Immunoglobulin fc region-binding protein

Abstract

Provided are a single-domain antibody capable of binding to an immunoglobulin Fc region, an immunoglobulin Fc region-binding protein containing one or more single-domain antibodies, and a fusion protein containing the single-domain antibody and the Fc region.

Description

Immunoglobulin Fc region binding protein Technical Field

[0001] The present disclosure relates to the field of biomedicine and discloses a binding protein targeting the Fc region of an immunoglobulin. Specifically, the present disclosure discloses a single-domain antibody targeting the Fc region of an immunoglobulin, a fusion protein comprising the single-domain antibody, and its medical uses. Background Art

[0002] Immunoglobulin G (IgG) is the most abundant immunoglobulin in human serum and extracellular fluids. It is the primary effector molecule of systemic humoral immune responses and plays a key role in mediating protection against pathogens and promoting allergic and inflammatory responses by recruiting immune system components to tissues, mucous membranes, and skin surfaces. However, high levels of pathogenic IgG can also induce a variety of pathologies, such as autoimmune diseases, inflammatory diseases, and other diseases characterized by overexpression of IgG.

[0003] Most IgG (IgG1, 2, and 4) in human serum have a relatively long half-life, approximately 22-23 days, compared to other immunoglobulins. This long half-life is primarily dependent on IgG binding to the neonatal Fc receptor (FcRn) under acidic conditions, which protects it from degradation in lysosomes. IgG can be taken up by vascular endothelial cells and enter the cell via pinocytosis. Subsequently, IgG binds to FcRn in a pH-dependent manner within endosomes (pH 6.0-6.5) to form an IgG-FcRn complex. Antibodies not bound to FcRn fuse with lysosomes and are degraded by proteases within the lysosome. When the IgG-FcRn complex circulates to the cell surface, it rapidly dissociates from FcRn under a slightly neutral environment (pH 7.0-7.4) and re-enters the circulation.

[0004] Eliminating overexpressed IgG is an effective approach for treating or alleviating autoimmune diseases. Given the crucial role of FcRn in extending the half-life of IgG, scientists currently primarily develop drugs by antagonizing FcRn. For example, WO2015100299 discloses an Fc fragment variant that reduces serum IgG levels by antagonizing FcRn, preventing its binding to IgG. WO2015167293 discloses antibodies targeting FcRn, which are used to treat autoimmune diseases by competitively inhibiting IgG binding to FcRn.

[0005] Patent applications CN109776681A, CN102399288A, CN103204937A, etc. disclose single-domain antibodies against immunoglobulin Fc, but they are mainly used as secondary antibody reagents and do not have the therapeutic effect of reducing serum IgG.

[0006] Therefore, there is still a need to develop novel binding proteins targeting the IgG Fc fragment for the treatment of IgG-related diseases.

[0007] Summary of the Invention

[0008] The present disclosure provides single-domain antibodies that bind to the Fc region of an immunoglobulin, immunoglobulin Fc region-binding proteins comprising a plurality of such single-domain antibodies, fusion proteins comprising the single-domain antibodies or the immunoglobulin Fc region-binding proteins and an Fc region fused thereto, multispecific antibodies comprising the single-domain antibodies, the immunoglobulin Fc region-binding proteins, and the fusion proteins, nucleic acid molecules, expression vectors, cells, and compositions encoding the same, and therapeutic methods utilizing the same.

[0009] In a first aspect, the present disclosure provides a single domain antibody that binds to an immunoglobulin Fc region, the single domain antibody comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3.

[0010] In some embodiments, the single domain antibody has at least one of the following characteristics: a) binding to human IgG Fc with a KD value of less than 1×10-7 M; b) blocking the interaction between IgG and FcRn; and c) reducing the level of immunoglobulins (e.g., IgG) in a mammal.

[0011] In some embodiments, the immunoglobulin Fc region is derived from IgG. In some embodiments, the immunoglobulin Fc region is derived from IgG1, IgG2, or IgG4.

[0012] In some embodiments, the immunoglobulin Fc region is derived from human, mouse, rat, rabbit, or monkey immunoglobulin. In some embodiments, the immunoglobulin Fc region is derived from human IgG1.

[0013] In some embodiments, the single domain antibody is a camelid antibody, a humanized antibody, or a chimeric antibody.

[0014] In some embodiments, the single-domain antibody comprises CDR3 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody further comprises CDR1 and CDR2 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody comprises: (a) a CDR1 comprising the sequence of SEQ ID NO: 49, a CDR2 comprising the sequence of SEQ ID NO: 50; and a CDR3 comprising the sequence of SEQ ID NO: 51, as determined according to the Kabat definition scheme; (b) a CDR1 comprising the sequence of SEQ ID NO: 52, a CDR2 comprising the sequence of SEQ ID NO: 53; and a CDR3 comprising the sequence of SEQ ID NO: 54, as determined according to the AbM definition scheme; (c) a CDR1 comprising the sequence of SEQ ID NO: 55, a CDR2 comprising the sequence of SEQ ID NO: 56; and a CDR3 comprising the sequence of SEQ ID NO: 57, as determined according to the Chothia definition scheme; or (d) a CDR1 comprising the sequence of SEQ ID NO: 58, a CDR2 comprising the sequence of SEQ ID NO: 59; and a CDR3 comprising the sequence of SEQ ID NO: 60, as determined according to the IMGT definition scheme. In some embodiments, the single-domain antibody comprises the sequence of SEQ ID NO: 4. In some embodiments, the single domain antibody comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0015] In some other embodiments, the single-domain antibody comprises: (1) CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:1; (2) CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:2; (3) CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:3; (4) CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:5; (5) CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:6; (6) CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:7; or (7) CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:8.

[0016] In some embodiments, in the single-domain antibodies of the present disclosure, the single-domain antibody comprises: (1a) a CDR1 comprising the sequence of SEQ ID NO: 13, a CDR2 comprising the sequence of SEQ ID NO: 14; and a CDR3 comprising the sequence of SEQ ID NO: 15, as determined according to the Kabat definition scheme; (1b) a CDR1 comprising the sequence of SEQ ID NO: 16, a CDR2 comprising the sequence of SEQ ID NO: 17; and a CDR3 comprising the sequence of SEQ ID NO: 18, as determined according to the AbM definition scheme; (1c) a CDR1 comprising the sequence of SEQ ID NO: 19, a CDR2 comprising the sequence of SEQ ID NO: 20; and a CDR3 comprising the sequence of SEQ ID NO: 21, as determined according to the Chothia definition scheme; or (1d) a CDR1 comprising the sequence of SEQ ID NO: 22, a CDR2 comprising the sequence of SEQ ID NO: 23; and a CDR3 comprising the sequence of SEQ ID NO: 24, as determined according to the IMGT definition scheme.

[0017] In some specific embodiments, in the single-domain antibodies of the present disclosure, the single-domain antibody comprises: (2a) a CDR1 comprising the sequence of SEQ ID NO: 25, a CDR2 comprising the sequence of SEQ ID NO: 26; and a CDR3 comprising the sequence of SEQ ID NO: 27, as determined according to the Kabat definition scheme; (2b) a CDR1 comprising the sequence of SEQ ID NO: 28, a CDR2 comprising the sequence of SEQ ID NO: 29; and a CDR3 comprising the sequence of SEQ ID NO: 30, as determined according to the AbM definition scheme; (2c) a CDR1 comprising the sequence of SEQ ID NO: 31, a CDR2 comprising the sequence of SEQ ID NO: 32; and a CDR3 comprising the sequence of SEQ ID NO: 33, as determined according to the Chothia definition scheme; or (2d) a CDR1 comprising the sequence of SEQ ID NO: 34, a CDR2 comprising the sequence of SEQ ID NO: 35; and a CDR3 comprising the sequence of SEQ ID NO: 36, as determined according to the IMGT definition scheme.

[0018] In some specific embodiments, in the single-domain antibodies of the present disclosure, the single-domain antibody comprises: (3a) a CDR1 comprising the sequence of SEQ ID NO: 37, a CDR2 comprising the sequence of SEQ ID NO: 38; and a CDR3 comprising the sequence of SEQ ID NO: 39, as determined according to the Kabat definition scheme; (3b) a CDR1 comprising the sequence of SEQ ID NO: 40, a CDR2 comprising the sequence of SEQ ID NO: 41; and a CDR3 comprising the sequence of SEQ ID NO: 42, as determined according to the AbM definition scheme; (3c) a CDR1 comprising the sequence of SEQ ID NO: 43, a CDR2 comprising the sequence of SEQ ID NO: 44; and a CDR3 comprising the sequence of SEQ ID NO: 45, as determined according to the Chothia definition scheme; or (3d) a CDR1 comprising the sequence of SEQ ID NO: 46, a CDR2 comprising the sequence of SEQ ID NO: 47; and a CDR3 comprising the sequence of SEQ ID NO: 48, as determined according to the IMGT definition scheme.

[0019] In some specific embodiments, in the single-domain antibodies of the present disclosure, the single-domain antibody comprises: (4a) a CDR1 comprising the sequence of SEQ ID NO: 61, a CDR2 comprising the sequence of SEQ ID NO: 62; and a CDR3 comprising the sequence of SEQ ID NO: 63, as determined according to the Kabat definition scheme; (4b) a CDR1 comprising the sequence of SEQ ID NO: 64, a CDR2 comprising the sequence of SEQ ID NO: 65; and a CDR3 comprising the sequence of SEQ ID NO: 66, as determined according to the AbM definition scheme; (4c) a CDR1 comprising the sequence of SEQ ID NO: 67, a CDR2 comprising the sequence of SEQ ID NO: 68; and a CDR3 comprising the sequence of SEQ ID NO: 69, as determined according to the Chothia definition scheme; or (4d) a CDR1 comprising the sequence of SEQ ID NO: 70, a CDR2 comprising the sequence of SEQ ID NO: 71; and a CDR3 comprising the sequence of SEQ ID NO: 72, as determined according to the IMGT definition scheme.

[0020] In some specific embodiments, in the single-domain antibodies of the present disclosure, the single-domain antibody comprises: (5a) a CDR1 comprising the sequence of SEQ ID NO: 73, a CDR2 comprising the sequence of SEQ ID NO: 74; and a CDR3 comprising the sequence of SEQ ID NO: 75, as determined according to the Kabat definition scheme; (5b) a CDR1 comprising the sequence of SEQ ID NO: 76, a CDR2 comprising the sequence of SEQ ID NO: 77; and a CDR3 comprising the sequence of SEQ ID NO: 78, as determined according to the AbM definition scheme; (5c) a CDR1 comprising the sequence of SEQ ID NO: 79, a CDR2 comprising the sequence of SEQ ID NO: 80; and a CDR3 comprising the sequence of SEQ ID NO: 81, as determined according to the Chothia definition scheme; or (5d) a CDR1 comprising the sequence of SEQ ID NO: 82, a CDR2 comprising the sequence of SEQ ID NO: 83; and a CDR3 comprising the sequence of SEQ ID NO: 84, as determined according to the IMGT definition scheme.

[0021] In some specific embodiments, in the single-domain antibodies of the present disclosure, the single-domain antibody comprises: (6a) a CDR1 comprising the sequence of SEQ ID NO: 85, a CDR2 comprising the sequence of SEQ ID NO: 86; and a CDR3 comprising the sequence of SEQ ID NO: 87, as determined according to the Kabat definition scheme; (6b) a CDR1 comprising the sequence of SEQ ID NO: 88, a CDR2 comprising the sequence of SEQ ID NO: 89; and a CDR3 comprising the sequence of SEQ ID NO: 90, as determined according to the AbM definition scheme; (6c) a CDR1 comprising the sequence of SEQ ID NO: 91, a CDR2 comprising the sequence of SEQ ID NO: 92; and a CDR3 comprising the sequence of SEQ ID NO: 93, as determined according to the Chothia definition scheme; or (6d) a CDR1 comprising the sequence of SEQ ID NO: 94, a CDR2 comprising the sequence of SEQ ID NO: 95; and a CDR3 comprising the sequence of SEQ ID NO: 96, as determined according to the IMGT definition scheme.

[0022] In some specific embodiments, in the single-domain antibodies of the present disclosure, the single-domain antibodies comprise: (7a) a CDR1 comprising the sequence of SEQ ID NO: 97, a CDR2 comprising the sequence of SEQ ID NO: 98; and a CDR3 comprising the sequence of SEQ ID NO: 99, as determined according to the Kabat definition scheme; (7b) a CDR1 comprising the sequence of SEQ ID NO: 100, a CDR2 comprising the sequence of SEQ ID NO: 101; and a CDR3 comprising the sequence of SEQ ID NO: 102, as determined according to the AbM definition scheme; (7c) a CDR1 comprising the sequence of SEQ ID NO: 103, a CDR2 comprising the sequence of SEQ ID NO: 104; and a CDR3 comprising the sequence of SEQ ID NO: 105, as determined according to the Chothia definition scheme; or (7d) a CDR1 comprising the sequence of SEQ ID NO: 106, a CDR2 comprising the sequence of SEQ ID NO: 107; and a CDR3 comprising the sequence of SEQ ID NO: 108. The CDR3 of the sequence of NO:108 was determined according to the IMGT definition scheme.

[0023] In some embodiments, the single domain antibody comprises the sequence of any one of SEQ ID NOs: 1-3 and 5-8.

[0024] In a second aspect, the present disclosure provides an immunoglobulin Fc region binding protein comprising 1 to 10 single domain antibodies according to the first aspect of the present disclosure.

[0025] In some embodiments, the immunoglobulin Fc region binding protein comprises 1-10, 2-8, 2-7, 2-6, 3-10, 3-8, 3-7, or 3-6 of the single domain antibodies, preferably, it comprises 4-6 of the single domain antibodies, and most preferably, it comprises 5 of the single domain antibodies.

[0026] In some embodiments, the single domain antibodies are identical, partially identical, or different from each other.

[0027] In some embodiments, the immunoglobulin Fc region binding protein comprises five identical single domain antibodies.

[0028] In some embodiments, at least one of the single domain antibodies in the immunoglobulin Fc region binding protein comprises the VHH set forth in SEQ ID NO: 4. In some embodiments, the immunoglobulin Fc region binding protein comprises five single domain antibodies comprising the VHH set forth in SEQ ID NO: 4.

[0029] In some embodiments, the single domain antibodies are directly connected or indirectly connected via a linker. In some embodiments, the linker between the single domain antibodies is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(SGG) n S (SEQ ID NO: 114), (GGGS) n (SEQ ID NO: 115), (GGSG) n (SEQ ID NO: 116), (GGGGS) n (SEQ ID NO: 117), (SGGGG) n (SEQ ID NO: 118), (GGGGSGG) n (SEQ ID NO: 119), (GGGGSGG) n (SEQ ID NO: 120), (GGGGSGGS) n (SEQ ID NO: 121), (GGGGSGGGS) n (SEQ ID NO: 122), (PGGGS) n (SEQ ID NO: 123), (AGGGS) n (SEQ ID NO: 124), (GGGSE) n (SEQ ID NO: 125), (GGGGA) n (SEQ ID NO: 126), (AP) n 、(GAP) n 、(EAAAK) n (SEQ ID NO:127), (ASTKGPSVFPLAGAP) n (SEQ ID NO:128), (ASTKGPSVPLAGGG) n (SEQ ID NO:129), (AKTTPKLEEGEFSEAR) n (SEQ ID NO:130), (AKTTPKLEEGEFSEARV) n (SEQ ID NO: 131), (AKTTPKLGG) n (SEQ ID NO: 132), (ADAAP) n (SEQ ID NO:133), (ADAAPTVSIFPP) n (SEQ ID NO: 134), (AKTTPP) n(SEQ ID NO:135), (AKTTPPSVTPLAP) n (SEQ ID NO: 136), (AKTTAP) n (SEQ ID NO:137), (AKTTAPSVYPLAP) n (SEQ ID NO: 138), (ASTKGP) n (SEQ ID NO: 139) and (ASTKGPSVFPLAP) n (SEQ ID NO: 140), wherein n is an integer from 1 to 10. In some embodiments, the polypeptide linker is selected from one or more of the following amino acid sequences: GS, GGGGGG (SEQ ID NO: 141), GGGS (SEQ ID NO: 142), GGGGS (SEQ ID NO: 143), GGGGSGGGS (SEQ ID NO: 144), GAP, and ASTKGPSVFPLAGAP (SEQ ID NO: 145). In some embodiments, the immunoglobulin Fc region binding protein comprises the amino acid sequence of SEQ ID NO: 11 or 12.

[0030] In a third aspect, the present disclosure provides a fusion protein comprising the single domain antibody according to the first aspect of the present disclosure or the immunoglobulin Fc region binding protein according to the second aspect of the present disclosure.

[0031] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region.

[0032] In some specific embodiments, the immunoglobulin Fc region in the fusion protein is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, IgG2, IgG3 or IgG4; more preferably the Fc region of human IgG1.

[0033] In some specific embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation that weakens the binding of the immunoglobulin Fc region in the fusion protein to the single domain antibody. In some specific embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation that weakens the binding between the immunoglobulin Fc region in the fusion protein and FcRn.

[0034] In some specific embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 314. In some embodiments, the mutation at amino acid position 314 is L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W, or L314Y, preferably L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W, or L314Y. 4F, L314G, L314H, L314I, L314K, L314M, L314P, L314R, L314S, L314V, L314W or L314Y; more preferably L314A, L314S, L314G, L314R, L314K, L314W or L314Y; even more preferably L314A, L314R, L314S or L314W.

