Human serum albumin nano antibody and application thereof

Abstract

The invention relates to a human serum albumin nano antibody and application thereof, the nano antibody comprises CDR1-CDR3, according to the definition of a Kabat numbering system, the amino acid sequence of the CDR1 is HZ1Z2Z3Z4, the amino acid sequence of the CDR2 is SISGSSDTLYADSVKG or a variant thereof, and the amino acid sequence of the CDR3 is IGGY1Y2X1Y3N1; wherein X1 is selected from S or H; y1 is selected from A or S; y2 is selected from L, A or H; y3 is selected from R, A, V, W, S, T, H, K, Y, M, Q, G or L; n1 is selected from S or H; z1 is selected from F or H; z2 is selected from G or H; z3 is selected from M or H; z4 is selected from S or H.

Description

Technical Field

[0001] The present application relates to nanobodies against human serum albumin and their applications. Background Art

[0002] The neonatal Fc receptor (FcRn) is a specific receptor for immunoglobulin G (IgG) and human serum albumin (HSA), binding to both in a pH-dependent manner, enabling IgG and HSA to avoid lysosomal degradation and have a long plasma half-life. Immunoblotting and surface plasmon resonance studies have shown that IgG and HSA bind non-cooperatively to different sites on FcRn, and the affinity of FcRn for HSA and IgG decreases by 200-fold from acidic to neutral pH. Although the characteristics of the interaction between FcRn and IgG and HSA are different, the pH-dependent binding mechanism is generally similar. HSA and IgG can be recycled and released extracellularly through pH-dependent binding to FcRn, thus having a long plasma half-life.

[0003] The binding of FcRn to HSA has a profound impact on basic physiology. HSA accounts for approximately 80% of the total plasma proteins, mediating fundamental life functions, including maintaining the circulating plasma colloid osmotic pressure, regulating the pH of the plasma, and having a transport function, transporting numerous molecules throughout the body, including biological products, toxins, drugs, and therapeutic proteins.

[0004] Anti-HSA antibodies are often genetically engineered to be linked to bioactive proteins to form fusion proteins for the production of long-acting biologics. During the treatment of some diseases, the effective concentration of the therapeutic active molecule in human serum needs to be controlled within a reasonable range. For example, when administering growth hormone to patients with growth hormone deficiency or insulin to diabetic patients. Therefore, when using long-acting biologics based on anti-HSA antibodies, in addition to considering the half-life of the drug molecule, the effective concentration of the drug active molecule under physiological conditions (pH 7.4) also needs to be considered. In many cases, due to steric hindrance after binding to HSA, the activity of the active group of the anti-HSA antibody fusion protein will be reduced; while after dissociation from HSA, the activity of the active group of the free fusion protein will be restored. Therefore, by regulating the binding affinity between the anti-HSA antibody and HSA, the effective concentration of the anti-HSA antibody fusion protein drug molecule in serum can be effectively controlled. Summary of the Invention

[0005] Most of the anti-HSA antibodies or antigen-binding fragments known in the art bind to HSA with high affinity under physiological conditions (pH 7.4) in a non-pH-dependent manner, and may block the activity of the anti-HSA antibody fusion protein after binding, resulting in a low serum concentration of the active free anti-HSA antibody fusion protein. To improve the effective release of the anti-HSA antibody fusion protein drug molecule in the blood, the usual approach is to reduce the affinity of the anti-HSA antibody under physiological conditions (pH 7.4). However, to maintain the effect of the anti-HSA antibody in prolonging the drug half-life, it is necessary to ensure the high affinity of the antibody under pH 6.0 conditions. To achieve the above two goals, a pH-dependent anti-HSA antibody with lower affinity at pH 7.4 and higher affinity at pH 6.0 needs to be obtained, so as to develop a long-acting biopreparation fused with the pH-dependent anti-HSA antibody, in order to optimize the half-life and effective concentration of the drug molecule in the serum.

[0006] In the first aspect of the present application, a pH-dependent anti-human serum albumin nanobody or its antigen-binding fragment is provided. The human serum albumin nanobody includes CDR1-CDR3, defined according to the Kabat numbering system. The amino acid sequence of CDR1 is HZ1Z2Z3Z4, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG or its variant, and the amino acid sequence of CDR3 is IGGY1Y2X1Y3N1; wherein, X1 is selected from S or H; Y1 is selected from A or S; Y2 is selected from L, A or H; Y3 is selected from R, A, V, W, S, T, H, K, Y, M, Q, G or L; N1 is selected from S or H; Z1 is selected from F or H; Z2 is selected from G or H; Z3 is selected from M or H; Z4 is selected from S or H.

[0007] In the second aspect of the present application, a multispecific antibody is provided, which comprises the nanobody or its antigen-binding fragment, and another antibody or its antigen-binding fragment, or an antibody analog.

[0008] In the third aspect of the present application, a fusion protein is provided, which comprises the nanobody or its antigen-binding fragment, and another bioactive polypeptide.

[0009] In the fourth aspect of the present application, an isolated nucleic acid molecule is provided, which encodes the nanobody or its antigen-binding fragment, the multispecific antibody or the fusion protein.

[0010] In the fifth aspect of the present application, a vector is provided, which comprises the isolated nucleic acid molecule.

[0011] In the sixth aspect of the present application, a host cell is provided, which comprises the isolated nucleic acid molecule and / or the vector.

