Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/2221—Relaxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/64—Relaxins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• the present disclosure belongs to the field of biomedicine, and specifically relates to a fusion protein comprising relaxin or its analogues and a serum albumin binding domain, and its medical use for treating diseases (eg, heart failure). And, it relates to a serum albumin-binding molecule, which contains an immunoglobulin single variable domain (such as VHH) capable of binding to serum albumin, has good plasma stability, and can be used to extend the plasma half-life of a drug.
• a serum albumin-binding molecule which contains an immunoglobulin single variable domain (such as VHH) capable of binding to serum albumin, has good plasma stability, and can be used to extend the plasma half-life of a drug.
• Relaxin an important hormone during pregnancy, was first discovered by Frederick Hisaw in 1926. It is a peptide hormone and belongs to the insulin superfamily. Relaxin contains seven polypeptide members in humans, including relaxin 1 (RLN-1 or H1), relaxin 2 (RLN-2 or H2), and relaxin 3 (RLN-3 or H3). These relaxins have low primary sequence homology but similar tertiary structures. Among them, relaxin 2 is the main relaxin in human circulation and is encoded by the RLN2 gene. Relaxin is expressed in the form of a precursor hormone (the A and B chains are linearly connected through C peptide). In the body, the C peptide is hydrolyzed by endonucleases to release mature relaxin.
• Natural mature relaxin 2 contains two polypeptide chains A and B. They are covalently linked through disulfide bonds between the two chains to form a heterodimer. There is also a pair of disulfide bonds within the A chain (Schwabe, et al.Science .1977,197,914-915).
• Relaxin 2 is a pleiotropic hormone, and its signaling is mainly achieved through the receptor RXFP1 (also known as LGR7).
• RXFP1 is a G protein-coupled receptor (GPCR), which is rich in leucine repeat units.
• GPCR G protein-coupled receptor
• RXFP2 also known as LGR8 also binds to relaxin 2.
• the combination of relaxin 2 and receptors can activate a variety of cascade signals, including activation of cAMP and phosphoinositide 3-kinase pathways, activation of nitric oxide signaling pathways, etc.
• the binding of relaxin 2 to the receptor can also inhibit the phosphorylation of Smad2/3, thereby inhibiting the production of extracellular matrix and collagen.
• relaxin 2 has physiological effects such as promoting vasodilation, anti-inflammation, reducing vascular resistance and increasing arterial compliance, promoting angiogenesis and remodeling, regulating extracellular matrix, and anti-fibrosis, and is beneficial to heart failure and myocardial fibrosis.
• the half-life (t1 /2 ) of relaxin 2 is very short, and the half-life (t1/2) of natural relaxin 2 in the body is only a few minutes, which brings challenges to relaxin as a therapeutic drug.
• the natural recombinant relaxin 2 (serelaxin) that is already on the market requires continuous intravenous infusion for clinical treatment, which causes inconvenience to patients and also shows short-lived efficacy.
• researchers have modified relaxin 2 to increase its half-life (t 1/2 ), such as introducing PEG, fatty acid chains, etc. through chemical coupling, or integrating proteins, peptides, or Fc with extended half-life through recombinant methods.
• Albumin is the most abundant protein in plasma, accounting for 40-60% of plasma proteins. Albumin can bind many endogenous and exogenous ligands and is a natural "multifunctional carrier" in the body. Albumin is similar to immunoglobulin G (IgG) and remains in the body for a relatively long time. It has been reported that the half-life of albumin in the human body is approximately 19-21 days (Berson, S.A. et al, J.Clin.Invest.1953, 32,746-768), this is mainly because: the molecular weight of albumin is 66.5KD, and its molecular hydrodynamic radius exceeds the glomerular filtration threshold of 3.5-6nm (Lin, J.H. Curr. Drug Metab.
• Albumin can bind to FcRn, and this binding is pH-dependent. After endocytosis into the endosome, the binding between the two is stronger at the acidic pH of the endosome (pH 5.5 or 6.0), thereby protecting albumin from immunity. Due to the degradation of lysosomes, as FcRn is transported to the cell surface, albumin dissociates from FcRn under neutral physiological pH conditions, thereby achieving recycling. This principle is similar to the recycling of IgG, and albumin is recycled. The protein and IgG bind to two independent sites on FcRn and do not affect each other.
• the present disclosure provides novel structures of single domain antibodies that bind serum albumin, which can significantly increase the in vivo half-life of pharmaceutical active ingredients.
• the single-domain antibody binding to serum albumin of the present disclosure is fused to relaxin or its analogues as the active ingredient into a fusion protein, the in vivo half-life of relaxin or its analogues can be significantly increased.
• the fusion protein provided by the present disclosure has high activity, high stability, easy production, and excellent clinical application potential.
• the present disclosure provides a serum albumin-binding molecule, which contains an immunoglobulin single variable domain that specifically binds serum albumin, has good plasma stability, and can be used to extend the plasma half-life of a drug.
• the present disclosure provides serum albumin binding molecules.
• the serum albumin binding molecule comprises an immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 as described in any one of 1)-5) below:
• CDR1 is X 1 SCX 2 G (SEQ ID NO: 64), where X 1 is T or N, and X 2 is M or L;
• CDR2 is X 3 IYX 4 VGGSTFYX 5 X 6 SVKG (SEQ ID NO: 58 ) , where X 3 is S or T , X 4 is T or M,
• CDR3 is GSPARCX 7 _ _
• one or more of the above-mentioned X 1 to X 9 may be conservative amino acid mutations of its optional amino acids.
• the above-mentioned CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, the CDR is defined according to the Kabat numbering system.
• the serum albumin-binding molecule includes an immunoglobulin single variable domain
• the immunoglobulin single variable domain includes any one or more of CDR1, CDR2, and CDR3 in 1)-5) above. (for example, 2 or 3) combinations.
• the serum albumin binding molecule comprises an immunoglobulin single variable domain, which contains a CDR1 with 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the foregoing CDR1s; and/ Or, the CDR2 included in the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared with any of the aforementioned CDR2s; and/or, the immunoglobulin single variable domain
• the CDR3 contained in the variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared with any of the aforementioned CDR3s.
• the amino acid mutations in CDR1, CDR2 and/or CDR3 are conservative substitutions.
• the aforementioned immunoglobulin single variable domain is of camel origin.
• the aforementioned immunoglobulin single variable domain is modified by any of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerism ation, or a combination thereof.
• the framework region in the aforementioned humanized immunoglobulin single variable domain is derived from the human heavy chain variable region germline gene IGHV3-23 or IGVH3-66.
• the aforementioned humanized immunoglobulin single variable domain contains amino acid mutations at at least one of the following positions: 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83 and 84 positions (position based on EU numbering system);
• amino acid mutation selected from: 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q or 84P;
• amino acid mutation combinations selected from any of the following:
• the serum albumin-binding molecule provided by the present disclosure comprises an immunoglobulin single variable domain comprising or being any of SEQ ID NO: 6 or 7 or having Amino acid sequences with at least 80%, at least 90% sequence identity.
• the immune The immunoglobulin single variable domain is of camel origin.
• the serum albumin binding molecule provided by the present disclosure includes an immunoglobulin single variable domain, which includes or is any one of SEQ ID NOs: 18-27 or with An amino acid sequence having at least 80%, at least 90% sequence identity.
• the immunoglobulin single variable domain is humanized.
• At least 90% (sequence) identity encompasses 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;
• at least 80% (sequence) identity encompasses 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 range between any two of the foregoing values, including integers and decimals.
• the immunoglobulin single variable domain in the serum albumin-binding molecule includes three complementarity determining regions CDR1, CDR2 and CDR3 and four FRs, in the following order from the amino terminus to the carboxyl terminus. Arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
• the aforementioned serum albumin-binding molecule is an antibody that binds serum albumin or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or an antigen-binding fragment thereof.
• the aforementioned antibody is a camel antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
• the aforementioned antigen-binding fragment is an sdAb, a bispecific antibody, or a multispecific antibody.
• the aforementioned immunoglobulin single variable domain is VHH.