[0035] In some specific embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 311. In some embodiments, the mutation at amino acid position 311 is Q311A, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, or Q311Y, preferably Q311D or Q311E, and more preferably Q311D.

[0036] In some specific embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 253 and / or amino acid position 254. In some embodiments, the mutation at amino acid position 253 and / or amino acid position 254 is selected from the group consisting of: I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, I253Y, S254A, S254D, S254E, S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W, S254Y , I253A / S254A, I253G / S254A, I253A / S254G, I253E / S254D, I253E / S254E or I253D / S254E, preferably I253A, I253D, I253E, I253G, I253N, I253Q, I253S, S254A, S254G 4D, S254E, S254G, S254N, S254Q, I253A / S254A, I253G / S254A, I253A / S254G, I253E / S254D, I253E / S254E or I253D / S254E, more preferably I253A / S254A or I253E / S254D.

[0037] Furthermore, in some embodiments, the immunoglobulin Fc region in the fusion protein further comprises a mutation at amino acid position 435 and / or amino acid position 436. In some embodiments, the mutation at amino acid position 435 and / or amino acid position 436 is selected from the following mutations: H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W, H435Y, Y436A, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, 6N, Y436Q, Y436R, Y436S, Y436T, Y436V, Y436W, H435A / Y436A, H435A / Y436D, H435A / Y436S, H435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E or H435M / Y436Q, preferably H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W, H435Y, Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q, Y436N, H435A / Y436A, H435A / Y436S, H435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E or H435M / Y436Q; more preferably H435A, H435S, H435G, H435M, H435L, H435Y, H435I, Y436S, Y436A, Y436G, Y436D, Y 436E, Y436Q, Y436N, H435A / Y436A, H435A / Y436S, H435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E or H435M / Y436Q, more preferably H435A, H435S, H435M, H435A / Y436A, H435A / Y436S, H435M / Y436Q, H435M / Y436D, H435A / Y436D or H435M / Y436E.

[0038] In some preferred embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation selected from the group consisting of: L314A / H435A, L314S / H435S, L314S / H435A / Y436S, L314R / N315R, L314R / N315M, L314R / H435A, L314R / H435M, L314A / H435M, L314R / H435M / Y436S 436Q, L314A / H435M / Y436D, I253E / S254D / H435A / Y436A, I253E / S254D / H435A / Y436D, L314R / H435M / Y436D, I253E / S254D / H435M / Y436D, I253E / S254D / H435M / Y436Q, L314W / H435M / Y436D L314W / H435M / Y436E, L314W / H435M / Y436Q, L314A / H435M / Y436E, L314A / H435M / Y436Q, Q311D / H435M / Y436E, or Q311D / H435M / Y436D.

[0039] In some embodiments, the immunoglobulin Fc region in the fusion protein further comprises a mutation that enhances the interaction between the immunoglobulin Fc region in the fusion protein and FcγRIIb. In some embodiments, the mutations that enhance the interaction between the immunoglobulin Fc region and FcγRIIb in the fusion protein are one or more mutations selected from the group consisting of E233D, G237D, P238D, T265A, H268D, K290A, R255A, E258A, S267A, S267E, P271G, E272A, N276A, D280A, H285A, N286A, T307A, L309A, N315A, K326A, L328E, L328F, A330R, P331A, S337A, A378Q, E430A, H268A, R301A, R301M, K322A, K334R, and K334N. In some embodiments, the mutation that enhances the interaction between the immunoglobulin Fc region and FcγRIIb in the fusion protein is a combination of mutations selected from the following: S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233D / A330R, E233D / P238D, G237D / P23 8D, P238D / H268D, P238D / P271G, P238D / A330R, E233D / P238D / A330R, E233D / P271G / A330R, G237D / H268D / P271G, G237D / P271G / A330R, S239D / A330L / I332E, G236A / S239D / I332E, E233D / P238D / P 271G / A330R, G237D / P238D / H268D / P271G, E233D / H268D / P271G / A330R, G237D / P238D / P271G / A33 0R, G237D / H268D / P271G / A330R, E233D / G237D / H268D / P271G / A330R, E233D / P238D / H268D / P271G / A330R, G237D / P238D / H268D / P271G / A330R or E233D / G237D / P238D / H268D / P271G / A330R, preferably, the mutation that enhances the interaction between the immunoglobulin Fc region and FcγRIIb in the fusion protein is S267E / L328F or E233D / G237D / P238D / H268D / P271G / A330R.

[0040] In some specific embodiments, the immunoglobulin Fc variant in the fusion protein comprises: (a) mutations S267E / L328F, and mutations L314A / H435M / Y436D; (b) mutations S267E / L328F, and mutations L314A / H435M / Y436E; (c) mutations S267E / L328F, and mutations L314W / H435M / Y436D; (d) mutation S267E / L328F, and mutation L314W / H435M / Y436E; (e) mutation S267E / L328F, and mutation L314R / H435M / Y436Q; (f) mutation S267E / L328F, and mutation I253E / S254D / H435A / Y436D; (g) mutation S267E / L328F, and mutation I253E / S254D / H435A / Y436A; (h) mutation E233D / G237D / P238D / H268D / P271G / A330R, and mutation L314A / H435M / Y436D; (i) mutation E233D / G237D / P238D / H268D / P271G / A330R, and mutation L314A / H435M / Y436E; (j) mutation E233D / G237D / P238D / H268D / P271G / A330R, and mutation L314A / H435M / Y436E; (k) mutation E233D / G237D / P238D / H268D / P271G / A330R, and mutation L314A / H435M / Y436E. 1G / A330R, and mutations L314W / H435M / Y436E; (l) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436Q; (m) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436D; or (n) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435A / Y436A.

[0041] In a fourth aspect, the present disclosure provides a multispecific antibody comprising the single domain antibody of the first aspect of the present disclosure, the immunoglobulin Fc region binding protein of the second aspect of the present disclosure, or the fusion protein of the third aspect of the present disclosure, and one or more additional antigen binding regions, wherein the one or more additional antigen binding regions bind to different antigens or different epitopes of the same antigen as the single domain antibody, the immunoglobulin Fc region binding protein or the fusion protein.

[0042] In the fifth aspect, the present disclosure provides a conjugate comprising the single domain antibody described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, or the multispecific antibody described in the fourth aspect of the present disclosure, and the conjugate further comprises a molecule conjugated to the single domain antibody, the immunoglobulin Fc region binding protein, the fusion protein, or the multispecific antibody.

[0043] In a sixth aspect, the present disclosure provides a nucleic acid molecule encoding the single domain antibody described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, or the multispecific antibody described in the fourth aspect of the present disclosure.

[0044] In a seventh aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule according to the sixth aspect of the present disclosure operably linked to an expression regulatory element.

[0045] In an eighth aspect, the present disclosure provides a cell comprising the nucleic acid molecule of the sixth aspect of the present disclosure and / or transformed with the expression vector of the seventh aspect of the present disclosure.

[0046] In a ninth aspect, the present disclosure provides a composition comprising the single-domain antibody of the first aspect of the present disclosure, the immunoglobulin Fc region binding protein of the second aspect of the present disclosure, the fusion protein of the third aspect of the present disclosure, the multispecific antibody of the fourth aspect of the present disclosure, the conjugate of the fifth aspect of the present disclosure, the nucleic acid molecule of the sixth aspect of the present disclosure, the expression vector of the seventh aspect of the present disclosure, or the cell of the eighth aspect of the present disclosure. In some embodiments, the composition of the present disclosure is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier and / or excipient.

[0047] In the tenth aspect, the present disclosure provides a kit comprising the single domain antibody described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, the multispecific antibody described in the fourth aspect of the present disclosure, the conjugate described in the fifth aspect of the present disclosure, the nucleic acid molecule described in the sixth aspect of the present disclosure, the expression vector described in the seventh aspect of the present disclosure, or the cell described in the eighth aspect of the present disclosure, and / or the composition described in the ninth aspect of the present disclosure.

[0048] In the eleventh aspect, the present disclosure provides a method for treating and / or preventing immunoglobulin-mediated diseases and / or symptoms, comprising administering to a subject in need thereof an effective amount of the single domain antibody of the first aspect of the disclosure, the immunoglobulin Fc region binding protein of the second aspect of the disclosure, the fusion protein of the third aspect of the disclosure, the multispecific antibody of the fourth aspect of the disclosure, the conjugate of the fifth aspect of the disclosure, the nucleic acid molecule of the sixth aspect of the disclosure, the expression vector of the seventh aspect of the disclosure, or the cell of the eighth aspect of the disclosure, and / or the composition of the ninth aspect of the disclosure. In some preferred embodiments, the immunoglobulin-mediated disease and / or symptom is an IgG-mediated disease and / or symptom.

[0049] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0051] Figure 1 shows the blocking ability of anti-Fc single-domain antibodies on the binding of IgG1-Fc to FcRn, where ○ represents iFc02, □ represents iFc21, △ represents iFc102, ◇ represents iFc106, ● represents iFc231, ■ represents iFc284S, and ▲ represents iFc347.

[0052] Figure 2 shows the blocking ability of anti-Fc single-domain antibodies on the binding of IgG1-Fc to FcRn, where ○ represents iFc02, □ represents iFc21, △ represents iFc62, ◇ represents iFc102, ● represents iFc106, ■ represents iFc231, and ▲ represents iFc347.

[0053] Figure 3 shows the blocking ability of humanized anti-Fc single-domain antibodies on the binding of Fc to FcRn, where ○ and □ represent the blocking ability of huFc106v1 and huFc106v2 on the binding of IgG1-Fc to FcRn, respectively; △ and ◇ represent the blocking ability of huFc106v1 and huFc106v2 on the binding of KN002 to FcRn, respectively.

[0054] FIG4 shows the effect of a single intraperitoneal injection of multivalent anti-Fc single domain antibodies on endogenous IgG levels in BALB / c mice.

[0055] Figure 5 shows the effect of a single intraperitoneal or subcutaneous injection of multivalent anti-Fc single domain antibodies on endogenous IgG levels in CD1 mice, with ARGX-113 as a control.

[0056] Figure 6 shows the effect of a single subcutaneous injection of multivalent anti-Fc single domain antibodies on endogenous IgG levels in CD1 mice, with ARGX-113 as a control.

[0057] Figures 7a and 7b show the blocking ability of humanized anti-Fc single-domain antibody-Fc fusion protein on the binding of Fc to FcRn, wherein □ and △ in Figure 7a represent the blocking ability of huFc106v1-FcEF-314A-435MD and huFc106v2-FcEF-314A-435MD on the binding of KN002 to FcRn, respectively, and ○ and ◇ represent the blocking ability of huFc106v1 and huFc106v1 on the binding of KN002 to FcRn, respectively; ○ and □ in Figure 7b represent the blocking ability of huFc106v2-FcEF-314W-435MD and huFc106v2 on the binding of KN002 to FcRn, respectively.

[0058] FIG8 shows the effect of a single intraperitoneal injection of humanized anti-Fc single domain antibody-Fc fusion protein on the endogenous IgG level in BALB / c mice.

[0059] FIG9 shows the effect of a single intraperitoneal injection of humanized anti-Fc single domain antibody-Fc fusion protein on the endogenous IgG level in C57BL6 mice, with Batoclimab as a control.

[0060] Figure 10 shows the effects of a single intravenous or subcutaneous injection of humanized anti-Fc single domain antibody or humanized anti-Fc single domain antibody-Fc fusion protein on the endogenous IgG level in BALB / c mice. DETAILED DESCRIPTION

[0061] Definition of terms

[0062] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd edition), Gower Medical Publishing, London, New York (1989), as well as the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, which will be understood by those skilled in the art. Reference is also made, for example, to standard manuals, the above-mentioned general prior art and other references cited therein.

[0063] Unless otherwise indicated, the terms "antibody" or "immunoglobulin", which are used interchangeably herein, whether referring to heavy chain antibodies or conventional four-chain antibodies, are used as a general term to include full-length antibodies, individual chains thereof, and all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively).

[0064] In addition, the term "sequence" used herein (for example, in terms such as "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited interpretation is required herein.

[0065] The term "immunoglobulin" generally refers to a protein consisting essentially of one or more polypeptides encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. In general, immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). The NH2-terminus (about 110 amino acids) of the full-length immunoglobulin "light chain" (about 25KD and 214 amino acids) is encoded by the variable region gene, and the COOH-terminus is encoded by the kappa or lambda constant region gene. Natural immunoglobulins essentially consist of two Fab molecules and an Fc region connected via an immunoglobulin hinge region.

[0066] As used herein, the term "immunoglobulin Fc region" refers to a domain derived from the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody and typically includes a hinge region and two constant domains (a CH2 domain and a CH3 domain). The Fc region can be a native sequence Fc region or a variant Fc region, such as the amino acid sequence shown in any one of SEQ ID NOs: 109-112, which correspond to the Fc sequences of human IgG1, IgG2, IgG3, and IgG4, respectively.

[0067] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and / or the domain.

[0068] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.

[0069] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antibody for an antigen by having an antigen-binding site.

[0070] As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without being paired with other immunoglobulin variable domains. One example of an immunoglobulin single variable domain within the meaning of the present disclosure is a "domain antibody," such as an immunoglobulin single variable domain VH (VH domain) and an immunoglobulin single variable domain VL (VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") derived from Camelidae, as defined below.

[0071] As used herein, the term "single domain antibody," "sdAb," or "nanobody" refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). Single domain antibodies are capable of binding to an antigen alone without the need for pairing with a corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelid heavy chain antibodies (HCAbs), and their heavy chain variable domains are referred to herein as "VHHs." Camelid single domain antibodies are one of the smallest known antigen-binding antibody fragments.

[0072] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment and VHH antibody, is the variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., an "antibody lacking a light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in conventional four-chain antibodies (which is referred to herein as a "VH domain") and the light chain variable domain present in conventional four-chain antibodies (which is referred to herein as a "VL domain"). The VHH domain specifically binds an epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and efficient antigen-recognition unit formed by a single immunoglobulin domain.

[0073] For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used for the VHH domain of Camelidae can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0074] Alternative methods for numbering the amino acid residues of VH domains are known in the art and can be similarly applied to VHH domains. For example, Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs represent a compromise between the Kabat hypervariable regions and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residues of the CDRs from each method are described below:

[0075] The antibody CDRs can also be IMGT-CDRs, which is a CDR definition based on the IMGT antibody encoding, which was obtained by integrating structural information from over 5,000 sequences. In the IMGT VH CDR encoding, CDR1: 27-38; CDR2: 56-65; CDR3: 105-117.

[0076] It should be noted, however, that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0077] For example, the CDRs may comprise "extended CDRs", e.g., 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2), and 93-102, 94-102, or 95-102 (HCDR3) in VH.

[0078] The total number of amino acid residues in a VHH domain will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0079] Other structural characteristics and functional properties of VHH domains and polypeptides containing them can be summarized as follows:

[0080] VHH domains (which are naturally "designed" to functionally bind to antigens in the absence of, and without interacting with, light chain variable domains) can be used as single, relatively small, functional antigen-binding structural units, domains, or polypeptides. This distinguishes VHH domains from the VH and VL domains of conventional four-chain antibodies, which are generally unsuitable for practical application as single antigen-binding proteins or immunoglobulin single variable domains by themselves, but need to be combined in some form or another to provide a functional antigen-binding unit (e.g., in the form of conventional antibody fragments such as Fab fragments; or in the form of scFvs consisting of a VH domain covalently linked to a VL domain).

[0081] Due to these unique properties, the use of VHH domains—alone or as part of a larger polypeptide—offers a number of significant advantages over the use of conventional VH and VL domains, scFvs, or conventional antibody fragments (e.g., Fab- or F(ab')2-fragments): only a single domain is required to bind antigen with high affinity and selectivity, thereby eliminating the need for the presence of two separate domains and the need to ensure that the two domains are in the proper spatial conformation and configuration (e.g., scFvs generally require the use of specially designed linkers); VHH domains can be expressed from a single gene and do not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats (formatting); VHH domains are highly soluble and have no tendency to aggregate; VHH domains They are highly stable to heat, pH, proteases, and other denaturants or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thereby saving costs, time, and the environment. VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production. VHH domains are relatively small compared to conventional four-chain antibodies and their antigen-binding fragments (approximately 15 kDa, or 1 / 10 the size of conventional IgG), and therefore exhibit higher tissue penetration and can be administered at higher doses than conventional four-chain antibodies and their antigen-binding fragments. VHH domains can exhibit so-called cavity-binding properties (particularly due to their extended CDR3 loop compared to conventional VH domains), allowing them to reach targets and epitopes that are inaccessible to conventional four-chain antibodies and their antigen-binding fragments. Studies have shown that the extended CDR3 portion of VHH plays a major role in the binding of VHH to antigens (see WO2018133873).

[0082] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0083] Camelidae-derived VHH domains can be "humanized" (also referred to herein as "sequence optimization"; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as removal of potential post-translational modification sites) by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in a conventional human four-chain antibody VH domain. The humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be accomplished using protein surface amino acid humanization methods and / or humanized universal framework CDR grafting, for example, as exemplified in the Examples.

[0084] In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the present disclosure) can bind to. Specificity can be determined based on the affinity and / or avidity of an antigen binding protein. Avidity, represented by the dissociation equilibrium constant (KD) of an antigen and an antigen binding protein, is a measure of the binding strength between an epitope and an antigen binding site on an antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (or, affinity can also be expressed as an association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be measured in a known manner depending on the specific antigen of interest. Avidity is a measure of the binding strength between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and a related antigen. Avidity is related to both the affinity between the antigen binding site on its antigen binding protein and the number of related binding sites present on the antigen binding protein.