[0012] The seventh aspect of the present application provides a pharmaceutical composition, which comprises the nanobody or its antigen-binding fragment, the multispecific antibody, the fusion protein, the isolated nucleic acid molecule, the vector or the host cell, and a pharmaceutically acceptable carrier and / or excipient.

[0013] The eighth aspect of the present application provides the use of the nanobody or its antigen-binding fragment for prolonging the in vivo half-life of an active agent linked thereto. Brief Description of the Drawings

[0014] The subject matter of the present application can be understood by reading the following description of non-limiting examples and referring to the drawings, wherein:

[0015] Figures 1A - 1F Binding curves of some anti-HSA nanobodies of the examples of the present application at pH 6.0 and pH 7.4 are shown;

[0016] Figures 2A - 2J Fortebio detection results of some anti-HSA nanobodies of the examples of the present application are shown. Detailed Description of the Embodiments

[0017] Definition

[0018] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.

[0019] Generally, the nomenclature used herein and the laboratory procedures of biochemistry, biology, molecular biology, cell biology, immunology and pharmacology described herein are well known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0020] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric protein of approximately 150,000 daltons with the same structural features, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a plurality of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0021] As used herein, the terms "nanobody" and "single domain antibody (sdAb or VHH)" have the same meaning and refer to the variable region of a cloned antibody heavy chain, and a nanobody consisting of only one heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region of the heavy chain (CH1), the variable region of the antibody heavy chain is cloned, and a nanobody (VHH) consisting of only one heavy chain variable region is constructed.

[0022] As a novel small molecule antibody fragment, the nanobody / single domain antibody is obtained by cloning the variable region of the heavy chain of the naturally occurring heavy chain antibody in camels. The nanobody (Nb) has excellent biological properties, with a molecular weight of 12 - 15 kDa, which is one-tenth of that of a complete antibody. It has good tissue penetration, high specificity, and good water solubility. Due to its special structural properties, it combines the advantages of traditional antibodies and small molecule drugs, and almost perfectly overcomes the defects of traditional antibodies such as long development cycle, low stability, and harsh storage conditions. It has gradually become a new emerging force in the new generation of antibody therapy and shows broad application prospects in immunoassay and treatment.

[0023] The nanobody basically consists of 4 framework regions (FRs) and 3 complementarity-determining regions (CDRs). The 3 complementarity-determining regions are CDR1 - CDR3 respectively. The antigen-binding fragment contains at least a part of the nanobody, and this part is sufficient to endow the fragment with the ability to specifically bind serum albumin. The nanobody or its antigen-binding fragment specifically binds to human serum albumin.

[0024] Unless otherwise specified, the numbering of residues in the immunoglobulin heavy chain herein refers to the EU index numbering of Kabat et al. Kabat is an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0025] "Framework" or "FR" residues are variable domain residues other than the CDR residues defined herein.

[0026] The present invention

[0027] Through extensive and in-depth research and a large number of screenings, the present applicant has successfully obtained a pH-dependent anti-human serum albumin nanobody. The experimental results show that the anti-human serum albumin nanobody obtained in this application has different affinities for human serum albumin under the conditions of pH 6.0 and pH 7.4, and thus has pH dependence.

[0028] For the existing anti-human serum albumin nanobodies, no obvious pH dependence is shown when binding to human serum albumin. After the anti-HSA antibodies without pH dependence are constructed into fusion proteins together with active pharmacophore groups, they always tightly bind to human serum albumin under the human pH environment (pH 7.4). The binding of human serum albumin may cause steric hindrance of the pharmacophore groups and reduce their biological activities. The pH-dependent anti-human serum albumin nanobody can have a high affinity for human serum albumin under acidic conditions (pH 5 to pH 6.5) and a low affinity for human serum albumin under neutral conditions (pH 7 to pH 8), so as to achieve the dynamic release of active pharmacophore groups in the human body, enabling the fusion protein drug molecules to have both a long half-life and maintain an effective concentration in the serum.

[0029] Nanobody

[0030] In the first aspect of this application, there is provided an anti-human serum albumin nanobody or its antigen-binding fragment, and the nanobody is substantially composed of 4 framework regions and 3 complementarity-determining regions. The antigen-binding fragment contains at least a part of the nanobody, and this part is sufficient to endow the fragment with the ability to specifically bind serum albumin. The nanobody or its antigen-binding fragment specifically binds to human serum albumin.

[0031] In certain embodiments, the nanobody includes CDR1-CDR3, defined according to the Kabat numbering system. The amino acid sequence of CDR1 is HZ1Z2Z3Z4, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG or its variant, and the amino acid sequence of CDR3 is IGGY1Y2X1Y3N1; wherein, X1 is selected from S or H; Y1 is selected from A or S; Y2 is selected from L, A or H; Y3 is selected from R, A, V, W, S, T, H, K, Y, M, Q, G or L; N1 is selected from S or H; Z1 is selected from F or H; Z2 is selected from G or H; Z3 is selected from M or H; Z4 is selected from S or H.

[0032] In certain embodiments, the variant has 1-3 amino acid substitutions, deletions, additions, or any combination thereof compared to SISGSGSDTLYADSVKG. In certain embodiments, the nanobody or antigen-binding fragment thereof comprising the above substitutions, deletions, or additions retains the immunogenicity against humans and / or the activity of specifically binding to human serum albumin of the nanobody or antigen-binding fragment thereof from which it is derived.