• the aforementioned immunoglobulin single variable domain is a humanized VHH.
• the aforementioned immunoglobulin single variable domain is an affinity matured VHH.
• serum albumin binding molecules comprise one or more (eg, 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, said immunoglobulin
• the single variable domains can be the same or different, and any two immunoglobulin single variable domains can be connected directly or through a linker.
• serum albumin binding molecules are provided that bind or compete for binding to the same epitope as the immunoglobulin single variable domains of the disclosure described above.
• serum albumin binding molecules are provided that block the binding of the aforementioned immunoglobulin single variable domains of the present disclosure to serum albumin.
• serum albumin binding molecules are provided whose binding to serum albumin is blocked by the immunoglobulin single variable domains of the present disclosure as described above.
• the aforementioned serum albumin-binding molecule also includes an Fc region of an immunoglobulin, such as an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4; or a histidine tag, such as (His) 6 tag or (His) 8 tag.
• an Fc region of an immunoglobulin such as an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4
• a histidine tag such as (His) 6 tag or (His) 8 tag.
• Fc regions useful in the present disclosure can be from immunoglobulins of different subtypes, e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM.
• the Fc region includes the hinge region or a portion of the hinge region, the CH2 region, and the CH3 region of the constant region.
• the aforementioned immunoglobulin single variable domain and the Fc region or histidine tag can be connected by a linker.
• the linker may be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure.
• the linker is a flexible linker, such as 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 combinations thereof.
• the linker is GGGGSGGGS (SEQ ID NO:72).
• the serum albumin binding molecule provided by the present disclosure includes or is an amino acid sequence shown in SEQ ID NO: 15 or 16 or has at least 80%, at least 90% sequence identity therewith.
• the immunoglobulin single variable domain in the aforementioned serum albumin-binding molecule specifically binds serum albumin
• the serum albumin can be rodent serum albumin (e.g., mouse serum albumin (mSA)), or primate serum albumin (e.g., cynomolgus serum albumin (cySA), human serum albumin (HSA)).
• the serum albumin is HSA.
• the serum albumin-binding molecule of the present disclosure may comprise any of the aforementioned complete immunoglobulin single variable domains; it may also comprise a functional part of any of the aforementioned immunoglobulin single variable domains or its functional parts.
• Variants 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.
• the variant of the functional portion of the immunoglobulin single variable domain may be a CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2- The serum albumin binding function of FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, or FR1-CDR1-FR2-CDR2-FR3-CDR3 and having at least 80% or at least 90% sequence homology therewith
• a polypeptide that retains the serum albumin binding function of CDR3 and has a certain sequence homology with it such as a polypeptide with at least 80% or at least 90% sequence homology with any of the above-mentioned CDR3s.
• the immunoglobulin single variable domain in the serum albumin-binding molecules provided by the present disclosure, can be used as a carrier for extending half-life, covalently or non-covalently linked to any other macromolecule or small molecule compound. .
• the serum albumin binding molecules of the present disclosure comprise one or more therapeutic or diagnostic agents, wherein the therapeutic or diagnostic agent is covalent or non-covalent with the immunoglobulin single variable domain. Valence connection.
• the therapeutic or diagnostic agent is selected from the group consisting of therapeutic or diagnostic proteins, nucleic acids, and small molecule compounds.
• the present disclosure provides a serum albumin binding molecule or an immunoglobulin monomer therein.
• a variable domain is defined as ⁇ 1 ⁇ 10 -7 M, such as ⁇ 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 -9 M, ⁇ 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 - K D values of 10 M, ⁇ 3 ⁇ 10 -10 M, ⁇ 2 ⁇ 10 -10 M, ⁇ 1 ⁇ 10 -10 M bind serum albumin, such as human serum albumin.
• the affinity K D value of the serum albumin-binding molecule provided by the present disclosure for human serum albumin is 10 nM-100 nM, for example, 10 nM-50 nM.
• the detection method of the K D value is commonly used in the art, for example, it is provided in Example 4 of the present disclosure.
• the serum albumin binding molecule provided by the present disclosure has an activity selected from at least one of the following:
• the present disclosure provides serum albumin binding molecules that retain the advantageous properties of single domain antibodies (eg, VHH) and have an extended lifetime in the circulation of an individual. As a result, such molecules are able to circulate in a subject's serum for several days, thereby reducing the frequency of treatment, inconvenience to the subject, and cost of treatment.
• single domain antibodies eg, VHH
• the half-life of a serum albumin binding molecule of the present disclosure can be controlled by the number of single variable domains present in the molecule that specifically bind serum proteins.
• serum albumin-binding molecules of the present disclosure promote binding of serum albumin to FcRn in vivo and/or in vitro.
• the present disclosure provides fusion proteins comprising a serum albumin binding domain comprising at least one immunoglobulin single variable domain and relaxin or an analog thereof.
• the domain is any of the immunoglobulin single variable domains of the present disclosure described above.
• the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 according to any one of the following 1)-5):
• CDR1 is X 1 SCX 2 G (SEQ ID NO: 64), where X 1 is T or N, and X 2 is M or L;
• CDR2 is X 3 IYX 4 VGGSTFYX 5 X 6 SVKG (SEQ ID NO: 58 ) , where X 3 is S or T , X 4 is T or M,
• CDR3 is GSPARCX 7 _ _
• one or more of the above-mentioned X 1 to X 9 may be conservative amino acid mutations of its optional amino acids.
• the above-mentioned CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, the CDR is defined according to the Kabat numbering system.
• the aforementioned immunoglobulin single variable domain is modified by any of the following: camel origin, or humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation and/ or reduce antibody isomerization, or a combination thereof.
• the framework region in the humanized immunoglobulin single variable domain is derived from the human heavy chain variable region germline gene IGHV3-23 or IGVH3-66.
• the humanized immunoglobulin single variable domain comprises an amino acid mutation at at least one of the following positions: 20, 23, 27, 29, 30, 37, 44, 45, 47 , 49, 74, 78, 83 and 84 bits (position based on EU numbering system);
• amino acid mutation selected from: 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q or 84P;
• amino acid mutation combinations selected from any of the following:
• the immunoglobulin single variable domain comprises or is an amino acid sequence shown in any one of SEQ ID NO: 6, 18-27, or has at least 80%, at least 90% sequence identity therewith.
• the immunoglobulin single variable domain comprises or is an amino acid sequence shown in SEQ ID NO: 7 or has at least 80%, at least 90% sequence identity thereto.
• the immunoglobulin single variable domain is a VHH, such as a camel-derived, humanized VHH.
• the relaxin or analog thereof in the fusion protein includes an A chain and a B chain.
• the A chain has the amino acid sequence shown in any one of SEQ ID NO: 31, 50, 52, 54, 56, or has at least 90% the same amino acid sequence as any one of SEQ ID NO: 31, 50, 52, 54, 56 Amino acid sequences with sequence identity, or one or more amino acids compared to any of SEQ ID NO: 31, 50, 52, 54, 56
• the amino acid sequence is the addition, deletion, insertion or replacement of amino acids.
• the A chain can be SEQ ID NO: 31 or 50, with 1, 2, 3 or 4 amino acids deleted at the N-terminus, or 1, 2, 3 or 4 amino acids added. .
• the B chain has the amino acid sequence shown in any one of SEQ ID NO: 32, 51, 53, 55, and 57, or is at least 90% identical to any one of SEQ ID NO: 32, 51, 53, 55, and 57.
• the A chain and B chain of relaxin or its analog are any combination of the above.
• the relaxin or analog thereof comprises an A chain and a B chain combination of any one of 1)-7) below:
• DSWMEEVIKLCGRDLVRAQIAICGMSTWS (SEQ ID NO: 57).
• the fusion protein includes one of the aforementioned immunoglobulin single variable domains and one of the aforementioned relaxin or an analog thereof. In some embodiments, the fusion protein includes 1 aforementioned immunoglobulin single variable domain and 2 aforementioned relaxin or analogs thereof. In some embodiments, the fusion protein includes 2 of the aforementioned immunoglobulin single variable domains and 1 of the aforementioned relaxin or an analog thereof. In some embodiments, the fusion protein includes 2 of the aforementioned immunoglobulin single variable domains and 2 of the aforementioned relaxin or analogs thereof.