[0085] As used herein, the term "immunoglobulin Fc region binding protein" refers to any protein that can specifically bind to the Fc region of an immunoglobulin, which can be an endogenous immunoglobulin of a mammal, such as an endogenous immunoglobulin of a human or mouse; in particular, an endogenous IgG1 of a mammal, such as an endogenous IgG1 of a human or mouse. Immunoglobulin Fc region binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.

[0086] Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue of similar chemical structure and which has little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art. For example, conservative amino acid substitutions are preferably substitutions of an amino acid within the following groups (i) to (v) by another amino acid residue within the same group: (i) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is substituted by Met, Leu or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; Val is substituted by Ile or Leu.

[0087] As used herein, "mutation" or "amino acid mutation" generally includes any type of change or modification to a sequence (nucleic acid or amino acid sequence), including deletion, truncation, inactivation, destruction, substitution or translocation of an amino acid or nucleotide. In the present application, an amino acid mutation can be an amino acid substitution, and the term "substitution" generally refers to replacing at least one amino acid residue in a predetermined parent amino acid sequence with a different "replacement" amino acid residue. The one or more replaced amino acid residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code). AxxxB is generally used herein to represent an amino acid mutation, for example "S267E", where A represents the parent amino acid residue, B represents the replaced amino acid residue, and xxx represents the mutated amino acid residue numbering. If not otherwise specified, the EU index of Kabat is used herein for numbering.

[0088] As used herein, "linker" generally refers to an amino acid sequence that connects two heterologous polypeptides or fragments thereof. In this application, a linker can be an amino acid sequence that covalently links polypeptides to form a fusion polypeptide. The length of the linker can be 1-20 amino acids in length. The linker can be flexible or rigid.

[0089] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program from the NCBI database can be used to determine identity. For the determination of sequence identity, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0090] In this application, the term "isolated" generally refers to a biological material (e.g., a virus, nucleic acid, or protein) that is substantially free of components that are typically associated with or interact with the biological material in its naturally occurring environment. The isolated biological material optionally includes additional materials that are not found in the biological material in its natural environment (e.g., nucleic acid or protein). In this application, when referring to proteins, "isolated" generally refers to the separation and separation of the molecule from the entire organism in which the molecule is found naturally occurring, or the substantial absence of other biological macromolecules of the same type. When referring to nucleic acid molecules, it is completely or partially separated from the sequence to which it is naturally bound, or the nucleic acid has a heterologous sequence to which it is bound, or the nucleus is separated from the chromosome.

[0091] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component with which it is normally associated in that source or medium (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) compared to its natural biological source and / or the reaction medium or culture medium from which it was obtained. In particular, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. An "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique (e.g., a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).

[0092] As used herein, "effective amount" means an amount of the immunoglobulin Fc region binding protein of the present disclosure or a pharmaceutical composition comprising the same that can result in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of damage or disability caused by disease affliction.

[0093] "IgG-mediated diseases and / or conditions" refer to conditions caused or characterized by elevated levels or activity of immunoglobulin G (IgG), either as monomers or in immune complexes containing IgG. Typical "IgG-mediated diseases and / or conditions" include autoimmune diseases, inflammatory diseases, and allergic diseases.

[0094] In this application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or backbone portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen binding protein to the antigen. Antigen binding proteins can typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen binding activity.

[0095] In this application, the terms "polypeptide" or "protein" are used interchangeably and generally refer to polymers of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term may also include modified amino acid polymers, for example, by adding sugar residues to form glycoproteins or modified by phosphorylation. Polypeptides and proteins can be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein, or molecules having one or more amino acids deleted, added and / or substituted from the native sequence. The terms "polypeptide" and "protein" particularly include sequences of one or more amino acids deleted, added and / or substituted from the antigen-binding proteins described herein.

[0096] In this application, the term "conjugate" generally refers to a substance formed by linking an antigen binding protein with other active agents, which may be small molecule active agents, such as therapeutic agents, imaging probes, or spectroscopic probes.

[0097] In this application, the term "nucleic acid" molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.

[0098] In this application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used for inserting DNA or RNA into a cell, a vector primarily used for replicating DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0099] In this application, the term "cell" generally refers to an individual cell, cell line or cell culture that may contain or have contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that can express an antigen-binding protein as described herein. The cell may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be identical in morphology or genome to the original parent cell, but may be able to express the antibody or antigen-binding fragment thereof as described herein. The cell may be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

[0100] As used herein, the term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0101] In this application, the term "treatment" generally refers to the desire to change the natural course of the individual being treated, and can be a clinical intervention to achieve prevention or treatment or performed during clinical pathological processes. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some cases, antigen binding proteins (e.g., antibodies against specific antigens of the present application) can be used to delay disease development or slow disease progression.

[0102] In this application, the term "administering" generally refers to a method of administering a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be performed by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if desired for local treatment) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0103] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0104] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0105] Detailed Description of the Invention

[0106] The present disclosure utilizes phage display technology to screen and obtain single-domain antibodies (VHHs) against the Fc region of immunoglobulins and their derivative proteins. These single-domain antibodies (VHHs) and their derivative proteins can bind to the Fc region of immunoglobulins with high affinity, blocking the binding of IgG to FcRn, preventing IgG from being recycled via FcRn in the body, thereby reducing IgG levels in the body and achieving the purpose of treating and / or preventing diseases and / or symptoms mediated by IgG.

[0107] Single domain antibodies

[0108] In one aspect, the present disclosure relates to a single domain antibody that binds to an immunoglobulin Fc region, the single domain antibody comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3.

[0109] The single domain antibody disclosed herein can specifically bind to the Fc region of an immunoglobulin. Specifically, the single domain antibody has at least one of the following characteristics: a) a KD value for binding to human IgG Fc is less than 1×10 -7 M; b) blocking the interaction between IgG and FcRn; c) reducing IgG levels in mammals.

[0110] In some embodiments, the single domain antibody binds to human IgG Fc with a KD value of less than 1×10 -7 M, preferably less than 5×10 -8 M, or less than 1×10 -8 M.

[0111] In some embodiments, the single-domain antibody blocks the interaction between IgG and FcRn. The phrase "the single-domain antibody blocks the interaction between IgG and FcRn" herein means that the single-domain antibody described herein is capable of substantially preventing IgG from binding to FcRn. For example, this is characterized by a response value of less than 0.1 as measured by biofilm interferometry (BLI).

[0112] In some embodiments, the single domain antibodies of the present disclosure reduce the level of immunoglobulins (e.g., IgG) in a mammal. In some embodiments, the single domain antibodies of the present disclosure reduce the level of immunoglobulins (e.g., IgG, e.g., the sum of IgG1, IgG2, and IgG4) in a mammal by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% compared to not taking the single domain antibody.

[0113] The immunoglobulin Fc region as a target can be derived from a mammalian immunoglobulin, such as IgG, IgA, IgM, IgD, and IgE. The immunoglobulin Fc region as a target can be derived from human, mouse, rat, rabbit, or monkey IgG (including IgG1, IgG2, IgG3, and / or IgG4), and in particular can be human IgG1, IgG2, and / or IgG4.

[0114] In some embodiments, the single domain antibody is a single domain antibody of a Camelidae HCAb or a humanized single domain antibody.

[0115] In some embodiments, the single domain antibody can be a single domain antibody derived from HCAb of camel, llama, alpaca, dromedary, llama, guanaco and Bactrian camel. In some embodiments, the single domain antibody can be a humanized single domain antibody obtained by humanizing the single domain antibody derived from HCAb of Camelidae, such as camel, llama, alpaca, dromedary, llama, guanaco and Bactrian camel, for example, a method of protein surface amino acid humanization (resurfacing) and / or a humanized universal framework CDR transplantation method (CDR grafting to a universal framework) to complete, for example, as illustrated in the Examples.

[0116] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 4.

[0117] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 4. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 4. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 4 according to different definition schemes are: (a) SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, determined according to the Kabat definition scheme; (b) SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, determined according to the AbM definition scheme; (c) SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, determined according to the Chothia definition scheme; or (d) SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, determined according to the IMGT definition scheme.

[0118] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0119] In some embodiments, the single-domain antibody is a humanized single-domain antibody. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9 or 10. In some embodiments, the single-domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9 or 10. In some embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO: 9 or 10. In some embodiments, the single-domain antibody consists of the amino acid sequence shown in SEQ ID NO: 9 or 10.

[0120] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 1. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 1. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 1. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 1.

[0121] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 1, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 1. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 1, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 1. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 1, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 1. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 1, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 1. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 1 according to different definition schemes are: (a) SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, determined according to the Kabat definition scheme; (b) SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, determined according to the AbM definition scheme; (c) SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, determined according to the Chothia definition scheme; or (d) SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, determined according to the IMGT definition scheme.

[0122] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0123] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 2. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 2. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 2. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 2.

[0124] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 2, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 2. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 2, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 2. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 2, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 2. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 2, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 2. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 2 according to different definition schemes are: (a) SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, determined according to the Kabat definition scheme; (b) SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, determined according to the AbM definition scheme; (c) SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, determined according to the Chothia definition scheme; or (d) SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, determined according to the IMGT definition scheme.

[0125] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0126] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 3. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 3. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 3. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 3.

[0127] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 3, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 3. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 3, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 3. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 3, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 3. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 3, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 3. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 3 according to different definition schemes are: (a) SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, determined according to the Kabat definition scheme; (b) SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, determined according to the AbM definition scheme; (c) SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, determined according to the Chothia definition scheme; or (d) SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, determined according to the IMGT definition scheme.

[0128] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0129] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 5. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 5. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 5. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 5.

[0130] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 5, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 5. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 5, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 5. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 5, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 5. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 5, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 5. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:5 according to different definition schemes are: (a) SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, determined according to the Kabat definition scheme; (b) SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, determined according to the AbM definition scheme; (c) SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, determined according to the Chothia definition scheme; or (d) SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, determined according to the IMGT definition scheme.

[0131] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0132] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 6. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 6. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 6. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 6.

[0133] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 6, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 6. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 6, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 6. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 6, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 6. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 6, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 6. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:6 according to different definition schemes are: (a) SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, determined according to the Kabat definition scheme; (b) SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:78, determined according to the AbM definition scheme; (c) SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, determined according to the Chothia definition scheme; or (d) SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, determined according to the IMGT definition scheme.

[0134] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 6.

[0135] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 7. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR3 of the VHH set forth in SEQ ID NO: 7. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 7. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 7.

[0136] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR1 of the VHH set forth in SEQ ID NO: 7, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR2 of the VHH set forth in SEQ ID NO: 7. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR1 of the VHH set forth in SEQ ID NO: 7, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR2 of the VHH set forth in SEQ ID NO: 7. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 7, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 7. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 7, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 7. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:7 according to different definition schemes are: (a) SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, determined according to the Kabat definition scheme; (b) SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, determined according to the AbM definition scheme; (c) SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93, determined according to the Chothia definition scheme; or (d) SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96, determined according to the IMGT definition scheme.

[0137] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0138] In some embodiments, the single-domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 8. In some embodiments, the single-domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH set forth in SEQ ID NO: 8. In some embodiments, the single-domain antibody comprises a CDR3 comprising the CDR3 of the VHH set forth in SEQ ID NO: 8. In some embodiments, the single-domain antibody comprises a CDR3 consisting of the CDR3 of the VHH set forth in SEQ ID NO: 8.

[0139] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 8, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 8. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 8, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 8. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 8, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 8. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 8, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 8. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:8 according to different definition schemes are: (a) SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, determined according to the Kabat definition scheme; (b) SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102, determined according to the AbM definition scheme; (c) SEQ ID NO:103, SEQ ID NO:104 and SEQ ID NO:105, determined according to the Chothia definition scheme; or (d) SEQ ID NO:106, SEQ ID NO:107 and SEQ ID NO:108, determined according to the IMGT definition scheme.

[0140] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 8.

[0141] In some embodiments, the single domain antibody is a humanized single domain antibody.

[0142] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9 or 10. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9 or 10. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 9 or 10. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 9 or 10.

[0143] In some embodiments, the single domain antibody of the present disclosure comprises a sequence represented by the following formula ([ ] indicates the type of amino acid that can be selected at that position): QVQL[QV]ESGGG[SL]VQ[SP]GGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVAAICSDGKTTYADSVKGRFTIS[QR]D[GN][AS]KNTLYLQMNSL[KR][PA]EDTA[MV]YYCAAGVKQPRYSGGYYVIWDGDFTYWGQGT[QL]VTVSS (SEQ ID NO: 113).

[0144] Immunoglobulin Fc region binding protein

[0145] In a second aspect, the present disclosure provides an immunoglobulin Fc region binding protein comprising 1 to 10 single domain antibodies according to the first aspect of the present disclosure.

[0146] In some embodiments, the immunoglobulin Fc region binding protein comprises 2-10, 2-9, 2-8, 2-7, 2-6, 3-10, 3-9, 3-8, 3-7, 3-6, 4-10, 4-9, 4-8, 4-7, or 4-6 single domain antibodies according to the first aspect of the disclosure. In a specific embodiment, the immunoglobulin Fc region binding protein of the present disclosure comprises 5 single domain antibodies according to the first aspect of the disclosure.

[0147] When comprising a plurality of immunoglobulin single variable domains, the binding protein further has improved properties compared to a protein comprising a single immunoglobulin single variable domain, for example, the KD value of the binding protein to human IgG1-Fc is less than 1×10- 9 M, preferably less than 1×10- 10 M, more preferably less than 1×10 -11 M.

[0148] In some embodiments, the single domain antibodies are identical, partially identical, or different from each other.

[0149] In some embodiments, all single domain antibodies in the immunoglobulin Fc region binding protein are identical, for example, all are iFc106 VHH.

[0150] In some embodiments, the single domain antibodies in the immunoglobulin Fc region binding protein are partially identical, meaning that the immunoglobulin Fc region binding protein comprises a plurality of identical single domain antibodies and one or more other single domain antibodies that are different therefrom. For example, the immunoglobulin Fc region binding protein comprises 2, 3, or 4 iFc106VHHs and further comprises one or more of iFc02, IFc21, iFc102, IFc231, IFc284S, IFc307, and IFc347.

[0151] In some embodiments, each single domain antibody in the immunoglobulin Fc region binding protein is different. For example, the immunoglobulin Fc region binding protein comprises five single domain antibodies, namely iFc106 VHH, iFc02 VHH, IFc 21 VHH, iFc102 VHH, and IFc231 VHH.

[0152] In some embodiments, at least one of the single domain antibodies in the immunoglobulin Fc region binding protein comprises the VHH set forth in SEQ ID NO: 4. In some embodiments, the immunoglobulin Fc region binding protein comprises five single domain antibodies comprising the VHH set forth in SEQ ID NO: 4.

[0153] In some embodiments, the single domain antibodies in the immunoglobulin Fc region binding protein are directly connected or indirectly connected via a linker.

[0154] In some embodiments, the single domain antibodies in the immunoglobulin Fc region binding protein are connected via a polypeptide linker.

[0155] In some specific embodiments, the polypeptide linker is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(SGG) n S (SEQ ID NO: 114), (GGGS) n (SEQ ID NO: 115), (GGSG) n (SEQ ID NO: 116), (GGGGS) n (SEQ ID NO: 117), (SGGGG) n (SEQ ID NO: 118), (GGGGSGG) n (SEQ ID NO: 119), (GGGGSGG) n (SEQ ID NO: 120), (GGGGSGGS) n (SEQ ID NO: 121), (GGGGSGGGS) n (SEQ ID NO: 122), (PGGGS) n (SEQ ID NO: 123), (AGGGS) n (SEQ ID NO: 124), (GGGSE) n (SEQ ID NO: 125), (GGGGA) n (SEQ ID NO: 126), (AP) n 、(GAP) n 、(EAAAK) n (SEQ ID NO:127), (ASTKGPSVFPLAGAP) n (SEQ ID NO:128), (ASTKGPSVPLAGGG) n (SEQ ID NO:129), (AKTTPKLEEGEFSEAR) n (SEQ ID NO:130), (AKTTPKLEEGEFSEARV) n (SEQ ID NO: 131), (AKTTPKLGG) n(SEQ ID NO: 132), (ADAAP) n (SEQ ID NO:133), (ADAAPTVSIFPP) n (SEQ ID NO: 134), (AKTTPP) n (SEQ ID NO:135), (AKTTPPSVTPLAP) n (SEQ ID NO: 136), (AKTTAP) n (SEQ ID NO:137), (AKTTAPSVYPLAP) n (SEQ ID NO: 138), (ASTKGP) n (SEQ ID NO: 139) and (ASTKGPSVFPLAP) n (SEQ ID NO: 140), wherein n is an integer from 1 to 10.

[0156] In some embodiments, the linker is further selected from one or more of the following amino acid sequences: GS, GGGGGG (SEQ ID NO: 141), GGGS (SEQ ID NO: 142), GGGGS (SEQ ID NO: 143), GGGGSGGGS (SEQ ID NO: 144), GAP, and ASTKGPSVFPLAGAP (SEQ ID NO: 145).

[0157] For example, the immunoglobulin Fc region binding protein comprises five single domain antibodies, and from the N-terminus to the C-terminus, each single domain antibody has a linker combination shown in Table 1:

[0158] Table 1. Linker combinations

[0159] In some embodiments, the immunoglobulin Fc region binding protein comprises the amino acid sequence shown in SEQ ID NO: 11 or 12.