[0033] In certain embodiments, the variant has 1-2 amino acid substitutions compared to SISGSGSDTLYADSVKG.

[0034] In certain embodiments, the variant has 1 amino acid substitution and is substituted with H compared to SISGSGSDTLYADSVKG.

[0035] In certain embodiments, the amino acid sequence of the variant is: SISY4SGSDTLYADSVKG, SISGSGSY5TLYADSVKG, SISGSGSDY6LYADSVKG, SISGSGSDTLY7ADSVKG, SISGSGSDTLYAY8SVKG, SISGSGSDTLYADSY9KG, SISGSGSDTLYADSVY 10 G, or SISGSGSDTLYADSVKY 11 ; wherein, Y4 is selected from D, G, A, S, or N; Y5 is selected from D, G, A, I, F, Y, S, T, M, Q, K, R, V, L, E, or N; Y6 is selected from T, H, G, A, V, L, I, F, Y, M, Q, D, E, K, R, or N; Y7 is selected from Y, H, G, A, V, L, I, F, W, S, T, C, M, Q, D, E, K, R, or N; Y8 is selected from D, H, G, A, V, L, I, F, Y, W, S, T, M, N, Q, E, K, or R; Y9 is selected from V, H, G, A, L, F, Y, W, S, T, N, Q, D, E, K, M, or R; Y 10 is selected from K, H, G, A, V, L, I, F, Y, W, S, T, C, M, N, Q, D, E, or R; Y 11 is selected from G or H.

[0036] In certain embodiments, the amino acid sequence of CDR1 is SFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHLS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLGADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLFADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVAG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLGADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLFADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVAG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLGADSVKG, and the amino acid sequence of CDR3 is IGGSLHLS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLFADSVKG, and the amino acid sequence of CDR3 is IGGSLHLS.In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVAG, and the amino acid sequence of CDR3 is IGGSLHLS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSVTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSLTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSETLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISDSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSMKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGALSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGALHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSASRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSAHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSVS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHVS.In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSQS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHQS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGHDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGHDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDHLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDHLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDVLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDVLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDFLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDFLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDYLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDYLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS.In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDQLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDQLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDKLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDKLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDRLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDRLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSLKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSLKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSWKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSWKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKH, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKH, and the amino acid sequence of CDR3 is IGGSLHRS.In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYATSVKG, and the amino acid sequence of CDR3 is IGGSLSRS. In certain embodiments, the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYATSVKG, and the amino acid sequence of CDR3 is IGGSLHRS. In certain embodiments, the nanobody comprises FR1-FR4, wherein the amino acid sequence of FR1 is EVQLVESGGGLVQPGNSLRLSCAASGFTFS; the amino acid sequence of FR2 is WVRQAPGKGLEWVS; the amino acid sequence of FR3 is RFTISRDNAKTTLYLQMNSLRPEDTAVYYCT; the amino acid sequence of FR4 is SQGTLVTVSS.

[0037] In certain embodiments, the substitutions described in any of the above embodiments are conservative substitutions.

[0038] Multispecific antibody

[0039] In a second aspect of the present application, there is provided a multispecific antibody comprising the anti-HSA single-domain antibody or an antigen-binding fragment thereof described in the first aspect of the present application, and another antibody or an antigen-binding fragment thereof.

[0040] In certain embodiments, the said another antibody or an antigen-binding fragment thereof has a binding specificity different from that of the aforementioned anti-HSA single-domain antibody or an antigen-binding fragment thereof. In certain embodiments, the said another antibody or an antigen-binding fragment thereof does not bind serum albumin.

[0041] In certain embodiments, the multispecific antibody comprises one anti-HSA single-domain antibody or an antigen-binding fragment thereof, and one another antibody or an antigen-binding fragment thereof, or an antibody mimetic.

[0042] In certain embodiments, the multispecific antibody comprises one anti-HSA nanobody or an antigen-binding fragment thereof, and two another antibodies or an antigen-binding fragments thereof.

[0043] In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetravalent antibody.

[0044] Fusion protein

[0045] In a third aspect of the present application, there is provided a fusion protein comprising the anti-HSA nanobody or an antigen-binding fragment thereof described in the first aspect, and another bioactive polypeptide.

[0046] In certain embodiments, the bioactive polypeptide is selected from granulocyte colony-stimulating factor, colony-stimulating factor, G protein-coupled receptor, IL-15, IL15RD, an IL15RD and IL-15 fusion protein, human growth hormone, cytokine-binding protein, protein A, allergy inhibitor, necroglycan, immunotoxin, lymphotoxin, tumor suppressor, transforming growth factor, α1-antitrypsin, α-lactalbumin, apolipoprotein-E, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, bone stimulating protein, calcitonin, atrial natriuretic peptide, cartilage-inducing factor, elcatonin, connective tissue activating factor, tissue factor pathway inhibitor, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, autotoxin, lactoferrin, myostatin, or a member of the heat shock protein family.