• the fusion protein includes polypeptides as shown in any one of Formula (I) to Formula (IV) or any combination: BL 1 -AL 2 -VHH Formula (I) AL 1 -BL 2 -VHH formula (II) VHH-L 2 -BL 1 -A Formula (III) VHH-L 1 -AL 1 -B Formula (III)
• A is any of the aforementioned relaxin A chains
• B is any of the aforementioned relaxin B chains
• L 1 and L 2 are linkers, which may or may not exist independently.
• 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 from 1 to 10, such as 3, 4, 5, 6, 7, 8.
• L 1 comprises an amino acid sequence set forth in, or having at least 90% sequence identity with, any of SEQ ID NOs: 33-35.
• L 2 includes the amino acid sequence of ( G Integers from 1 to 10, such as 3, 4, 5, 6, 7, 8.
• L 2 comprises the amino acid sequence set forth in SEQ ID NO: 49 or having at least 90% sequence identity thereto.
• L 2 comprises 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 an amino acid sequence having at least 90% sequence identity therewith.
• the fusion protein is a single chain protein.
• the fusion protein is a multimer, such as a dimeric protein.
• Fusion protein comprising a serum albumin binding domain comprising an immunoglobulin single variable domain and relaxin or an analog thereof, the immunoglobulin single variable domain comprising SEQ ID CDR1, CDR2 and CDR3 as shown in NO:8, 9 and 10, or as in SEQ ID NO: CDR1, CDR2 and CDR3 shown in 11, 12 and 13;
• the relaxin or its analog includes the A chain and B chain shown in SEQ ID NO: 31, 32, or as shown in SEQ ID NO: 50, 51 A chain and B chain as shown, or A chain and B chain as shown in SEQ ID NO: 52, 53, or A chain and B chain as shown in SEQ ID NO: 54, 55, or as SEQ ID NO: 31 and 53, or A chain and B chain as shown in SEQ ID NO: 52 and 32, or A chain and B chain as shown in SEQ ID NO: 56 and 57.
• Fusion protein comprising a serum albumin binding domain comprising an immunoglobulin single variable domain and relaxin or an analog thereof, the immunoglobulin single variable domain comprising SEQ ID
• the amino acid sequence shown in any one of NO: 6 and 18-27 or having at least 90% sequence identity with it, or the amino acid sequence shown in SEQ ID NO: 7 or having at least 90% sequence identity with it; described Relaxin or its analogues include A chain and B chain as shown in SEQ ID NO: 31, 32, or A chain and B chain as shown in SEQ ID NO: 50, 51, or as SEQ ID NO: 52, A chain and B chain shown in 53, or A chain and B chain shown in SEQ ID NO: 54, 55, or A chain and B chain shown in SEQ ID NO: 31, 53, or SEQ ID A chain and B chain shown in NO: 52 and 32, or A chain and B chain shown in SEQ ID NO: 56 and 57.
• fusion proteins comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 36-43 or having at least 90% sequence identity thereto.
• the binding affinity of the fusion proteins of the present disclosure to the RXFP1 receptor is comparable to the binding affinity of native human relaxin 2 (SEQ ID NO: 31 and 53). In other embodiments, the fusion proteins of the present disclosure have a binding affinity to the RXFP1 receptor that is less than the binding affinity to native human relaxin 2 (SEQ ID NOs: 31 and 53). In other embodiments, the fusion proteins of the present disclosure have a binding affinity to the RXFP1 receptor that is greater than the binding affinity to native human relaxin 2 (SEQ ID NOs: 31 and 53).
• the fusion proteins of the present disclosure significantly increase compared to native human relaxin 2 (SEQ ID NO: 31 and 53), such as having an in vivo half-life (t 1/2 ) comparable to that of serum albumin in different species. From a dozen hours to a dozen days.
• the fusion proteins of the present disclosure bind HSA with a KD value of ⁇ 1 ⁇ 10 ⁇ 7 M, for example, ⁇ 1 ⁇ 10 ⁇ 8 M.
• the fusion proteins of the present disclosure do not block the binding of serum albumin to FcRn in vivo and/or in vitro.
• Fusion proteins of the present disclosure encompass pharmaceutically acceptable salts thereof.
• polynucleotides encoding serum albumin binding molecules of the present disclosure are provided.
• polynucleotides encoding fusion proteins of the present disclosure are provided.
• the polynucleotide can be RNA, DNA or cDNA. According to some embodiments of the disclosure, the polynucleotides of the disclosure are isolated polynucleotides.
• the polynucleotides of the present disclosure may also be in the form of a vector, may be present in a vector, and/or may be part of a vector, such as a plasmid, cohesive plasmid, YAC, or viral vector.
• the vector may be, inter alia, an expression vector, i.e. Vectors for expression of serum albumin binding molecules in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system).
• the expression vector typically contains at least one polynucleotide of the present disclosure operably linked to one or more suitable expression control elements (eg, promoter, enhancer, terminator, etc.). Selection of the elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the serum albumin-binding molecules of the present disclosure are, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.
• polynucleotides of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA technology) based on the amino acid sequence information of the polypeptides of the present disclosure, and/or can be isolated from suitable natural sources. .
• host cells are provided that express or are capable of expressing one or more serum albumin binding molecules of the present disclosure, and/or recombinant host cells containing polynucleotides or vectors of the present disclosure.
• the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
• Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strain, Streptomyces strain, Staphylococcus strain and Lactococcus strain) cells.
• Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
• Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strain, Streptomyces strain, Staphylococcus strain and Lactococcus strain
• Fungal cells include, for example, cells of species of the genus Trichoderma, Neurospora, and Aspergillus; or include cells of the genus Saccharomyces (such as Saccharomyces cerevisiae), Schizosaccharomyces pombe Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen Cells of species of the genus Hansenula.
• Saccharomyces such as Saccharomyces cerevisiae
• Schizosaccharomyces pombe Schizosaccharomyces such as Schizosaccharomyces pombe
• Pichia such as Pichia pastoris and Pichia methanolica
• mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
• Amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins may also be used with the present disclosure.
• the cells of the present disclosure cannot develop into completed plant or animal individuals.
• the present disclosure provides methods for preparing the serum albumin binding molecules or fusion proteins of the present disclosure, comprising:
• Serum albumin binding molecules or fusion proteins of the present disclosure may be produced intracellularly (eg, in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, followed by isolation from the host cell and optionally further purified; or it can be produced extracellularly (eg in the medium in which the host cells are grown), followed by isolation from the medium and optionally further purified.
• Recombinant immunoglobulin expression vectors can stably transfect cells.
• Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region.
• Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors to produce antibodies.
• the culture medium secreting antibodies can be purified and collected using conventional techniques.
• Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
• the obtained product needs to be frozen immediately, such as -70°C, or freeze-dried.
• 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.
• the composition is a pharmaceutical composition containing a prophylactically or therapeutically effective amount of the serum albumin binding molecule or fusion protein of the present disclosure as described above, and/or encoding the serum albumin binding molecule or The polynucleotide of the fusion protein, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
• the pharmaceutical composition unit dose may contain 0.01 to 99% by weight of serum albumin binding molecules or fusion proteins.
• the unit dose of the pharmaceutical composition contains the serum albumin binding molecule or fusion protein in an amount of 0.1-2000 mg; in some specific embodiments, it is 1-1000 mg.
• kits or products which comprise any of the aforementioned serum albumin-binding molecules of the present disclosure and/or nucleic acid molecules encoding the serum albumin-binding molecules of the present disclosure.
• the present disclosure provides compositions for detecting serum albumin, the compositions comprising a serum albumin binding molecule of the present disclosure.
• the present disclosure also provides methods, systems or devices for detecting serum albumin in vivo or in vitro, comprising using the serum albumin binding molecules of the present disclosure.