[0160] As an exemplary embodiment, the immunoglobulin Fc region binding protein of the present disclosure comprises 3-6 identical single domain antibodies. In some embodiments, the single domain antibody comprises a CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH shown in SEQ ID NO: 4. In some embodiments, the single domain antibody comprises a CDR3 consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the CDR3 of the VHH shown in SEQ ID NO: 4. In some embodiments, the single domain antibody comprises a CDR3 comprising the CDR3 of the VHH shown in SEQ ID NO: 4. In some embodiments, the single domain antibody comprises a CDR3 consisting of the CDR3 of the VHH shown in SEQ ID NO: 4.

[0161] In some embodiments, the single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 4. The single-domain antibodies of the present disclosure further comprise, in addition to the CDR3 described above, a CDR1 and a CDR2, wherein the CDR1 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CDR2 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 comprises the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 comprises the CDR2 of the VHH set forth in SEQ ID NO: 4. In some embodiments, the single-domain antibody further comprises, in addition to the CDR3 described above, CDR1 and CDR2, wherein the CDR1 consists of the CDR1 of the VHH set forth in SEQ ID NO: 4, and the CDR2 consists of the CDR2 of the VHH set forth in SEQ ID NO: 4. The sequences of CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 4 according to different definition schemes are: (a) SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, determined according to the Kabat definition scheme; (b) SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, determined according to the AbM definition scheme; (c) SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, determined according to the Chothia definition scheme; or (d) SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, determined according to the IMGT definition scheme.

[0162] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0163] In some embodiments, the single domain antibody is a humanized single domain antibody.

[0164] In some embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9 or 10. In some embodiments, the single domain antibody consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9 or 10. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 9 or 10. In some embodiments, the single domain antibody consists of the amino acid sequence set forth in SEQ ID NO: 9 or 10.

[0165] In some embodiments, the single domain antibody of the present disclosure comprises a sequence represented by the following formula ([ ] indicates the type of amino acid that can be selected at that position): QVQL[QV]ESGGG[SL]VQ[SP]GGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVAAICSDGKTTYADSVKGRFTIS[QR]D[GN][AS]KNTLYLQMNSL[KR][PA]EDTA[MV]YYCAAGVKQPRYSGGYYVIWDGDFTYWGQGT[QL]VTVSS (SEQ ID NO: 113).

[0166] Fusion protein

[0167] In a third aspect, the present disclosure provides a fusion protein comprising the single domain antibody according to the first aspect of the present disclosure or the immunoglobulin Fc region binding protein according to the second aspect of the present disclosure.

[0168] In some embodiments, in addition to the single domain antibody of the first aspect of the disclosure or the immunoglobulin Fc region binding protein of the second aspect of the disclosure, the fusion protein further comprises one or more other polypeptides.

[0169] In some embodiments, the single domain antibody of the first aspect of the present disclosure or the immunoglobulin Fc region binding protein of the second aspect of the present disclosure may be located at the N-terminus of the one or more other polypeptides. In some embodiments, the single domain antibody of the first aspect of the present disclosure or the immunoglobulin Fc region binding protein of the second aspect of the present disclosure may be located at the C-terminus of the one or more other polypeptides.

[0170] In some specific embodiments, in addition to the single domain antibody described in the first aspect of the present disclosure or the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein further comprises an immunoglobulin Fc region.

[0171] In some embodiments, the immunoglobulin Fc region in the fusion protein can be a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region in the fusion protein can be the Fc region of human IgG1, IgG2, IgG3, or IgG4. In preferred embodiments, the immunoglobulin Fc region in the fusion protein is the Fc region of human IgG1. In some embodiments, the immunoglobulin Fc region in the fusion protein is the Fc region derived from human IgG1.

[0172] In some embodiments, the immunoglobulin Fc region in the fusion protein may be an Fc region derived from human IgG, comprising a mutation that weakens the binding of the immunoglobulin Fc region in the fusion protein to the single-domain antibody and / or comprising a mutation that weakens the binding between the immunoglobulin Fc region in the fusion protein and FcRn.

[0173] In some embodiments, the single-domain antibody described in the first aspect of the present disclosure or the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure may be located at the N-terminus of the immunoglobulin Fc region in the fusion protein. In some embodiments, the single-domain antibody described in the first aspect of the present disclosure or the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure may be located at the N-terminus or C-terminus of the one or more other polypeptides, preferably at the N-terminus of the one or more other polypeptides.

[0174] In some embodiments, the immunoglobulin Fc region in the fusion protein has an amino acid residue mutation compared to the wild-type immunoglobulin Fc region, and the mutation causes the fused immunoglobulin Fc region to have an altered effector function and / or an altered binding ability to the single-domain antibody.

[0175] As previously mentioned, the single-domain antibody of the first aspect of the present disclosure and the immunoglobulin Fc region-binding protein of the second aspect of the present disclosure can specifically bind to the immunoglobulin Fc region. When the single-domain antibody or immunoglobulin Fc region-binding protein further comprises an immunoglobulin Fc region fused thereto, interactions between the single-domain antibody and the fused immunoglobulin Fc region between different molecules are undesirable, which may reduce the stability of the molecule and the therapeutic effect.

[0176] In some embodiments, the immunoglobulin Fc region in the fusion proteins of the present disclosure comprises mutations that reduce its interaction with the single domain antibody.

[0177] In some embodiments, the immunoglobulin Fc region in the fusion protein of the present disclosure comprises a mutation that weakens its interaction with FcRn.

[0178] For example, in some embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 311 or 314; the amino acid positions are numbered using the EU index as in Kabat.

[0179] In some embodiments, the immunoglobulin Fc region in the fusion protein specifically comprises a mutation selected from the group consisting of L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W, L314Y, Q311A, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, and Q311Y. Preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from L314A, L314F, L314G, L314H, L314I, L314K, L314M, L314P, L314R, L314S, L314V, L314W, L314Y, Q311D or Q311E; more preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from L314A, L314S, L314G, L314R, L314K, L314W, L314Y, Q311D or Q311E; further preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from L314A, L314R, L314W or Q311D.

[0180] In some embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 315.

[0181] For example, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from the group consisting of N315A, N315D, N315E, N315F, N315G, N315H, N315I, N315K, N315L, N315M, N315P, N315Q, N315R, N315S, N315T, N315V, N315W, and N315Y. Preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from the group consisting of N315R and N315M.

[0182] In other embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 253 and / or amino acid position 254.

[0183] In some embodiments, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from the group consisting of: I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, I253Y, S254A, S254D, S254E , S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W, S254Y, I253A / S254A, I253G / S254A, I253A / S254G, I253E / S254D, I253E / S254E, or I253D / S254E.

[0184] Preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from I253A, I253D, I253E, I253G, I253N, I253Q, I253S, S254A, S254D, S254E, S254G, S254N, S254Q, I253A / S254A, I253G / S254A, I253A / S254G, I253E / S254D, I253E / S254E or I253D / S254E; more preferably, the fusion protein The immunoglobulin Fc region in the fusion protein may comprise a mutation selected from I253A, I253G, I253S, I253SE, I253D, S254A, S254G, S254D, S254E, I253A / S254A, I253A / S254G, I253D / S254E, I253E / S254D or I253E / S254E; further preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from I253A / S254A or I253E / S254D.

[0185] In other embodiments, the immunoglobulin Fc region in the fusion protein may further comprise mutations at amino acid positions 435 and / or 436. The mutations at amino acid positions 435 and / or 436 may function independently or in combination with the aforementioned amino acid mutations at other positions.

[0186] In some embodiments, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from the group consisting of: H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W, H435Y, Y436A, Y436 D. Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y43 6Q, Y436R, Y436S, Y436T, Y436V, Y436W, H435A / Y436A, H435A / Y436S, H 435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E or H435M / Y436Q.

[0187] Preferably, the immunoglobulin Fc region in the fusion protein may comprise a member selected from the group consisting of H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W, H435Y, Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q, Y436N, H435A / Y436A, H435A / Y436S, H435A / Y436G, H435M / Y436D, H435M / Y436Q, H435M / Y436E or H435M / Y436E More preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from H435A, H435S, H435G, H435M, H435L, H435Y, H435I, Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q, Y436N, H435A / Y436A, H435A / Y436S, H435A / Y436G, H435M / Y436D, H435M / Y436Q, H435M / Y436E or H435M / Y436N. Further preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from H435A, H435M, H435M / Y436D, H435M / Y436Q, H435M / Y436E or H435A / Y436A.

[0188] In some embodiments, the immunoglobulin Fc region in the fusion protein may comprise a combination of mutations selected from the group consisting of L314A / H435A, L314S / H435S, L314S / H435A / Y436S, L314R / N315R, L314R / N315M, L314R / H435A, L314R / H435M, L314A / H435M, L314 R / H435M / Y436Q, L314A / H435M / Y436D, I253E / S254D / H435A / Y436A, I253E / S254D / H435 A / Y436D, L314R / H435M / Y436D, I253E / S254D / H435M / Y436D, I253E / S254D / H435M / Y436Q , L314W / H435M / Y436D L314W / H435M / Y436E, L314W / H435M / Y436Q, L314A / H435M / Y436E, L314A / H435M / Y436Q, Q311D / H435M / Y436E, or Q311D / H435M / Y436D. Preferably, the immunoglobulin Fc region in the fusion protein may comprise a combination of mutations selected from the group consisting of L314A / H435A, L314R / H435A, L314R / H435M, L314A / H435M, L314A / H435M / Y436D, L314A / H435M / Y436E, L314R / H435M / Y436 Preferably, the immunoglobulin Fc region in the fusion protein comprises a combination of mutations selected from the group consisting of L314A / H435M / Y436D, L314A / H435M / Y436E, L314W / H435M / Y436D, and L314W / H435M / Y436E.

[0189] In addition, in some embodiments, the immunoglobulin Fc region in the fusion protein may further comprise a mutation that modulates its interaction with an Fcγ receptor, such as FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a) and / or FcγRIIIb (CD16b). In some embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation that enhances its interaction with FcγRIIb.

[0190] The mutation that enhances the interaction with FcγRIIb can be any mutation or combination of mutations known in the prior art, such as those described in Rena Liu, Antibodies 2020, 9, 64; Chu, SY, Mol. Immunol. 2008, 45, 3926-3933; Zhang, D., J. Biol. Chem. 2016, 291, 27134-27146; Dahan, R., Cancer Cell 2016, 29, 820-831; Mimoto, F., Protein Eng. Des. Sel. 2013, 26, 589-598; Robert L. Shields, J. Biol. Chem. 2001, 276: 6591-6604.

[0191] In some embodiments, the immunoglobulin Fc region in the fusion protein further comprises one or more mutations selected from the group consisting of E233D, G237D, P238D, T265A, H268D, K290A, R255A, E258A, S267A, S267E, P271G, E272A, N276A, D280A, H285A, N286A, T307A, L309A, N315A, K326A, L328E, L328F, A330R, P331A, S337A, A378Q, E430A, H268A, R301A, R301M, K322A, K334R, and K334N.

[0192] In some embodiments, the immunoglobulin Fc region in the fusion protein comprises a combination of mutations selected from the group consisting of S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233D / A330R, E233D / P238D, G237D / P238D, P238D / H268D, P238D / P271G, P238D / A330R, E233D / P238D / A330R, E 233D / P271G / A330R, G237D / H268D / P271G, G237D / P271G / A330R, S239D / A330L / I3 32E, G236A / S239D / I332E, E233D / P238D / P271G / A330R, G237D / P238D / H268D / P27 1G, E233D / H268D / P271G / A330R, G237D / P238D / P271G / A330R, G237D / H268D / P271 G / A330R, E233D / G237D / H268D / P271G / A330R, E233D / P238D / H268D / P271G / A330R , G237D / P238D / H268D / P271G / A330R or E233D / G237D / P238D / H268D / P271G / A330R.

[0193] In some specific embodiments, the immunoglobulin Fc region in the fusion protein comprises a mutation selected from the group consisting of P238D, S267E, L328F, A330R, S267E / L328F, G237D / P238D / H268D / P271G / A330R, or E233D / G237D / P238D / H268D / P271G / A330R. Preferably, the fused immunoglobulin Fc region comprises a mutation selected from the group consisting of S267E / L328F (hereinafter abbreviated as EF) or E233D / G237D / P238D / H268D / P271G / A330R (hereinafter abbreviated as V12).

[0194] In some embodiments, the immunoglobulin Fc region in the fusion protein comprises a combination of a mutation that enhances interaction with FcγRIIb and the aforementioned amino acid mutations. For example, the immunoglobulin Fc region in the fusion protein may comprise a mutation selected from Table 2:

[0195] Table 2. Types of mutations in the immunoglobulin Fc region of the fusion protein

[0196] In some specific embodiments, the immunoglobulin Fc region in the fusion protein may comprise a mutation combination selected from EF / L314A / H435M / Y436E, EF / L314A / H435M / Y436D, EF / L314W / H435M / Y436E, EF / L314W / H435M / Y436D, V12 / L314A / H435M / Y436E, V12 / L314A / H435M / Y436D, V12 / L314W / H435M / Y436E or V12 / L314W / H435M / Y436D. Preferably, the immunoglobulin Fc region in the fusion protein may comprise a mutation combination selected from EF / L314A / H435M / Y436D or EF / L314W / H435M / Y436D.

[0197] Multispecific antibodies

[0198] In a fourth aspect, the present disclosure provides a multispecific antibody comprising the single domain antibody of the first aspect of the present disclosure, the immunoglobulin Fc region binding protein of the second aspect of the present disclosure, or the fusion protein of the third aspect of the present disclosure, and one or more additional antigen binding regions, wherein the one or more additional antigen binding regions bind to different antigens or different epitopes of the same antigen as the single domain antibody, the immunoglobulin Fc region binding protein or the fusion protein.

[0199] Conjugate

[0200] In the fifth aspect, the present disclosure provides a conjugate comprising the single domain antibody described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, or the multispecific antibody described in the fourth aspect of the present disclosure, and the conjugate further comprises a molecule conjugated to the single domain antibody, the immunoglobulin Fc region binding protein, the fusion protein, or the multispecific antibody.

[0201] Nucleic acids, vectors, and recombinant cells

[0202] In a sixth aspect, the present disclosure relates to a nucleic acid molecule encoding the single domain antibody of the first aspect of the present disclosure, the immunoglobulin Fc region binding protein of the second aspect of the present disclosure, the fusion protein of the third aspect of the present disclosure, or the multispecific antibody of the fourth aspect of the present disclosure. The nucleic acid of the present disclosure may be RNA, DNA, or cDNA. According to one embodiment of the present disclosure, the nucleic acid of the present disclosure is a substantially isolated nucleic acid.

[0203] The nucleic acids of the present disclosure can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the present disclosure given herein, and / or can be isolated from suitable natural sources.

[0204] In a seventh aspect, the present disclosure relates to a vector comprising the nucleic acid molecule according to the sixth aspect of the present disclosure.

[0205] The nucleic acid described in the sixth aspect of the present disclosure may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, a cosmid or a YAC. The vector may be, in particular, an expression vector, i.e., a vector for expressing the single domain antibody, immunoglobulin Fc region binding protein, fusion protein or multispecific antibody described in the present disclosure in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common sense for those skilled in the art to select the elements and their sequences for expression in a specific host. Specific examples of regulatory elements and other elements useful or necessary for the expression of the immunoglobulin Fc region binding protein of the present disclosure, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0206] In an eighth aspect, the present disclosure relates to a recombinant host cell that expresses or is capable of expressing one or more of the single domain antibodies described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, or the multispecific antibody described in the fourth aspect of the present disclosure, and / or contains the nucleic acid described in the sixth aspect of the present disclosure or the vector described in the seventh aspect of the present disclosure. Preferred host cells of the present disclosure are bacterial cells, fungal cells, or mammalian cells.

[0207] Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0208] Suitable fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0209] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0210] However, the present disclosure may also utilize amphibian cells, insect cells, and the like, and any other cells known in the art for expressing heterologous proteins.

[0211] The single domain antibodies, immunoglobulin Fc region binding proteins, fusion proteins, or multispecific antibodies of the present disclosure can be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in the cells described above, followed by isolation from the host cells and optionally further purification; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), followed by isolation from the culture medium and optionally further purification.

[0212] Methods and reagents for recombinantly producing polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods for producing the single domain antibodies, immunoglobulin Fc region binding proteins, fusion proteins, or multispecific antibodies of the present disclosure are well known to those skilled in the art.

[0213] Composition

[0214] In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing one or a combination of single domain antibodies, immunoglobulin Fc region binding proteins, fusion proteins, multispecific antibodies, conjugates, nucleic acid molecules, expression vectors or cells of the present disclosure formulated together with a pharmaceutically acceptable carrier.

[0215] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0216] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition that produces a therapeutic effect. Typically, based on 100%, this amount ranges from about 0.01% to about 99% of the active ingredient, e.g., about 0.1% to about 70%, or about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0217] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present disclosure being administered, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being administered, the duration of treatment, other drugs, compounds, and / or materials being administered in combination with the particular composition being administered, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0218] The compositions of the present disclosure can be administered by one or more methods well known in the art through one or more routes of administration. It will be understood by those skilled in the art that the route of administration and / or mode are different depending on the desired result. Preferred routes of administration for immunoglobulin Fc region binding proteins of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and topical administration, typically injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0219] Optionally, the pharmaceutical composition may comprise, in addition to the single domain antibody, immunoglobulin Fc region binding protein, fusion protein, multispecific antibody, conjugate, nucleic acid molecule, expression vector, cell, or composition described herein, other therapeutic agents formulated together in the pharmaceutical composition.