[0047] In certain embodiments, the anti-HSA nanobody or its antigen-binding fragment has a high affinity for human serum albumin under acidic pH conditions (pH 5 to pH 6.5) and a low affinity for human serum albumin under neutral pH conditions (pH 7 to pH 8), thereby achieving the dynamic release of the active pharmacophore in the human body, enabling the fusion protein drug molecule to have both a long in vivo half-life and maintain an effective concentration in the serum. The long in vivo half-life is relative to the in vivo half-life of the additional bioactive polypeptide in the absence of the anti-HSA nanobody or its antigen-binding fragment.

[0048] Preparation of antibody and fusion protein

[0049] The anti-HSA nanobody, multispecific antibody or fusion protein of the present application can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, a DNA molecule encoding the nanobody, multispecific antibody or fusion protein of the present application is obtained by chemical synthesis or PCR amplification. The obtained DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cell is cultured under specific conditions to express the nanobody, multispecific antibody or fusion protein of the present application.

[0050] The antigen-binding fragments of the present application can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Additionally, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab’ fragments can be directly obtained from host cells; Fab’ fragments can be chemically conjugated to form F(ab’)2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). Additionally, Fv, Fab or F(ab’)2 fragments can also be directly isolated from the culture broth of recombinant host cells. The preparation of the nanobody or its antigen-binding fragment of the present application can also refer to Muyldermans, Reviews in Molecular Biotechnology 74, 277-302 (2001), J. Sambrook & D. Russell, Molecular Cloning: A Laboratory Munual (3rd Edition), Cold Spring Harbor Laboratory Press, 2000, or its Chinese translation published by Science Press of China. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0051] Nucleic acid molecule

[0052] In the fourth aspect of the present application, an isolated nucleic acid molecule is provided, which contains a nucleotide sequence encoding the nanobody or its antigen-binding fragment described in the first aspect, a nucleotide sequence encoding the multispecific antibody described in the second aspect, or a nucleotide sequence encoding the fusion protein described in the third aspect. According to the codon degeneracy known in the art, in certain embodiments, the nucleotide sequence can be replaced according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0053] Vector

[0054] In the fifth aspect of the present application, a vector is provided, which comprises the isolated nucleic acid molecule described in the fourth aspect. In certain embodiments, the vector of the present application is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing the nanobody of the present application or its antigen-binding fragment, multispecific antibody or fusion protein in a subject (such as a mammal, such as a human).

[0055] Host cell

[0056] In the sixth aspect of the present application, a host cell is provided, which comprises the nucleic acid molecule of the fourth aspect and / or the vector of the fifth aspect. The host cell can be a eukaryotic cell (such as a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (such as Escherichia coli). Suitable eukaryotic cells include but are not limited to NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include but are not limited to Sf9 cells. In certain embodiments, the host cell is Escherichia coli.

[0057] Use and pharmaceutical composition

[0058] In the seventh aspect of the present application, a pharmaceutical composition is provided, which comprises the nanobody of the first aspect or its antigen-binding fragment, the multispecific antibody of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the host cell of the sixth aspect; and a pharmaceutically acceptable carrier and / or excipient.

[0059] In certain embodiments, the pharmaceutical composition of the present application comprises the nanobody of the present application or its antigen-binding fragment, and an active agent linked to the nanobody or its antigen-binding fragment.

[0060] In certain embodiments, the active agent is another antibody or its antigen-binding fragment, or an antibody analogue. In certain embodiments, the pharmaceutical composition comprises the multispecific antibody of the present application.

[0061] In certain embodiments, the active agent is an additional bioactive polypeptide. In certain embodiments, the bioactive polypeptide is selected from granulocyte colony-stimulating factor, colony-stimulating factor, G protein-coupled receptor, IL-15, IL15RD, IL15RD and IL-15 fusion protein, human growth hormone, cytokine binding protein, protein A, allergy suppressor, necrotizing glycoprotein, immunotoxin, lymphotoxin, tumor suppressor, transforming growth factor, α1-antitrypsin, α-lactalbumin, apolipoprotein-E, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, bone stimulating protein, calcitonin, atrial natriuretic peptide, cartilage-inducing factor, elcatonin, connective tissue activating factor, tissue factor pathway inhibitor, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, autotoxin, lactoferrin, myostatin, or a member of the heat shock protein family. In certain embodiments, the pharmaceutical composition comprises the fusion protein of the present application.

[0062] In certain embodiments, the pharmaceutical composition of the present application comprises the vector or host cell of the present application, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the host cell comprises the isolated nucleic acid molecule or vector as described above.

[0063] Therefore, the anti-HSA nanobody or antigen-binding fragment of the present application can be used to improve the therapeutic effect of long-acting protein drugs, and has great clinical value.

[0064] The embodiments of the present application will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present application and should not be regarded as limiting the scope of the present application. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in the art (for example, referring to "Molecular Cloning: A Laboratory Manual", Third Edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through market purchase.