• an in vitro detection method, system or device may, for example, comprise (1) contacting a sample with a serum albumin binding molecule of the present disclosure; (2) detecting complex; and/or (3) contacting a reference sample (e.g., a control sample) with the antibody; and (4) determining the extent of complex formation between the antibody and the sample by comparison with the reference sample.
• a change eg, a statistically significant change
• a change in complex formation in a sample or subject as compared to a control sample or subject indicates the presence of serum albumin in the sample.
• in vivo detection methods, systems or devices may include: (1) administering a serum albumin binding molecule of the present disclosure to a subject; and (2) detecting a combination of the serum albumin binding molecule of the present disclosure and the subject the formation of complexes between them. Detection can include determining where or when the complex is formed.
• Antibodies that bind serum albumin-binding molecules can be labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound antibodies. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
• Complex formation between serum albumin binding molecules of the present disclosure and serum albumin can be detected by measuring or visualizing the antibodies that bind or do not bind to the serum albumin binding molecules.
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• tissue immunohistochemistry a sample is analyzed for the presence of serum albumin by a competitive immunoassay using standards labeled with a detectable substance and unlabeled serum albumin binding molecules of the present disclosure.
• the living sample to be detected or measured may be tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.
• the serum albumin binding molecules of the present disclosure may be labeled with a fluorophore or chromophore for detection purposes.
• the present disclosure provides a method for extending the plasma half-life of a therapeutic or diagnostic agent, which includes covalently or non-conjugating a serum albumin binding molecule of the present disclosure or an immunoglobulin single variable domain thereof with the therapeutic or diagnostic agent.
• Covalent connection The method for ligation is known in the art, or may be any future method, for example, it may be fused by chemical coupling, fused at the DNA level, fused at the mRNA level, or fused at the protein level, etc.
• the therapeutic or diagnostic molecule can be a therapeutic target, diagnostic target.
• the target can be any protein, peptide, nucleic acid, oligonucleotide, sugar, polysaccharide, glycoprotein of therapeutic or diagnostic use. 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.
• the plasma half-life of a therapeutic or diagnostic agent linked to a serum albumin binding molecule of the present disclosure or an immunoglobulin single variable domain thereof is increased by at least 1.5 times, preferably at least 2 times, compared to before linking, For example, at least 5 times, such as at least 10 times or more than 20 times; for example, compared with before connection, the increased plasma half-life can be greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, or even greater than 24, 48 or 72 hours.
• the present disclosure provides methods of treating and/or preventing disease comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of a serum albumin binding molecule or fusion protein of the present disclosure.
• the present disclosure also provides a serum albumin binding molecule or fusion protein for use in a method of treating and/or preventing disease, the method comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of a serum albumin binding molecule of the present disclosure.
• molecules or fusion proteins are also provided.
• the present disclosure also provides the use of a serum albumin binding molecule in the preparation of a medicament for the treatment and/or prevention of disease.
• the disease is a fibrotic disease or a cardiovascular disease.
• the cardiovascular disease is heart failure (heart failure) and myocardial hypertrophy.
• Figure 1A to Figure 1C ELISA binding experiment results of serum albumin binding molecules.
• Figure 1A shows the results of an ELISA binding experiment between anti-HSA antibodies and human serum albumin (HSA).
• Figure 1B shows the ELISA binding experiment results of anti-HSA antibody binding to monkey serum albumin (CySA).
• Figure 1C shows the results of an ELISA binding experiment of anti-HSA antibodies binding to mouse serum albumin (mSA).
• Figure 2A and Figure 2B show the experimental results of the effect of anti-HSA antibodies on the binding of FcRn and HSA.
• Figure 3 shows the drug-time curve in plasma after intravenous administration of anti-HSA antibody in mouse pharmacokinetics study.
• Figure 4A to Figure 4B show the results of pharmacokinetic studies of anti-HSA antibodies in mice.
• Figure 4A shows the comparison results of calculating the T 1/2 parameters of anti-HSA antibodies in plasma using a non-compartmental model.
• Figure 4B shows the Cl_obs(mL/(min*kg)) parameter comparison results.
• Figure 5 is a schematic structural diagram of the fusion protein (formula (III)) of the present disclosure.
• Figure 6A shows the results of testing the in vitro activity of the fusion protein in THP1 cells under HSA-free conditions.
• Figure 6B shows the results of testing the in vitro activity of the fusion protein in THP1 cells under the condition that the system contains 1% HSA.
• Figure 7A shows the plasma drug time curve of the fusion protein in mouse model.
• Figure 7B shows the plasma drug time curve of the fusion protein in rat model.
• Figure 8 shows the heart weight/tibia length ratio of the mouse ISO cardiac hypertrophy model in the experiment on the effect of fusion protein on mouse cardiac hypertrophy.
• Antibody or “immunoglobulin” is used as a general term whether it refers to a heavy chain antibody or a conventional tetrapeptide chain antibody consisting of two heavy chains and two light chains connected by interchain disulfide bonds. To include full-length antibodies, individual chains thereof, and all parts, domains or fragments thereof (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH/VL domains).
• Sequence (for example in the terms “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence” etc.) is generally understood to include both the relevant amino acid sequence , also includes nucleic acid sequences or nucleotide sequences encoding said sequences, unless the disclosure requires further limited interpretation.
• a “domain” of a polypeptide or protein refers to a folded protein structure that can function independently of the rest of the protein maintain its three-level structure. Generally speaking, a domain is responsible for a single functional property of a protein, and in many cases can be added, removed, or transferred to other proteins without losing the functionality of the remainder of the protein and/or the domain.
• Immunoglobulin domain refers to the globular region of an antibody chain.
• Immunoglobulin variable domain refers to a structure basically consisting of “framework region 1" (FR1), “framework region 2" (FR2), “framework region 3” (FR3), and “framework region 4" (FR4). , and a domain composed of "complementarity determining region 1" (CDR1), “complementarity determining region 2" (CDR2), and “complementarity determining region 3” (CDR3). Therefore, the general structure (or sequence) of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain has an antigen-binding site that confers specificity for the antigen.
• Antibody framework (FR) refers to the portion of a variable domain that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain.
• Immunoglobulin single variable domain is generally used to refer to a structure that can function without interaction with other variable domains (e.g., without the need between the VH and VL domains of conventional four-chain monoclonal antibodies).
• an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that forms a functional antigen binding site.
• immunoglobulin single variable domains include Nanobodies (including VHH, humanized VHH and/or camelized VH, such as camelized human VH), IgNAR, domains, as VH domains or derived from VH domain (single domain) antibodies (such as dAbs TM ) and (single domain) antibodies that are VL domains or derived from VL domains (such as dAbs TM ).
• Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains, such as VH or VHH domains, are generally preferred.
• One specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
• VHH domain also known as heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment, VHH antibody, Nanobody, is called “heavy chain antibody” (i.e., “antibody lacking light chain”)
• Antigen-binding variable domains of immunoglobulins Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature363 , 446-448(1993)).
• VHH domain is used to distinguish the variable domain from the VH and VL found in conventional tetrapeptide chain structured antibodies.
• the VHH domain specifically binds the epitope without the need for other antigen-binding domains (for the VH or VL domain in conventional tetrapeptide chain-structured antibodies, the epitope is recognized by the VL domain together with the VH domain).
• the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
• the terms "heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH domain”, “VHH domain”, “ VHH antibody fragment”, “VHH antibody”, as well as” "Nanobody”("Nanobody” is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably.
• VHH domain includes, but is not limited to, natural antibodies produced by camelids, and may also be antibodies produced by camelids. Later humanized, or obtained through phage display technology screening. The total number of amino acid residues in the VHH domain will usually be in the range of 110 to 120, often between 112 and 115. However, it should be noted Smaller and longer sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind specific antigens or epitopes have been previously disclosed in: R.
• the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one according to the Kabat numbering or multiple positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows).
• the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
• Other numbering systems or coding conventions include Chothia, IMGT, and AbM.
• Humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. It can overcome the strong immune response induced by chimeric antibodies carrying a large number of non-human protein components. In order to avoid a decrease in activity while reducing immunogenicity, a minimum of reverse mutations can be performed on the variable region of the fully human antibody to maintain activity.
• Examples of "humanization” include converting a VHH domain derived from the Camelidae family by replacing the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in a human conventional tetrapeptide chain structure antibody VH domain.
• a humanized VHH domain may contain one or more fully human framework sequences and, in some embodiments, may contain human framework sequences of IGHV3.
• Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework grafting method (CDR grafting to a universal framework), that is, CDR "grafting” to other "scaffolds” (including but not limited to human scaffolds or non-immunoglobulin scaffold). Scaffolds and techniques suitable for such CDR transplantation are known in the art.
• the germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database and in Kabat, E.A.
• humanized antibodies of the present disclosure also include humanized antibodies that further undergo affinity maturation of CDRs by phage display.
• a minimum of reverse mutation or back mutation can be performed on the human antibody variable region framework sequence to maintain activity.
• Epitope or the term “antigenic determinant” used interchangeably refers to any antigenic determinant on an antigen to which an antibody binds.
• Antigenic determinants typically comprise chemically active surface groups of the molecule, such as amino acids or sugar side chains, and often have specific three-dimensional structural characteristics and/or specific charge characteristics.
• an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear ” epitope or “conformational” epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).
• linear epitopes can be determined by, for example, the simultaneous synthesis of a large number of peptides corresponding to parts of a protein molecule on a solid support and allowing the peptides to remain attached to the support while still attached to the support. Antibody response.
• 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.
• Conformational epitopes can also be identified by determining the spatial configuration of amino acids, such as by, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
• Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen.
• a high-throughput method to obtain antibodies binding to the same epitope based on their cross-competition is described in international patent application WO03/48731. Therefore, antibodies and antigen-binding fragments thereof that compete for binding to the same epitope on serum albumin as the antibody molecules of the present disclosure can be obtained using conventional techniques known to those skilled in the art.
• 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 (eg, a serum albumin-binding molecule of the present disclosure) can bind.
• the specificity of an antigen-binding protein can be determined based on its affinity and/or avidity.
• Affinity expressed by the dissociation equilibrium constant (K D ) of an antigen and an antigen-binding protein, is a measure of the strength of the binding between an epitope and the antigen-binding site on the antigen-binding protein: the smaller the K D value, the better the epitope binds to the antigen.
• affinity is a measure of the strength of the binding between an antigen-binding protein (eg, an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and the antigen of interest. Affinity is related to both the affinity for the antigen-binding site on its antigen-binding protein and the number of relevant binding sites present on the antigen-binding protein.
• an antigen-binding protein eg, an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same
• Affinity is related to both the affinity for the antigen-binding site on its antigen-binding protein and the number of relevant binding sites present on the antigen-binding protein.
• “Serum albumin binding domain” means any polypeptide capable of specifically binding to serum albumin or an epitope thereof.
• "Serum albumin-binding molecule” means any molecule that can specifically bind to serum albumin or its epitope, including but not limited to proteins and polypeptides.
• Serum albumin-binding molecules may include antibodies or antigen-binding fragments thereof as defined in the present disclosure directed against serum albumin or epitopes thereof, or conjugates or fusion proteins comprising said antibodies, antigen-binding fragments thereof.
• the antigen-binding fragment is, for example, sdAb, bispecific antibody, or multispecific antibody.
• Serum albumin-binding molecules of the present disclosure may comprise at least one (eg, 2, 3, 4, or more) immunoglobulin single variable domains (eg, VHH) that bind serum albumin.
• Serum albumin-binding molecules of the present disclosure may also comprise, in addition to immunoglobulin single variable domains, linkers and/or moieties having the function of effector molecules, including but not limited to diagnostic or therapeutic agents, e.g., anti-tumor agents. agents, immunomodulators, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.
• an "affinity matured" serum albumin antibody e.g., VHH
• an "affinity matured" serum albumin antibody that has one or more changes in one or more CDRs that results in an increased affinity for serum albumin compared to its parent anti-serum albumin antibody.
• Increase. Affinity matured anti-serum albumin antibodies can be prepared by methods known in the art, for example, as described by 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.
• Reverse mutation refers to the mutation of the amino acid residues in the FR region of the human antibody to the amino acid residues at the corresponding positions of the original antibody. This is usually done to avoid the decrease in activity caused by the humanized antibody while reducing the immunogenicity. , the variable region of the humanized antibody can be subjected to minimal reverse mutation to maintain the activity of the antibody.
• the serum albumin binding molecules of the present disclosure will preferably be from 10 "7 to 10 "10 mol/L (M), more preferably from 10 "8 to 10" 10 mol/L as measured in the Biacore or KinExA or Fortibio assays , even more preferably a dissociation constant (K D ) of 10 -9 to 10 -10 or lower, and/or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, even more preferably Binds the antigen to be bound (i.e., serum albumin) with a binding constant (K a ) of at least 10 ⁇ 10 M. Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
• Specific binding of an antigen or epitope by an antigen-binding protein can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays described in this disclosure ( Examples include radioimmunoassay (RIA), enzyme immunoassay (ELISA) and sandwich competitive assays.
• SPR surface plasmon resonance
• RIA radioimmunoassay
• ELISA enzyme immunoassay
• sandwich competitive assays sandwich competitive assays.
• inhibitortion or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking.
• “Homology” and “identity” in this disclosure refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
• the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
• sequences are optimally aligned, if 6 of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 of 100 positions in the two sequences match or homologous, then the two sequences are 95% homologous. In general, comparisons are made when aligning two sequences yields the greatest percent homology.
• Constant amino acid mutation or “conservative (amino acid) substitution” refers to the conservative amino acid substitution of one or more amino acid residues in a protein or polypeptide.
• the chemical structure of the amino acid residue before substitution is similar to that of the amino acid residue after substitution. , and the extraction has little or essentially no impact on the function, activity or other biological properties of the protein or polypeptide.
• the conservative amino acid substitution is well known in the art.
• the conservative amino acid substitution is preferably one amino acid in the following groups (i)-(v) replaced by another amino acid residue in the same group:
• Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Replacement; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
• Nucleic acid “nucleic acid molecule” or “polynucleotide” are used interchangeably in this disclosure to refer to any single- or double-stranded DNA molecule or RNA molecule and, in the case of single-stranded, its complementary sequence
• the molecule is preferably double-stranded DNA.
• a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
• the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
• the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
• the vectors of the present disclosure are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors) or can be integrated into the host cell's genome upon introduction, thereby subsequently The host genome is replicated together (e.g., non-episomal mammalian vectors).
• Host cell includes any cell or cell culture that is or has been the recipient of a vector for incorporating a nucleic acid insert.
• Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell.
• Host cells include cells transfected and/or transformed in vivo with the nucleic acids of the present disclosure.
• Cell “cell line,” and “cell culture” are used interchangeably, and all such designations include their progeny. It should also be understood that all offspring are unlikely to be exactly the same in terms of DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened in the originally transformed cells are included.
• “Pharmaceutical composition” means containing one or more fusion proteins, serum albumin-binding Mixtures of molecules, polynucleotides and other components such as physiologically/pharmaceutically acceptable carriers, diluents, buffers or excipients.
• the purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.
• administering when applied to an animal, human, subject, cell, tissue, organ or biological fluid, means the administration of an exogenous drug, therapeutic, diagnostic or composition to the animal, Contact with persons, subjects, cells, tissues, organs or biological fluids.
• administering may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental procedures.
• Treatment of cells includes contact of reagents with the cells, and contact of the reagents with a fluid, wherein the fluid is in contact with the cells.
• administering also mean treating a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell.
• Treatment when applied to human, veterinary medicine or research subjects means therapeutic treatment, prophylactic or prophylactic measures, research and diagnostic applications.
• Treat means administering an internal or external therapeutic agent (eg, a fusion protein of the present disclosure) to a subject having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect.
• a therapeutic agent is administered to a subject or population in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms, or to inhibit the progression of such symptoms to any clinically measurable extent.
