Relaxin or a fusion protein of an analog thereof and its pharmaceutical use

Abstract

The present disclosure relates to a fusion protein of relaxin or an analog thereof and its pharmaceutical use. Specifically, the present disclosure relates to a fusion protein comprising relaxin or an analog thereof and a serum albumin binding domain, and its pharmaceutical use for treating diseases (such as heart failure). Further, the present disclosure relates to a serum albumin binding molecule comprising an immunoglobulin single variable domain (such as VHH) capable of binding to serum albumin, having good plasma stability, and being usable for extending the plasma half-life of a drug.

Description

Technical Field

[0001] This disclosure claims the priority of a Chinese patent application (application number CN202210491720.7) filed on May 7, 2022.

[0002] This disclosure belongs to the field of biopharmaceuticals. Specifically, it relates to a fusion protein comprising relaxin or an analog thereof and a serum albumin binding domain, and its pharmaceutical use for treating diseases (such as heart failure). It also relates to a serum albumin binding molecule comprising an immunoglobulin single variable domain (such as VHH) capable of binding to serum albumin, having good plasma stability, and being usable for extending the plasma half-life of a drug.

Background Art

[0003] Relaxin was first discovered in 1926 by Frederick Hisaw as an important hormone during pregnancy, and it is a polypeptide hormone belonging to the insulin superfamily. In humans, relaxin includes seven polypeptide members, including relaxin 1 (RLN-1 or H1), relaxin 2 (RLN-2 or H2), and relaxin 3 (RLN-3 or H3). These relaxins have low homology in the primary sequence but have similar tertiary structures. Among them, relaxin 2 is the main relaxin in human circulation, encoded by the RLN2 gene. Relaxin is expressed in the form of a precursor hormone (where the A chain and the B chain are linearly linked via a C peptide), and is hydrolyzed by an endonuclease in the body to remove the C peptide, releasing mature relaxin. Naturally mature relaxin 2 contains two polypeptide chains, A and B, which are covalently bonded via an interchain disulfide bond to form a heterodimer, and the A chain further has an internal pair of disulfide bonds (Schwabe, et al. Science. 1977, 197, 914 - 915).

[0004] Relaxin 2 is a multifunctional hormone whose signal transduction is mainly realized by the receptor RXFP1 (also called LGR7). RXFP1 is a G protein-coupled receptor (GPCR) rich in leucine repeat units, and RXFP2 (also called LGR8) also binds to relaxin 2. The binding of relaxin 2 to the receptor can activate multiple cascade signals, including the activation of the cAMP and phosphoinositide 3-kinase pathways and the activation of the nitric oxide signal transduction pathway. The binding of relaxin 2 to the receptor can further inhibit the production of extracellular matrix and collagen by inhibiting the phosphorylation of Smad2 / 3. Studies have shown that relaxin 2 has physiological effects such as promoting vasodilation, anti-inflammation, reducing vascular resistance and increasing arterial compliance, promoting angiogenesis and remodeling, regulating the extracellular matrix, and anti-fibrosis, and plays a beneficial role in heart failure and myocardial fibrosis (Chiara Sassoli, et al. Current Molecular Medicine 2022, 22, 196-208).

[0005] Relaxin 2 has a very short half-life (t 1 / 2 ), and the half-life (t1 / 2) of natural relaxin 2 in the body is only a few minutes, which poses a problem for relaxin as a therapeutic agent. The already commercially available natural recombinant relaxin 2 (serelaxin) requires clinically continuous intravenous infusion treatment, which brings inconvenience to patients and also shows short efficacy. Researchers have modified relaxin 2 to improve its half-life (t 1 / 2 ), for example, introducing PEG, fatty acid chains, etc. by chemical coupling methods, or fusing proteins, polypeptides or Fc with extended half-lives by recombinant methods.

[0006] Albumin is the most abundant protein in plasma, accounting for 40% - 60% of plasma proteins. Albumin can bind to many endogenous and exogenous ligands and is a natural "multifunctional vector" in the body. Similar to immunoglobulin G (IgG), albumin has a relatively long retention time in the body. It is reported that the half-life of albumin in the human body is about 19 - 21 days (Berson, S. A. et al, J. Clin. Invest. 1953, 32, 746 - 768). The main reason is that the molecular weight of albumin is 66.5 KD and the hydrodynamic radius of its molecule exceeds the glomerular filtration threshold of 3.5 - 6 nm (Lin, J. H. Curr. Drug Metab. 2009, 10, 661 - 691). Albumin can bind to FcRn, and this binding has pH-dependent characteristics. After endocytosing and entering the endosome, the binding between the two becomes relatively strong at the acidic pH (pH 5.5 or 6.0) of the endosome, so albumin is protected from degradation by lysosomes. As FcRn is transferred to the cell surface, under neutral physiological pH conditions, albumin dissociates from FcRn to achieve recycling. This principle is similar to the recycling of IgG, and albumin and IgG bind to two independent sites on FcRn respectively and do not affect each other.

[0007] The present disclosure provides a single-domain antibody with a new structure that binds to serum albumin, which can significantly improve the in vivo half-life of a drug active ingredient. For example, after fusing the single-domain antibody that binds to serum albumin of the present disclosure with relaxin or its analog as the active ingredient to form a fusion protein, the in vivo half-life of relaxin or its analog can be significantly improved. The fusion protein provided by the present disclosure has high activity, high stability, is easily produced, and has excellent potential for clinical use.

Summary of the Invention

[0008] The present disclosure provides a serum albumin-binding molecule that includes an immunoglobulin single variable domain that specifically binds to serum albumin, has good plasma stability, and can be used to extend the plasma half-life of a drug. Serum albumin-binding molecule

[0009] The present disclosure provides a serum albumin-binding molecule.

[0010] In some embodiments, the serum albumin-binding molecule includes an immunoglobulin single variable domain, and the immunoglobulin single variable domain is any one of the following 1) to 5), namely, 1) CDR1, CDR2, and CDR3 in the amino acid sequence represented by any one of SEQ ID NOs: 6, 18 to 27, 2) CDR1, CDR2, and CDR3 in the amino acid sequence represented by SEQ ID NO: 7, 3) CDR1, CDR2, and CDR3 represented by SEQ ID NOs: 8, 9, and 10, respectively, 4) CDR1, CDR2, and CDR3 represented by SEQ ID NOs: 11, 12, and 13, respectively, and 5) CDR1 is X 1 SCX 2 G (SEQ ID NO: 64), wherein X 1 is T or N, X 2 is M or L, including CDR1, CDR2, and CDR3 described in any one of the above items, CDR2 is X 3 IYX 4 VGGSTFYX 5 X 6 SVKG (SEQ ID NO: 58), wherein X 3 is S or T, X 4 is T or M, X 5 is T or A, X 6 is N or D, CDR3 is GSPARCX 7 X 8 RDPTTFX 9 Y (SEQ ID NO: 59), wherein X 7 is R or L, X 8 is L or R, X 9 is D or N, Among them, the above X 1 ~X 9 One or more of them may be conservative amino acid mutations of the selected amino acids.

[0011] The above CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. For example, the above CDRs are defined according to the Kabat numbering system.

[0012] In some embodiments, the serum albumin-binding molecule comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises any one or more (e.g., two or three) combinations of CDR1, CDR2, and CDR3 among the above 1) to 5).

[0013] In some embodiments, the serum albumin-binding molecule comprises an immunoglobulin single variable domain, and CDR1 contained therein has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any one of the above CDR1s, and / or CDR2 contained in the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any one of the above CDR2s, and / or CDR3 contained in the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any one of the above CDR3s. In some specific embodiments, the amino acid mutations in CDR1, CDR2 and / or CDR3 are conservative substitutions.

[0014] In some embodiments, the above immunoglobulin single variable domain is derived from camel.

[0015] In some embodiments, the above immunoglobulin single variable domain is modified by any one of humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof.

[0016] In some embodiments, the framework region in the humanized immunoglobulin single variable domain is derived from the germline gene IGHV3-23 or IGHV3-66 of the human heavy chain variable region.

[0017] In some specific embodiments, the humanized immunoglobulin single variable domain contains amino acid mutations at at least one of the positions 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83, and 84 (positions based on the EU numbering system), for example, contains at least one amino acid mutation selected from 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q, or 84P, For example, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, L20V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, A23V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S74A, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, L78V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S49A, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, R83Q, A84P, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, A84P, S30L, V37F, G44E, L45R, W47G, A84P, or S30L, V37F, G44E, L45R, W47G, and contains any one combination of amino acid mutations selected from.

[0018] In some embodiments, the serum albumin-binding molecule provided by the present disclosure includes an immunoglobulin single variable domain, and the immunoglobulin single variable domain includes an amino acid sequence having at least 80% or at least 90% sequence identity with any one of SEQ ID NO: 6 or 7, or is the amino acid sequence. In some embodiments, the immunoglobulin single variable domain is derived from a camel.

[0019] In some embodiments, the serum albumin-binding molecule provided by the present disclosure includes an immunoglobulin single variable domain, and the immunoglobulin single variable domain includes an amino acid sequence represented by any one of SEQ ID NOs: 18 to 27 or having at least 80% or at least 90% sequence identity with it, or is the amino acid sequence. In some embodiments, the immunoglobulin single variable domain is humanized.

[0020] In the present disclosure, "at least 90% (sequence) identity" covers at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity, and "at least 80% (sequence) identity" covers at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity, and the ranges between any two of the above numerical values, including integers and decimals.

[0021] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding molecule provided by the present disclosure includes three complementarity determining regions CDR1, CDR2, and CDR3, and four FRs, and in the order from the amino terminus to the carboxy terminus, they are arranged as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0022] In some embodiments, the serum albumin binding molecule provided by the present disclosure is an antibody that binds to serum albumin or an antigen-binding fragment thereof, or is a complex or fusion protein containing the above antibody or its antigen-binding fragment.

[0023] In some embodiments, the antibody is a camel antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0024] In some embodiments, the antigen-binding fragment is an sdAb or a bispecific antibody or a multispecific antibody.

[0025] In some embodiments, the immunoglobulin single variable domain is a VHH.

[0026] In some embodiments, the immunoglobulin single variable domain is a humanized VHH.

[0027] In some embodiments, the immunoglobulin single variable domain is an affinity-matured VHH.

[0028] In some embodiments, a serum albumin binding molecule containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the above immunoglobulin single variable domains is provided. The above immunoglobulin single variable domains may be the same or different, and any two immunoglobulin single variable domains may be directly linked or linked via a linker.

[0029] In some embodiments, provided are serum albumin binding molecules that bind to or competitively bind to the same epitope as the immunoglobulin single variable domain of the present disclosure.

[0030] In some embodiments, provided are serum albumin binding molecules that block the binding of the immunoglobulin single variable domain of the present disclosure to serum albumin.

[0031] In some embodiments, provided are serum albumin binding molecules in which the binding to serum albumin is blocked by the immunoglobulin single variable domain of the present disclosure.

[0032] In some embodiments, the serum albumin binding molecule provided by the present disclosure further includes an Fc region of an immunoglobulin such as an Fc region selected from human IgG1, IgG2, IgG3, IgG4, or (His) 6 tag or (His) 8 tag and includes a histidine tag such as that.

[0033] The Fc region usable in the present disclosure may be derived from immunoglobulins of different isotypes such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 isotype), IgA1, IgA2, IgD, IgE, or IgM. In some embodiments, the Fc region includes the hinge region or a part of the hinge region of the constant region, and the CH2 region and the CH3 region.

[0034] In some embodiments, between the immunoglobulin single variable domain and the Fc region or the histidine tag may be linked by a linker. The linker may have a length of 1 to 20 or more amino acids and may be a non-functional amino acid sequence without a secondary or higher structure. For example, the linker is a flexible linker, for example, G 4 S (SEQ ID NO: 67), GS, GAP, (G 4 S) 2 (SEQ ID NO: 68), (G 4 S) 3 (SEQ ID NO: 69), (G 4 S)4 (SEQ ID NO: 70), (G 4 S) 5 (SEQ ID NO: 71), ASGS, G 3 S or a combination thereof. In some embodiments, the linker is GGGGSGGGS (SEQ ID NO: 72).

[0035] In some embodiments, the serum albumin-binding molecule provided by the present disclosure comprises an amino acid sequence represented by SEQ ID NO: 15 or 16 or having at least 80% or at least 90% sequence identity thereto, or is the above amino acid sequence.

[0036] In some embodiments, the immunoglobulin single variable domain in the above serum albumin-binding molecule specifically binds to serum albumin, and the above serum albumin may be rodent serum albumin (e.g., mouse serum albumin (mSA)) or primate serum albumin (e.g., cynomolgus monkey serum albumin (cySA), human serum albumin (HSA)). In some specific embodiments, the above serum albumin is HSA.