[0065] Example. pH-dependent anti-HSA nanobody

[0066] 1. Construct an expression vector of an anti-HSA nanobody with a single-site mutation

[0067] The amino acid sequence of the unmodified anti-HSA nanobody is as follows:

[0068] EVQLVESGGGLVQPGNSLRLSCAASGFTFS SFGMS WVRQAPGKGL EWVS SISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCT IGGSLSRS SQGTLVTVSS。

[0069] Nucleotide sequence of the unmodified anti-HSA nanobody:

[0070] GAGGTTCAGCTGGTAGAGTCAGGAGGCGGTCTGGTTCAGCCAGGTAACTCTTTGAGATTGTCCTGCGCTGCTTCAGGCTTCACCTTCAGTTCTTTTGGTATGTCTTGGGTTAGGCAAGCTCCTGGTAAGGGTTTGGAATGGGTATCATCAATAAGTGGATCAGGATCTGACACTTTGTATGCCGACAGTGTTAAGGGAAGATTCACCATATCAAGAGATAACGCCAAGACTACCCTGTATTTGCAAATGAACTCTTTGAGACCTGAGGACACTGCCGTTTATTACTGCACCATTGGTGGATCTTTGTCCCGATCCTCTCAAGGAACCCTGGTCACCGTTTCTTCT。

[0071] In this example, site-directed mutagenesis technology was used to construct mutants. Two fragments with mutation sites were obtained by two rounds of PCR using two primers with mutation sites plus two outer primers, and then the mutant gene fragment could be obtained by PCR using the two outer primers with the left and right fragments as templates. The two outer primers were HSAuni-F2 and HSAuni-R. The two primers with mutation sites for each mutation site are shown in Table 1.

[0072] Sequence of HSAuni-F2:

[0073] 5’-CGGCGGCTCGAGAAAAGAGAGGTTCAGCTGGTAGAGTC-3’;

[0074] Sequence of HSAuni-R:

[0075] 5’-ACGAGAAGAAACAAAATGAC-3’。

[0076] The nucleotide sequence of the unmodified anti-HSA nanobody was artificially synthesized and inserted into the multiple cloning site of the pPICZαA plasmid to construct the anti-HSA-pPICZαA plasmid, and then sequenced.

[0077] According to the principle of overlapping PCR, using the correctly sequenced anti-HSA-pPICZαA plasmid as a template, and the reverse primer among the two primers with mutation sites shown in Table 1 and HSAuni-F2, PCR was carried out through the following program and system. System 1: In a 25 μl system, it contained 22 μl of TSINGKE TSE101 Gold Mix (green) (Beijing Tsingke Biotechnology Co., Ltd.), 1 μl of each 10 μM primer, and 1 μl of anti-HSA-pPICZαA plasmid. PCR Program 1: Pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s, extension at 72 °C for 10 s, for a total of 30 cycles; extension at 72 °C for 2 min, incubation at 4 °C.

[0078] Using the anti-HSA-pPICZαA plasmid as a template, and the forward primer among the two primers with mutation sites shown in Table 1 and HSAuni-R, PCR was carried out through the following program and system. System 2: In a 25 μl system, it contained 22 μl of TSINGKE TSE101 Gold Mix (green), 1 μl of each 10 μM primer, and 1 μl of anti-HSA-pPICZαA plasmid. PCR Program 2: Pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s, extension at 72 °C for 10 s, for a total of 30 cycles; extension at 72 °C for 2 min, incubation at 4 °C.

[0079] Using the above two PCR amplification products as templates, and HSAuni-F2 and HSAuni-R primers, PCR was carried out through the following program and system. System 3: In a 51 μl system, it contained 45 μl of TSINGKE TSE101 Gold Mix (green), 2 μl of each 10 μM primer, and 1 μl of each of the two gene fragments. PCR Program 3: Pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s, extension at 72 °C for 10 s, for a total of 30 cycles; extension at 72 °C for 2 min, incubation at 4 °C. The target fragment with corresponding single-site mutations was amplified.

[0080] 2. Construction of an expression vector for anti-HSA nanobody with multiple-site mutations

[0081] Except for the two primers with mutation sites and the template, the anti-HSA nanobody with multiple-site mutations was prepared according to the method for preparing the anti-HSA nanobody with single-site mutations described above. The primers and templates used are shown in Table 2 and Table 3.

[0082] 3. Expression of the target protein

[0083] The target fragments with corresponding mutation sites were respectively digested with restriction enzymes Xho I / Xba I, and ligated into pPICZαA digested with Xho I / Xba I. The ligation products were transformed into Top10F strain and cultured on LB plates containing 25 μg / mL bleomycin. Single colonies were picked for colony PCR verification, and positive clones were verified by sequencing.

[0084] The plasmid with correct sequencing was transformed into the expression host Pichia Pastoris X-33 by electroporation. The transformation products were spread on YPDS plates (10 g yeast extract, 20 g tryptone, 20 g glucose, 182.17 g sorbitol dissolved in 1 L ddH2O, added with 15 g agar powder) containing bleomycin (100 μg / mL), and cultured inverted at 30 °C for 2 - 4 days until single colonies grew. Single colonies were picked and inoculated into 50 mL YPG liquid medium (10 g yeast extract, 20 g tryptone, 10 mL glycerol dissolved in 890 mL ddH2O, added with 100 mL potassium phosphate buffer at pH 6.0), and cultured at 30 °C and 220 rpm until the OD 600 reached 3 - 4. The bacterial solution was centrifuged at 3000 rpm for 5 min, the supernatant was removed to collect the cells, and all the cells were resuspended in 50 mL BMMY liquid medium (10 g yeast extract, 20 g tryptone dissolved in 800 mL ddH2O, added with 100 mL potassium phosphate buffer at pH 6.0, 100 mL 10× yeast nitrogen base without amino acids, 2 mL 500× biotin and 5 mL methanol), and induced to culture at 28 °C and 220 rpm for 48 h, and 250 μL methanol was added every 24 h. 1 mL of the bacterial solution induced for 48 h was taken for SDS PAGE electrophoresis (the separation gel concentration was 12%) verification.