• the amount of therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
• Whether disease symptoms have been alleviated may be assessed by any of the clinical tests commonly used by physicians or other health care professionals to assess the severity or progression of symptoms. Although embodiments of the present disclosure (e.g., treatments or articles of manufacture) may not be effective in alleviating each target disease symptom, the results may be determined according to any statistical test known in the art, such as the Student's t-test, Chi-square test, Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should alleviate the symptoms of the target disease in a statistically significant number of patients.
• any statistical test known in the art such as the Student's t-test, Chi-square test, Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should alleviate the symptoms of the target disease in a statistically significant number of patients.
• Subject and “patient” in this disclosure refer to mammals, especially primates, especially humans.
• HSA human serum albumin
• the binding region between albumin and FcRn is located in the third domain of the albumin molecule.
• an albumin molecule was designed that lacks some amino acids of the third domain at the C-terminus (and the Avi-his tag is fused at the C-terminus). :
• Rat (Rat) serum albumin full-length amino acid sequence is
• TMB chromogenic solution for color development After washing again, add TMB chromogenic solution for color development, terminate with 1M sulfuric acid, and read the absorbance value at OD450nm wavelength using a SpectraMax M5 microplate reader. The titer was detected after 1:51200-fold dilution. If the titer is qualified, camel peripheral blood is collected for bank construction.
• PBMC peripheral blood lymphocytes
• RNA 600 ⁇ L of enzyme-removed deionized water was reconstituted to obtain RNA, which was reverse transcribed to obtain cDNA for phage library construction.
• HSA protein that lacks some amino acids of the C-terminal third domain (also known as truncated HSA, 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. Screening is performed to obtain antibodies with affinity for HSA.
• Example 2 The 57 specific sequences obtained through phage library screening in Example 2 were classified and sorted, 19 of them were cloned, and the phage supernatant ELISA binding of these 19 anti-HSA antibodies was tested, among which #7 and The OD450 values of clone #9 are shown in Table 1.
• CDR1 is X 1 SCX 2 G, where X 1 is T or N and X 2 is M or L;
• CDR2 is X 3 IYX 4 VGGSTFYX 5 X 6 SVKG (SEQ ID NO: 58 ) , where X 3 is S or T , X 4 is T or M,
• CDR3 is GSPARCX 7 _ _
• the VHH sequence is fused to human IgG1-Fc (CH2-CH3) and constructed into the PTT5 expression vector.
• the sequence of the connected human IgG1-Fc is as follows:
• the following is the full sequence of the VHH sequence fused to the human Fc (CH2-CH3) segment.
• the single underline is the human IgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 14), and the double underline is the linker sequence.
• the full sequence is as follows:
• the #7aHSA and #9a anti-HSA antibodies fused with human IgG1-Fc were transiently expressed and ProteinA affinity purified in HEK293 cells respectively. It was then detected through ELISA binding experiment and Biacore protein interaction experiment.
• 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).
• the negative control is PBS
• the positive control uses ALB8 fused with human IgG1-Fc (the variable region sequence comes from the sequence of ALB8 in Ablynx patent publication WO 2008/028977 A2). The sequence is as follows:
• the single underline is the Fc region of IgG1, and the double underline is the linker region).
• PBST pH 7.4, 0.05% Tween-20
• affinity constants of anti-HSA antibodies fused with human IgG1Fc and human, monkey, and mouse albumin were also measured using a Biacore T200 (GE Healthcare) instrument.
• protein A was covalently coupled to the CM5 S series chip (GE, Cat. 29-1049-88) using an amino coupling kit (GE, Cat. BR-1000-50), and the anti-HSA antibody was captured through affinity to protein A conjugated on the chip, and then flow different concentrations of human, monkey, and mouse albumin on the chip surface, and use Biacore instrument to detect the reaction signal in real time to obtain the binding and dissociation curve.
• BIAevaluation version 4.1 and GE software are used to perform the Langmuir 1:1 binding model. Fit to obtain the affinity constant.
• the buffer used in the experiment was HBS-EP solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20, pH 7.4, GE, Cat.#BR-1006-69). After each cycle, the chip was regenerated using pH 1.5 glycine-hydrochloric acid (GE, Cat. #BR-1003-54). The affinity results are shown in Table 4.
• the specific humanized sequence obtained is as follows, the CDR region is underlined, and the encoding rule is Kabat encoding:
• Example 5 Use the method in Example 5 to construct a fusion protein of the humanized anti-HSA antibody and the Fc (CH2-CH3) segment of hIgG1 or a protein fused with a 6His tag.
• the single underline is the sequence of the hIgG1-Fc (CH2-CH3) segment (SEQ ID NO: 14), the italics are the 6His tag sequence, and the double underline is the linker sequence.
• the protein sequence is as follows (taking #7_hu_1-hlgG1Fc and #7_hu_10-6His as examples, the same applies to other humanized anti-HSA antibodies).
• #7_hu_10-6His is the C-terminal fusion of #7_Hu_10 (SEQ ID NO: 26) The same goes for other antibodies.
• the plasmid was transiently transfected into HEK293 cells. After 5 days of cell culture, the expression supernatant was collected, centrifuged at 4000 rpm for 15 minutes to remove the cells, the supernatant was harvested and filtered with a 0.45 ⁇ M filter, and then used a Protein A column or a nickel column as routine in the field. The method was used for purification and separation. After detection, the anti-HSA antibody of the present disclosure is obtained.
• the affinity constants of the humanized anti-HSA antibody fused with the 6His tag and human, monkey, and mouse albumin were measured by Biacore T200 (GE Healthcare) instrument.
• Biacore T200 GE Healthcare
• the amino coupling kit GE, Cat.BR-1000-50
• His capture kit GE, Cat.28-9950-56
• the surface response value reaches about 10000RU, and is blocked with ethanolamine for use
• the humanized anti-HSA antibody fused with the 6His tag is captured on the anti-his chip through affinity, and then flows over the chip surface
• the Biacore instrument was used to detect the reaction signal in real time to obtain the binding and dissociation curve.
• BIAevaluation version 4.1 and GE software were used to fit the Langmuir 1:1 binding model to obtain the affinity constant.
• the buffer used in the experiment was HBS-EP solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20, pH 7.4, GE, Cat.#BR-1006-69). After each cycle, use pH 1.5 glycine-hydrochloride (GE, Cat.# BR-1003-54) to regenerate the chip.
• the affinity results of some humanized antibodies are shown in Table 6. The results show that the affinity of the antibodies obtained by screening in the present disclosure to HSA is equivalent to or slightly better than that of the positive control.
• HEK293E cells were used to transiently express the anti-HSA antibody 7_Hu_10-6his. After nickel column affinity purification, its affinity constant with human, monkey, mouse, rat, dog, and rabbit albumin was measured by Biacore T200 (GE Healthcare) instrument.
• the buffer used in the experiment is HBS-EP solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20, pH 7.4, GE, Cat.#BR-1006-69). After each cycle, the chip was regenerated using pH 1.5 glycine-hydrochloric acid (GE, Cat. #BR-1003-54). The affinity results are shown in Table 7.
• Example 7 Effect of anti-HSA antibodies on FcRn and biotin-HSA binding in cells stably expressing hFcRn
• the blocking effect of anti-HSA antibodies on the binding of HSA to FcRn was measured by the amount of fluorescence of random biotinylated HSA bound to the surface of HEK293 cells stably expressing hFcRn mutants (293-hFcRn-mut cells). Changes are detected.
• This experiment used HEK293 cells stably expressing hFcRn mutants (L320A, L321A), which can be better positioned and maintained on the cell membrane surface. Cells expressing hFcRn mutants were incubated with different concentrations of anti-HSA antibodies and Biotin-HSA, and then SA-FITC secondary antibodies were added for incubation. The weakening of the secondary antibody fluorescence signal (MFI) was used as a measure of the effect of anti-HSA antibodies on biotin-HSA. The strength of the blocking effect of HSA binding to FcRn.