[0037] In some alternative forms, the serum albumin-binding molecule of the present disclosure may comprise any one of the above complete immunoglobulin single variable domains, or a functional portion or a variant thereof of any one of the above immunoglobulin single variable domains, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0038] In some embodiments, variants of the functional portion of the above immunoglobulin single variable domain are CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, or FR1-CDR1-FR2-CDR2-FR3-CDR3, which retain the serum albumin binding function and may be polypeptides having at least 80% or at least 90% sequence homology thereto. For example, they may be polypeptides that retain the serum albumin binding function of CDR3 and have a certain sequence homology thereto. For example, they may be polypeptides having at least 80% or at least 90% sequence homology with any one of the above CDR3s.

[0039] In some embodiments, in the serum albumin binding molecule provided by the present disclosure, the above immunoglobulin single variable domain can be covalently or non-covalently bound to any other macromolecule or small molecule compound as a vector for extending the half-life.

[0040] In some embodiments, the serum albumin binding molecule of the present disclosure includes one or more therapeutic or diagnostic agents that are covalently or non-covalently bound to the above immunoglobulin single variable domain.

[0041] In some embodiments, the above therapeutic or diagnostic agent is selected from therapeutic or diagnostic proteins, nucleic acids, and small molecule compounds.

[0042] In some embodiments, the serum albumin binding molecule provided by the present disclosure or the immunoglobulin single variable domain therein is ≤ 1×10 -7 M, for example ≤ 2×10 -8 M, ≤ 1×10 -8 M, ≤ 9×10 -9 M, ≤ 8×10 -9 M, ≤ 7×10 -9 M, ≤ 6×10 -9 M, ≤ 5×10 -9 M, ≤ 4×10 -9 M, ≤ 3×10 -9M, ≤ 2×10 -9 M, or ≤ 1×10 -9 M, or ≤ 9×10 -10 M, ≤ 8×10 -10 M, ≤ 7×10 -10 M, ≤ 6×10 -10 M, ≤ 5×10 -10 M, ≤ 4×10 -10 M, ≤ 3×10 -10 M, ≤ 2×10 -10 M, ≤ 1×10 -10 The K of M D value binds to serum albumin, such as human serum albumin.

[0043] In some embodiments, the serum albumin-binding molecule provided by the present disclosure has an affinity K for human serum albumin D value of 10 - 100 nM, such as 10 - 50 nM. The above K D value detection method is well used in this field, for example, the one provided in Example 4 of the present disclosure.

[0044] In some embodiments, the serum albumin-binding molecule provided by the present disclosure (a) ≤ 1×10 -7 M, for example, ≤ 1×10 -8 M of K D value binds to HSA, (b) in vivo and / or in vitro, no blockade of the binding between serum albumin such as HSA and FcRn such as human FcRn is detected, (c) when including a therapeutic agent or a diagnostic agent, extending the plasma half-life of the above therapeutic agent or diagnostic agent, has at least one activity selected from.

[0045] In some embodiments, the serum albumin-binding molecule provided by the present disclosure retains the advantageous properties of single-domain antibodies (e.g., VHH) and has an extended lifespan in the circulation of an individual. Therefore, such molecules can circulate in the serum of a subject for several days, reducing the frequency of treatment and the inconvenience to the subject, and reducing the treatment cost.

[0046] In some embodiments, the half-life of the serum albumin-binding molecule of the present disclosure can be controlled by the number of single variable domains that specifically bind to serum proteins present in the molecule.

[0047] In some embodiments, the serum albumin-binding molecule of the present disclosure promotes the binding of serum albumin and FcRn in vivo and / or in vitro. Fusion protein

[0048] The present disclosure provides a fusion protein comprising a serum albumin-binding domain and relaxin or an analog thereof, wherein the serum albumin-binding domain comprises at least one immunoglobulin single variable domain, and the immunoglobulin single variable domain is any one of the immunoglobulin single variable domains of the present disclosure.

[0049] In some embodiments, the immunoglobulin single variable domain is any one of the following 1) to 5), namely, 1) CDR1, CDR2 and CDR3 in the amino acid sequence represented by any one of SEQ ID NOs: 6, 18 to 27, 2) CDR1, CDR2 and CDR3 in the amino acid sequence represented by SEQ ID NO: 7, 3) CDR1, CDR2 and CDR3 represented by SEQ ID NOs: 8, 9 and 10, 4) CDR1, CDR2 and CDR3 represented by SEQ ID NOs: 11, 12 and 13, and 5) CDR1 is X 1 SCX 2 G (SEQ ID NO: 64), wherein X 1 is T or N, X 2 is M or L, comprises CDR1, CDR2 and CDR3 described in any one of the above items, CDR2 is X 3 IYX 4 VGGSTFYX 5 X 6 SVKG (SEQ ID NO: 58), wherein X 3 is S or T, X 4 is T or M,5 is T or A, X 6 is N or D, CDR3 is GSPARCX 7 X 8 RDPTTFX 9 is Y (SEQ ID NO: 59), wherein X 7 is R or L, X 8 is L or R, X 9 is D or N, wherein said X 1 ~X 9 one or more of which may be conservative amino acid mutations of the optional amino acids.

[0050] The above CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, the above CDRs are defined according to the Kabat numbering system.

[0051] In some embodiments, the above immunoglobulin single variable domain is modified by any one selected from camel-derived or humanized, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation and / or reduction of antibody isomerization, or a combination thereof.

[0052] In some embodiments, the framework region in the above humanized immunoglobulin single variable domain is derived from the germline gene IGHV3-23 or IGHV3-66 of the human heavy chain variable region.

[0053] In some specific embodiments, the above humanized immunoglobulin single variable domain contains amino acid mutations at at least one of the positions 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83 and 84 (positions based on the EU numbering system), for example, contains at least one amino acid mutation selected from 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q or 84P, for example, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, L20V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, A23V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S74A, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, L78V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S49A, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, R83Q, A84P, F27N, F29Y, S30L, V37F, G44E, L45R, W47G, A84P, S30L, V37F, G44E, L45R, W47G, A84P, or S30L, V37F, G44E, L45R, W47G, comprises any one combination of amino acid mutations selected therefrom.

[0054] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence represented by any one of SEQ ID NOs: 6, 18 - 27, or has at least 80%, at least 90% sequence identity with it, or is the amino acid sequence.

[0055] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence represented by SEQ ID NO: 7, or has at least 80%, at least 90% sequence identity with it, or is the amino acid sequence.

[0056] In some embodiments, the immunoglobulin single variable domain is a VHH, for example, a VHH derived from a camel or a humanized VHH.

[0057] In some embodiments, relaxin or an analog thereof in the fusion protein comprises an A chain and a B chain.

[0058] In some embodiments, the A chain has an amino acid sequence represented by any one of SEQ ID NOs: 31, 50, 52, 54, 56, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 31, 50, 52, 54, 56, or an amino acid sequence having one or more amino acid additions, deletions, insertions, or substitutions with respect to any one of SEQ ID NOs: 31, 50, 52, 54, 56. For example, the A chain may have 1, 2, 3, or 4 amino acids deleted from the N-terminus of SEQ ID NO: 31 or 50, or 1, 2, 3, or 4 amino acids added.

[0059] In some embodiments, the B chain has an amino acid sequence represented by any one of SEQ ID NOs: 32, 51, 53, 55, 57, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 32, 51, 53, 55, 57, or an amino acid sequence having one or more amino acid additions, deletions, insertions, or substitutions with respect to any one of SEQ ID NOs: 32, 51, 53, 55, 57. For example, the B chain may have 1 amino acid deleted from the N-terminus of SEQ ID NO: 51.

[0060] In some embodiments, the A chain and the B chain of the relaxin or an analog thereof are any of the above combinations.

[0061] In some embodiments, the relaxin or an analog thereof is any of the following 1) to 7), namely, 1) an A chain represented by SEQ ID NO: 31 and a B chain represented by SEQ ID NO: 32, 2) an A chain represented by SEQ ID NO: 50 and a B chain represented by SEQ ID NO: 51, 3) an A chain represented by SEQ ID NO: 52 and a B chain represented by SEQ ID NO: 53, 4) an A chain represented by SEQ ID NO: 54 and a B chain represented by SEQ ID NO: 55, 5) an A chain represented by SEQ ID NO: 31 and a B chain represented by SEQ ID NO: 53, 6) The A chain represented by SEQ ID NO: 52, the B chain represented by SEQ ID NO: 32, and 7) The A chain represented by SEQ ID NO: 56 and the B chain represented by SEQ ID NO: 57, contains any one of the combinations of the A chain and the B chain.

[0062] > Human RLN2 A chain QLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 31) > Human RLN2 B chain (delB1) SWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 32) > Human RLN1 A chain PYVALFEKCCLIGCTKRSLAKYC (SEQ ID NO: 50) > Human RLN1 B chain VAAKWKDDVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 51) > Human RLN2 A chain (delA1-4) ALANKCCHVGCTKRSLARFC (SEQ ID NO: 52) > Human RLN2 B chain DSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 53) > Human RLN3 A chain DVLAGLSSSCCKWGCSKSEISSLC (SEQ ID NO: 54) > Human RLN3 B chain RAAPYGVRLCGREFIRAVIFTCGGSRW (SEQ ID NO: 55) > Human RLN2 A chain (Z 10 LYS) X 10 LYSALANKCCHVGCTKRSLARFC, wherein X 10 is Q or D (SEQ ID NO: 56) > Human RLN2 B chain (mutation from E to D) DSWMEEVIKLCGRDLVRAQIAICGMSTWS (SEQ ID NO: 57).

[0063] In some embodiments, the fusion protein comprises one of the above immunoglobulin single variable domains and one of the above relaxin or an analog thereof. In some embodiments, the fusion protein comprises one of the above immunoglobulin single variable domains and two of the above relaxin or an analog thereof. In some embodiments, the fusion protein comprises two of the above immunoglobulin single variable domains and one of the above relaxin or an analog thereof. In some embodiments, the fusion protein comprises two of the above immunoglobulin single variable domains and two of the above relaxin or an analog thereof.

[0064] In some embodiments, the fusion protein is represented by Formula (I) to Formula (IV), that is, B-L 1 -A-L 2 -VHH Formula (I) A-L 1 -B-L 2 -VHH Formula (II) VHH-L 2 -B-L 1 -A Formula (III), and VHH-L 1 -A-L 1 -B Formula (IV) is represented by any one of them or contains a polypeptide of any combination, wherein A is any one of the above relaxin A chains, B is any one of the above relaxin B chains, and L 1 and L 2 are linkers, and each may or may not exist independently.

[0065] In some embodiments, L 1 comprises an amino acid sequence selected from (GGGGQ)n (SEQ ID NO: 73), (GGGQ)n, (GGGGS)n, (PGPQ)n, and (PGPA)n, where n is selected from integers of 1 to 10, for example, 3, 4, 5, 6, 7, 8.

[0066] In some embodiments, L 1It contains an amino acid sequence represented by any one of SEQ ID NOs: 33 to 35 or having at least 90% sequence identity thereto.

[0067] In some embodiments, L 2 is (G X S Y ) Z and contains the amino acid sequence thereof, where X, Y, and Z are independently selected from integers from 1 to 10. For example, L 2 is (GGGGS)z, where Z is selected from integers from 1 to 10, such as 3, 4, 5, 6, 7, 8.

[0068] In some embodiments, L 2 contains an amino acid sequence represented by SEQ ID NO: 49 or having at least 90% sequence identity thereto.

[0069] In some embodiments, L 2 is any one selected from KR, KRKPTGYGSRKKR (SEQ ID NO: 65), KRKPTGYGSRKR (SEQ ID NO: 66), KRGGGPRR (SEQ ID NO: 60), KRGGGPKR (SEQ ID NO: 61), KRKPTGYGSKR (SEQ ID NO: 62), KRSLKR (SEQ ID NO: 63) or contains an amino acid sequence having at least 90% sequence identity thereto.

[0070] In some embodiments, the fusion protein is a single-chain protein.

[0071] In some embodiments, the fusion protein is a multimer, for example, a dimer protein.

[0072] Exemplary embodiments are shown below.

[0073] A fusion protein comprising a serum albumin binding domain and relaxin or an analog thereof, wherein the serum albumin binding domain comprises an immunoglobulin single variable domain, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 represented by SEQ ID NOs: 8, 9 and 10, or CDR1, CDR2 and CDR3 represented by SEQ ID NOs: 11, 12 and 13, and the relaxin or an analog thereof comprises an A chain and a B chain represented by SEQ ID NOs: 31, 32, or an A chain and a B chain represented by SEQ ID NOs: 50, 51, or an A chain and a B chain represented by SEQ ID NOs: 52, 53, or an A chain and a B chain represented by SEQ ID NOs: 54, 55, or an A chain and a B chain represented by SEQ ID NOs: 31, 53, or an A chain and a B chain represented by SEQ ID NOs: 52, 32, or an A chain and a B chain represented by SEQ ID NOs: 56, 57.