[0220] Reagent test kit

[0221] In the tenth aspect, the present disclosure provides a kit comprising the single domain antibody described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, the multispecific antibody described in the fourth aspect of the present disclosure, the conjugate described in the fifth aspect of the present disclosure, the nucleic acid molecule described in the sixth aspect of the present disclosure, the expression vector described in the seventh aspect of the present disclosure, or the cell described in the eighth aspect of the present disclosure, and / or the composition described in the ninth aspect of the present disclosure.

[0222] The kits of the present disclosure may comprise the single domain antibodies, immunoglobulin Fc region binding proteins, fusion proteins, multispecific antibodies, conjugates, nucleic acid molecules, expression vectors, cells, or compositions described herein in a single common container. Optionally, the kits of the present disclosure may further comprise one or more other therapeutic agents in other containers.

[0223] Treat and / or prevent disease

[0224] On the other hand, the present disclosure also provides a method for treating and / or preventing a disease, in particular, treating and / or preventing an IgG-mediated disease and / or symptom, comprising administering to a subject in need thereof an effective amount of the single domain antibody of the first aspect of the present disclosure, the immunoglobulin Fc region binding protein of the second aspect of the present disclosure, the fusion protein of the third aspect of the present disclosure, the multispecific antibody of the fourth aspect of the present disclosure, the conjugate of the fifth aspect of the present disclosure, the nucleic acid molecule of the sixth aspect of the present disclosure, the expression vector of the seventh aspect of the present disclosure, or the cell of the eighth aspect of the present disclosure, and / or the composition of the ninth aspect of the present disclosure. The IgG-mediated diseases and / or symptoms include autoimmune diseases, inflammatory diseases, and allergic diseases.

[0225] In other embodiments, the present disclosure also provides the use of the single-domain antibody described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, the multispecific antibody described in the fourth aspect of the present disclosure, the conjugate described in the fifth aspect of the present disclosure, the nucleic acid molecule described in the sixth aspect of the present disclosure, the expression vector described in the seventh aspect of the present disclosure, or the cell described in the eighth aspect of the present disclosure, and / or the composition described in the ninth aspect of the present disclosure in the preparation of a drug for treating and / or preventing IgG-mediated diseases and / or symptoms.

[0226] In other embodiments, the present disclosure also provides the single domain antibody described in the first aspect of the present disclosure, the immunoglobulin Fc region binding protein described in the second aspect of the present disclosure, the fusion protein described in the third aspect of the present disclosure, the multispecific antibody described in the fourth aspect of the present disclosure, the conjugate described in the fifth aspect of the present disclosure, the nucleic acid molecule described in the sixth aspect of the present disclosure, the expression vector described in the seventh aspect of the present disclosure, or the cell described in the eighth aspect of the present disclosure, and / or the composition described in the ninth aspect of the present disclosure, which can be used to treat and / or prevent IgG-mediated diseases and / or symptoms.

[0227] In another aspect, the present disclosure provides a drug delivery device that can be used to administer the single-domain antibody, immunoglobulin Fc region binding protein, fusion protein, multispecific antibody, conjugate, nucleic acid molecule, expression vector, cell, or composition described herein.

[0228] The present disclosure also provides the following non-limiting embodiments:

[0229] 1. An immunoglobulin Fc region binding protein comprising one or more immunoglobulin single variable domains, wherein the immunoglobulin single variable domain comprises a CDR3 selected from a VHH shown in SEQ ID NO: 1-8.

[0230] 2. The immunoglobulin Fc region binding protein according to embodiment 1, wherein the immunoglobulin is derived from IgG, IgA, IgM, IgD or IgE.

[0231] 3. The immunoglobulin Fc region binding protein according to embodiment 2, wherein the immunoglobulin is derived from IgG1, IgG2, IgG3 or IgG4.

[0232] 4. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 3, wherein the immunoglobulin Fc region is derived from human, mouse, rat, rabbit or monkey immunoglobulin.

[0233] 5. The immunoglobulin Fc region binding protein according to embodiment 4, wherein the immunoglobulin Fc is derived from human IgG1.

[0234] 6. The immunoglobulin Fc region binding protein according to embodiment 1, which has at least one of the following characteristics: a) a KD value for binding to human IgG Fc is less than 1×10 -7 M; b) blocking the interaction between IgG and FcRn; c) reducing IgG levels in mammals.

[0235] 7. The immunoglobulin Fc region binding protein according to any one of embodiments 1-6, wherein the immunoglobulin single variable domain further comprises a CDR1 and a CDR2 selected from the VHHs shown in SEQ ID NOs: 1-8.

[0236] 8. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 7, wherein the CDR is a Kabat CDR, an AbM CDR, a Chothia CDR or an IMGT CDR.

[0237] 9. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1.

[0238] 10. The immunoglobulin Fc region binding protein according to embodiment 9, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NO: 13-15, SEQ ID NO: 16-18, SEQ ID NO: 19-21, SEQ ID NO: 22-24.

[0239] 11. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 2.

[0240] 12. According to the immunoglobulin Fc region binding protein according to embodiment 11, the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 2 are selected from any one of the following groups: SEQ ID NO: 25-27, SEQ ID NO: 28-30, SEQ ID NO: 31-33, SEQ ID NO: 34-36.

[0241] 13. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 3.

[0242] 14. The immunoglobulin Fc region binding protein according to embodiment 13, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 3 are selected from any one of the following groups: SEQ ID NO: 37-39, SEQ ID NO: 40-42, SEQ ID NO: 43-45, SEQ ID NO: 46-48.

[0243] 15. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 4.

[0244] 16. The immunoglobulin Fc region binding protein according to embodiment 15, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 4 are selected from any one of the following groups: SEQ ID NO: 49-51, SEQ ID NO: 52-54, SEQ ID NO: 55-57, SEQ ID NO: 58-60.

[0245] 17. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 5.

[0246] 18. The immunoglobulin Fc region binding protein according to embodiment 17, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 5 are selected from any one of the following groups: SEQ ID NO: 61-63, SEQ ID NO: 64-66, SEQ ID NO: 67-69, SEQ ID NO: 70-72.

[0247] 19. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 6.

[0248] 20. The immunoglobulin Fc region binding protein according to embodiment 19, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 6 are selected from any one of the following groups: SEQ ID NO: 73-75, SEQ ID NO: 76-78, SEQ ID NO: 79-81, SEQ ID NO: 82-84.

[0249] 21. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 7.

[0250] 22. According to the immunoglobulin Fc region binding protein according to embodiment 21, the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 7 are selected from any one of the following groups: SEQ ID NO: 85-87, SEQ ID NO: 88-90, SEQ ID NO: 91-93, SEQ ID NO: 94-96.

[0251] 23. The immunoglobulin Fc region binding protein according to any one of embodiments 1 to 8, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 8.

[0252] 24. The immunoglobulin Fc region binding protein according to embodiment 23, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 8 are selected from any one of the following groups: SEQ ID NO: 97-99, SEQ ID NO: 100-102, SEQ ID NO: 103-105, SEQ ID NO: 106-108.

[0253] 25. The immunoglobulin Fc region binding protein according to any one of embodiments 1-8, wherein the immunoglobulin single variable domain comprises a VHH selected from any one of SEQ ID NOs: 1-8.

[0254] 26. The immunoglobulin Fc region binding protein according to any one of embodiments 1-25, comprising 1-10 immunoglobulin single variable domains.

[0255] 27. The immunoglobulin Fc region binding protein of embodiment 26, comprising 2, 3, 4, 5, 6, 7 or 8 immunoglobulin single variable domains.

[0256] 28. The immunoglobulin Fc region binding protein according to embodiment 27, wherein the immunoglobulin single variable domains are identical, partially identical or different from each other.

[0257] 29. The immunoglobulin Fc region binding protein according to any one of embodiments 26-28, comprising 5 immunoglobulin single variable domains.

[0258] 30. The immunoglobulin Fc region binding protein of embodiment 29, wherein the immunoglobulin single variable domains are identical to each other.

[0259] 31. The immunoglobulin Fc region binding protein according to embodiment 30, wherein the immunoglobulin single variable domain comprises the VHH shown in SEQ ID NO: 4.

[0260] 32. The immunoglobulin Fc region binding protein according to any one of embodiments 27-31, wherein the immunoglobulin single variable domains are directly connected to each other or indirectly connected via a linker.

[0261] 33. The immunoglobulin Fc region binding protein according to embodiment 32, wherein the immunoglobulin single variable domains are indirectly connected via a linker; preferably via a flexible polypeptide linker.

[0262] 34. The immunoglobulin Fc region binding protein according to embodiment 33, wherein the linker is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(SGG) n S (SEQ ID NO: 114), (GGGS) n (SEQ ID NO: 115), (GGSG) n (SEQ ID NO: 116), (GGGGS) n (SEQ ID NO: 117), (SGGGG) n (SEQ ID NO: 118), (GGGGSGG) n (SEQ ID NO: 119), (GGGGSGG) n (SEQ ID NO: 120), (GGGGSGGS) n (SEQ ID NO: 121), (GGGGSGGGS) n (SEQ ID NO: 122), (PGGGS) n (SEQ ID NO: 123), (AGGGS)n (SEQ ID NO: 124), (GGGSE) n (SEQ ID NO: 125), (GGGGA) n (SEQ ID NO: 126), (AP) n 、(GAP) n 、(EAAAK) n (SEQ ID NO:127), (ASTKGPSVFPLAGAP) n (SEQ ID NO:128), (ASTKGPSVPLAGGG) n (SEQ ID NO:129), (AKTTPKLEEGEFSEAR) n (SEQ ID NO:130), (AKTTPKLEEGEFSEARV) n (SEQ ID NO: 131), (AKTTPKLGG) n (SEQ ID NO: 132), (ADAAP) n (SEQ ID NO:133), (ADAAPTVSIFPP) n (SEQ ID NO: 134), (AKTTPP) n (SEQ ID NO:135), (AKTTPPSVTPLAP) n (SEQ ID NO: 136), (AKTTAP) n (SEQ ID NO:137), (AKTTAPSVYPLAP) n (SEQ ID NO: 138), (ASTKGP) n (SEQ ID NO: 139) and (ASTKGPSVFPLAP) n (SEQ ID NO: 140), wherein n=1-10.

[0263] 35. The immunoglobulin Fc region binding protein of embodiment 34, wherein the linker is selected from one or more of the following amino acid sequences: GS, GGGGGG (SEQ ID NO: 141), GGGS (SEQ ID NO: 142), GGGGS (SEQ ID NO: 143), GGGGSGGGS (SEQ ID NO: 144), GAP, and ASTKGPSVFPLAGAP (SEQ ID NO: 145).

[0264] 36. The immunoglobulin Fc region binding protein according to embodiment 35, which comprises the amino acid sequence shown in SEQ ID NO: 11 or 12.

[0265] 37. An immunoglobulin Fc region binding protein according to any one of embodiments 1-36, which comprises a humanized VHH.

[0266] 38. According to the immunoglobulin Fc region binding protein of embodiment 37, the humanized VHH is selected from SEQ ID NO: 9 or 10.

[0267] 39. The immunoglobulin Fc region binding protein according to any one of embodiments 1-38, further comprising an immunoglobulin Fc region fused to an immunoglobulin single variable domain; preferably a human immunoglobulin Fc region, such as the Fc region of human IgG1, IgG2, IgG3 or IgG4; more preferably the Fc region of human IgG1.

[0268] 40. The immunoglobulin Fc region binding protein according to embodiment 39, wherein the fused immunoglobulin Fc region comprises a mutation that weakens the interaction between the immunoglobulin Fc region and the immunoglobulin single variable domain.

[0269] 41. The immunoglobulin Fc region binding protein according to embodiment 39 or 40, wherein the fused immunoglobulin Fc region comprises a mutation that weakens its interaction with FcRn.

[0270] 42. The immunoglobulin Fc region binding protein according to embodiment 40 or 41, wherein the fused immunoglobulin Fc region comprises a mutation at amino acid position 311 or 314.

[0271] 43. The immunoglobulin Fc region binding protein according to embodiment 42, wherein the fused immunoglobulin Fc region comprises a mutation selected from the group consisting of: L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S , L314T, L314V, L314W, L314Y, Q311A, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W, or Q311Y.

[0272] 44. The immunoglobulin Fc region binding protein according to embodiment 40 or 41, wherein the fused immunoglobulin Fc region comprises a mutation at amino acid position 253 and / or amino acid position 254.

[0273] 45. The immunoglobulin Fc region binding protein according to embodiment 44, wherein the fused immunoglobulin Fc region comprises a mutation selected from the group consisting of: I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, I253Y, S254A, S254D , S254E, S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W, S254Y, I253A / S254A, I253G / S254A, I253A / S254G, I253E / S254D, I253E / S254E, or I253D / S254E.

[0274] 46. ​​The immunoglobulin Fc region binding protein according to any one of embodiments 40-45, wherein the fused immunoglobulin Fc region further comprises a mutation at amino acid position 435 and / or amino acid position 436.

[0275] 47. The immunoglobulin Fc region binding protein according to embodiment 46, wherein the fused immunoglobulin Fc region comprises a mutation selected from the group consisting of: H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W, H435Y, Y436A, Y436D, Y436E, Y436A, Y436D, Y436E, Y436A, Y436D, Y436E, Y436A, Y436D, Y436E, Y436A, Y436D, Y436E, Y436A, Y436D, Y436E, Y436A, Y436D, Y436E, Y436 36F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436Q, Y436R, Y436S, Y436T, Y436V, Y436W, H435A / Y436A, H435A / Y436S, H435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E, or H435M / Y436Q.

[0276] 48. The immunoglobulin Fc region binding protein according to any one of embodiments 40-47, wherein the fused immunoglobulin Fc region further comprises a mutation that enhances its interaction with FcγRIIb.

[0277] 49. The immunoglobulin Fc region binding protein according to embodiment 48, wherein the fused immunoglobulin Fc region comprises one or more mutations selected from the group consisting of E233D, G237D, P238D, T265A, H268D, K290A, R255A, E258A, S267A, S267E, P271G, E272A, N276A, D280A, H285A, N286A, T307A, L309A, N315A, K326A, L328E, L328F, A330R, P331A, S337A, A378Q, E430A, H268A, R301A, R301M, K322A, K334R and K334N.

[0278] 50. The immunoglobulin Fc region binding protein according to embodiment 49, wherein the fused immunoglobulin Fc region comprises a combination of mutations selected from the group consisting of S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233D / A330R, E ... 233D / P238D, G237D / P238D, P238D / H268D, P238D / P271G, P238D / A330R, E233D / P23 8D / A330R, E233D / P271G / A330R, G237D / H268D / P271G, G237D / P271G / A330R, S239D / A330L / I332E, G236A / S239D / I332E, E233D / P238D / P271G / A330R, G237D / P238D / H2 68D / P271G, E233D / H268D / P271G / A330R, G237D / P238D / P271G / A330R, G237D / H268D / P271G / A330R, E233D / G237D / H268D / P271G / A330R, E233D / P238D / H268D / P271G / A330R, G237D / P238D / H268D / P271G / A330R, or E233D / G237D / P238D / H268D / P271G / A330R

[0279] 51. A conjugate comprising the immunoglobulin Fc region binding protein according to any one of embodiments 1-50.

[0280] 52. A nucleic acid molecule encoding the immunoglobulin Fc region binding protein of any one of embodiments 1-50.

[0281] 53. An expression vector comprising the nucleic acid molecule of embodiment 52 operably linked to an expression control element.

[0282] 54. A recombinant cell comprising the nucleic acid molecule of embodiment 53 and / or transformed with the expression vector of embodiment 53, and capable of expressing the immunoglobulin Fc region binding protein.

[0283] 55. A pharmaceutical composition comprising the immunoglobulin Fc region binding protein of any one of embodiments 1-50 or the conjugate of embodiment 51, and a pharmaceutically acceptable carrier.

[0284] 56. A kit comprising the immunoglobulin Fc region binding protein of any one of embodiments 1-50, the conjugate of embodiment 51 and / or the pharmaceutical composition of embodiment 55.

[0285] 57. A method for treating and / or preventing IgG-mediated diseases and / or symptoms, comprising administering to a subject in need thereof an effective amount of the immunoglobulin Fc region binding protein of any one of embodiments 1-50, the conjugate of embodiment 51, or the pharmaceutical composition of embodiment 55.

[0286] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0287] Example

[0288] Example 1 Screening of heavy chain single domain antibodies targeting Fc

[0289] 1.1 Library Construction

[0290] Before immunization, 50 mL of peripheral blood was collected from camels into a vacuum tube, and the supernatant was used as pre-immune serum. For the initial immunization, a healthy camel was selected. HER3-Fc was used as the antigen and mixed evenly with an equal volume of complete Freund's adjuvant to fully emulsify the protein. The mixture was then injected subcutaneously at multiple sites. For subsequent immunizations, equal amounts of antigen were mixed evenly with an equal volume of incomplete Freund's adjuvant. Immunizations were administered every two weeks for a total of six times. At the end of the final immunization, 50 mL of peripheral blood was collected from the camel into a vacuum tube, and the supernatant was used as post-immune serum.

[0291] PBMCs were isolated by density gradient centrifugation, and total RNA was extracted using an RNA extraction kit provided by QIAGEN. The extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRAND SUPERMIX kit according to the manufacturer's instructions, and the nucleic acid fragment encoding the variable region of the heavy chain antibody was amplified by nested PCR.