• the specific experimental methods are as follows:
• Result analysis Based on the fluorescence signal value (MFI), determine whether the binding of the anti-HSA antibody to HSA blocks the binding of HSA to FcRn. If the fluorescence signal decreases, it means that the binding is blocked.
• MFI fluorescence signal value
• #7_Hu_10-6his and #7_Hu_10 do not block the binding of HSA to FcRn (detected at a concentration of 1 mg/mL).
• anti-HSA antibody of the present disclosure does not affect the pH-dependent binding of FcRn to HSA and can well maintain the FcRn-mediated serum albumin recycling mechanism.
• anti-HSA antibodies (#7_Hu_6-6his; #7_Hu_7-6his; #7_Hu_10-6his), positive control antibodies (ALB8-6his) and isotype control antibodies (Isotype, non-HSA binding VHH molecules) were detected in CD-1
• the pharmacokinetic characteristics were studied in male mice (Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.). Three mice in each group were administered via tail vein injection at a dose of 4 mg/kg on day 0, before administration, and at 5 minutes, 1 hour, 7 hours, 24 hours, 48 hours, 72 hours, Blood samples were collected at 96 hours, 120 hours, and 144 hours, and EDTA anticoagulation was used and centrifuged to prepare plasma for pharmacokinetic determination.
• PK determination Use 5 ⁇ g/mL anti-his antibody (Thermo, MAI-21315-1mg) to coat, add a volume of 100 ⁇ L/well to a 96-well enzyme plate (Corning, Cat No. 9018), and place it at 4°C overnight for 16 -20 hours. After discarding the liquid, wash the plate three times with PBST (pH 7.4, 0.05% Tween-20) buffer, and then add Casin Blocker (Thermo, 37528) blocking solution (300 ⁇ L/well), incubate in a 37°C incubator for 2 hours for blocking.
• PBST pH 7.4, 0.05% Tween-20
• each anti-HSA antibody #7_Hu_6-6his, #7_Hu_7-6his, and #7_Hu_10-6his of the present disclosure has a similar pK curve, and the half-life is within the half-life range of mouse serum albumin, and is better than the positive control ALB8-6his The clearance rate of the antibody is also lower than that of the ALB8-6his antibody.
• the relaxin A chain and B chain are connected through a linker to form a single-chain relaxin molecule.
• the single-chain relaxin molecule is further fused with an anti-HSA single-domain antibody to form a fusion protein.
• the structural diagram is shown in Figure 5.
• connector 1 can select connectors a, b, and c, where a is a flexible connector and b and c are rigid connectors.
• the positive control 0054 sequence in this example comes from WO2021022139A1 Example 7.
• the DNA sequence encoding the fusion protein (SEQ ID NO: 36-44) was codon optimized, synthesized and cloned into the pTT5 expression vector, and the expression plasmid was transiently transfected into expiCHO (ThermoFisher, Cat No. A29127) cells using ExpiFectamine CHO Transfection Kit. (ThermoFisher, Cat No.A29133), transfection was performed according to the instructions of the kit. Using the "High protocol" in the instructions, the cells were placed in a 32°C shaker for suspension and shaking culture the next day after transfection (5% CO 2 , 110 rpm, 75% humidity). On days 10-12 after transfection, collect the cell culture supernatant, centrifuge at 4000 rpm for 20 minutes, take the supernatant and filter it with a 0.45uM filter.
• A3 affinity chromatography captures the fusion protein: Centrifuge the supernatant of the cells expressing the fusion protein at high speed to collect the supernatant, and filter it with a 0.22um filter membrane.
• the A3 (JSR Life Sciences, Cat No. BP-AMS-A3-0100) affinity column was regenerated with 5 column volumes of 0.1 M NaOH (Sigma, Cat No. 71687), and then with 5 column volumes of 1 ⁇ PBS ( pH7.4) (Sangon Biotech (Shanghai) co., Ltd.: Cat No. E607016-0500) cleaning balance. Load the filtered supernatant at a low flow rate in order to combine with the affinity column. Control the flow rate to make the retention time about 1 minute or longer.
• Example 10 Affinity determination of fusion protein and serum albumin of different species
• the affinity of 0051 to human, monkey, rat, mouse, dog, pig, rabbit and bovine serum albumin was determined using Biacore T200 (GE Healthcare) instrument.
• the positive control is 0054.
• human albumin HSA Sigma, Cat No. 126658
• monkey albumin Abcam, Cat No. ab184894
• rat albumin Abcam, Cat No. ab198656
• mouse albumin Abcam, Cat No. ab1832278
• rabbit albumin Abcam, Cat No. ab188055
• bovine albumin Sigma, Cat No. B2064
• the S series CM5 sensor chip (GE, Cat No. 29-1049-88) was tested for anti-VHH antibody (MonoRab TM Rabbit Anti-Camelid VHH Cocktail) using an amino coupling kit (GE, Cat No. BR-1000-50). , Genescript, Cat No. A02014) coupling.
• the antibody was diluted to 10 ⁇ g/mL using 10 mM sodium acetate pH 4.5 (GE, Cat No. BR-1003-50).
• the CM5 chip is activated by EDC/NHS, the diluted anti-VHH antibody flows through it to make the surface response value reach about 5000RU, and then it is blocked with ethanolamine and set aside for use.
• Albumins of various genera were serially diluted using 1XHBS-EP (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% Surfactant P20).
• Use conjugated anti-VHH antibody chip Capture the fusion protein molecules to be tested, with a response value of about 30RU, and then flow serum albumin of different concentrations on the chip surface for 3 minutes, with a flow rate of 50 ⁇ L/min and a dissociation time of 1-10 minutes (different albumin species have different dissociation times) ), use 10mM glycine-hydrochloride PH1.5 (GE, Cat No. BR-1003-54) to regenerate the chip after each cycle.
• 1XHBS-EP 10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% Surfactant P20.
• CM5 sensor chip GE, Cat. 29-1049-88
• Various albumins were diluted to 2 ⁇ g/mL.
• EDC/NHS EDC/NHS
• diluted albumin flows through it to make the surface response value reach about 200 (180 to 230) RU, and then it is blocked with ethanolamine.
• the blank control channel also requires EDC/NHS activation and ethanolamine blocking.
• Molecules 0051 and 0054 were serially diluted using 1XHBS-EP (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% Surfactant P20): 2000, 666.66, 222.22, 74.074, 24.69, 8.23, 2.74, 0.91, 0.305nM .
• Samples of different concentrations of 0051 and 0054 flowed through the albumin coupling channel and blank channel for 3 minutes respectively, with a flow rate of 50 ⁇ L/min and a dissociation time of 4 minutes. After each cycle, 10mM glycine-hydrochloride pH 1.5 (GE, Cat. #BR-1003-54) Regenerate the chip.
• Biacore T200 detects the reaction signal in real time to obtain the binding and dissociation curves. Select the appropriate concentration point for the obtained data and use the BIAevaluation version 4.1 software to fit it with the Langmuir 1:1 binding model to obtain the affinity value.
• the affinity of the 0051 molecule to human, monkey, and rat albumin is comparable to that of the positive control molecule 0054, and the affinity to mouse and canine albumin is weaker than that of the positive control molecule 0054 (data not shown).
• Table 11 exemplifies the results of HSA, rat, and mouse.
• Example 11 In vitro cell biological activity of fusion protein
• THP-1 is an endogenously expressed RXFP1 receptor in human peripheral blood mononuclear cells (Procell, Cat No. CL-0233). Relaxin binds to the receptor on THP-1 cells and induces THP-1 cells to produce cAMP. . Therefore, in this example, the in vitro activity of the fusion protein was determined by detecting the production of cAMP.
• This example uses the homogeneous time-resolved fluorescence (HTRF) cAMP-Gs Dynamic kit (Cisbio, Cat No. 62AM4PEB) and determines cAMP levels based on the manufacturer's protocol. 0054, 800814 (from WO2015067113A1) and 0060 (WO2021255127A1, RELAX0023) were used as positive controls.