[0074] A fusion protein comprising a serum albumin binding domain and relaxin or an analog thereof, wherein the serum albumin binding domain comprises an immunoglobulin single variable domain, the immunoglobulin single variable domain comprises an amino acid sequence represented by any one of SEQ ID NOs: 6, 18 - 27 or having at least 90% sequence identity therewith, or an amino acid sequence represented by SEQ ID NO: 7 or having at least 90% sequence identity therewith, and the relaxin or an analog thereof comprises an A chain and a B chain represented by SEQ ID NOs: 31, 32, or an A chain and a B chain represented by SEQ ID NOs: 50, 51, or an A chain and a B chain represented by SEQ ID NOs: 52, 53, or an A chain and a B chain represented by SEQ ID NOs: 54, 55, or an A chain and a B chain represented by SEQ ID NOs: 31, 53, or an A chain and a B chain represented by SEQ ID NOs: 52, 32, or an A chain and a B chain represented by SEQ ID NOs: 56, 57.

[0075] In some embodiments, provided is a fusion protein comprising an amino acid sequence represented by any one of SEQ ID NOs: 36 - 43 or having at least 90% sequence identity therewith.

[0076] In some embodiments, the binding affinity of the fusion protein of the present disclosure for the RXFP1 receptor is equivalent to the binding affinity of native human relaxin 2 (SEQ ID NOs: 31 and 53). In some other embodiments, the binding affinity of the fusion protein of the present disclosure for the RXFP1 receptor is less than the binding affinity of native human relaxin 2 (SEQ ID NOs: 31 and 53). In some other embodiments, the binding affinity of the fusion protein of the present disclosure for the RXFP1 receptor is greater than the binding affinity of native human relaxin 2 (SEQ ID NOs: 31 and 53).

[0077] In some embodiments, the fusion protein of the present disclosure has a significantly increased in vivo half-life (t 1 / 2 ) equivalent to that of serum albumin in the body, for example, ranging from a dozen hours to a dozen days in different species, compared to native human relaxin 2 (SEQ ID NOs: 31 and 53).

[0078] In some embodiments, the fusion protein of the present disclosure binds to HSA with a K -7 value of ≤ 1×10 -8 M, for example, ≤ 1×10 D M.

[0079] In some embodiments, the fusion protein of the present disclosure does not block the binding of serum albumin to FcRn in vivo and / or in vitro.

[0080] The fusion protein of the present disclosure covers its pharmaceutically acceptable salts. Polynucleotide and vector

[0081] In some embodiments, a polynucleotide encoding the serum albumin-binding molecule of the present disclosure is provided.

[0082] In some embodiments, a polynucleotide encoding the fusion protein of the present disclosure is provided.

[0083] The above polynucleotide may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the polynucleotide of the present disclosure is an isolated polynucleotide.

[0084] The polynucleotide of the present disclosure may be in the form of a vector, may be present in a vector, and / or may be part of a vector, which vector is, for example, a plasmid, cosmid, YAC, or viral vector. The vector may particularly be an expression vector, i.e., a vector that enables the expression of the serum albumin-binding molecule in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector usually contains at least one polynucleotide of the present disclosure, which is operably linked to one or more appropriate expression regulatory elements (e.g., promoter, enhancer, terminator, etc.). Selecting the above elements and their sequences for expression in a specific host is common general knowledge in the art. Regulatory elements and other elements useful or essential for the expression of the serum albumin-binding molecule of the present disclosure are, for example, a promoter, enhancer, terminator, integration factor, selection marker, leader sequence, reporter gene.

[0085] The polynucleotide of the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA technology) based on the information about the amino acid sequence of the polypeptide of the present disclosure, and / or may be isolated from an appropriate natural source. Host cell

[0086] In some embodiments, a host cell that expresses or is capable of expressing one or more serum albumin-binding molecules of the present disclosure, and / or a recombinant host cell containing the polynucleotide or vector of the present disclosure is provided.

[0087] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0088] Bacterial cells include, for example, cells of Gram-negative strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0089] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0090] Exemplarily, mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0091] The present disclosure may use amphibian cells, insect cells, plant cells, and any other cells for expressing heterologous proteins in this field.

[0092] The cells of the present disclosure cannot develop into a complete plant or animal individual. Production or preparation method

[0093] The present disclosure provides a method for preparing the serum albumin-binding molecule or fusion protein of the present disclosure, which - culturing the host cells of the present disclosure under conditions that enable the expression of the serum albumin-binding molecule or fusion protein of the present disclosure; - recovering the serum albumin-binding molecule or fusion protein expressed in the host cells from the culture; - optionally, further purifying and / or modifying the serum albumin-binding molecule or fusion protein of the present disclosure; and comprising.

[0094] The serum albumin-binding molecule or fusion protein of the present disclosure may be produced in an intracellular manner (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, then isolated from the host cells, and optionally further purified, or may be produced in an extracellular manner (e.g., in the medium in which the host cells are cultured), then isolated from the medium, and optionally further purified.

[0095] Methods and reagents for producing recombinant polypeptides or proteins, such as certain suitable expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, culture conditions, etc. are known in the art. Similarly, techniques for isolating and purifying target proteins applicable to the production of binding molecules or antibodies according to the present disclosure are known to those skilled in the art. Methods for producing and purifying antibodies are well known in the prior art and can be found, for example, in the Antibody Engineering Laboratory Manual of Cold Spring Harbor (Chapters 5-8 and 15). The engineered antibodies of the present disclosure may be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains may be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can stably transfect cells. Mammalian expression systems will cause glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones can be obtained by expressing antibodies specific for human antigens. Positive clones are expanded in a serum-free medium in a bioreactor to produce antibodies. The culture broth in which the antibody is secreted can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and multimers may be removed by conventional methods such as molecular sieving and ion exchange. The resulting product must be immediately frozen at -70°C etc. or lyophilized.

[0096] However, the serum albumin binding molecules or fusion proteins of the present disclosure may also be obtained by other methods of producing proteins known in the art, such as chemical synthesis including solid-phase or liquid-phase synthesis. Composition

[0097] In some embodiments, the composition is a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of the serum albumin-binding molecule or fusion protein of the present disclosure as described above, and / or a polynucleotide encoding the serum albumin-binding molecule or fusion protein, and one or more pharmaceutically acceptable vectors, diluents, buffers or excipients.

[0098] In some specific embodiments, the unit dose of the pharmaceutical composition may contain 0.01-99% by weight of the serum albumin-binding molecule or fusion protein. In some other specific embodiments, the amount of the serum albumin-binding molecule or fusion protein contained in the unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments it is 1-1000 mg. Reagent kit and detection

[0099] The present disclosure provides a reagent kit or product comprising any serum albumin-binding molecule of the present disclosure as described above and / or a nucleic acid molecule encoding the serum albumin-binding molecule of the present disclosure.

[0100] The present disclosure provides a composition for detecting serum albumin, the composition comprising the serum albumin-binding molecule of the present disclosure. The present disclosure further provides a method, system or device for detecting serum albumin in vivo or in vitro, which includes using the serum albumin-binding molecule of the present disclosure.

[0101] In some embodiments, the in vitro detection method, system or device may include, for example, (1) contacting a sample with the serum albumin-binding molecule of the present disclosure, (2) detecting a complex formed between the serum albumin-binding molecule of the present disclosure and the sample, and / or (3) contacting a reference sample (e.g., a control sample) with an antibody, and (4) comparing with the reference sample to confirm the degree of complex formation between the antibody and the sample. For example, a change in complex formation (e.g., a statistically significant change) in the sample or subject compared to that in the control sample or subject indicates the presence of serum albumin in the sample.

[0102] In some other embodiments, the in vivo detection method, system or device may include: (1) administering the serum albumin binding molecule of the present disclosure to a subject; and (2) detecting the complex formation between the serum albumin binding molecule of the present disclosure and the subject. The detection may include identifying the location or time of complex formation. The antibody that binds to the serum albumin binding molecule may be directly or indirectly labeled with a detectable substance to facilitate the detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. By measuring or visualizing the antibody that binds or does not bind to the serum albumin binding molecule, the complex formation between the serum albumin binding molecule of the present disclosure and serum albumin can be detected. Conventional detection assays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry, can be used. In some embodiments, the presence of serum albumin in a sample is analyzed by a competitive immunoassay, which uses a standard substance labeled with a detectable substance and the serum albumin binding molecule of the present disclosure that is not labeled. The biological sample to be detected or measured may be tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.

[0103] In some embodiments, for the purpose of detection, the serum albumin binding molecule of the present disclosure may be labeled with a fluorophore or a chromophore. Method for treating and preventing diseases and pharmaceutical use

[0104] The present disclosure provides a method for prolonging the plasma half-life of a therapeutic or diagnostic agent, which includes covalently or non-covalently binding the serum albumin binding molecule of the present disclosure or its immunoglobulin single variable domain to the therapeutic or diagnostic agent. The methods for conjugation are known in the art or may be any future methods, such as fusing it by chemical coupling, fusing at the DNA level, fusing at the mRNA level, or fusing at the protein level.

[0105] In some embodiments, the therapeutic or diagnostic molecule may be a therapeutic target or a diagnostic target. The target may be any protein, peptide, nucleic acid, oligonucleic acid, sugar, polysaccharide, glycoprotein for therapy or diagnosis. Examples include, but are not limited to, receptors, receptor ligands, viral coat proteins, immune system proteins, hormones, enzymes, antigens, cell signaling proteins or fragments thereof.

[0106] In some embodiments, the plasma half-life of the therapeutic or diagnostic agent linked to the serum albumin-binding molecule or its immunoglobulin single variable domain of the present disclosure is at least 1.5-fold, preferably at least 2-fold, for example at least 5-fold, for example at least 10-fold or 20-fold or more improved compared to before linkage. For example, the increased plasma half-life may exceed 1 hour compared to before linkage, preferably exceed 2 hours, more preferably exceed 6 hours, for example exceed 12 hours, or even exceed 24, 48 or 72 hours.

[0107] The present disclosure provides a method for treating and / or preventing a disease, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of the serum albumin-binding molecule or fusion protein of the present disclosure.

[0108] The present disclosure further provides a method of a serum albumin-binding molecule or fusion protein for treating and / or preventing a disease, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of the serum albumin-binding molecule or fusion protein of the present disclosure.

[0109] The present disclosure further provides the use of a serum albumin-binding molecule in the preparation of a medicament for treating and / or preventing a disease.

[0110] In some embodiments, the disease is a fibrotic disease or a cardiovascular disease.

[0111] In some specific embodiments, the cardiovascular disease is heart failure (HF), myocardial hypertrophy.

Brief Description of the Drawings

[0112]

Figure 1

Figure 2

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Figure 4

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Figure 6

Figure 7

Figure 8

Modes for Carrying Out the Invention

[0113] Definitions To facilitate a better understanding of the present disclosure, several technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art.

[0114] The three-letter and one-letter codes for amino acids used in the present disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0115] "Antibody" or "immunoglobulin" is used as a general term that refers to a heavy-chain antibody, or a normal tetrapeptide-chain antibody in which two heavy chains and two light chains are linked by interchain disulfide bonds, and includes full-length antibodies, their individual chains, and all of their parts, domains or fragments (including, but not limited to, antigen-binding domains or fragments such as VHH domains or VH / VL domains).

[0116] "Sequence" (as in terms such as "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include not only the relevant amino acid sequence, but also the nucleic acid sequence or nucleotide sequence encoding the above sequence, unless further limited interpretation is required in the present disclosure.

[0117] The "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of other parts of the protein. Generally, a domain bears a single functional property of the protein and can often be added, removed, or transferred to other proteins without loss of the function of other parts and / or domains of the protein.

[0118] "Immunoglobulin domain" refers to the globular region of an antibody chain.

[0119] The "immunoglobulin variable domain" basically consists of "framework region 1" (FR1), "framework region 2" (FR2), "framework region 3" (FR3), and "framework region 4" (FR4), and "complementary determining region 1" (CDR1), "complementary determining region 2" (CDR2), and "complementary determining region 3" (CDR3). Therefore, the general structure (or sequence) of the variable domain may be shown as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Since the variable domain has an antigen-binding site, specificity for the antigen is conferred.

[0120] The "antibody framework (FR)" refers to a part of the variable domain that is used as a stent for the antigen-binding loops (CDRs) of the variable domain.

[0121] An "immunoglobulin single variable domain" is usually used to refer to an immunoglobulin variable domain (which may be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site when it does not interact with other variable domains (for example, when there is no VH / VL interaction required between the VH and VL domains of a normal four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including camelized VH such as VHH, humanized VHH, and / or camelized human VH), IgNAR, domains, antibodies as VH domains or derived from VH domains (single domain antibodies, e.g., dAbs TM ) and antibodies as VL domains or derived from VL domains (single domain antibodies, e.g., dAbs TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are usually preferred. A specific example of an immunoglobulin single variable domain is the "VHH domain" (or abbreviated as "VHH") defined as follows.