[0292] The target heavy chain single domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pMECS using restriction endonucleases PstI and NotI (purchased from NEB). The product was then electroporated into E. coli electrocompetent cells TG1 to construct an anti-HER3-Fc single domain antibody phage display library and assay the library. The library size was calculated to be 1.5×10 8 To test the insertion rate of the library, 50 clones were randomly selected for sequencing. 50 clones had the correct exogenous fragment insertion, a 100% accuracy rate. Analysis and alignment of the DNA and amino acid sequences of the sequenced clones confirmed that all sequences were VHH sequences, with an estimated diversity of over 95%.

[0293] 1.2 Selection of heavy chain single domain antibodies targeting Fc

[0294] First round of panning: Screening was performed using Fc protein (prepared in-house, sequence referenced from the Uniprot database, accession number P01857; the last 227 amino acids were selected, the same below). Plates were coated with 10 μg / well of Fc protein and incubated at 4°C overnight. The next day, after blocking with 2% skim milk for 2 hours at room temperature, 100 μL of phage (approximately 7 × 10^11 cfu / well, from the display library described in Example 1.1, previously screened with CB negative technology to eliminate nonspecific binding) was added and allowed to react for 2 hours at room temperature. The plates were then washed 25 times with PBST20 (PBS containing 0.05% Tween 20) to remove unbound phage. A 56 μg / mL stock solution of schuFcRn-Chis (single-chain human FcRn with a C-terminal His tag) or sFcRn (soluble FcRn), diluted to 100 μg / mL in PBST20, pH 6.0, was added for competition. The supernatant was removed, and the cells were washed 25 times with PBST20. Finally, the phages specifically bound to Fc were dissociated with glycine (100 mM, pH 2.0) to obtain the phages of the first round of panning.

[0295] Second round of panning: Plates were coated with muFc-containing fusion proteins Control-1 (10 μg / mL) and Control-2 (50 μg / mL). Under similar conditions, 100 μL of phage (from the first round of panning, previously screened with CB negative screen to remove nonspecific binding) was added for selection. Next, 100 μL of schuFcRn-Chis diluted to 100 μg / mL in PBST20 (pH 6.0) was added for competition. The supernatant was removed, and the plate was washed 25 times with PBST20. Finally, specifically bound phage were dissociated with glycine (100 mM, pH 2.0) and neutralized with Tris-HCl (pH 9.0).

[0296] Phages obtained from the second round of panning were used to infect E. coli TG1 in logarithmic phase, diluted to an appropriate concentration, and plated onto LB-Amp plates. 380 single clones were selected and inoculated into 96-well plates. The seed solution was cultured overnight. The next day, a 1% inoculation ratio was added to 1 mL deep-well plates and cultured at 37°C to an OD600 of 0.8-1.0. IPTG was then added to 1 mM and expression was induced overnight at 25°C.

[0297] The bacterial culture fluid induced for expression overnight was centrifuged, the supernatant was discarded, and lysis buffer was added to the bacteria for overnight lysis. Centrifuge the next day, and the supernatant was used for ELISA detection. Plates were coated with Fc, Control-1, Control-2, HER3-muFc and HER3-Chis at 4°C overnight, and the obtained bacterial lysate supernatant was added and reacted at room temperature for 2 hours. After washing, the secondary antibody Goat anti-HA tag HRP (purchased from abcam) was added and reacted at room temperature for 2 hours. After washing, TMB colorimetric solution was added, and the absorbance values ​​at 450nm and 650nm were read. The absorbance value at 450nm was subtracted from the absorbance value at 650nm to obtain the final absorbance value. The clones that bound to the proteins Fc and muFc used in the first round of panning and did not bind to HER3-Chis were selected for sequencing to obtain the sequence of the antibody that specifically bound to Fc. The OD values ​​of some clones that specifically bound to Fc are shown in Table 3.

[0298] Table 3

[0299] Example 2 Identification of the function of anti-Fc single domain antibodies

[0300] 2.1 Detection of affinity of anti-Fc single domain antibody to human IgG1-Fc

[0301] The binding kinetics of anti-Fc single-domain antibodies to human IgG1-Fc were measured using biomembrane interferometry (BLI). Human IgG1-Fc was immobilized on an AHC sensor at pH 7.4. The anti-Fc single-domain antibodies were then diluted to eight concentrations and added to the AHC sensor to bind to the immobilized human IgG1-Fc. The equilibrium dissociation constant (KD) was calculated using Octet data analysis software. The results are shown in Table 4.

[0302] Table 4

[0303] The binding kinetics of anti-Fc single-domain antibodies to human IgG1-Fc were measured using biomembrane interferometry (BLI). Human IgG1-Fc was immobilized on an AHC sensor at pH 6.0. The anti-Fc single-domain antibodies were then diluted to seven concentrations and added to the AHC sensor for binding to the immobilized human IgG1-Fc. The equilibrium dissociation constant (KD), association rate (ka), and dissociation rate (kdis) were calculated using Octet data analysis software. The results are shown in Table 5.

[0304] Table 5

[0305] 2.2 Detection of the blocking ability of anti-Fc single domain antibodies on the binding of Fc to FcRn

[0306] (1) 5 μg / mL of the Fc used in the first round of panning, i.e., IgG1-Fc, was coated on a 96-well plate. Anti-Fc single-domain antibodies (iFc 02-Chis, iFc 21-Chis, iFc 102-Chis, iFc 106-Chis, iFc 231-Chis, iFc 284S-Chis, or iFc 347-Chis) were diluted 3-fold starting from 6500 nM to 11 concentration points, and added to the wells for incubation along with biotin-mFcRn (biotin-conjugated mouse FcRn). Streptavidin (HRP) secondary antibody was then added for incubation, and finally, the plate was developed with a high-sensitivity TMB colorimetric solution for 8 minutes and the OD value was read. Softmax Pro software was used to process the data and plot the results, as shown in Figure 1.

[0307] (2) A 96-well plate was coated with 2 μg / mL IgG1-Fc. Each anti-Fc single-domain antibody (iFc 02-Chis, iFc 21-Chis, iFc 102-Chis, iFc 106-Chis, iFc 231-Chis, iFc 284S-Chis, or iFc 347-Chis) was diluted 4-fold starting from 7000 nM to 11 concentration points. The plate was then incubated with schuFcRn-biotin (biotin-conjugated single-chain human FcRn) and then incubated with streptavidin (HRP) secondary antibody. Finally, the plate was developed with a high-sensitivity TMB colorimetric solution for 5 minutes and the OD value was read. The data were processed and plotted using Softmax Pro software. The results are shown in Figure 2.

[0308] (3) The biomembrane interferometry (BLI) technique was used to detect the ability of anti-Fc single domain antibodies to block the binding of KN002 and human FcRn. KN002 is an adalimumab biosimilar with an IgG1 subtype Fc. The protein containing human FcRn, Human FCGRT&B2M (Acro biosystems, catalog number FCM-H8286), was directly immobilized on a streptavidin sensor. Then, 1000 nM of anti-Fc single domain antibody and 100 nM of KN002 were mixed, and the mixture was added to the sensor to bind to the immobilized human FcRn protein. The response value was used to determine whether the anti-Fc single domain antibody could block the binding of KN002 and human FcRn. A response value < 0.1 was considered to indicate no binding. The results are shown in Table 6. These single domain antibodies can all block the binding of KN002 (human IgG1) to human FcRn.

[0309] Table 6

[0310] Example 3 Humanization of anti-Fc single domain antibody

[0311] The humanization method uses the protein surface amino acid humanization (resurfacing) method and VHH humanization universal framework transplantation method (CDR grafting to a universal framework) to complete.

[0312] First, we obtained the universal humanized VHH framework hNbBcII10FGLA (PDB ID: 3EAK), designed by Cécile Vincke et al. based on sequence homology. This framework is based on the nanobody NbBcII10 antibody (PDB ID: 3DWT). We then used computer simulation techniques to model and calculate the relative solvent accessibility of the amino acids on the framework.

[0313] The specific steps for the universal framework transplantation method for VHH humanization are as follows: A highly homologous human antibody sequence is obtained through IMGT. The target sequence is then humanized using the universal humanized VHH framework hNbBcII10FGLA (PDB ID: 3EAK) as a reference. Using the highly homologous framework as a framework template, the CDRs are replaced with the CDR regions of the target antibody strain. Based on the modeling, non-surface amino acids on the framework are then backmutated to complete the humanization of the target antibody.

[0314] The antibody strain iFc106 was humanized to obtain humanized variants huFc106v1 and huFc106v2. The amino acid sequences of huFc106v1 and huFc106v2 are SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0315] Example 4 Identification of the Function of Humanized Anti-Fc Single Domain Antibodies

[0316] 4.1 Detection of affinity of humanized anti-Fc single domain antibody to human IgG1-Fc

[0317] The binding kinetics of humanized anti-Fc single-domain antibodies huFc106v1 and huFc106v2 to human IgG1-Fc were examined using biomembrane interferometry (BLI) technology and compared with two batches of non-humanized anti-Fc single-domain antibody iFc106-Chis. Human IgG1-Fc was directly immobilized onto an AHC sensor. The anti-Fc single-domain antibodies (huFc106v1, huFc106v2, and two batches of iFc106-Chis) were then diluted to seven concentrations and added to the AHC sensor to bind to the immobilized human IgG1-Fc. The equilibrium dissociation constant (KD), association rate (ka), and dissociation rate (kdis) were calculated using Octet data analysis software. The results are shown in Table 7. The affinity of the humanized anti-Fc single-domain antibodies for human IgG1-Fc was similar to that of their non-humanized single-domain antibodies.

[0318] Table 7

[0319] 4.2 Detection of the blocking ability of humanized anti-Fc single domain antibodies on Fc binding to FcRn

[0320] (1) The ability of humanized anti-Fc single domain antibodies huFc106v1 and huFc106v2 to block the binding of KN002 (containing IgG1-Fc) to human FcRn was detected by biomembrane interferometry (BLI) technology, and compared with the non-humanized anti-Fc single domain antibody iFc106-Chis. The human FcRn protein Human FCGRT&B2M was directly immobilized on a streptavidin sensor, and then 1000nM of the anti-Fc single domain antibody and 100nM of KN002 were mixed. The mixture was added to the streptavidin sensor to bind to the immobilized human FcRn protein. The response value was used to determine whether the anti-Fc single domain antibody could block the binding of KN002 to human FcRn. The results are shown in Table 8. Both the humanized anti-Fc single domain antibody and its corresponding non-humanized anti-Fc single domain antibody had significant blocking activity.

[0321] Table 8

[0322] (2) 96-well plates were coated with 2ug / mL IgG1-Fc or KN002, respectively. Humanized anti-Fc single-domain antibodies (huFc106v1 or huFc106v2) were diluted 3-fold from 20,000nM to 10 concentration points, added to the wells and incubated with schuFcRn-biotin, and then incubated with streptavidin (HRP) secondary antibody. The plates were then washed 3 times with PBST20 and finally developed with high-sensitivity TMB colorimetric solution to read the OD value. Softmax Pro software was used to process the data and plot the graphs. As shown in Figure 3, both humanized anti-Fc single-domain antibodies huFc106v1 and huFc106v2 could block the binding of IgG1-Fc to human schuFcRn-Chis and the binding of KN002 to human schuFcRn-Chis.

[0323] Example 5 Preparation of multivalent anti-Fc single domain antibodies

[0324] The concatenated sequences of multiple anti-Fc single-domain antibody segments were gene synthesized, with flexible linkers inserted between the segments. The resulting DNA fragments were cloned into conventional mammalian expression vectors to obtain recombinant plasmids for expressing multivalent anti-Fc single-domain antibodies in mammals. This resulted in recombinant plasmids capable of expressing the multivalent anti-Fc single-domain antibodies iFc106-C12GAP-penta (SEQ ID NO: 11) and iFc106-G4S-penta (SEQ ID NO: 12).

[0325] The recombinant plasmid obtained above was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmid was diluted with Freestyle 293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each plasmid / PEI mixture was added to the HEK293 cell suspension and placed in a 37°C, 5% CO2 suspension culture. Afterwards, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target multivalent anti-Fc single-domain antibody. The protein purity was assessed by SDS-PAGE. The results showed that the purity of each protein after purification by Protein A affinity chromatography was greater than 95%.

[0326] Example 6 Identification of the Function of Multivalent Anti-Fc Single Domain Antibodies

[0327] 6.1 Detecting the affinity of multivalent Fc single-domain antibodies to human IgG1-Fc

[0328] The binding kinetics of the iFc106-penta series to human IgG1-Fc were examined using biomembrane interferometry (BLI) and compared with the single-domain antibody iFc106-Chis. Human IgG1-Fc was directly immobilized onto an AHC sensor. Anti-Fc single-domain antibodies (iFc106-C12GAP-penta, iFc106-G4S-penta, or iFc106-Chis) were then diluted to seven concentrations and added to the AHC sensor to bind to the immobilized human IgG1-Fc. The equilibrium dissociation constant (KD), association rate (ka), and dissociation rate (kdis) were calculated using Octet data analysis software. The results are shown in Table 9. The multivalent anti-Fc single-domain antibodies exhibited higher affinity for human IgG1-Fc than the monovalent anti-Fc single-domain antibodies.

[0329] Table 9

[0330] 6.2 Detection of the Blocking Ability of Multivalent Anti-Fc Single Domain Antibodies on Fc Binding to FcRn

[0331] Biomembrane interferometry (BLI) was used to test the ability of multivalent anti-Fc single-domain antibodies to block the binding of KN002 (containing human IgG1-Fc) to human FcRn. The human FcRn protein, Human FCGRT&B2M, was directly immobilized onto a streptavidin sensor. Then, 1000 nM of the anti-Fc single-domain antibody and 100 nM of KN002 were mixed, and the mixture was allowed to bind to the immobilized human FcRn protein. The response value was used to determine whether the anti-Fc single-domain antibody could block the binding of KN002 to human FcRn. As shown in Table 10, the multivalent anti-Fc single-domain antibody significantly attenuated the binding of human IgG1 to human FcRn.

[0332] Table 10

[0333] 6.3 Detecting the in vivo efficacy of multivalent anti-Fc single-domain antibodies

[0334] (1) Thirty BALB / c mice were randomly divided into five groups of six mice each based on body weight. The day of group administration was designated D0. PBS, iFc106-C12GAP-penta 40 mg / kg and 180 mg / kg, and iFc106-G4S-penta 40 mg / kg and 180 mg / kg were administered intraperitoneally as a single dose. Serum samples were collected on D1, D2, D3, D4, D6, D7, D14, and D21 for endogenous IgG level detection. As shown in Figure 4, the multivalent anti-Fc single domain antibody significantly reduced the level of endogenous IgG in BALB / c mice.

[0335] (2) 24 CD1 mice, half male and half female, were randomly divided into 4 groups according to body weight, with 6 mice in each group. The day of group administration was marked as D0, and PBS, iFc106-C12GAP-penta 4.6 mg / kg, and ARGX-113 (prepared by referring to the sequence of efgartigimod alfa in WHO Drug Information, Vol. 31, No. 3, 2017. "Recommended INN: List 78") 3.2 mg / kg were administered intraperitoneally or subcutaneously. Serum samples were collected before administration on D0 and on D1, D2, D3, D4, and D7 after administration for endogenous IgG level detection. As shown in Figure 5, the multivalent anti-Fc single domain antibody significantly reduced the level of endogenous IgG in CD1 mice and was superior to ARGX-113 of equimolar mass.

[0336] (3) Eighteen CD1 mice, half male and half female, were randomly divided into three groups of six mice based on body weight. The day of group administration was designated as D0. PBS, iFc106-C12GAP-penta 4.6 mg / kg, and ARGX-113 3.2 mg / kg were administered subcutaneously as a single dose. Serum samples were collected before administration on D0 and on D1, D2, D3, D4, and D7 after administration for endogenous IgG levels. The results are shown in Figure 6.

[0337] Example 7 Screening of Fc variants that do not bind to anti-Fc single domain antibodies and FcRn

[0338] To obtain Fc fragments suitable for fusion with anti-Fc single-domain antibodies, human IgG1-Fc variants that do not bind to anti-Fc single-domain antibodies were screened. To prevent the Fc single-domain antibody-Fc fusion protein from recycling via FcRn after binding to IgG in vivo, thereby reducing IgG levels in the body, IgG1-Fc variants that do not bind to FcRn were also screened. Some Fc variants incorporate the S267E / L328F mutation or the E233D / G237D / P238D / H268D / P271G / A330R mutation (hereinafter abbreviated as FcEF or FcV12), which enhance Fc binding to FcγRIIb (CD32b).

[0339] (1) Identification of the binding ability of Fc variants to anti-Fc single domain antibodies

[0340] A 96-well plate was coated with 5 μg / mL iFc106-Chis. Fc and its variants were diluted to various concentrations and added to the wells for incubation. Secondary antibodies were then added for incubation. Finally, the plate was developed with a high-sensitivity TMB colorimetric solution for 30 minutes and the OD values ​​were read. The results are shown in Tables 11-13.

[0341] In Table 9, numbers + single letters are used to indicate the mutation sites and the mutated amino acids. Mutations at adjacent positions are marked with only the numbers of the starting positions. For example, "Fc-253AA-435AA" represents an Fc variant comprising the I253A / S254A / H435A / Y436A mutation combination relative to the sequence of SEQ ID NO: 109, and the same applies below.

[0342] Table 11

[0343] Table 12

[0344] Table 12 continued

[0345] Table 12 continued

[0346] Table 13

[0347] It can be seen that each Fc variant exhibits different degrees of binding ability to the anti-Fc single domain antibody.

