.4 mg / kg / dose via SC, the effects of this dose were judged to be harmful. The effects of these doses were judged to be harmless because the severity of the findings was mild and there were no effects on the health and welfare of animals administered 100 mg / kg / dose via SC or 198.4 mg / kg / dose via IV. Therefore, the no-observed-adverse-effect level (NOAEL) is 100 mg / kg / dose via SC injection and 198.4 mg / kg / dose via IV injection. The SC dose level corresponds to the mean maximum blood concentration (Cmax) of 964 mg / L observed on Day 43 of the administration period and the area under the blood concentration-time curve (AUCtau or AUC0-72) value of 2290 mg / L over the dosing interval on Day 22 of the administration period (it should be noted that AUC0-72 on Day 43 could not be calculated because the sacrificial death at the end of the treatment period occurred one day after the administration on Day 43). The IV dose level corresponds to the mean maximum blood concentration (Cmax) of 7100 mg / L observed on Day 43 of the administration period and the area under the blood concentration-time curve (AUCtau or AUC0-72) over the dosing interval. tau or AUC 0-72 This corresponds to a value of 10800 mg x day / L. [Example 15]

[0293] Subcutaneous and / or intravenous dose range and pharmacodynamic studies to determine the toxicity, toxicogenicity, and toxicological effects of HL161ANS in cynomolgus monkeys. The objective of this study was to evaluate the toxicity of HL161ANS when administered to cynomolgus monkeys for at least 6 weeks by subcutaneous (SC) injection twice a week or intravenous (IV, continuous bolus) injection once a week, and to determine its toxicological (TK) and pharmacodynamic (PD) properties.

[0294] Male and female cynomolgus monkeys were assigned to six groups and administered the doses shown in the following table. The animals were administered intravenously and / or subcutaneously once or twice a week at a volume of 1 mL / kg / dose. The control medium / diluent was HL161ANS preparation buffer.

[0295] [Table 25]

[0296] Toxicity assessment was based on mortality, clinical findings, body weight, food intake, skin findings, immunotoxicology, and clinical and anatomical pathology. Blood samples were collected for toxicological evaluation and anti-drug antibody analysis.

[0297] No deaths were associated with HL161ANS, and no clinical or cutaneous findings, changes in body weight, weight gain, or food intake, organ weight differences, macroscopic findings, or microscopic findings associated with HL161ANS were observed.

[0298] The clinical and chemical effects associated with HL161ANS observed in animals administered 100 mg / kg / dose intravenously or via seroconjunctival administration were limited to minimal to mild decreases in albumin concentrations observed in individual animals. Albumin decreases were more consistently observed in animals administered 100 mg / kg / dose via seroconjunctival administration from Day 15 or 22 to 50, but this was primarily observed in animals administered 100 mg / kg / dose intravenously on Day 36 and Day 43 of the administration period. The mechanism of albumin decreases was not understood.

[0299] In conclusion, male and female cynomolgus monkeys were administered either HL161ANS buffer (medium control) by subcutaneous injection twice a week and intravenously once a week, or HL161ANS at doses of 3, 10, or 100 mg / kg / dose intravenously once a week, or HL161ANS at doses of 100 mg / kg / dose twice a week by subcutaneous injection. HL161ANS was well tolerated at all dose levels and routes of administration. No deaths associated with HL161ANS occurred, and no clinical findings, changes in body weight, body weight gain, and food intake, organ weight differences, macroscopic findings, or microscopic findings associated with HL161ANS were observed. No clinical or pathological effects associated with HL161ANS were observed in animals receiving up to 100 mg / kg / dose intravenously. In animals administered 100 mg / kg / dose via SC, the clinical and pathological effects associated with HL161ANS were limited to minimal to mild decreases in albumin concentration from Day 15 or 22 to Day 50. Based on these findings, HL161ANS at 100 mg / kg / dose, the highest dose level administered intravenously or subcutaneously, is considered the maximum tolerated dose (MTD). [Example 16]

[0300] A Phase 1 trial to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of HL161ANS in healthy participants. This example describes a phase 1 randomized, placebo-controlled, double-blind, sequential parallel-group trial investigating the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple escalating doses of HL161ANS in healthy adult male participants and non-pregnancy-possible (NCBP) adult female participants. Participants joined a single dosing cohort.

[0301] There are six single-dose escalation (SAD) cohorts [100 mg, 300 mg, 600 mg, and 1,200 mg intravenously (IV); and 300 mg and 600 mg subcutaneously (SC)]. There are also two multi-dose escalation (MAD) cohorts [300 mg and 600 mg SC once weekly (QW)] and, as appropriate, two MAD cohorts (150 mg and 450 mg SC QW). Each MAD cohort receives HL161ANS or placebo for 4 weeks. Two sentinel subjects in each cohort receive the treatment (one active and one placebo).

[0302] Each SAD cohort consists of 6 participants with HL161ANS and 2 participants with placebo, while each of the four MAD cohorts consists of 10 participants with HL161ANS and 2 participants with placebo. Therefore, this trial has a maximum total of 96 participants (76 with HL161ANS and 20 with placebo).

[0303] The selected dose must not exceed the fixed dose of 1,530 mg IV for SAD and 800 mg SC for MAD.

[0304] Participants will be screened, their eligibility assessed, and randomized to the trial. In the SAD cohort, participants will be randomized into six sequential dosing cohorts, with eight participants per cohort. In each SAD cohort, two participants will be randomized in a 1:1 ratio to receive either HL161ANS or placebo as a sentinel dose. Then, if HL161ANS is well tolerated in each sentinel participant, the remaining six participants will be randomized in a 5:1 ratio to receive either HL161ANS or placebo.