[0122] The "VHH domain" is a heavy chain single domain antibody, VHH, V HHA domain, also called a VHH antibody fragment, VHH antibody, or nanobody, 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 VH and VL present in antibodies with a normal tetrapeptide chain structure. The VHH domain specifically binds to an epitope without requiring other antigen-binding domains (for the VH or VL domain in an antibody with a normal tetrapeptide chain structure, the epitope is recognized by both the VL domain and the VH domain). The VHH domain is a small, stable, and efficient antigen recognition unit formed by a single immunoglobulin domain. "Heavy-chain single-domain antibody", "VHH domain", "VHH", "V HHThe terms "domain", "VHH antibody fragment", "VHH antibody", "Nanobody®" and "Nanobody® domain" (where "Nanobody" is a trademark of Ablynx N.V., Ghent, Belgium) may be used interchangeably. The "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, may be humanized after antibody production by camelids, or may be screened by phage display technology. The total number of amino acid residues in the VHH domain is usually in the range of 110 to 120, and often between 112 and 115. However, it should be noted that relatively small and relatively long sequences can also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the literature of 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.

[0123] As is known in the art for VH and VHH domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions based on Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering). This generally means that Kabat-based numbering may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or rules include Chothia, IMGT, AbM.

[0124] "Humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the framework of the human antibody variable region. It can overcome the strong immune response induced by chimeric antibodies having a large amount of non-human protein components. In order to avoid the reduction in activity associated with the decrease in immunogenicity, the activity can be maintained by introducing the least number of back mutations to the above-mentioned complete human antibody variable region. Examples of "humanization" include the replacement of 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 corresponding positions in the VH domain of the normal human tetrapeptide chain structure antibody, so that it can be "humanized". The humanized VHH domain may include one or more complete human framework region sequences, and in some specific embodiments, it may include the human framework region sequence of IGHV3. The humanization method is, for example, the antibody humanization universal framework transplantation method (CDR grafting to a universal framework) of "resurfacing" the amino acids on the protein surface and "transplanting" the CDR to other "stents" (including but not limited to human stents or non-immunoglobulin stents). The stents and techniques suitable for the above CDR transplantation are known in the art. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the VBase human germline sequence database and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies of the present disclosure further include humanized antibodies with affinity maturation for the CDR presented by phage. Also, in order to avoid the reduction in activity associated with the decrease in immunogenicity, the activity can be maintained by introducing the least number of back mutations or revertant mutations to the above-mentioned human antibody variable region framework sequence.

[0125] One of ordinary skill in the art can determine the amino acid sequence boundaries of antibody CDRs by any one of a number of known numbering schemes, which include the numbering schemes described in Kabat et al., supra (the "Kabat" numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme), MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme), Lefran et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme) and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), which are incorporated by reference in their entireties, respectively.

[0126] The term "epitope" or, interchangeably, "antigenic determinant" refers to any antigenic determinant in an antigen to which an antibody binds. Antigenic determinants usually include chemically active surface groups of molecules such as, for example, amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and / or specific charge characteristics. For example, an epitope usually contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation and may be a "linear" epitope or a "conformational" epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996). In a linear epitope, all of the interaction sites between the antigen and the interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the antigen. In a conformational epitope, the interaction sites exist across amino acid residues that are separated from each other. Many epitope mapping techniques well known in the art can be used to identify the epitopes of a given antigen. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996). For example, a linear epitope can be determined by a method such as simultaneously synthesizing a large number of peptides corresponding to each part of a protein molecule on a solid support and reacting these peptides with an antibody while still bound to the support. These techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871, Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002, Geysen et al., (1986) Molec. Immunol. 23:709-715. A conformational epitope may be identified, for example, by determining the spatial arrangement of amino acids by X-ray crystallography and two-dimensional nuclear magnetic resonance. Antibodies against binding to the same epitope can be competitively screened using conventional techniques known to those skilled in the art.For example, by performing competition and cross-competition studies, antibodies that compete or cross-compete with each other to bind to an antigen can be obtained. A high-throughput method for obtaining antibodies that bind to the same epitope by such cross-competition is described in International Patent Application WO03 / 48731. Therefore, using the ordinary techniques known to those skilled in the art, antibodies and antigen-binding fragments thereof that compete with the antibody molecules according to the present disclosure and bind to the same epitope in serum albumin can be obtained.

[0127] Generally, the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein (e.g., the serum albumin-binding molecule of the present disclosure) can bind. The specificity can be confirmed based on the affinity and / or avidity of the antigen-binding molecule. The dissociation equilibrium constant (K D ) of the antigen and the antigen-binding protein indicates a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein. The smaller the K D value, the stronger the binding strength between the epitope and the antigen-binding protein (or the affinity may be indicated by the binding constant (K D ) which is 1 / K a ). As is known to those skilled in the art, for specific antigens of interest, the affinity can be measured by known methods. Affinity is a measure of the binding strength between an antigen-binding protein (e.g., an immunoglobulin, antibody, single variable domain of an immunoglobulin, or a polypeptide containing the same) and a related antigen. Affinity is related to the affinity for the antigen-binding site on the antigen-binding protein and the number of related binding sites present on the antigen-binding protein.

[0128] "Serum albumin binding domain" refers to any polypeptide capable of specifically binding to serum albumin or an epitope thereof. "Serum albumin binding molecule" refers to a molecule capable of specifically binding to serum albumin or an epitope thereof, including, but not limited to, proteins and polypeptides. Serum albumin binding molecules include antibodies or antigen-binding fragments thereof as defined in the present disclosure against serum albumin or an epitope thereof, or may include complexes of the above antibodies, antigen-binding fragments thereof, or fusion proteins. The antigen-binding fragment is, for example, an sdAb or a bispecific antibody, a multispecific antibody. The serum albumin binding molecule of the present disclosure may include at least one (e.g., two, three, four or more) immunoglobulin single variable domains (e.g., VHHs) that bind to serum albumin. In addition to the immunoglobulin single variable domain, the serum albumin binding molecule of the present disclosure may also include a linker and / or a portion having the function of an effector molecule, and the effector molecule includes, but is not limited to, a diagnostic agent or a therapeutic agent, such as an anti-tumor agent, an immunomodulator, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0129] An "affinity matured" serum albumin antibody (e.g., VHH) has one or more changes in one or more CDRs which increase its affinity for serum albumin compared to the parent anti-serum albumin antibody. Affinity matured anti-serum albumin antibodies can be prepared, for example, by the methods described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813., Shier et al., 1995, Gene 169:147-155, Yelton et al., 1995, Immunol. 155:1994-2004, Jackson et al., 1995, J. Immunol. 154(7):3310-9, and Hawkins et al., 1992, J. MoI. Biol. 226(3):889896, KS Johnson & RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press. 1996.

[0130] "Back mutation" refers to the mutation of amino acid residues in the FR region derived from a human antibody to amino acid residues at the corresponding positions in the original antibody. In order to avoid the reduction in activity that usually occurs along with the reduction in immunogenicity of a humanized antibody, the activity of the antibody can be maintained by performing the minimum number of back mutations on the variable region of the humanized antibody.

[0131] Typically, the serum albumin binding molecules of the present disclosure have a binding affinity of preferably 10 -7 ~10 -10 moles / liter (M), more preferably 10 -8 ~10 -10 moles / liter, and even more preferably 10 -9 ~10 -10 or less dissociation constant (K D ), and / or at least 10 -7M, preferably at least 10 -8 M, more preferably at least 10 -9 M, even more preferably at least 10 -10 The binding constant (K a ) binds to the antigen to be bound (i.e., serum albumin). Any K -4 value greater than 10 D is generally considered to exhibit non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any suitable known method, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (ELISA), and sandwich competitive assays) described in the present disclosure.

[0132] The terms "inhibit" or "block" may be used interchangeably and cover both partial and complete inhibition / blocking.

[0133] In the present disclosure, "homology" and "identity" refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two sequences being compared are each occupied by the same base or amino acid monomer subunit, for example, when each position of two DNA molecules is occupied by adenine, the molecules are identical at that position. The percentage homology between two sequences is a function of dividing the number of matching or identical positions shared by the two sequences by the number of positions being compared and multiplying by 100. For example, when performing optimal alignment of sequences, if 6 out of 10 positions in the two sequences match or are identical, the two sequences are 60% homologous, and if 95 out of 100 positions in the two sequences match or are identical, the two sequences are 95% homologous. Generally, the two sequences are compared when aligned to obtain the maximum percentage homology.

[0134] "Conservative amino acid mutation" or "conservative (amino acid) substitution" means that one or more amino acid residues of a protein or polypeptide are subjected to conservative amino acid substitutions, and the chemical structures of the amino acid residues before and after substitution are similar, and the influence of the substitution on the function, activity and other biological characteristics of the protein or polypeptide is relatively small or basically none. The above conservative amino acid substitutions are known in the art. For example, in the conservative amino acid substitution, it is preferred that one amino acid in the following groups (i) to (v) is substituted by another amino acid residue in the same group: (i) Relatively small 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) Relatively large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys, and (v) Aromatic residues: Phe, Tyr and Trp.

[0135] Particularly preferably, in the conservative amino acid substitution, 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, and Val is substituted by Ile or Leu.

[0136] "Nucleic acid", "nucleic acid molecule" or "polynucleotide" may be used interchangeably in the present disclosure, and refers to any single-stranded or double-stranded DNA molecule or RNA molecule, and in the case of single-stranded, the molecule of its complementary sequence, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is said to be "effectively linked". For example, when a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the above coding sequence.

[0137] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA ring to which other DNA segments can be linked. In another embodiment, the vector is a viral vector, among which another DNA segment can be linked to the viral genome. The vectors in the present disclosure can self-replicate in the host cells into which they are introduced (for example, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or after being introduced into the host cells, they can be integrated into the genome of the host cells and replicated together with the host genome (for example, non-episomal mammalian vectors).

[0138] The expressions "cell", "cell line" and "cell culture" used in the present disclosure may be used interchangeably, and these names all include progeny. Therefore, "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of passages. Also, it should be understood that due to intentional or unintentional mutations, all progeny may not be exactly the same in DNA content. Mutant progeny having the same function or biological activity as those screened from the original transformed cells are included. When referring to different names, it will be clear from the context.

[0139] "Host cell" includes each cell or cell culture, which may be a receptor for a vector for integrating a nucleic acid insert fragment or already be such. A host cell includes the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny are not necessarily exactly the same as the original parental cell (in terms of morphology or genomic DNA complement). A host cell includes cells transfected and / or transformed in vivo by the nucleic acids according to the present disclosure. "Cell", "cell line" and "cell culture" may be used interchangeably, and all such names include their progeny. Also, it should be understood that due to intentional or unintentional mutations, all progeny may not be exactly the same in DNA content. Mutant progeny having the same function or biological activity as those screened from the first transformed cell are included.

[0140] "Pharmaceutical composition" refers to a mixture containing one or more of the fusion proteins, serum albumin-binding molecules, polynucleotides and other components described in the present disclosure, and the other components are, for example, physiologically / pharmaceutically acceptable vectors, diluents, buffers or excipients. A pharmaceutical composition is for promoting administration to a living body, contributing to the absorption of the active ingredient and further exerting biological activity.

[0141] "Administer", "administration" and "treatment", when used in relation to an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, the human, the subject, the cell, the tissue, the organ or the biological fluid. "Administer", "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes the contact of a reagent with the cell and the contact of the reagent with a fluid, wherein the fluid contacts the cell. "Administer", "administration" and "treatment" also refer to treating a cell in vitro and ex vivo with a reagent, a diagnostic, a binding composition or through another type of cell. "Treatment" refers to therapeutic treatment, prophylaxis or preventive measures, research and diagnostic use when applied to a human, veterinary or research subject.

[0142] "Treatment" refers to administering an oral or topical therapeutic agent (e.g., a fusion protein of the present disclosure) to a subject, where the subject has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the patient or population being treated is administered the therapeutic agent in an amount effective to alleviate one or more disease symptoms, thereby inducing resolution of these symptoms or inhibiting these symptoms from progressing to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any particular disease symptom (also referred to as the "therapeutically effective amount") can vary depending on various factors such as the patient's disease state, age and weight, and the ability of the agent to produce the required therapeutic effect in the patient. Whether the disease symptoms have been reduced can be evaluated by any clinical detection method commonly used by a physician or other professional healthcare provider to assess the severity or progression of the symptoms. Embodiments of the present disclosure (e.g., treatment methods or products) may be ineffective in alleviating the respective target disease symptoms, but should be confirmed to reduce the target disease symptoms in a statistically significant number of patients by any statistical testing method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0143] "Optional" or "optionally" means that the event or situation described thereafter may or may not occur, and this description includes both the case where the event or situation occurs and the case where it does not occur. "And / or" should be regarded as specifically indicating whether each of the two specified features or components has or does not have the other. Accordingly, the term "and / or" as used in the phrase "A and / or B" in the present disclosure includes "A and B", "A or B", "A" (alone), and "B" (alone). Unless otherwise specified in the context, throughout the specification and claims, words such as "comprising", "having", "containing", etc. should not be construed in an exclusive or exhaustive sense, but rather in an inclusive sense, that is, in the sense of "including but not limited to".

[0144] The "subject" and "patient" in the present disclosure refer to mammals, particularly primates, especially humans.