[0348] (2) Identification of Fc variants' ability to bind to FcRn

[0349] The binding kinetics of Fc and its Fc variants to human FcRn were examined using biomembrane interferometry (BLI). Human FcRn protein was directly immobilized onto the HIS1K sensor. 10,000 nM of Fc variants were added to the immobilized human FcRn protein for binding. Binding was assessed based on the response. The results, shown in Table 14, demonstrate significantly reduced binding between the Fc variants and human FcRn.

[0350] Table 14

[0351] (3) Identification of the binding ability of Fc variants to FcγRIIb

[0352] To investigate changes in the binding ability of Fc variants to FcγRs, particularly FcγRIIb, the binding kinetics of Fc and its variants to human FcγRs were examined using biomembrane interferometry (BLI). Human FcγR protein was directly immobilized onto the HIS1K sensor. Fc and its variants were then diluted to various concentrations and allowed to bind to the immobilized human FcγR protein. Octet data analysis software was used to calculate the equilibrium dissociation constant (KD), or to assess binding affinity based on response values. The results are shown in Tables 15-17.

[0353] Table 15

[0354] Table 16

[0355] Table 17

[0356] As can be seen, after introducing the EF or V12 mutation combination, the resulting Fc variant's binding ability to CD32b (FcγRIIb) increased, while its binding to CD16a and CD32a weakened. Further addition of the disclosed mutation combination maintained the aforementioned trend in FcγR binding ability.

[0357] Example 8 Preparation of humanized anti-Fc single domain antibody-Fc fusion protein

[0358] A DNA sequence comprising the coding sequences of the humanized sequences huFc106v1 and huFc106v2 in Example 3 and a DNA fragment encoding human IgG1-Fc or its variants fused thereto was designed and obtained, and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing a humanized anti-Fc single-domain antibody-Fc fusion protein in mammals.

[0359] The constructed vector obtained above was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each group of plasmid / PEI mixtures was added to the HEK293 cell suspension and placed at 37°C, 5% CO2 for suspension culture. Afterwards, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target humanized anti-Fc single domain antibody-Fc fusion protein. The protein was examined for purity by SDS-PAGE. After one-step purification, the SDS purity of each protein was greater than 95%.

[0360] Example 9 Identification of the Function of Humanized Anti-Fc Single Domain Antibody-Fc Fusion Protein

[0361] 9.1 Detecting the affinity of humanized anti-Fc single domain antibody-Fc fusion protein to human IgG1

[0362] The binding kinetics of humanized anti-Fc single domain antibody-Fc fusion protein and KN002 (human IgG1) were detected by biomembrane interferometry (BLI) technology. KN002 protein was directly immobilized on the FAB2G sensor, and anti-Fc single domain antibody-Fc fusion protein and immobilized KN002 protein were added for binding. Octet data analysis software was used to calculate the equilibrium dissociation constant (KD), association rate (ka) and dissociation rate (kdis). The results are shown in Table 18. The affinity of the three humanized anti-Fc single domain antibody-Fc fusion proteins to human IgG1 was 10 -11 ~10 -10 M.

[0363] Table 18

[0364] Table 18 continued

[0365] The binding kinetics of humanized anti-Fc single domain antibody-Fc fusion protein and KN002 (human IgG1) under different pH conditions were detected by biomembrane interferometry (BLI) technology. KN002 protein was directly immobilized on the FAB2G sensor. At pH 6.0 or pH 7.4, anti-Fc single domain antibody-Fc fusion protein and immobilized KN002 protein were added for binding. Octet data analysis software was used to calculate the equilibrium dissociation constant (KD), association rate (ka) and dissociation rate (kdis). The results are shown in Table 19. Under acidic and neutral conditions, the affinity of the two humanized anti-Fc single domain antibody-Fc fusion proteins to human IgG1 was 10 -11 ~10 -10 M.

[0366] Table 19

[0367] 9.2 Detection of the Blocking Ability of Humanized Anti-Fc Single Domain Antibody-Fc Fusion Protein on Fc Binding to FcRn

[0368] A 96-well plate was coated with 2 μg / mL KN002 (human IgG1). A humanized anti-Fc single domain antibody-Fc fusion protein was diluted fourfold starting at 8000 nM and added to the wells along with schuFcRn-biotin for incubation. Streptavidin (HRP) secondary antibody was then added for incubation. Finally, the OD values ​​were read using a highly sensitive TMB colorimetric solution. Softmax Pro software was used to process and plot the data. As shown in Figure 7, both humanized anti-Fc single domain antibody-Fc fusion proteins exhibited significant blocking activity, outperforming the monovalent humanized anti-Fc single domain antibody.

[0369] 9.3 Detecting the affinity of humanized anti-Fc single domain antibody-Fc fusion protein and FcγR

[0370] The binding kinetics of the humanized anti-Fc single-domain antibody-Fc fusion protein to human FcγRs were examined using biomembrane interferometry (BLI). The biotinylated humanized anti-Fc single-domain antibody-Fc fusion protein was directly immobilized onto a streptavidin sensor (streptavidin sensor), and human FcγR protein was added for binding. The equilibrium dissociation constant (KD), association rate (ka), and dissociation rate (kdis) were calculated using Octet data analysis software. The results are shown in Table 20. The humanized anti-Fc single-domain antibody-Fc fusion protein significantly increased its affinity for CD32b, while its affinity for CD32a and CD64 did not change significantly.

[0371] Table 20

[0372] 9.4 Detecting the affinity of humanized anti-Fc single domain antibody-Fc fusion protein to different IgG subtypes

[0373] The binding kinetics of humanized anti-Fc single-domain antibody-Fc fusion protein to different IgG subtypes were detected using biomembrane interferometry (BLI) technology. The biotinylated humanized anti-Fc single-domain antibody-Fc fusion protein was directly immobilized on a streptavidin sensor, and 1000nM of different IgG subtypes was added for binding. KN012 is a denosumab biosimilar containing human IgG2 Fc, and KN056 is a fusion protein with human IgG4 Fc, the sequence of which is from international patent application WO2023280133. Human IgG3-Fc was purchased from Sino Biological (Cat. No. 13906-HNAH). Octet data analysis software was used to calculate the equilibrium dissociation constant (KD), and binding was determined based on the response value and KD value. The results are shown in Table 21. The humanized Fc single-domain antibody-Fc fusion protein can bind to IgG1, IgG2, and IgG4, but not to IgG3.

[0374] Table 21

[0375] 9.5 Detecting the in vivo efficacy of humanized anti-Fc single domain antibody-Fc fusion protein

[0376] (1) Twenty-four female BALB / c mice were randomly divided into four groups of six mice each based on body weight. The day of group administration was designated as D0. PBS, huFc106v1-FcEF-314A-435MD 3 mg / kg, huFc106v2-FcEF-314A-435MD 3 mg / kg, and huFc106v2-FcEF-314W-435MD 3 mg / kg were administered intraperitoneally as a single dose. Serum samples were collected before administration on D0 and on D1, D2, D3, D4, and D7 after administration for endogenous IgG level detection. As shown in Figure 8, the humanized anti-Fc single domain antibody-Fc fusion protein significantly reduced the endogenous IgG level in BALB / c mice.

[0377] (2) Thirty-six female C57BL6 mice were randomly divided into six groups according to body weight, with six mice in each group. The day of group administration was designated as D0. PBS, huFc106v2-FcEF-314A-435MD 5 mg / kg, huFc106v2-FcEF-314A-435MD 20 mg / kg, huFc106v2-FcEF-314W-435MD 5 mg / kg, huFc106v2-FcEF-314W-435MD 20 mg / kg, and Batoclimab 36 mg / kg (prepared with reference to the sequence in WHO Drug Information, Vol. 34, No. 1, 2020. “Recommended INN: List 83”) were administered intraperitoneally once. Serum samples were collected before administration on D0 and on D1, D2, D3, D4, and D7 after administration for the detection of endogenous IgG levels. As shown in FIG9 , the humanized anti-Fc single domain antibody-Fc fusion protein significantly reduced the endogenous IgG in C57BL6 mice, and the effect was better than that of equimolar Batoclimab.

[0378] (3) 48 BALB / c mice, half male and half female, were randomly divided into 7 groups according to body weight, with 6 mice in each group. The day of group administration was marked as D0, and PBS, huFc106v2-FcEF-314A-435MD 5 mg / kg, huFc106v2-FcEF-314W-435MD 5 mg / kg, and huFc106v2-Chis 2 mg / kg were administered intravenously or subcutaneously. Serum samples were collected before administration on D0 and on D1, D2, D3, D4, and D7 after administration for endogenous IgG level detection. The results are shown in Figure 10. Both humanized anti-Fc single domain antibody and Fc fusion protein have a significant effect in reducing endogenous IgG in BALB / c mice, and are superior to monovalent humanized anti-Fc single domain antibody.

[0379] Sequences used in this disclosure

[0380] iFc02(SEQ ID NO:1)

[0381] iFc21(SEQ ID NO:2)

[0382] iFc102(SEQ ID NO:3)

[0383] iFc106(SEQ ID NO:4)

[0384] iFc231(SEQ ID NO:5)

[0385] iFc284S(SEQ ID NO:6)

[0386] iFc307(SEQ ID NO:7)

[0387] iFc347(SEQ ID NO:8)

[0388] huFc106v1(SEQ ID NO:9)

[0389] huFc106v2(SEQ ID NO:10)

[0390] iFc106-C12GAP-penta(SEQ ID NO:11)

[0391] iFc106-G4S-penta(SEQ ID NO:12)

[0392] IgG1-Fc(SEQ ID NO:109)

[0393] IgG2-Fc(SEQ ID NO:110)

[0394] IgG3-Fc (SEQ ID NO: 111)

[0395] IgG4-Fc (SEQ ID NO: 112)

[0396] Single domain antibody general sequence ([] refers to the type of amino acid that can be selected at that position)

[0397] Peptide linker

[0398] (GS) n

[0399] (GG) n

[0400] (GGS) n

[0401] (SGG) n S (SEQ ID NO: 114)

[0402] (GGGS) n (SEQ ID NO: 115)

[0403] (GGSG) n (SEQ ID NO: 116)

[0404] (GGGGS) n (SEQ ID NO: 117)

[0405] (SGGGG) n (SEQ ID NO: 118)

[0406] (GGGGSGG) n (SEQ ID NO: 119)

[0407] (GGGGSGG) n (SEQ ID NO: 120)

[0408] (GGGGSGGS) n (SEQ ID NO: 121)

[0409] (GGGGSGGGS) n (SEQ ID NO: 122)

[0410] (PGGGS) n (SEQ ID NO: 123)

[0411] (AGGGS) n (SEQ ID NO:124)

[0412] (GGGSE) n (SEQ ID NO:125)

[0413] (GGGGA) n (SEQ ID NO:126)

[0414] (AP) n

[0415] (GAP) n

[0416] (EAAAK) n (SEQ ID NO:127)

[0417] (ASTKGPSVFPLAGAP) n (SEQ ID NO:128)

[0418] (ASTKGPSVFPLAGGG) n (SEQ ID NO:129)

[0419] (AKTTPKLEEGEFSEAR) n (SEQ ID NO:130)

[0420] (AKTTPKLEEGEFSEARV) n (SEQ ID NO:131)

[0421] (AKTTPKLGG) n (SEQ ID NO:132)

[0422] (ADAAP) n (SEQ ID NO:133)

[0423] (ADAAPTVSIFPP) n (SEQ ID NO:134)

[0424] (AKTTPP) n (SEQ ID NO:135)

[0425] (AKTTPPSVTPLAP) n (SEQ ID NO:136)

[0426] (AKTTAP) n (SEQ ID NO:137)

[0427] (AKTTAPSVYPLAP) n (SEQ ID NO: 138)

[0428] (ASTKGP) n (SEQ ID NO: 139)

[0429] (ASTKGPSVFPLAP) n (SEQ ID NO: 140)

[0430] Peptide linker

[0431] GS

[0432] GGGGGG (SEQ ID NO: 141)

[0433] GGGS (SEQ ID NO: 142)

[0434] GGGGS (SEQ ID NO: 143)

[0435] GGGGSGGGS (SEQ ID NO: 144)

[0436] GAP

[0437] ASTKGSVFPLAGAP(SEQ ID NO:145)

Claims

1. A single-domain antibody, which binds to an immunoglobulin Fc region, and which comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3.

2. The single domain antibody according to claim 1, wherein the single domain antibody has at least one of the following characteristics: a) a KD value for binding to human IgG Fc is less than 1×10 -7 M; b) blocking the interaction between IgG and FcRn; and c) reducing the level of immunoglobulins (such as IgG) in a mammal.

3. The single domain antibody according to claim 1 or 2, wherein the immunoglobulin Fc region is derived from IgG. The single domain antibody according to claim 3 , wherein the immunoglobulin Fc region is derived from IgG1, IgG2 or IgG4.

5. The single-domain antibody according to any one of claims 1 to 4, wherein the immunoglobulin Fc region is derived from human, mouse, rat, rabbit or monkey immunoglobulin. The single-domain antibody according to claim 5 , wherein the immunoglobulin Fc region is derived from human IgG1.

7. The single domain antibody according to any one of claims 1 to 6, wherein the single domain antibody is a camelid antibody, a humanized antibody, or a chimeric antibody.

8. The single-domain antibody according to any one of claims 1 to 7, wherein the single-domain antibody comprises CDR3 in the VHH shown in SEQ ID NO:

4.

9. The single-domain antibody according to claim 8, wherein the single-domain antibody further comprises CDR1 and CDR2 in the VHH shown in SEQ ID NO:

4.

10. The single domain antibody according to claim 7, wherein the single domain antibody comprises: (a) a CDR1 comprising the sequence of SEQ ID NO:49, a CDR2 comprising the sequence of SEQ ID NO:50; and a CDR3 comprising the sequence of SEQ ID NO:51, as determined according to the Kabat definition scheme; (b) a CDR1 comprising the sequence of SEQ ID NO:52, a CDR2 comprising the sequence of SEQ ID NO:53; and a CDR3 comprising the sequence of SEQ ID NO:54, as determined according to the AbM definition scheme; (c) a CDR1 comprising the sequence of SEQ ID NO:55, a CDR2 comprising the sequence of SEQ ID NO:56; and a CDR3 comprising the sequence of SEQ ID NO:57, as determined according to the Chothia definition scheme; or (d) a CDR1 comprising the sequence of SEQ ID NO:58, a CDR2 comprising the sequence of SEQ ID NO:59; and a CDR3 comprising the sequence of SEQ ID NO:60, as determined according to the IMGT definition scheme.

11. The single domain antibody according to any one of claims 8 to 10, wherein the single domain antibody comprises the sequence of SEQ ID NO:

4.

12. The single domain antibody according to any one of claims 8 to 10, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:

10.

13. The single domain antibody according to any one of claims 1 to 7, wherein the single domain antibody comprises: (1) CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1; (2) CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 2; (3) CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 3; (4) CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:5; (5) CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:6; (6) CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:7; or (7) CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:

8.