[0305] In the MAD cohort, participants will be randomized into up to four sequential cohorts (two planned and two as needed), with 12 participants per cohort. In each MAD cohort, two participants will be randomized in a 1:1 ratio to receive sentinel HL161ANS or placebo. If HL161ANS is well tolerated in each sentinel participant, the remaining 10 participants will then be randomized in a 9:1 ratio to receive either HL161ANS or placebo.

[0306] The objectives and evaluation items are shown in Table 25.

[0307] [Table 26]

Claims

1. A light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 1, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and Heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO:

7. An anti-FcRn antibody or an antigen-binding fragment thereof, comprising An anti-FcRn antibody or antigen-binding fragment thereof, wherein the amino acid at N3 in the amino acid sequence of SEQ ID NO: 1 is substituted with Ser(S) or Gln(Q), and / or the amino acid at C2 in the amino acid sequence of SEQ ID NO: 5 is substituted with Ser(S) or Tyr(Y).

2. The anti-FcRn antibody or antigen-binding fragment according to claim 1, wherein the heavy chain variable region comprises a framework consisting of FR1 of the amino acid sequence of SEQ ID NO: 15, FR2 of the amino acid sequence of SEQ ID NO: 16, FR3 of the amino acid sequence of SEQ ID NO: 17, and FR4 of the amino acid sequence of SEQ ID NO:

18.

3. An anti-FcRn antibody or an antigen-binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 8, An anti-FcRn antibody or antigen-binding fragment thereof, wherein the amino acid at N25 in the amino acid sequence of SEQ ID NO: 4 is substituted with Ser(S) or Gln(Q), and the amino acid at C32 in the amino acid sequence of SEQ ID NO: 8 is substituted with Ser(S) or Tyr(Y).

4. A light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 9, LCDR2 containing the amino acid sequence of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence of SEQ ID NO: 3, and Heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7 An anti-FcRn antibody or an antigen-binding fragment containing this antibody.

5. (i) VL containing the amino acid sequence of SEQ ID NO: 10 and VH containing the amino acid sequence of SEQ ID NO: 12, or (ii) VL containing the amino acid sequence of SEQ ID NO: 14 and VH containing the amino acid sequence of SEQ ID NO: 12 An anti-FcRn antibody or antigen-binding fragment according to any one of claims 1 to 4, comprising:

6. The anti-FcRn antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the anti-FcRn antibody comprises an Fc region, and the Fc region is an IgG1 Fc region or an IgG4 Fc region.

7. The anti-FcRn antibody or antigen-binding fragment according to claim 6, wherein the Fc region is an IgG1 Fc region containing amino acid substitutions of Leu234Ala and Leu235Ala.

8. (i) a light chain region comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain region comprising the amino acid sequence of SEQ ID NO: 22 or 23, or (ii) Light chain region containing the amino acid sequence of SEQ ID NO: 24 and heavy chain region containing the amino acid sequence of SEQ ID NO: 22 or 23 An anti-FcRn antibody or antigen-binding fragment according to any one of claims 1 to 4, comprising:

9. An anti-FcRn antibody according to any one of claims 1 to 4, or a polynucleotide encoding the antigen-binding fragment thereof.

10. A recombinant expression vector comprising the polynucleotide described in claim 9.

11. Host cells transformed with the recombinant expression vector described in claim 10.

12. The host cells described in claim 11 are cultured to produce antibodies, The generated antibody is isolated and purified to recover an antibody that specifically binds to FcRn. A method for producing an anti-FcRn antibody or an antigen-binding fragment thereof, including the above.

13. A pharmaceutical composition comprising an anti-FcRn antibody or an antigen-binding fragment according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for treating an autoimmune disease, comprising an anti-FcRn antibody or an antigen-binding fragment according to any one of claims 1 to 4.

15. The pharmaceutical composition according to claim 14, wherein the autoimmune disease is an autoimmune disease selected from the group consisting of autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus deciduousis, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, and membranous nephropathy.

16. A pharmaceutical composition comprising an anti-FcRn antibody or an antigen-binding fragment according to any one of claims 1 to 4 for use in a method of treating an autoimmune disease in a subject requiring treatment, wherein the method comprises administering the anti-FcRn antibody or the antigen-binding fragment to a subject, and the subject may be a human.

17. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is selected from the group consisting of autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus deciduousis, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid of pregnancy, mucosal pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, and membranous nephropathy.

18. The pharmaceutical composition according to claim 16, wherein the administration is parenteral.

19. The pharmaceutical composition according to claim 18, wherein the administration is subcutaneous or intravenous.

20. The pharmaceutical composition according to claim 16, wherein the administration is performed in doses of 300 mg to 2400 mg.

21. The pharmaceutical composition according to claim 16, wherein the administration is once a week, once every two weeks, once every three weeks, or once a month.

22. The pharmaceutical composition according to claim 16, wherein the administration is a subcutaneous dose of 300 mg to 900 mg once a week.

23. The pharmaceutical composition according to claim 16, wherein administration is a subcutaneous administration once every two weeks at a dose of 300 mg to 1800 mg.

24. The pharmaceutical composition according to claim 16, wherein administration does not cause a decrease in the blood albumin level of a subject by more than 5% or more than 10% compared to the blood albumin level before administration of the anti-FcRn antibody or antigen-binding fragment.

25. The pharmaceutical composition according to claim 16, wherein administration does not cause an increase in blood total cholesterol or low-density lipoprotein (LDL) levels in a subject by more than 5% or more than 10% compared to the blood total cholesterol or low-density lipoprotein (LDL) levels before administration of the anti-FcRn antibody or antigen-binding fragment.

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