[0145] Ordinal numbers such as "first", "second", "L1", "L2", etc. in the present disclosure are merely used to distinguish different elements, steps, and technical features, and are not intended to limit quantity, level, grade, or order. Examples

[0146] Hereinafter, the present disclosure will be further described in conjunction with examples, but these examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure usually follow normal conditions, such as those in the Cold Spring Harbor Antibody Technology Experimental Manual, Molecular Cloning Manual, etc., or the conditions proposed by the raw material or commercial manufacturer. Reagents for which specific sources are not specified are ordinary commercially available reagents.

[0147] Example 1. Preparation of Serum Albumin Antigen and Protein for Detection 1. Design and Expression of Human Serum Albumin: Using human serum albumin (HSA) as a template, the amino acid sequence of the protein for detection with some C-terminal amino acids deleted was designed.

[0148] Full-length amino acid sequence of human serum albumin:

Chem.

[0149] The albumin and FcRn binding regions are distributed in the third domain of the albumin molecule, and an albumin molecule with some amino acids deleted from a part of the third domain at the C-terminus was designed based on the molecular simulation structure (simultaneously, an Avi-his tag was fused to the C-terminus):

Chem.

[0150] 2. Full-length amino acid sequence of monkey serum albumin:

Chem.

[0151] 3. Full-length amino acid sequence of mouse serum albumin:

Chem.

[0152] 4. Full-length amino acid sequence of rat serum albumin:

Chem.

[0153] Camel immunization Inner Mongolia native Bactrian camels were immunized using serum albumin extracted from commercially available human blood (Sigma, Cat No. 126658). 5 mL of camel serum before immunization was collected and the serum was isolated. After mixing Freund's complete or incomplete adjuvant and the antigen at a volume ratio of 1:1, subcutaneous immunization was performed at multiple sites on the camel (the immunization dose was 100 μg of protein per animal per time). Booster immunization was performed once every two weeks, and the titer was measured after 4 immunizations. Plates (Costar, Cat.No.9018) were coated with 100 μL / well of 5 μg / mL human serum albumin (HSA) and left overnight at 4 °C. The next day, after washing 3 times with 300 μL / well of PBST (0.05% Tween 20), 4% skim milk powder was added and blocking was performed at 37 °C for 2 h. After washing, serially diluted immune camel serum was added and incubated at 37 °C for 1 h. The negative controls were pre-immune serum and blank PBS solution serially diluted in the same way. After incubation, washing was performed 3 times with PBST, and a polyclonal antibody of goat anti-camel Fc labeled with horseradish peroxidase (Thermo, Cat No.A16060, diluted at 1:5000) was added and incubated at 37 °C for 1 h. After washing again, TMB chromogenic solution was added for color development, stopped with 1 M sulfuric acid, and the absorbance value was read at a wavelength of OD450nm using a SpectraMax M5 plate reader. The titer was detected after dilution at 1:51200 times. When the titer passed, camel peripheral blood was collected to construct a library.

[0154] Establishment of phage library 100 - 200 mL of camel peripheral blood was taken, and peripheral blood lymphocytes (PBMC) were separated using the Ficoll method, with the cell count being 1.2×10 8 cells, and resuspended by adding Trizol reagent (1×10 7Trizol (in cells / mL), the cells were lysed, left standing on ice for 5 min, centrifuged at 13,000 rpm for 3 min, the supernatant was taken, the precipitate was discarded, 1 / 5 volume of chloroform was added, shaken vigorously for 30 - 60 s, left standing in an ice bath for 2 min, centrifuged at 13,000 rpm for 10 min, the upper aqueous layer was aspirated into a new 1.5 mL tube, an equal volume of isopropanol was added, mixed uniformly, left standing at -20 °C for 30 min, centrifuged at 13,000 rpm for 10 min, the supernatant was removed, the precipitate was retained, pre-cooled 75% ethanol was added to wash the precipitate, left standing at room temperature for 5 - 10 min, 600 μL of deionized water free of RNase was added to redissolve, RNA was obtained, reverse transcribed to obtain cDNA, and a phage library was constructed.

[0155] Screening of the phage library An HSA protein with a partial amino acid deletion in the third domain at the C-terminus (also called truncated HSA, and the sequence is shown in SEQ ID NO: 2) was expressed and purified in HEK293 cells, and this protein was used to screen the phage library. Antibodies with affinity for HSA were obtained by screening.

[0156] 10 μg of the above-mentioned truncated HSA-avi-biotin protein was bound to 1 mg of Dynabeads M-280 streptavidin (Invitrogen, Cat No. 11206D), left standing at room temperature for 1 hour, then washed 3 times with 1×PBS, blocked with 2% skim milk at room temperature for 2 hours, a camel heavy chain single domain antibody phage display library was added, and allowed to act at room temperature for 1 hour. Washed 9 times with PBST (0.05% Tween-20) solution to remove unbound phages. Phages specifically binding to truncated HSA were eluted with 1 mg / mL trypsin, and Escherichia coli TG1 in the logarithmic growth phase was infected to produce and purify phages for the next screening. After repeating the same screening process 1 - 2 times, positive clone sequences were enriched.

[0157] Identification by phage ELISA Monoclonal colonies were selected from the clones obtained from the final screening, packaged into single-chain antibodies of phage, and used in phage ELISA tests. Human, monkey, and mouse serum albumin (HSA, CySA, mSA) at 2 μg / mL each was coated on ELISA plates overnight, blocked with 4% skim milk at 37°C for 1 hour, then blocked phage supernatant using 2% skim milk was added and allowed to act at room temperature for 1 hour, washed 3 times with PBST (0.05% Tween 20), and detected by adding anti-M13 HRP. Clones with OD450 values exceeding 0.5 for all three albumins in the ELISA binding test were sequenced, and 50 specific sequences were obtained. Only some antibodies that simultaneously showed high OD450 values against human and monkey serum albumin were sequenced, and another 7 new specific sequences were obtained. Phage was screened to obtain 57 new specific sequences.

[0158] Example 3. Construction of Antibody Mammalian Cell Expression Vector The 57 specific sequences obtained by screening with the phage library in Example 2 were classified and sorted, 19 of these sequences were cloned, and the ELISA binding of the phage supernatants of these 19 anti-HSA antibodies was tested. Among them, the OD450 values of #7 and #9 clones are as shown in Table 1.

[0159]

Table 1

Chem.

Chem.

[0160] In the sequences of SEQ ID NOs: 6 and 7, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italicized sequences in the sequences are FR sequences, and the underlined sequences are CDR1, CDR2, and CDR3 sequences, respectively. The anti-HSA antibodies provided by the present disclosure are summarized as shown in Table 2 according to the CDR sequences encoded according to Kabat.

[0161] [Table 2]

[0162] Therefore, the CDRs of the following anti-HSA antibodies are provided: CDR1 is X 1 SCX 2 G, wherein X 1 is T or N, X 2 is M or L, CDR2 is X 3 IYX 4 VGGSTFYX 5 X 6 SVKG (SEQ ID NO: 58), wherein X 3 is S or T, X 4 is T or M, X 5 is T or A, X 6 is N or D, CDR3 is GSPARCX 7 X 8 RDPTTFX 9 Y (SEQ ID NO: 59), wherein X 7 is R or L, X 8 is L or R, X 9 is D or N.

[0163] The VHH sequence was fused to human IgG1-Fc (CH2-CH3) and constructed into a PTT5 expression vector, and the sequence of the ligated human IgG1-Fc is as follows: >human IgG1-Fc [Chemical formula] The following is the complete sequence of the VHH sequence fused to the human Fc (CH2-CH3) segment. The single underline indicates the human IgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 14), and the double underline indicates the linker sequence. The complete sequence is as follows: >#7aHSA-IgG1Fc:

Chem.

[0164] TIFF2025517100000012.tif52148

[0165] Example 4. Expression, Screening, and Detection of Anti-HSA Antibodies #7aHSA and #9a anti-HSA antibodies fused with human IgG1-Fc were transiently expressed and Protein A affinity purified in HEK293 cells, respectively. Subsequently, detection was performed by ELISA binding experiments and Biacore protein interaction experiments.

[0166] The in vitro binding abilities of #7aHSA and #9a anti-HSA antibodies to human, monkey, and mouse serum albumins were detected by ELISA binding experiments. The test antigens were human serum albumin (Sigma, Cat No. 126658), monkey serum albumin (Abcam, Cat No. ab184894), and mouse serum albumin (Abcam, Cat No. ab183228). Among them, the negative control was PBS, and the positive control was ALB8 fused with human IgG1-Fc (the variable region sequence was derived from the sequence of ALB8 in Ablynx patent disclosure WO 2008 / 028977 A2), and the sequence was as follows: >ALB8-IgG1-Fc

Chem.

[0167] Human, monkey, and mouse albumin were diluted to 1 μg / mL with PBS buffer at pH 7.4, and a 96-well microplate (Corning, Cat No. 9018) was added at a volume of 100 μL / well and left at 4°C overnight for 16 - 20 hours. After discarding the liquid, the plate was washed 3 times with PBST (pH 7.4, 0.05% Tween-20) buffer, and then 2% non-fat dry milk blocking solution (300 μL / well) diluted with PBS buffer was added and incubated at 37°C for 1 hour for blocking. After the blocking was completed, the blocking solution was discarded, and the plate was washed 3 times with PBST buffer. Then, an anti-HSA antibody with an initial concentration of 2 μg / mL was added, diluted to 11 gradients at a 3-fold ratio with PBS buffer, and incubated at 25°C for 1 hour. After the incubation was completed, the reaction solution in the plate was discarded, and the plate was washed 3 times with PBST. Then, 100 μL of a secondary antibody of goat anti-human IgG Fc labeled with HRP (Jackson immu, 109-035-190) (diluted 1:5000) was added to each well and incubated at 25°C for 1 hour. After the plate was washed 3 times with PBST, 100 μL of TMB chromogenic substrate was added to develop color, the reaction was stopped using 1 M sulfuric acid, the absorbance value was read at OD450nm using a SpectraMax M5 plate reader, and the absorbance value and antibody concentration were fitted and plotted with 4 parameters using GraphPad prism. The results are as shown in Figures 1A - 1C, and the binding EC 50 value of the antibody to the antigen was calculated, and the results are as shown in Table 3.

[0168]

Table 3

[0169] Furthermore, the affinity constants of the anti-HSA antibody fused with human IgG1Fc to human, monkey, and mouse albumin (HSA, CySA, mSA) were measured using a Biacore T200 (GE Healthcare) instrument. First, protein A was covalently coupled to a CM5 S series chip (GE, Cat.29-1049-88) using an amine coupling kit (GE, Cat.BR-1000-50). The anti-HSA antibody was affinity captured on the chip coupled with protein A, and then human, monkey, and mouse albumin at different concentrations was flowed over the chip surface. A binding-dissociation curve was obtained by detecting the reaction signal in real time using the Biacore instrument, and it was fitted with a Langmuir 1:1 binding model using BIAevaluation version 4.1, GE software to obtain the affinity constant. The buffer used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4, GE, Cat.#BR-1006-69). After the end of each cycle, the chip was regenerated using glycine-hydrochloric acid at pH 1.5 (GE, Cat.#BR-1003-54). The affinity results are as shown in Table 4.

[0170]

Table 4

[0171] Example 5. Humanization modification of anti-HSA antibody By performing three-dimensional structure homology modeling on the selected specific anti-HSA antibody molecule #7aHSA, in accordance with the results of comparison with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, the heavy chain variable region germline gene IGHV3-23 with high homology to the screened antibody was selected as a template, and the CDRs of the single-domain antibody derived from camel were transplanted into the corresponding human template to form a variable region sequence with the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The three-dimensional structure of the transplanted single-domain antibody was simulated and analyzed again. Specific sites that affect the CDR region structure in the FR region were reverted to mutations. The modified antibody has higher stability. Among them, the amino acid residues are determined and annotated by the Kabat numbering system. Among them, more human germline sequences are retained in the FRs of #7_Hu-9 and #7_Hu-10 so as to have lower immunogenic mutations.

[0172] The design of the reverted mutation sites and mutation methods is as shown in Table 5.

[0173]

Table 5

[0174] The specific sequences of the humanized ones obtained are as follows. The CDR regions are underlined, and the numbering rule is Kabat numbering: >#7_Hu_1

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0175] Using the method in Example 5, a chimeric anti-HSA antibody and hIgG1 Fc (CH2-CH3) segment fusion protein, or a protein fused with a 6His tag was constructed. The single underline is the hIgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 14), the italic is the 6His tag sequence, and the double underline is the linker sequence. The protein sequences are as follows (taking #7_hu_1-hIgG1Fc and #7_hu_10-6His as examples, and the same applies to other chimeric anti-HSA antibodies). According to the antibody numbering rule, #7_hu_10-6His is the one with TIFF2025517100000027.tif562 fused to the C-terminus of #7_Hu_10 (SEQ ID NO: 26), and the same applies to other antibodies.

[0176] >#7_hu_1-hIgG1Fc:

Chem.

Chem.

Chem.