[0085] Table 1

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] Table 2

[0096]

[0097]

[0098] Table 3

[0099]

[0100]

[0101] 4. Purification

[0102] First, capture with MEP hypercel packing material, and then perform fine purification with a SOURCE 15S chromatography column to obtain a sample with a purity of 95%.

[0103] Experimental steps:

[0104] Step 1: Initial purification - capture with MEP hypercel packing material

[0105] Adjust the pH of the fermentation broth to pH 6.0, centrifuge at 8000 rpm for 15 min, collect the supernatant, dilute it 1-fold with ultrapure water, and then load it onto the column.

[0106]

[0107] Purification steps: Equilibrate the chromatography column with equilibration buffer 1 for 3 - 4 CV, add the above-treated sample to the chromatography column, then re-equilibrate with equilibration buffer 1 for 1 - 2 CV, re-equilibrate with equilibration buffer 2 for 1 - 2 CV until the baseline becomes flat, elute the target protein with elution buffer at pH 2.8, collect the eluate, and obtain the target protein with an electrophoretic purity of about 70%, containing a small amount of pigment.

[0108] Step 2: Fine purification - SOURCE 15S chromatography column

[0109] Sample treatment: Dilute the sample from the initial purification in Step 1 1-fold with equilibration buffer 1 and then load it onto the column.

[0110] Buffer Buffer component Use Equilibration buffer 1 20 mM sodium acetate, pH 4.5 Column equilibration Elution buffer 30 mM sodium chloride, 20 mM sodium acetate, pH 4.5 Elute the target protein

[0111] Purification steps: Equilibrate the chromatography column with equilibration buffer 1 for 3 - 4 CV, add the treated sample to the chromatography column, then re-equilibrate with equilibration buffer 1 for 1 - 2 CV until the baseline becomes flat, elute the target protein with elution buffer, collect the eluate, and obtain the target protein with an electrophoretic and HPLC purity of about 95%.

[0112] Sequence the obtained target protein. The CDR sequences of the modified anti-HSA nanobody are shown in Table 4. Determine the affinity of the modified anti-HSA nanobody by ELISA method.

[0113] Table 4

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] 5. Determine the affinity

[0120] Determine the affinity under the conditions of pH 7.4 and pH 6.0 respectively. Under the condition of pH 7.4, the pH values of the sample diluent, washing solution and antibody diluent are 7.4; under the condition of pH 6.0, the pH values of the sample diluent, washing solution and antibody diluent are 6.0.

[0121] Dilute HSA (Heyuan, HYC002M01) with PBS (pH 7.4) to 0.5 μg / ml, coat overnight at 4°C, wash 3 times with the washing solution, block at 37°C for 2 h, and wash 3 times. Dilute the prepared anti-HSA nanobody with the sample diluent (PBST) to 20000 ng / ml, and then dilute it 11 gradients by 3-fold to 0.11 ng / ml. Divide the wells of the ELISA plate into groups, with each antibody corresponding to a group, 12 wells in each group. Add 100 μl of anti-HSA nanobody with different dilutions to each well, shake well, and incubate at 37°C for 1 h. Wash 3 times with the washing solution. Dilute Anti-Camelid VHH Cocktail-HRP (GenScript, A02016) with the antibody diluent PBST to 1:5000, add 100 μl to each well, incubate at 37°C for 1 h, and wash 3 times. Add TMB chromogenic solution (SinoBiological, SEKCR01) until the color develops clearly, terminate the reaction with 1N HCl, and read the OD value at a wavelength of 450 nm with an ELISA reader. Use GraphPad Prism 5.0 software to fit the logarithmic concentration and OD value to a four-parameter curve, and compare the differences between the fitted curves of the modified anti-HSA nanobody and the unmodified HSA (HSA1). The binding activity results of the modified anti-HSA nanobody and HSA are as follows:

[0122] Mutants that result in the loss of binding activity include HSA5, HSA6, HSA7, HSA20, HSA21, HSA22, HSA23, HSA24, HSA25, HSA26, HSA27, HSA28, HSA29, HSA30, HSA31, HSA32, HSA33, HSA34, HSA35, HSA36, HSA37, HSA38, HSA39, HSA40, HSA41, HSA42, HSA43, HSA44, HSA45, HSA46, HSA47, HSA48, HSA49, HSA50, HSA51, HSA52, HSA53, HSA57, HSA59, HSA62, HSA63, HSA68, HSA72, HSA74, HSA76, HSA77, HSA79, HSA80, HSA83, HSA84, HSA87, HSA88, HSA89, HSA92, HSA93, HSA94, HSA95, HSA96, HSA97, HSA98, HSA99, HSA100, HSA101, HSA102, HSA103, HSA168, HSA194, HSA195, HSA196, HSA197, HSA198, HSA199, HSA201, HSA202, HSA204, HSA206, HSA207, HSA208.