14. The single domain antibody according to any one of claims 1 to 7, wherein the single domain antibody comprises: (1a) a CDR1 comprising the sequence of SEQ ID NO: 13, a CDR2 comprising the sequence of SEQ ID NO: 14; and a CDR3 comprising the sequence of SEQ ID NO: 15, as determined according to the Kabat definition scheme; (1b) a CDR1 comprising the sequence of SEQ ID NO: 16, a CDR2 comprising the sequence of SEQ ID NO: 17; and a CDR3 comprising the sequence of SEQ ID NO: 18, as determined according to the AbM definition scheme; (1c) a CDR1 comprising the sequence of SEQ ID NO: 19, a CDR2 comprising the sequence of SEQ ID NO: 20; and a CDR3 comprising the sequence of SEQ ID NO: 21, as determined according to the Chothia definition scheme; or (1d) a CDR1 comprising the sequence of SEQ ID NO: 22, a CDR2 comprising the sequence of SEQ ID NO: 23; and a CDR3 comprising the sequence of SEQ ID NO: 24, as determined according to the IMGT definition scheme; (2a) a CDR1 comprising the sequence of SEQ ID NO: 25, a CDR2 comprising the sequence of SEQ ID NO: 26; and a CDR3 comprising the sequence of SEQ ID NO: 27, as determined according to the Kabat definition scheme; (2b) a CDR1 comprising the sequence of SEQ ID NO:28, a CDR2 comprising the sequence of SEQ ID NO:29; and a CDR3 comprising the sequence of SEQ ID NO:30, as determined according to the AbM definition scheme; (2c) a CDR1 comprising the sequence of SEQ ID NO:31, a CDR2 comprising the sequence of SEQ ID NO:32; and a CDR3 comprising the sequence of SEQ ID NO:33, as determined according to the Chothia definition scheme; or (2d) a CDR1 comprising the sequence of SEQ ID NO:34, a CDR2 comprising the sequence of SEQ ID NO:35; and a CDR3 comprising the sequence of SEQ ID NO:36, as determined according to the IMGT definition scheme; (3a) a CDR1 comprising the sequence of SEQ ID NO: 37, a CDR2 comprising the sequence of SEQ ID NO: 38; and a CDR3 comprising the sequence of SEQ ID NO: 39, as determined according to the Kabat definition scheme; (3b) a CDR1 comprising the sequence of SEQ ID NO:40, a CDR2 comprising the sequence of SEQ ID NO:41; and a CDR3 comprising the sequence of SEQ ID NO:42, as determined according to the AbM definition scheme; (3c) a CDR1 comprising the sequence of SEQ ID NO:43, a CDR2 comprising the sequence of SEQ ID NO:44; and a CDR3 comprising the sequence of SEQ ID NO:45, as determined according to the Chothia definition scheme; or (3d) a CDR1 comprising the sequence of SEQ ID NO:46, a CDR2 comprising the sequence of SEQ ID NO:47; and a CDR3 comprising the sequence of SEQ ID NO:48, as determined according to the IMGT definition scheme; (4a) a CDR1 comprising the sequence of SEQ ID NO:61, a CDR2 comprising the sequence of SEQ ID NO:62; and a CDR3 comprising the sequence of SEQ ID NO:63, as determined according to the Kabat definition scheme; (4b) CDR1 comprising the sequence of SEQ ID NO: 64, CDR2 comprising the sequence of SEQ ID NO: 65; and a CDR3 comprising the sequence of SEQ ID NO:66, as determined according to the AbM definition protocol; (4c) a CDR1 comprising the sequence of SEQ ID NO:67, a CDR2 comprising the sequence of SEQ ID NO:68; and a CDR3 comprising the sequence of SEQ ID NO:69, as determined according to the Chothia definition scheme; or (4d) a CDR1 comprising the sequence of SEQ ID NO: 70, a CDR2 comprising the sequence of SEQ ID NO: 71; and a CDR3 comprising the sequence of SEQ ID NO: 72, as determined according to the IMGT definition scheme; (5a) a CDR1 comprising the sequence of SEQ ID NO: 73, a CDR2 comprising the sequence of SEQ ID NO: 74; and a CDR3 comprising the sequence of SEQ ID NO: 75, as determined according to the Kabat definition scheme; (5b) a CDR1 comprising the sequence of SEQ ID NO: 76, a CDR2 comprising the sequence of SEQ ID NO: 77; and a CDR3 comprising the sequence of SEQ ID NO: 78, as determined according to the AbM definition scheme; (5c) a CDR1 comprising the sequence of SEQ ID NO:79, a CDR2 comprising the sequence of SEQ ID NO:80; and a CDR3 comprising the sequence of SEQ ID NO:81, as determined according to the Chothia definition scheme; or (5d) a CDR1 comprising the sequence of SEQ ID NO: 82, a CDR2 comprising the sequence of SEQ ID NO: 83; and a CDR3 comprising the sequence of SEQ ID NO: 84, as determined according to the IMGT definition scheme; (6a) a CDR1 comprising the sequence of SEQ ID NO: 85, a CDR2 comprising the sequence of SEQ ID NO: 86; and a CDR3 comprising the sequence of SEQ ID NO: 87, as determined according to the Kabat definition scheme; (6b) a CDR1 comprising the sequence of SEQ ID NO:88, a CDR2 comprising the sequence of SEQ ID NO:89; and a CDR3 comprising the sequence of SEQ ID NO:90, as determined according to the AbM definition scheme; (6c) a CDR1 comprising the sequence of SEQ ID NO:91, a CDR2 comprising the sequence of SEQ ID NO:92; and a CDR3 comprising the sequence of SEQ ID NO:93, as determined according to the Chothia definition scheme; or (6d) a CDR1 comprising the sequence of SEQ ID NO:94, a CDR2 comprising the sequence of SEQ ID NO:95; and a CDR3 comprising the sequence of SEQ ID NO:96, as determined according to the IMGT definition scheme; or (7a) a CDR1 comprising the sequence of SEQ ID NO: 97, a CDR2 comprising the sequence of SEQ ID NO: 98; and a CDR3 comprising the sequence of SEQ ID NO: 99, as determined according to the Kabat definition scheme; (7b) a CDR1 comprising the sequence of SEQ ID NO: 100, a CDR2 comprising the sequence of SEQ ID NO: 101; and a CDR3 comprising the sequence of SEQ ID NO: 102, as determined according to the AbM definition scheme; (7c) a CDR1 comprising the sequence of SEQ ID NO: 103, a CDR2 comprising the sequence of SEQ ID NO: 104; and a CDR3 comprising the sequence of SEQ ID NO: 105, as determined according to the Chothia definition scheme; or (7d) CDR1 comprising the sequence of SEQ ID NO: 106, CDR2 comprising the sequence of SEQ ID NO: 107; and CDR3 comprising the sequence of SEQ ID NO: 108, as determined according to the IMGT definition scheme.

15. The single domain antibody according to claim 13 or 14, wherein the single domain antibody comprises the sequence of any one of SEQ ID NOs: 1-3 and 5-8.

16. An immunoglobulin Fc region binding protein comprising 1 to 10 single domain antibodies according to any one of claims 1 to 15.

17. The immunoglobulin Fc region binding protein according to claim 16, comprising 2-8, 2-7, 2-6, 3-10, 3-8, 3-7, or 3-6 of the single domain antibodies, preferably, comprising 4-6 of the single domain antibodies, and most preferably comprising 5 of the single domain antibodies.

18. The immunoglobulin Fc region binding protein according to claim 16 or 17, wherein the single domain antibodies are identical, partially identical or different from each other. The immunoglobulin Fc region binding protein according to any one of claims 16 to 18, comprising 5 single domain antibodies that are identical to each other.

20. The immunoglobulin Fc region binding protein according to any one of claims 16 to 19, wherein at least one of the single domain antibodies comprises the VHH shown in SEQ ID NO:

4.

21. The immunoglobulin Fc region binding protein according to any one of claims 16 to 20, comprising 5 single domain antibodies comprising VHH shown in SEQ ID NO:

4.

22. The immunoglobulin Fc region binding protein according to any one of claims 17 to 21, wherein the single domain antibodies are directly connected to each other or indirectly connected via a linker.

23. The immunoglobulin Fc region binding protein according to claim 22, wherein the single domain antibodies are indirectly connected via a linker, and the linker is a polypeptide linker.

24. The immunoglobulin Fc region binding protein according to claim 23, wherein the polypeptide linker is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(SGG) n S (SEQ ID NO: 114), (GGGS) n (SEQ ID NO: 115), (GGSG) n (SEQ ID NO: 116), (GGGGS) n (SEQ ID NO: 117), (SGGGG) n (SEQ ID NO: 118), (GGGGSGG) n (SEQ ID NO: 119), (GGGGSGG) n (SEQ ID NO: 120), (GGGGSGGS) n (SEQ ID NO: 121), (GGGGSGGGS) n (SEQ ID NO: 122), (PGGGS) n (SEQ ID NO: 123), (AGGGS) n (SEQ ID NO: 124), (GGGSE) n (SEQ ID NO: 125), (GGGGA) n (SEQ ID NO: 126), (AP) n 、(GAP) n 、(EAAAK) n (SEQ ID NO:127), (ASTKGPSVFPLAGAP) n (SEQ ID NO:128), (ASTKGPSVPLAGGG) n (SEQ ID NO:129), (AKTTPKLEEGEFSEAR) n (SEQ ID NO:130), (AKTTPKLEEGEFSEARV) n (SEQ ID NO: 131), (AKTTPKLGG) n (SEQ ID NO: 132), (ADAAP) n (SEQ ID NO:133), (ADAAPTVSIFPP) n (SEQ ID NO: 134), (AKTTPP) n (SEQ ID NO:135), (AKTTPPSVTPLAP) n (SEQ ID NO: 136), (AKTTAP) n (SEQ ID NO:137), (AKTTAPSVYPLAP) n (SEQ ID NO: 138), (ASTKGP) n (SEQ ID NO: 139) and (ASTKGPSVFPLAP) n (SEQ ID NO: 140), wherein n is an integer from 1 to 10.

25. The immunoglobulin Fc region binding protein according to claim 24, wherein the polypeptide linker is selected from one or more of the following amino acid sequences: GS, GGGGGG (SEQ ID NO: 141), GGGS (SEQ ID NO: 142), GGGGS (SEQ ID NO: 143), GGGGSGGGS (SEQ ID NO: 144), GAP and ASTKGPSVFPLAGAP (SEQ ID NO: 145).

26. The immunoglobulin Fc region binding protein according to claim 25, comprising the amino acid sequence shown in SEQ ID NO: 11 or 12.

27. A fusion protein comprising the single domain antibody according to any one of claims 1 to 15 or the immunoglobulin Fc region binding protein according to any one of claims 16 to 26. The fusion protein of claim 27 , further comprising an immunoglobulin Fc region.

29. The fusion protein according to claim 28, wherein the immunoglobulin Fc region in the fusion protein is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, IgG2, IgG3 or IgG4; more preferably the Fc region of human IgG1.

30. The fusion protein according to claim 28 or 29, wherein the immunoglobulin Fc region in the fusion protein comprises a mutation that weakens the binding of the immunoglobulin Fc region in the fusion protein to the single domain antibody.

31. The fusion protein according to any one of claims 28 to 30, wherein the immunoglobulin Fc region in the fusion protein comprises a mutation that weakens the binding between the immunoglobulin Fc region in the fusion protein and FcRn.

32. The fusion protein according to claim 30 or 31, wherein the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 314.

33. The fusion protein according to claim 32, wherein the mutation at amino acid position 314 is L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W, or L314Y, preferably L314 14A, L314F, L314G, L314H, L314I, L314K, L314M, L314P, L314R, L314S, L314V, L314W or L314Y; preferably L314A, L314S, L314G, L314R, L314K, L314W or L314Y; more preferably L314A, L314R, L314S or L314W.

34. The fusion protein according to claim 30 or 31, wherein the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 311.

35. The fusion protein according to claim 34, wherein the mutation at amino acid position 311 is Q311A, Q311D, Q311E, Q311F, Q311G, Q311H, Q311I, Q311K, Q311L, Q311M, Q311N, Q311P, Q311R, Q311S, Q311T, Q311V, Q311W or Q311Y, preferably Q311D or Q311E; most preferably Q311D.

36. The fusion protein according to claim 30 or 31, wherein the immunoglobulin Fc region in the fusion protein comprises a mutation at amino acid position 253 and / or amino acid position 254.

37. The fusion protein of claim 36, wherein the mutation at amino acid position 253 and / or amino acid position 254 is I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, I253Y, S254A, S254D, S254E, S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W, S254 Y, I253A / S254A, I253G / S254A, I253A / S254G, I253E / S254D, I253E / S254E or I 253D / S254E, preferably I253A, I253D, I253E, I253G, I253N, I253Q, I253S, S254A, S2 54D, S254E, S254G, S254N, S254Q, I253A / S254A, I253G / S254A, I253A / S254G, I253E / S254D, I253E / S254E or I253D / S254E, more preferably I253A / S254A or I253E / S254D.

38. The fusion protein according to any one of claims 30-37, wherein the immunoglobulin Fc region in the fusion protein further comprises a mutation at amino acid position 435 and / or amino acid position 436.

39. according to the fusion protein described in claim 38, the mutation of the amino acid at position 435 and / or position 436 is H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W, H435Y, Y436A, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436Q, Y436R , Y436S, Y436T, Y436V, Y436W, H435A / Y436A, H435A / Y436D, H435A / Y436S, H435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E or H435M / Y436Q, preferably H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W, H435Y, Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q, Y436N, H435A / Y436A, H435A / Y436S, H435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E or H435M / Y436Q; more preferably H435A, H435S, H435G, H435M, H435L, H435Y, H435I, Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q, Y436 N, H435A / Y436A, H435A / Y436S, H435S / Y436A, H435M / Y436A, H435M / Y436D, H435M / Y436E or H435M / Y436Q, and more preferably H435A, H435S, H435M, H435A / Y436A, H435A / Y436S, H435M / Y436Q, H435M / Y436D, H435A / Y436D or H435M / Y436E.

40. The fusion protein of any one of claims 30-39, wherein the immunoglobulin Fc region in the fusion protein comprises a mutation selected from the group consisting of: L314A / H435A, L314S / H435S, L314S / H435A / Y436S, L314R / N315R, L314R / N315M, L314R / H435A, L314R / H435M, L314A / H435M, L314R / H435M / Y436Q, L314A / H435M / Y436D, I253E / S254D / H435A / Y436A , I253E / S254D / H435A / Y436D, L314R / H435M / Y436D / I253E / S254D / H435M / Y436D, I253E / S254D / H435M / Y436Q, L314W / H435M / Y436D, L314W / H435M / Y436E, L314W / H435M / Y436Q, L314A / H435M / Y436E, L314A / H435M / Y436Q, Q311D / H435M / Y436E, or Q311D / H435M / Y436D. The fusion protein according to any one of claims 30 to 40, wherein the immunoglobulin Fc region in the fusion protein further comprises a mutation that enhances the interaction between the immunoglobulin Fc region in the fusion protein and FcγRIIb.

42. The fusion protein of claim 41, wherein the mutation that enhances the interaction between the immunoglobulin Fc region and FcγRIIb in the fusion protein is one or more mutations selected from the group consisting of E233D, G237D, P238D, T265A, H268D, K290A, R255A, E258A, S267A, S267E, P271G, E272A, N276A, D280A, H285A, N286A, T307A, L309A, N315A, K326A, L328E, L328F, A330R, P331A, S337A, A378Q, E430A, H268A, R301A, R301M, K322A, K334R, and K334N.

43. The fusion protein according to claim 41, wherein the mutation that enhances the interaction between the immunoglobulin Fc region and FcγRIIb in the fusion protein is a combination of mutations selected from the following: S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233D / A330R, E233D / P238D, G 237D / P238D, P238D / H268D, P238D / P271G, P238D / A330R, E233D / P238D / A330R, E233D / P271G / A330 R, G237D / H268D / P271G, G237D / P271G / A330R, S239D / A330L / I332E, G236A / S239D / I332E, E233D / P2 38D / P271G / A330R, G237D / P238D / H268D / P271G, E233D / H268D / P271G / A330R, G237D / P238D / P271G / A330R、G237D / H268D / P271G / A330R、E233D / G237D / H268D / P271G / A330R、E233D / P238D / H268D / P2 71G / A330R, G237D / P238D / H268D / P271G / A330R or E233D / G237D / P238D / H268D / P271G / A330R, preferably the mutation that enhances the interaction between the immunoglobulin Fc region and FcγRIIb in the fusion protein is S267E / L328F or E233D / G237D / P238D / H268D / P271G / A330R.

44. The fusion protein of claim 43, wherein the immunoglobulin Fc variant in the fusion protein comprises: (a) mutations S267E / L328F, and mutations L314A / H435M / Y436D; (b) mutations S267E / L328F, and mutations L314A / H435M / Y436E; (c) mutations S267E / L328F, and mutations L314W / H435M / Y436D; (d) mutations S267E / L328F, and mutations L314W / H435M / Y436E; (e) mutations S267E / L328F, and mutations L314R / H435M / Y436Q; (f) mutations S267E / L328F, and mutations I253E / S254D / H435A / Y436D; (g) mutations S267E / L328F, and mutations I253E / S254D / H435A / Y436A; (h) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314A / H435M / Y436D; (i) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314A / H435M / Y436E; (j) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314W / H435M / Y436D; (k) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314W / H435M / Y436E; (l) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436Q; (m) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436D; or (n) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435A / Y436A.

45. A multispecific antibody comprising the single domain antibody of any one of claims 1-15, the immunoglobulin Fc region binding protein of any one of claims 16-26, or the fusion protein of any one of claims 27-44, and one or more additional antigen binding regions, wherein the one or more additional antigen binding regions bind to different antigens or different epitopes of the same antigen as the single domain antibody, the immunoglobulin Fc region binding protein or the fusion protein.

46. ​​A conjugate comprising the single domain antibody of any one of claims 1-15, the immunoglobulin Fc region binding protein of any one of claims 16-26, the fusion protein of any one of claims 27-44, or the multispecific antibody of claim 45, wherein the conjugate further comprises a molecule conjugated to the single domain antibody, the immunoglobulin Fc binding protein, the fusion protein, or the multispecific antibody.

47. A nucleic acid molecule encoding the single domain antibody of any one of claims 1-15, the immunoglobulin Fc region binding protein of any one of claims 16-26, the fusion protein of any one of claims 27-44, or the multispecific antibody of claim 45.

48. An expression vector comprising the nucleic acid molecule of claim 47 operably linked to an expression regulatory element.

49. A cell comprising the nucleic acid molecule of claim 47 and / or transformed with the expression vector of claim 48.

50. A composition comprising the single domain antibody of any one of claims 1-15, the immunoglobulin Fc region binding protein of any one of claims 16-26, the fusion protein of any one of claims 27-44, the multispecific antibody of claim 45, the conjugate of claim 46, the nucleic acid molecule of claim 47, the expression vector of claim 48, or the cell of claim 49.

51. The composition according to claim 50, which is a pharmaceutical composition, optionally comprising a pharmaceutically acceptable carrier and / or excipient.

52. A kit comprising the single domain antibody of any one of claims 1-15, the immunoglobulin Fc region binding protein of any one of claims 16-26, the fusion protein of any one of claims 27-44, the multispecific antibody of claim 45, the conjugate of claim 46, the nucleic acid molecule of claim 47, the expression vector of claim 48, the cell of claim 49, and / or the composition of claim 50 or 51.

53. A method for treating and / or preventing immunoglobulin-mediated diseases and / or symptoms, comprising administering to a subject in need thereof an effective amount of the single domain antibody of any one of claims 1-15, the immunoglobulin Fc region binding protein of any one of claims 16-26, the fusion protein of any one of claims 27-44, the multispecific antibody of claim 45, the conjugate of claim 46, the nucleic acid molecule of claim 47, the expression vector of claim 48, the cell of claim 49, and / or the composition of claim 50 or 51, preferably the immunoglobulin-mediated diseases and / or symptoms are IgG-mediated diseases and / or symptoms.

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