[0177] The plasmid was transiently transfected into HEK293 cells, and the cells were cultured for 5 days. Then, the expression supernatant was collected, centrifuged at 4000 rpm for 15 minutes to remove the cells, and the supernatant was obtained and filtered using a 0.45 μM filter, and further purified and separated using conventional methods in this field, such as a Protein A column or a nickel column. Through detection, the anti-HSA antibody of the present disclosure was obtained.

[0178] Example 6. Measurement of the affinity of the humanized anti-HSA antibody for human, monkey, and mouse serum albumin The affinity constants of the humanized anti-HSA antibody fused with a 6His tag to human, monkey, and mouse albumin were measured using a Biacore T200 (GE Healthcare) instrument. First, the CM5 S series chip (GE, Cat.29-1049-88) was amino-coupled according to the instructions of the reagent kit using an amine coupling kit (GE, Cat.BR-1000-50) and a His capture reagent kit (GE, Cat.28-9950-56). After the surface response value reached approximately 10,000 RU and was blocked with ethanolamine, it was used to affinity capture the humanized anti-HSA antibody fused with a 6His tag to the anti-his chip. Then, human, monkey, and mouse albumin at different concentrations were flowed over the chip surface, and a binding dissociation curve was obtained by detecting the reaction signal in real time using the Biacore instrument. It was fitted with a Langmuir 1:1 binding model using BIAevaluation version 4.1, GE software to obtain the affinity constant. The buffer used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4, GE, Cat.#BR-1006-69). After the end of each cycle, the chip was regenerated using glycine-hydrochloric acid at pH 1.5 (GE, Cat.#BR-1003-54). The results of the affinity of some humanized antibodies are as shown in Table 6. As a result, it is shown that the antibodies obtained by screening in the present disclosure have an affinity for HSA equal to or slightly better than that of the positive control.

[0179]

Table 6

[0180] Using HEK293E cells, anti-HSA antibody 7_Hu_10-6his was transiently expressed and affinity purified using a nickel column. After that, the affinity constants for human, monkey, mouse, rat, dog, and rabbit albumin were measured using a Biacore T200 (GE Healthcare) instrument. First, using an amine coupling kit (GE, Cat. BR-1000-50) and a His capture reagent kit (GE, Cat. 28-9950-56), the CM5 S series chip (GE, Cat. 29-1049-88) was amino-coupled according to the reagent kit instructions. After the surface response value reached approximately 10,000 RU and was blocked with ethanolamine, the humanized anti-HSA antibody fused with a 6His tag was affinity captured on the anti-his chip. Then, different concentrations of human, monkey, mouse, rat, dog, and rabbit albumin were flowed over the chip surface, and the binding and dissociation curves were obtained by detecting the reaction signals in real time using the Biacore instrument. The curves were fitted using the Langmuir 1:1 binding model with BIAevaluation version 4.1, GE software to obtain the affinity constants. The buffer used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4, GE, Cat. #BR-1006-69). After each cycle, the chip was regenerated using glycine-hydrochloric acid at pH 1.5 (GE, Cat. #BR-1003-54). The affinity results are as shown in Table 7.

[0181]

Table 7

[0182] Example 7. Effect of anti-HSA antibody on the binding of FcRn and biotin-HSA in cells stably expressing hFcRn The blocking effect of anti-HSA antibodies on the binding of HSA to FcRn was detected by the change in the fluorescence intensity of antibodies against randomly biotinylated HSA bound to the surface of HEK293 cells (293-hFcRn-mut cells) stably expressing the hFcRn mutant. In this experiment, HEK293 cells stably expressing the hFcRn mutant (L320A, L321A) were used, and the hFcRn mutant could be relatively well localized and maintained on the cell membrane surface. Cells expressing the hFcRn mutant were incubated with different concentrations of anti-HSA antibodies and Biotin-HSA, and then SA-FITC secondary antibody was added for incubation. The degree of decrease in the secondary antibody fluorescence signal (MFI) was used to measure the strength of the blocking effect of anti-HSA antibodies on the binding of Biotin-HSA to FcRn. The specific experimental method is as follows.

[0183] After washing 293-hFcRn-mut cells twice with PBS at pH 5.5, 5×10 5 cells were added to each well in a U-bottom 96-well plate. After centrifugation to remove the supernatant, 90 μL of anti-HSA antibody diluted with PBS (pH 5.5) and 10 μL of Biotin-HSA (final concentration 250 μg / mL) were added to each well and co-incubated on ice for 40 minutes. After washing twice with 200 μL of PBS (pH 5.5), 100 μL of streptavidin-FITC secondary antibody (eBioscience, Cat No.11-4317-87) diluted with PBS (pH 5.5) was added to each well, with a dilution ratio of 1:200, and incubated on ice for 40 minutes. After washing twice with 200 μL of PBS (pH 5.5), 200 μL of PBS (pH 5.5) was added to each well, and the fluorescence value was measured using a flow cytometer (BD Biosciences, BD Accuri C6) and the FL1 channel. The data was analyzed using Flowjo software, and the analysis results were plotted using Prism6 software. The test results of the inhibitory effects of some antibodies are shown in Figures 2A and 2B.

[0184] Analysis of results: Based on the fluorescence signal value (MFI), it is determined whether the binding of the anti-HSA antibody to HSA blocks the binding of HSA to FcRn. If the fluorescence signal decreases, it indicates that the binding is blocked.

[0185] As shown in Figure 2A, using the biotin-HSA+FcRn stable transfected cells+SA-FITC group (non-blocking group) as the baseline and the biotin-HSA+HSA+FcRn stable transfected cells+SA-FITC group (HSA self-competing group) as the positive control, Nc is an isotype control antibody. The results show that the anti-HSA antibodies #7_Hu_6-6his and #7_Hu_7-6his of the present disclosure did not block the binding of HSA to FcRn.

[0186] As shown in Figure 2B, #7_Hu_10-6his and #7_Hu_10 did not block the binding of HSA to FcRn (detected at a concentration of 1 mg / mL).

[0187] This indicates that the anti-HSA antibodies of the present disclosure do not affect the pH-dependent binding of FcRn to HSA and can well maintain the FcRn-mediated serum albumin recycling mechanism.

[0188] Example 8. Pharmacokinetics of anti-HSA antibody in mice The pharmacokinetic characteristics of the anti-HSA antibodies (#7_Hu_6-6his, #7_Hu_7-6his, #7_Hu_10-6his) of the present disclosure, a positive control antibody (ALB8-6his), and an isotype control antibody (Isotype, a non-HSA-binding VHH molecule) were studied in CD-1 male mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). Three mice in each group were administered by tail vein injection at a dose of 4 mg / kg on day 0. Blood samples were collected before administration, and at 5 minutes, 1 hour, 7 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours after administration, anticoagulated using EDTA, centrifuged to prepare plasma, and used for pharmacokinetic measurement.

[0189] PK assay: Coated with 5 μg / mL anti-his antibody (Thermo, MAI-21315-1mg), added to a 96-well microplate (Corning, Cat No.9018) at a volume of 100 μL / well, and left at 4°C overnight for 16 - 20 hours. After discarding the liquid, the plate was washed 3 times with PBST (pH 7.4, 0.05% Tween-20) buffer, then Casin Blocker (Thermo, 37528) blocking solution (300 μL / well) was added and incubated at 37°C in an incubator for 2 hours for blocking. After the blocking was completed, the blocking solution was discarded, the plate was washed 3 times with PBST buffer, then mouse plasma at different time points appropriately diluted with diluent (Casin Blocker containing 1% CD1 mouse plasma) was added and incubated at 37°C for 1.5 hours. After the incubation was completed, the reaction solution in the plate was discarded, the plate was washed 3 times with PBST, 100 μL of secondary antibody of rabbit anti-camelid Cocktail VHH labeled with HRP (Genescript, A02016, without BSA) (diluted 1:5000) was added to each well, and incubated at 37°C for 1 hour. After the plate was washed 3 times with PBST, 100 μL of TMB chromogenic substrate was added to develop color, the reaction was stopped using 1M sulfuric acid, and the absorbance value was read at OD450nm using a SpectraMax M5 plate reader. The concentration of the VHH antibody in the plasma sample was calculated according to the standard curve, and the results are as shown in Figure 3. The calculated concentration was used with PK solver to calculate the half-life, and the results are as shown in Figure 4 and Table 8.

[0190]

Table 8

[0191] As a result, each anti-HSA antibody #7_Hu_6-6his, #7_Hu_7-6his, #7_Hu_10-6his of the present disclosure has a similar pK curve, the half-life is within the half-life range of mouse serum albumin, and is superior to the positive control ALB8-6his antibody, and the clearance is also lower than that of the ALB8-6his antibody.

[0192] Example 9. Design and Preparation of Fusion Protein The A-chain and B-chain of relaxin were linked via a linker to form a single-chain relaxin molecule. To improve the half-life, the single-chain relaxin molecule was further fused with a single-domain antibody against HSA to form a fusion protein, and the structural schematic diagram is as shown in Figure 5.

[0193] >Human RLN2 A-chain

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0194] Among them, the italic font represents the anti-HSA single domain antibody, the single underline represents linker 1, the double underline represents linker 2, the bold font represents the B chain of relaxin 2, and the italic bold font represents the A chain of relaxin 2. For linker 1, linker a, b, or c can be selected, where a is a flexible linker and b, c are rigid linkers.

[0195] The positive control 0054 sequence of this example is derived from Example 7 of WO2021022139A1. >0054 [Chemistry]

[0196] 1. Preparation of fusion protein: Codon optimization was performed on the DNA sequence encoding the fusion protein (SEQ ID NOs: 36-44), synthesized and cloned into the pTT5 expression vector, and the expression plasmid was transiently transfected into expiCHO (ThermoFisher, Cat No. A29127) cells. The ExpiFectamine CHO Transfection Kit (ThermoFisher, Cat No. A29133) was used for transfection according to the instructions of the reagent kit. The "High protocol" in the instructions was adopted, and the cells were placed on a shaker at 32 °C the day after transfection for suspension shaking culture (5% CO 2, 110 rpm, 75% humidity). On the 10th to 12th day after transfection, the supernatant of the cell culture medium was collected, centrifuged at 4000 rpm for 20 minutes, the supernatant was taken and filtered through a 0.45 μm filter for use.

[0197] 2. Purification of the fusion protein: (1) Capture of the fusion protein by A3 affinity chromatography: The cell culture supernatant expressing the fusion protein was centrifuged at high speed to collect the supernatant, which was filtered through a 0.22 μm filter membrane. The A3 (JSR Life Sciences, Cat No. BP-AMS-A3-0100) affinity column was regenerated with 0.1 M NaOH (Sigma, Cat No. 71687) which is 5 times the column volume, and then washed and equilibrated with 1×PBS (pH 7.4) (Sangon Biotech(Shanghai)co.,Ltd.: Cat No. E607016-0500) which is 5 times the column volume. To bind to the affinity column, the filtered supernatant was loaded at a low flow rate, the flow rate was controlled so that the retention time was about 1 min or more, and after the binding was completed, 1×PBS (pH 7.4) which is 5 times the column volume, 1×PBS (pH 7.4) which is 5 times the column volume, 0.8 M NaCl (Vetec, Cat No. V900058) and 1×PBS (pH 7.4) which is 5 times the column volume were used to wash the chromatography column until the ultraviolet absorption returned to the baseline level. The sample was eluted with 0.1 M Glycine (pH 3.0) (Sigma, Cat No. 410225) buffer, and the elution peak was collected by ultraviolet detection. The pH of the elution product was quickly adjusted to 7 - 8 with 1 M Tris-HCl (pH 7.5) (Vetec, Cat No. V900483) and stored temporarily. For the elution product, the solution can be replaced by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube, replacement of the solution with the desired buffer system, or replacement with the desired buffer system by molecular exclusion chromatography (for example, G-25 desalting).

[0198] (2) Purification of the fusion protein by hydrophobic chromatography: First, the sample was diluted with an equal volume of 20 mM Tris pH 7.5 (Vetec, Cat No. V900483), 1 M Na 2 SO4 (Sigma, Cat No. 239313). The hydrophobic chromatography column Phenyl 650M (Toyopearl, Cat No. 0014478) was regenerated with 0.5 M NaOH (Sigma, Cat No. 71687) at 5 column volumes, and then washed and equilibrated with 20 mM Tris pH 7.5 (Vetec, Cat No. V900483), 0.5 M Na 2 SO4 (Sigma, Cat No. 239313) at 10 column volumes. To bind to the hydrophobic column, the diluted sample was loaded at a low flow rate, the flow rate was controlled so that the retention time was about 2 min or more, and after the binding was completed, the chromatography column was washed with 20 mM Tris pH 7.5 (Vetec, Cat No. V900483), 0.5 M Na2SO4 (Sigma, Cat No. 239313) at 5 column volumes until the UV absorption returned to the baseline level. Gradient elution from 0% to 100% was performed with 20 mM Tris pH 7.5 (Vetec, Cat No. V900483) as the elution buffer, and the elution peak was detected and collected according to the purity of SEC-HPLC (Column: TSKgel G3000SWXL, 5 μm, Cat No. 008541). After two-step purification, the yield of the fusion protein is as shown in Table 9. For the elution product, the solution can be replaced by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube, replacement of the solution with the desired buffer system, or replacement with the desired buffer system by size exclusion chromatography (e.g., G-25 desalting).