[0123] Mutants with reduced binding activity include HSA10, HSA11, HSA58, HSA60, HSA61, HSA64, HSA65, HSA66, HSA67, HSA70, HSA71, HSA73, HSA75, HSA81, HSA82, HSA85, HSA86, HSA105, HSA107, HSA108, HSA109, HSA110, HSA112, HSA113, HSA115, HSA116, HSA118, HSA119, HSA120, HSA121, HSA122, HSA123, HSA124, HSA125, HSA126, HSA127, HSA128, HSA129, HSA130, HSA131, HSA132, HSA133, HSA134, HSA135, HSA136, HSA137, HSA138, HSA139, HSA140, HSA141, HSA142, HSA143, HSA144, HSA145, HSA146, HSA147, HSA148, HSA149, HSA150, HSA151, HSA152, HSA153, HSA154, HSA155, HSA156, HSA157, HSA158, HSA159, HSA160, HSA161, HSA162, HSA165, HSA166, HSA167, HSA169, HSA170, HSA171, HSA172, HSA173, HSA175, HSA176, HSA177, HSA178, HSA179, HSA180, HSA181, HSA182, HSA183, HSA184, HSA185, HSA187, HSA188, HSA189, HSA190, HSA191, HSA192, HSA193, HSA200, HSA203, HSA209, HSA210, HSA212, HSA213, HSA218, HSA219, HSA221, HSA222, HSA225, HSA226, HSA229, HSA230, HSA232, HSA233, HSA235, HSA236, HSA237, HSA238, HSA239, HSA240, HSA241, HSA242, HSA243, HSA244, HSA245.

[0124] Mutants with enhanced binding activity include HSA55, HSA56, HSA78, HSA117, HSA163, HSA164, HSA174, HSA205, HSA216, HSA217, HSA228, HSA234.

[0125] Mutants that do not significantly affect the reduction of binding activity include HSA8, HSA9, HSA54, HSA69, HSA104, HSA106, HSA111, HSA114, HSA186, HSA211, HSA214, HSA215, HSA220, HSA223, HSA224, HSA227, HSA231.

[0126] Among the mutants that cause a reduction in binding activity, compared with wild-type HSA1, the binding curves of HSA81, HSA93, HSA115, HSA116, HSA152, HSA153, HSA154, HSA155, HSA156, HSA157, HSA158, HSA159, HSA160, HSA241, HSA242, HSA243, HSA244, and HSA245 at pH 6.0 and pH 7.4 show a distinct separation, indicating pH-dependence. Figures 1A - 1F The binding curves of HSA81, HSA93, HSA94, HSA101, HSA102, HSA104, HSA106, HSA115, HSA116, HSA155, HSA156, HSA157, HSA158, HSA159, HSA160, HSA241, HSA244, and HSA245 at pH 6.0 and pH 7.4 are shown respectively.

[0127] 6. Fortebio Detection

[0128] Based on the ELISA analysis results, HSA1, HSA81, HSA93, HSA115, HSA155, HSA156, HSA157, HSA241, HSA242, HSA244, and HSA245 were selected for further Fortebio detection.

[0129] The test conditions for Fortebio are as follows:

[0130] Detection buffer: 2x PBST (0.05% Tween 20), pH 7.4 or pH 6.0; Instrument: Fortebio Qke; Bio-sensor: Octet SA Biosensors; Capture molecule: Biotinylated HSA (KACTUS, Cat. BSA-HM401B), concentration 3 μg / ml; Analyte: The engineered anti-HSA nanobody serially diluted 2-fold with the detection buffer, starting dose 5 μg / ml; Binding time: 180 s; Dissociation time: 180 s; Kinetic analysis was performed by global fitting using a 1:1 binding model.

[0131] As detected by Fortebio, the binding of HSA81, HSA93, HSA115, HSA155, HSA156, HSA157, HSA241, HSA242, HSA244, and HSA245 to HSA under the conditions of pH 7.4 and pH 6.0 is as Figures 2A - 2J shown. Compared with HSA, the KD values of the engineered anti-HSA nanobodies at pH 7.4 are significantly higher than those at pH 6.0, as shown in Table 6.

[0132] Table 6

[0133]

Claims

1. A human serum albumin nanobody or an antigen-binding fragment thereof, the nanobody comprising CDR1-CDR3, defined according to the Kabat numbering system, wherein the amino acid sequence of CDR1 is HZ1Z2Z3Z4, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG or a variant thereof, and the amino acid sequence of CDR3 is IGGY1Y2X1Y3N1; Among them, X1 is selected from S or H; Y1 is selected from A or S; Y2 is selected from L, A or H; Y3 is selected from R, A, V, W, S, T, H, K, Y, M, Q, G or L; N1 is selected from S or H; Z1 is selected from F or H; Z2 is selected from G or H; Z3 is selected from M or H; Z4 is selected from S or H.

2. The nanobody or antigen-binding fragment thereof according to claim 1, wherein, The variant has 1-3 amino acid substitutions, deletions, additions, or any combination thereof compared to SISGSGSDTLYADSVKG.

3. The nanobody or its antigen-binding fragment according to claim 1 or 2, wherein, The variant has 1-2 amino acid substitutions compared to SISGSGSDTLYADSVKG.

4. The nanobody or antigen-binding fragment thereof according to claim 3, wherein The variant has 1 amino acid substitution and is substituted with H compared to SISGSGSDTLYADSVKG.