• HTRF time-resolved fluorescence
• the experimental method is as follows: Cell THP-1 was cultured at 37°C and 5% CO2 , and the culture medium was RPMI1640+0.05mM b-mercaptoethanol+10%FBS+1% penicillin-streptomycin double antibody. On the day of the experiment, THP-1 cells in the logarithmic growth phase were collected by centrifugation and resuspended in serum-free medium. The cell density was adjusted to 4 ⁇ 10 6 cells/mL, and the cells were cultured in a volume of 50 ⁇ L per well (20,000 cells /well) were transferred to a 96-well assay plate (Cisbio, Cat No. 66PL96025).
• the fusion protein sample to be tested and 0054, 0060, and 800814 as positive controls were diluted 10 times in a separate U-bottom 96-well plate using Stimulation Buffer 1 in the kit, and transferred to a volume of 5 ⁇ L per well. In a 96-well assay plate containing THP-1 cells, incubate for 30 minutes at 37°C and 5% CO2 . Cells without adding the sample to be tested serve as a reference control.
• cAMP-d2 Dilute cAMP-d2 (acceptor fluorophore) and anti-cAMP-cryptat (donor fluorophore) using Lysis&Detection Buffer 1 in the kit, and add cAMP-d2 to the 96-well assay plate at a volume of 5 ⁇ L per well. In all wells except the blank control well, anti-cAMP-cyptat was then added to all wells and incubated at room temperature for 1 hour in the dark.
• Lipofectamine 2000 (Thermo Fisher, Cat No. 11668019) was used to transiently transfect CHO-K1 cells (CCTCC, Cat No. GDC0018).
• the CHO-K1 cells were resuspended in 6 ⁇ 10 5 cells/well in advance. In the well plate, incubate for 12 hours at 37°C and 5% CO2 and then remove the original culture medium. Add 1.5 mL of serum-free Ham's F-12K (Kaighn's) medium (Gibco, Cat No. 21127030). For cells in each well: Take two test tubes.
• test tube 1 dissolve 3 ⁇ g of the corresponding plasmid DNA (human, dog, cynomolgus monkey, rat, mouse RXFP1 receptor or human RXFP2 receptor DNA plasmid) in 250 ⁇ L Opti-MEM I minus In serum medium (Gibco, Cat No. 31985070), in test tube 2, dissolve 3 ⁇ L of Lipofectamine 2000 reagent in 250 ⁇ L Opti-MEM I reduced serum medium.
• plasmid DNA human, dog, cynomolgus monkey, rat, mouse RXFP1 receptor or human RXFP2 receptor DNA plasmid
• test tube 1 After incubating at room temperature for 5 minutes, add the reagent in test tube 1 to test tube 2, gently Mix well, incubate at room temperature for 20 minutes, add dropwise to a 6-well plate (500 ⁇ L/well), change to complete medium after 6 hours of transfection, continue to culture for 24 hours, and conduct subsequent experiments.
• 6-well plate 500 ⁇ L/well
• HTRF time-resolved fluorescence
• CHO-K1 cells transfected with human, dog, cynomolgus monkey, rat or mouse RXFP1 receptors were collected by centrifugation and resuspended in serum-free medium, and the cell density was adjusted to 4 ⁇ 10 6 cells/mL and transferred to a 96-well assay plate (Cisbio, Cat No. 66PL96025) in a volume of 50 ⁇ L per well (20,000 cells/well).
• Sample 0051 to be tested and 0054, 0060, and 800814 as positive controls were diluted 10 times in a separate U-bottom 96-well plate using Stimulation Buffer 1 in the kit, and transferred to a solution containing 5 ⁇ L per well.
• THP1 cells were cultured in a 96-well assay plate at 37°C and 5% CO for 30 minutes, and cells without the test sample were used as reference controls. Dilute cAMP-d2 (acceptor fluorophore) and anti-cAMP-cryptat (donor fluorophore) using Lysis&Detection Buffer 1 in the kit, and add cAMP-d2 to the 96-well assay plate at a volume of 5 ⁇ L per well. In all wells except the blank control well, anti-cAMP-cyptat was then added to all wells and incubated at room temperature for 1 hour in the dark. Then use 620nm and 665nm wavelengths for reading on Tecan (Infinite 200 PRO).
• mice Male SD rats, 4-6 weeks old, weighing 200-250 g, were purchased from Beijing Vitong Lever Experimental Animal Technology Co., Ltd. During the feeding period, they had free access to food and water, with a 12/12-hour light/dark cycle and a temperature of 16-26°C. To humidity 40-70%. Animals are adapted to feed for no less than 7 days after arrival at the facility. One day before the start of the experiment, the experimental animals were numbered and randomly divided into four groups, with three animals in each group. On the day of the experiment, the four groups of experimental animals were injected intravenously or subcutaneously with test drugs 0051 and 0054 respectively. The dosage was 4 mg/kg, and the injection volume was 5 mL/kg.
• blood was collected at various time points before administration and at 0.5, 1, 3, 6, 24, 48, 72, 96, 120, 168, 192 and 240 hours after administration.
• blood was collected before administration and at 3, 6, 24, 48, 72, 96, 120, 168, 192 and 240 hours after administration.
• centrifuge at 4500 rpm for 5 min. Take the supernatant in an EP tube and store it at -20°C. After the experiment, transfer to -80°C for storage.
• the concentrations of 0051 and 0054 drugs in plasma were detected using the ELISA method: 0.4 ⁇ g/mL Anti-Relaxin 2/RLN2 antibody (Abcam, Cat No. EPR21832-15) was used for coating, and a 96-well enzyme label was added at a volume of 100 ⁇ L/well. plate (Corning, Cat No. 9018) and place it at 4°C overnight for 16-20 hours. After discarding the liquid, wash the plate three times with PBST (pH 7.4, 0.05% Tween-20) buffer, add 4% bovine serum albumin solution (300 ⁇ L/well), and incubate at room temperature for 1 hour for blocking.
• PBST pH 7.4, 0.05% Tween-20
• TMB Sigma, Cat No. T0440
• chromogenic substrate for color development
• 1M sulfuric acid to terminate the reaction
• SpectraMax M5 microplate reader to read the absorbance value at OD450nm.
• the PK results in rats show that after IV administration, the AUC of 0054 is approximately 2 times that of 0051, the t1/2 difference between the two molecules is not significant, and the bioavailability of 0051 after SC administration is higher than that of 0054.
• the apparent distribution volume (Vdss) of 0051 is larger, indicating that the molecule has better tissue cell barrier permeability; in addition, 0051 has a longer apparent retention time (MRT), and it is expected that the molecule will perform in vivo
• MRT apparent retention time
• the drug effect lasts longer; in addition, the bioavailability (Bioavailability%) of 0051 is significantly higher than that of 0054 after subcutaneous injection of SC. Therefore, 0051 has better pharmacokinetic properties.
• a C57BL/6J mouse (Charles River Laboratories) cardiac hypertrophy model was prepared by subcutaneously implanting a mini-osmotic pump (Alzet 1002) and infusing isoproterenol (ISO, Sigma, Cat. #I5627), and dissolving ISO in a solution containing 0.002% Sodium ascorbate (Sigma, Cat.#11140) in PBS buffer (Servicebio, Cat.#G4202) to a concentration of 146mg/mL (calculated based on the average mouse weight of 25.8g), and an infusion concentration of 15mg/kg/ d. Continuous infusion for 14 days was used to prepare a pathological model. On the 14th day, the osmotic pressure pump was taken out. The model control group 1 was euthanized and the materials were collected. The remaining mice were randomly divided into groups. Subcutaneous injection of fusion protein or control molecules began. The dosage and grouping are as follows. Table 15.
• mice's body weight was measured after the animals were fasted for 6 hours, the mice were euthanized, and the heart weight and tibia length were measured. Cardiac hypertrophy is determined by the ratio of heart weight to tibia length. See Figure 8 and Table 16.
• Infusion of ISO into mice can significantly cause cardiac hypertrophy, and treatment with the fusion protein of the present disclosure can reverse ISO-induced cardiac hypertrophy to baseline levels, and the required dose is significantly lower than control 0060.

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