[0199]

Table 9

[0200] After two-step purification, a completely pure fusion protein is obtained, and the molecules of 0051 and 0053 of the present disclosure have a transient transfection yield in expiCHO that is twice that of the positive control molecule 0054 under the same conditions.

[0201] Example 10. Measurement of the affinity of the fusion protein for different species of serum albumin. The affinity of 0051 for human, monkey, rat, mouse, dog, pig, rabbit, and bovine serum albumin was measured using a Biacore T200 (GE Healthcare) instrument. The positive control was 0054. Experiments were conducted using human albumin HSA (Sigma, Cat No. 126658), monkey albumin (Abcam, Cat No. ab184894), rat albumin (Abcam, Cat No. ab198656), mouse albumin (Abcam, Cat No. ab183228), rabbit albumin (Abcam, Cat No. ab188055), and bovine albumin (Sigma, Cat No. B2064).

[0202] 1. Measurement of affinity using an anti-VHH antibody-coupled chip Using an amine coupling kit (GE, Cat No. BR-1000-50), the anti-VHH antibody (MonoRab (商標) Rabbit Anti-Camelid VHH Cocktail, Genescript, Cat No. A02014) was coupled to the S-series CM5 sensor chip (GE, Cat No. 29-1049-88). The antibody was diluted to 10 μg / mL using 10 mM sodium acetate pH 4.5 (GE, Cat No. BR-1003-50). After activating the CM5 chip with EDC / NHS, it was flowed through the diluted anti-VHH antibody so that the surface response value was approximately 5000 RU, and then blocked with ethanolamine and prepared for use.

[0203] Various kinds of albumin were serially diluted using 1×HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20). The fusion protein molecules waiting to be measured were captured using the coupled anti-VHH antibody chip, and the response value was about 30 RU. Then, different concentrations of serum albumin were flowed on the chip surface for 3 minutes at a flow rate of 50 μL / min and a dissociation time of 1 - 10 minutes (the dissociation time varies depending on the type of albumin). After each cycle, the chip was regenerated with 10 mM glycine-hydrochloric acid pH 1.5 (GE, Cat No. BR-1003-54). The binding and dissociation curves were obtained by detecting the reaction signals in real time with a Biacore T200 (GE). The obtained data was fitted using the Langmuir 1:1 binding model with the BIAevaluation version 4.1 software by selecting appropriate concentration points to obtain the affinity values, and the results are shown in Table 10.

[0204]

Table 10

[0205] The different fusion proteins designed in this disclosure have an affinity for human, monkey, rat, and bovine serum albumin equivalent to that of the positive control molecule 0054 (data not shown), and the binding results of HSA and rat albumin are exemplarily shown in Table 10.

[0206] 2. Measurement of Affinity by Albumin Coupling Chip Using an amine coupling kit (GE, Cat No. BR-1000-50), coupling of different species of serum albumin was performed on the S series CM5 sensor chip (GE, Cat. 29-1049-88). Various species of albumin were diluted to 2 μg / mL. After activating the CM5 chip with EDC / NHS, it was flowed through the diluted albumin so that the surface response value became about 200 (180 - 230) RU, and then blocked with ethanolamine. The blank control channel also needs to be activated with EDC / NHS and blocked with ethanolamine in the same manner.

[0207] For molecules 0051 and 0054, a series of gradient dilutions were performed using 1×HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20): 2000, 666.66, 222.22, 74.074, 24.69, 8.23, 2.74, 0.91, 0.305 nM. Samples with different concentrations of 0051 and 0054 were flowed through the albumin coupling channel and the blank channel for 3 minutes each, with a flow rate of 50 μL / min and a dissociation time of 4 minutes. After each cycle, the chip was regenerated using 10 mM glycine-hydrochloric acid pH 1.5 (GE, Cat. #BR-1003-54). By detecting the reaction signal in real time with a Biacore T200 (GE), a binding-dissociation curve was obtained. The obtained data was fitted using the BIAevaluation version 4.1 software with the Langmuir 1:1 binding model by selecting appropriate concentration points to obtain the affinity values.

[0208]

Table 11

[0209] The affinity of molecule 0051 for human, monkey, and rat albumin is equivalent to that of the positive control molecule 0054, and its affinity for mouse and dog albumin is weaker than that of the positive control molecule 0054 (data not shown). Table 11 exemplarily shows the results for HSA, rat, and mouse.

[0210] Example 11. Biological Activity of Cells In Vitro of the Fusion Protein 1. Biological Activity of the Fusion Protein in THP-1 Cells THP-1 is a human peripheral blood mononuclear cell (Procell, Cat No. CL-0233) and is an endogenously expressed RXFP1 receptor. When relaxin binds to the receptor in THP-1 cells, THP-1 cells are induced to produce cAMP. Therefore, in this example, the in vitro activity of the fusion protein was measured by detecting the production amount of cAMP. This example used a homogeneous time-resolved fluorescence (HTRF) cAMP-Gs Dynamic reagent kit (Cisbio, Cat No. 62AM4PEB) to measure the cAMP level based on the manufacturer's protocol. 0054, 800814 (by WO2015067113A1) and 0060 (WO2021255127A1, RELAX0023) were used as positive controls.

[0211] >A chain of 800814

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0212] The experimental method is as follows: THP-1 cells were cultured under the conditions of 37 °C and 5% CO 2 and the culture medium was RPMI1640 + 0.05 mM β-mercaptoethanol + 10% FBS + 1% penicillin-streptomycin double antibody. On the day of the experiment, after centrifuging and collecting THP-1 cells in the logarithmic growth phase, they were resuspended in serum-free medium, and the cell density was 4×106 Adjusted to cells / mL, and transferred to a 96-well measurement plate (Cisbio, Cat No.66PL96025) with a volume of 50 μL per well (20,000 cells / well). The fusion protein samples awaiting measurement and 0054, 0060, 800814 as positive controls were serially diluted 10-fold using Stimulation Buffer 1 in the reagent kit in individual U-bottom 96-well plates, and then transferred to the 96-well measurement plate containing THP-1 cells with a volume of 5 μL per well, and cultured at 37 °C, 5% CO 2 for 30 minutes under the conditions of, and cells without the sample awaiting measurement were used as a reference control. Dilute cAMP-d2 (receptor fluorophore) and anti-cAMP-cryptat (donor fluorophore) in the reagent kit using Lysis&Detection Buffer 1, add cAMP-d2 to all wells except the blank control wells in the 96-well measurement plate with a volume of 5 μL per well, then add anti-cAMP-cyptat to all wells, and incubate at room temperature in the dark for 1 hour. Then, read the values at wavelengths of 620 nm and 665 nm using Tecan (Infinite 200 PRO), show the signal intensity as the ratio of fluorescence intensity at 665 nm / fluorescence intensity at 620 nm × 10,000, perform non-linear fitting using Graphpad Prism to plot the cAMP concentration curve, and measure the EC 50 value (nM) at which the sample and control awaiting measurement induce THP-1 cells to produce cAMP. The results are shown in Table 12, Figure 6A and Figure 6B.

[0213]

Table 12

[0214] As a result, it has been shown that the in vitro activity of each fusion protein of the present disclosure is equivalent to that of the positive control molecule 0054.

[0215] 2. Biological Activity of Fusion Protein in CHO-K1 Cells Overexpressing RXFP Receptor (1) Preparation of CHO-K1 cells transiently transfected with RXFP1 and RXFP2 receptors CHO-K1 cells (CCTCC, Cat No. GDC0018) were transiently transfected using Lipofectamine 2000 (Thermo Fisher, Cat No. 11668019), and the CHO-K1 cells were resuspended in a 6-well plate at a concentration of 6×10 5 cells / well, and cultured at 37°C and 5% CO 2 for 12 hours. After that, the original medium was removed and 1.5 mL of serum-free Ham's F-12K (Kaighn's) medium (Gibco, Cat No. 21127030) was added. For the cells in each well, two test tubes were taken. In test tube 1, 3 μg of the corresponding plasmid DNA (DNA plasmid of human, dog, cynomolgus monkey, rat, mouse RXFP1 receptor or human RXFP2 receptor) was dissolved in 250 μL of Opti-MEM I serum-reduced medium (Gibco, Cat No. 31985070). In test tube 2, 3 μL of Lipofectamine 2000 reagent was dissolved in 250 μL of Opti-MEM I serum-reduced medium and incubated at room temperature for 5 minutes. Then, the reagent in test tube 1 was added to test tube 2, gently and uniformly mixed, incubated at room temperature for 20 minutes, added drop by drop to the 6-well plate (500 μL / well), transfected for 6 hours, then changed to complete medium and continued to culture for 24 hours, and subsequent experiments were carried out.

[0216] (2) In vitro activation effect of the fusion protein on its receptor In this example, a homogeneous time-resolved fluorescence (HTRF) cAMP-Gs Dynamic reagent kit (Cisbio, Cat No. 62AM4PEB) was used to measure the cAMP level according to the manufacturer's protocol. The specific method is as follows: On the day of the experiment, after collecting the CHO-K1 cells transfected with human, dog, cynomolgus monkey, rat or mouse RXFP1 receptor by centrifugation, they were resuspended in serum-free medium, and the cell density was adjusted to 4×10 6Adjusted to cells / mL, and transferred to a 96-well measurement plate (Cisbio, Cat No.66PL96025) at a volume of 50 μL per well (20,000 cells / well). Samples 0051 waiting for measurement and 0054, 0060, 800814 as positive controls were serially diluted 10-fold using Stimulation Buffer 1 in the reagent kit in individual U-bottom 96-well plates, and then transferred to the 96-well measurement plate containing THP-1 cells at a volume of 5 μL per well, and cultured at 37 °C and 5% CO 2 under the conditions for 30 minutes, with cells not containing the sample waiting for measurement as the reference control. Dilute cAMP-d2 (receptor fluorophore) and anti-cAMP-cryptat (donor fluorophore) in the reagent kit using Lysis&Detection Buffer 1, add cAMP-d2 to all wells except the blank control wells in the 96-well measurement plate at a volume of 5 μL per well, then add anti-cAMP-cyptat to all wells, and incubate at room temperature in the dark for 1 hour. Then, read the values at wavelengths of 620 nm and 665 nm using Tecan (Infinite 200 PRO), show the signal intensity as the ratio of fluorescence intensity at 665 nm / fluorescence intensity at 620 nm × 10,000, perform non-linear fitting using Graphpad Prism to plot the cAMP concentration curve, and the EC 50 value (μM) of the fusion protein and the control inducing CHO-K1 receptor overexpressing cells to produce cAMP was obtained. The results are shown in Table 13 and Figure 6B.

[0217]

Table 13

[0218] As a result, it has been shown that the in vitro activity of molecule 0051 is equivalent to that of control molecules 0054 and 0060.

[0219] Example 12. Pharmacokinetics of the fusion protein in rats

[0220] Male Sprague-Dawley rats, 4 - 6 weeks old and weighing 200 - 250 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. During the breeding period, they were allowed to freely ingest food and water, and were regulated under a 12 / 12-hour light / dark cycle, with the temperature at 16 - 26 °C and the relative humidity at 40 - 70%. After the animals arrived at the facility, adaptive breeding was carried out for more than 7 days. On the day before the experiment started, the experimental animals were numbered and randomly grouped, with a total of 4 groups, 3 animals in each group. On the day of the experiment, the test drugs 0051 and 0054 were intravenously injected or subcutaneously injected into the 4 groups of experimental animals respectively. The dosage was 4 mg / kg, and the injection volume was 5 mL / kg for both.

[0221] For the administration method by intravenous injection, blood samples were collected at each time point of 0.5, 1, 3, 6, 24, 48, 72, 96, 120, 168, 192, and 240 hours before and after administration. For the administration method by subcutaneous injection, blood samples were collected at each time point of 3, 6, 24, 48, 72, 96, 120, 168, 192, and 240 hours before and after administration. 0.03 mL of whole blood was collected from each animal, anticoagulated with EDTA-K2, and immediately centrifuged at 4500 rpm for 5 min after blood collection. The supernatant was taken into an EP tube and stored at -20 °C, and then transferred to -80 °C for storage after the experiment ended.