5. The nanobody or antigen-binding fragment thereof according to claim 3, wherein, The amino acid sequence of the variant is: SISY4SGSDTLYADSVKG, SISGSGSY5TLYADSVKG, SISGSGSDY6LYADSVKG, SISGSGSDTLY7ADSVKG, SISGSGSDTLYAY8SVKG, SISGSGSDTLYADSY9KG, or SISGSGSDTLYADSVY 10 G SISGSGSDTLYADSVKY 11 ; wherein, Y4 is selected from D, G, A, S, or N; Y5 is selected from D, G, A, I, F, Y, S, T, M, Q, K, R, V, L, E or N; Y6 is selected from T, H, G, A, V, L, I, F, Y, M, Q, D, E, K, R or N; Y7 is selected from Y, H, G, A, V, L, I, F, W, S, T, C, M, Q, D, E, K, R or N; Y8 is selected from D, H, G, A, V, L, I, F, Y, W, S, T, M, N, Q, E, K, or R; Y9 is selected from V, H, G, A, L, F, Y, W, S, T, N, Q, D, E, K, M or R; Y 10 selected from K, H, G, A, V, L, I, F, Y, W, S, T, C, M, N, Q, D, E or R; Y 11 Selected from G or H.

6. The nanobody or an antigen-binding fragment thereof according to claim 5, wherein, the amino acid sequence of CDR1 is SFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; the amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of the CDR3 is IGGSLHLS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLGADSVKG, and the amino acid sequence of the CDR3 is IGGSLSRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLFADSVKG, and the amino acid sequence of the CDR3 is IGGSLSRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSVAG, and the amino acid sequence of the CDR3 is IGGSLSRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLGADSVKG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLFADSVKG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSVAG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLGADSVKG, and the amino acid sequence of the CDR3 is IGGSLHLS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLFADSVKG, and the amino acid sequence of the CDR3 is IGGSLHLS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSVAG, and the amino acid sequence of the CDR3 is IGGSLHLS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSVTLYADSVKG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSLTLYADSVKG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSETLYADSVKG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISDSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSMKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGALSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGALHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGASASRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSAHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSVS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHVS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSQS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHQS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGHDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGHDTLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDHLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDHLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDVLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDVLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDFLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDFLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDYLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDYLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDQLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDQLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDKLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDKLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDRLYADSVKG, and the amino acid sequence of CDR3 is IGGSLSRS; The amino acid sequence of CDR1 is HFGMS, the amino acid sequence of CDR2 is SISGSGSDRLYADSVKG, and the amino acid sequence of CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSLKG, and the amino acid sequence of the CDR3 is IGGSLSRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSLKG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSWKG, and the amino acid sequence of the CDR3 is IGGSLSRS; or The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSWKG, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSVKH, and the amino acid sequence of the CDR3 is IGGSLSRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYADSVKH, and the amino acid sequence of the CDR3 is IGGSLHRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYATSVKG, and the amino acid sequence of the CDR3 is IGGSLSRS; The amino acid sequence of the CDR1 is HFGMS, the amino acid sequence of the CDR2 is SISGSGSDTLYATSVKG, and the amino acid sequence of the CDR3 is IGGSLHRS.

7. The nanobody or antigen-binding fragment thereof according to claim 6, wherein, The nanobody comprises FR1-FR4; The amino acid sequence of the FR1 is: EVQLVESGGGLVQPGNSLRLSCAASGFTFS; The amino acid sequence of the FR2 is: WVRQAPGKGLEWVS; The amino acid sequence of the FR3 is: RFTISRDNAKTTLYLQMNSLRPEDTAVYYCT; The amino acid sequence of the FR4 is SQGTLVTVSS.

8. A multispecific antibody comprising the nanobody according to any one of claims 1-7 or an antigen-binding fragment thereof, and another antibody or an antigen-binding fragment thereof.

9. A fusion protein comprising the nanobody according to any one of claims 1-7 or an antigen-binding fragment thereof, and another bioactive polypeptide.

10. The fusion protein according to claim 9, wherein, The bioactive polypeptide is selected from granulocyte colony-stimulating factor, colony-stimulating factor, G protein-coupled receptor, IL-15, IL15RD, the fusion protein of IL15RD and IL-15, human growth hormone, cytokine binding protein, protein A, allergy inhibitor, necroglycoprotein, immunotoxin, lymphotoxin, tumor suppressor, transforming growth factor, α1-antitrypsin, α-lactalbumin, apolipoprotein-E, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, bone stimulating protein, calcitonin, atrial natriuretic peptide, cartilage inducing factor, ecalcitonin, connective tissue activating factor, tissue factor pathway inhibitor, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, autotoxin, lactoferrin, myostatin, or a member of the heat shock protein family.

11. An isolated nucleic acid molecule encoding the nanobody or antigen-binding fragment thereof according to any one of claims 1-7, the multispecific antibody according to claim 8, or the fusion protein according to any one of claims 9-10.

12. A vector comprising the isolated nucleic acid molecule according to claim 11.

13. A host cell comprising the isolated nucleic acid molecule according to claim 11 and / or the vector according to claim 12.

14. A pharmaceutical composition comprising the nanobody or antigen-binding fragment thereof according to any one of claims 1-7, the multispecific antibody according to claim 8, the fusion protein according to any one of claims 9-10, the isolated nucleic acid molecule according to claim 11, the vector according to claim 12, or the host cell according to claim 13, and a pharmaceutically acceptable carrier and / or excipient.

15. Use of the nanobody or antigen-binding fragment thereof according to any one of claims 1-7 for prolonging the in vivo half-life of an active agent linked thereto.

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