[0222] The drug concentrations of 0051 and 0054 in plasma were detected using the ELISA method: Coated with 0.4 μg / mL of Anti-Relaxin 2 / RLN2 antibody (Abcam, Cat No. EPR21832-15), added to a 96-well microplate (Corning, Cat No. 9018) at a volume of 100 μL / well, and left at 4°C for 16 - 20 hours overnight. After discarding the liquid, the plate was washed 3 times with PBST (pH 7.4, 0.05% Tween-20) buffer, then 4% bovine serum albumin solution (300 μL / well) was added and incubated at room temperature for 1 hour for blocking. After the blocking was completed, the blocking solution was discarded, the plate was washed 3 times with PBST buffer, and rat plasma at different time points appropriately diluted using a diluent called Casin Blocker (Thermo, Cat No. 37528) containing 1% rat plasma was added and incubated at 37°C for 1 hour. After the incubation was completed, the reaction solution in the plate was discarded, the plate was washed 3 times with PBST, 100 μL of secondary antibody of rabbit anti-camelid Cocktail VHH labeled with HRP (Genescript, Cat No. A02016, without BSA) (diluted 1:5000) was added to each well, and incubated at 37°C for 1 hour. After the plate was washed 3 times with PBST, 100 μL of TMB (Sigma, Cat No. T0440) chromogenic substrate was added to develop color, the reaction was stopped using 1 M sulfuric acid, and the absorbance value was read at OD450nm using a SpectraMax M5 plate reader.

[0223] The data results were analyzed using Graphpad Prism software, the concentrations of 0051 and 0054 molecules in plasma samples were calculated according to the standard curve, and the rat results are as shown in Figure 7A. The calculated concentrations were used with PK solver to calculate the half-life, and the results are as shown in Figure 14. Furthermore, the pharmacokinetics of mice were detected in the same way, and as a result, as shown in Figure 7B, it is shown that the AUC exposure and t 1 / 2 of the 0051 molecule are equivalent to those of the positive control molecule 0054.

[0224]

Table 14

[0225] From the PK results of rats, after intravenous administration, the AUC of 0054 was about twice that of 0051, the difference in t1 / 2 between the two molecules was small, and after subcutaneous administration, the bioavailability of 0051 was shown to be higher than that of 0054. Compared with the 0054 molecule, 0051 has a larger apparent volume of distribution (Vdss), indicating that the molecule has better tissue cell barrier permeability. Furthermore, since 0051 has a longer apparent residence time (MRT), it is expected that the molecule will exert its pharmacological effect in the body for a longer time. Additionally, after subcutaneous administration by subcutaneous injection, the bioavailability (Bioavailability%) of 0051 is clearly higher than that of 0054. Therefore, 0051 has better pharmacokinetic properties.

[0226] Example 13. Effect of the fusion protein on mouse myocardial hypertrophy

[0227] A myocardial hypertrophy model of C57BL / 6J mice (Charles River Laboratories) was prepared by subcutaneously implanting a micro-osmotic pump (Alzet 1002) and injecting isoprenaline (ISO, Sigma, Cat.#I5627). ISO was dissolved in PBS buffer (Servicebio, Cat.#G4202) containing 0.002% sodium ascorbate (Sigma, Cat.#11140) to a concentration of 146 mg / mL (calculated based on the average body weight of mice of 25.8 g), and injected continuously at an injection concentration of 15 mg / kg / d for 14 days to prepare a pathological model. On the 14th day, the osmotic pump was removed, and model control group 1 was euthanized for sampling. The remaining mice were randomly grouped, and subcutaneous injection of the fusion protein or the control molecule was started. The dosage and grouping are as shown in Table 15.

[0228]

Table 15

[0229] At the end of the study, the animals were fasted for 6 hours, after which the mouse body weight was measured, and then the mice were euthanized, and the heart weight and tibia length were measured. The myocardial hypertrophy status was determined by the ratio of heart weight to tibia length. As shown in Figure 8 and Table 16.

[0230]

Table 16

[0231] The above measurement data are shown as mean ± standard deviation (mean±SD). For the comparison between two groups, the Student t test was adopted. *p<0.05, **p<0.01 vs the two model control groups, #p<0.05 vs the blank control group.

[0232] The injection of ISO into mice can significantly cause myocardial hypertrophy. By administering and treating with the fusion protein of the present disclosure, the myocardial hypertrophy caused by ISO can be restored to the baseline level, and the required dose is significantly lower than that of control 0060.

Claims

1. A fusion protein comprising a serum albumin binding domain and relaxin or an analog thereof, wherein the serum albumin binding domain comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises any one of 1) to 3), namely, 1) CDR1, CDR2 and CDR3 in the amino acid sequence represented by any one of SEQ ID NOs: 6 and 18 to 27, 2) CDR1, CDR2 and CDR3 in the amino acid sequence represented by SEQ ID NO: 7, and 3) CDR1, CDR2 and CDR3 of the amino acid sequences represented by SEQ ID NOs: 64, 58 and 59, respectively, comprising any one of CDR1, CDR2 and CDR3 selected therefrom, wherein the CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, preferably, the CDR1, CDR2 and CDR3 are represented by SEQ ID NOs: 8, 9 and 10, respectively, or represented by SEQ ID NOs: 11, 12 and 13, fusion protein.

2. The immunoglobulin single variable domain is modified by any one selected from camelization or humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof, preferably, the template of the framework region of the humanized modified human germline gene is derived from IGHV3-23, The fusion protein according to claim 1.

3. The immunoglobulin single variable domain contains an amino acid mutation at at least one of the positions 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83 and 84, where the positions are based on the EU numbering system, preferably, it contains at least one amino acid mutation selected from 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q or 84P, more preferably, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 20V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 74A, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 78V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 83Q, 84P, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 84P, 30L, 37F, 44E, 45R, 47G, 84P, or 30L, 37F, 44E, 45R, 47G, comprising any one combination of amino acid mutations selected from The fusion protein according to any one of claims 1 to 2.

4. The immunoglobulin single variable domain is comprising an amino acid sequence represented by any one of SEQ ID NO: 6, 18 to 27 or having at least 90% sequence identity thereto, or comprising an amino acid sequence represented by SEQ ID NO: 7 or having at least 90% sequence identity thereto, The fusion protein according to any one of the preceding claims.

5. The immunoglobulin single variable domain is VHH, The fusion protein according to any one of the preceding claims.

6. The relaxin or its analog comprises an A chain and a B chain, the A chain having an amino acid sequence represented by any one of SEQ ID NO: 31, 50, 52, 54, 56 or having at least 90% sequence identity thereto, and the B chain having an amino acid sequence represented by any one of SEQ ID NO: 32, 51, 53, 55, 57 or having at least 90% sequence identity thereto, Preferably, the A chain and the B chain are any one combination selected from the following 1) to 7), namely, 1) A chain represented by SEQ ID NO: 31 and B chain represented by SEQ ID NO: 32, 2) A chain represented by SEQ ID NO: 50 and B chain represented by SEQ ID NO: 51, 3) A chain represented by SEQ ID NO: 52 and B chain represented by SEQ ID NO: 53, 4) A chain represented by SEQ ID NO: 54 and B chain represented by SEQ ID NO: 55, 5) A chain represented by SEQ ID NO: 31 and B chain represented by SEQ ID NO: 53, 6) A chain represented by SEQ ID NO: 52 and B chain represented by SEQ ID NO: 32, and 7) A chain represented by SEQ ID NO: 56 and B chain represented by SEQ ID NO: 57, The fusion protein according to any one of the preceding claims.

7. Formula (I) to formula (IV), namely, comprising any one polypeptide represented by or any combination thereof, B-L １ -A-L ２ -VHH of formula (I) A-L １ -B-L ２ -VHh of formula (II) VHh-L ２ -B-L １ -A formula (III), and VHh-L １ -A-L １ -B formula (IV) wherein, A is the A chain of relaxin or its analog, B is the B chain of relaxin or its analog, The fusion protein according to claim 6. L １ and L ２ is a linker, L １ and L ２ each independently exists or does not exist,

8. The fusion protein according to claim 7. L １ is represented by an amino acid sequence selected from (GGGGQ)n, (GGGQ)n, (PGPQ)n, (PGPA)n, (G Ｘ S Ｙ ) Ｚ wherein n is selected from integers of 1 to 10, and X, Y, and Z are independently selected from integers of 1 to 10, L ２ (G Ｘ S Ｙ ) Ｚ , KR, and an amino acid sequence represented by any one of SEQ ID NOs: 60 to 63, 65 to 66, wherein X, Y, and Z are independently selected from integers of 1 to 10; Preferably, L １ contains an amino acid sequence represented by any one of SEQ ID NOs: 33 to 35, Preferably, L ２ contains the amino acid sequence represented by SEQ ID NO: 49,

9. ​ An amino acid sequence represented by any one of SEQ ID NOs: 36 to 43 or having at least 90% sequence identity thereto, The fusion protein according to any one of the preceding claims.

10. A single-chain protein, dimer or multimer protein, The fusion protein according to any one of the preceding claims.

11. A serum albumin-binding molecule comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is one of 1) to 3), namely, 1) CDR1, CDR2 and CDR3 in the amino acid sequence represented by any one of SEQ ID NOs: 6, 18 to 27, 2) CDR1, CDR2 and CDR3 in the amino acid sequence represented by SEQ ID NO: 7, and 3) CDR1, CDR2 and CDR3 of the amino acid sequences represented by SEQ ID NOs: 64, 58 and 59, respectively, Comprising any one of CDR1, CDR2 and CDR3 selected therefrom, Among them, the CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, Preferably, the CDR1, CDR2 and CDR3 are represented by SEQ ID NOs: 8, 9 and 10, respectively, or by SEQ ID NOs: 11, 12 and 13, Serum albumin-binding molecule.

12. The immunoglobulin single variable domain is modified by any one selected from camelization or humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof, Preferably, the template of the framework region of the humanized modified human germline gene is derived from IGHV3-23, The serum albumin-binding molecule according to claim 11.

13. The immunoglobulin single variable domain contains an amino acid mutation at least at one of the positions of 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83 and 84, The positions are based on the EU numbering system, Preferably, it contains at least one amino acid mutation selected from 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q or 84P, More preferably, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 20V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 74A, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 78V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 83Q, 84P, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 84P, 30L, 37F, 44E, 45R, 47G, 84P, or 30L, 37F, 44E, 45R, 47G, comprising any one combination of amino acid mutations selected from the serum albumin-binding molecule according to any one of claims 11 to 12.

14. The immunoglobulin single variable domain is comprising an amino acid sequence represented by any one of SEQ ID NO: 6, 18 to 27 or having at least 90% sequence identity therewith, or comprising an amino acid sequence represented by SEQ ID NO: 7 or having at least 90% sequence identity therewith, the serum albumin-binding molecule according to any one of claims 11 to 13.

15. The immunoglobulin single variable domain is VHH, the serum albumin-binding molecule according to any one of claims 11 to 14.

16. further comprising a human immunoglobulin Fc region or a histidine tag, preferably, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4, the serum albumin-binding molecule according to any one of claims 11 to 15.

17. further comprising one or more therapeutic or diagnostic agents, preferably, the therapeutic or diagnostic agent is covalently bound or fused to the immunoglobulin single variable domain, preferably, the therapeutic or diagnostic agent is any one selected from therapeutic or diagnostic proteins, polypeptides, nucleic acids, small molecule compounds, the serum albumin-binding molecule according to any one of claims 11 to 16.

18. (a) ≤ 1 × 10 －７ M, preferably ≤ 1 × 10 －８ The K of M Ｄ binds to human serum albumin at a value of (b) no blockade of the binding between serum albumin and FcRn is detected, and (c) when a therapeutic or diagnostic agent is included, extending the plasma half-life of the therapeutic or diagnostic agent, having at least one activity selected from the serum albumin-binding molecule according to any one of claims 11 to 17.

19. A polynucleotide encoding the fusion protein according to any one of claims 1 to 10 or the serum albumin-binding molecule according to any one of claims 11 to 18.

20. A vector comprising or expressing the polynucleotide according to claim 19.

21. A host cell comprising or expressing the vector according to claim 20.

22. A method for producing or preparing the fusion protein according to any one of claims 1 to 10 or the serum albumin-binding molecule according to any one of claims 11 to 18, comprising: culturing the host cell according to claim 22; recovering the fusion protein or serum albumin-binding molecule; optionally, purifying and / or modifying the fusion protein or serum albumin-binding molecule; A method comprising the steps of.

23. A method for prolonging the plasma half-life of a therapeutic or diagnostic agent, comprising: linking or fusing an immunoglobulin single variable domain defined in any one of claims 11 to 18 to the therapeutic or diagnostic agent; A method comprising the steps of.

24. A pharmaceutical composition comprising: the fusion protein according to any one of claims 1 to 10, the serum albumin-binding molecule according to any one of claims 11 to 18, the polynucleotide according to claim 19, or the vector according to claim 20; preferably further comprising one or more pharmaceutically acceptable vectors, diluents, buffers or excipients; A pharmaceutical composition.

25. Use of the fusion protein according to any one of claims 1 to 10 in the preparation of a medicament for treating and preventing diseases, preferably, the disease is a fibrotic disease or a cardiovascular disease, more preferably, the cardiovascular disease is heart failure or myocardial hypertrophy. Preferably, the disease is a fibrotic disease or a cardiovascular disease; More preferably, the cardiovascular disease is heart failure or myocardial hypertrophy. Use.

26. Use of the serum albumin-binding molecule according to any one of claims 11 to 18 in the preparation of a medicament for diagnosing, treating and preventing diseases.

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