Serum albumin binding molecules and uses thereof

Abstract

Provided are a serum albumin binding molecule and applications thereof. Specifically, provided are a nanobody or an antigen binding fragment thereof that binds to serum albumin, and applications of the nanobody or the antigen binding fragment thereof in preparing a drug and a detection reagent. The nanobody is modified by mutation, and has significantly improved serum albumin binding activity, and can significantly prolong the half-life of a protein drug, thereby providing a research and development basis for long-acting protein drug development.

Description

Serum albumin binding molecules and their applications Technical Field

[0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals, and more particularly to a serum albumin binding molecule and an application thereof. Background Art

[0002] Albumin is a single-chain polypeptide composed of 585 amino acids. The X-ray crystal structure of human serum albumin reveals a heart-shaped molecule composed primarily of α-helices and lacking β-sheets. These domains are organized into three homologous domains: DI, DII, and DIII. Each domain is divided into A and B subdomains (DIA, DIB, DIIA, DIIB, DIIIA, and DIIIB), connected by long, flexible loops. Albumin contains approximately 35% hydrophobic amino acids, which give it a strong hydration capacity. It also contains a high concentration of lysine and arginine, which impart a positive charge, allowing it to form salt bridges with anionic species and participate in the binding and transport of drugs and other molecules.

[0003] Albumin is the most abundant and structurally stable protein in human plasma, with a concentration of approximately 45 mg / ml (0.6 mM) and a circulation half-life of up to 20 days. Studies have shown that binding of nanobodies to human serum albumin increases their hydrodynamic radius and molecular weight, reduces glomerular filtration, and significantly prolongs their in vivo retention time. Furthermore, through FcRn-mediated recycling, their half-life can be extended from several hours to over three weeks.

[0004] Therefore, developing nanoantibodies targeting albumin and applying them to construct fusion proteins to extend the half-life of active drugs has become an important drug development strategy.

[0005] Summary of the Invention

[0006] The object of the present invention is to provide a binding molecule with high affinity to serum albumin, and the use of the binding molecule in the preparation of drugs and detection reagents.

[0007] In a first aspect of the present invention, there is provided a Nanobody or an antigen-binding fragment thereof that binds to serum albumin, wherein the Nanobody or the antigen-binding fragment thereof has an amino acid sequence selected from the group consisting of:

[0008] (Z1) a mutant sequence based on the amino acid sequence of SEQ ID NO: 1, wherein the mutation occurs at a position corresponding to SEQ ID NO: 1 selected from the group consisting of position 53, 54, 100, 101, 102, 103, 26, 31, 107, 112, or a combination thereof; or

[0009] (Z2) A mutant sequence based on the amino acid sequence shown in SEQ ID NO: 2, wherein the mutation occurs at a position corresponding to SEQ ID NO: 2 selected from the following group: position 99, 28, 30, 100, 103, 104, 110, or a combination thereof.

[0010] In another preferred embodiment, the antibody or antigen-binding fragment thereof has the following amino acid sequence: a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation is selected from the following group: S53 (A / R), G54 (A / R), S100G, G101S, D102 (N / R), G103A, G26A, R31S, T56R, C112S, or a combination thereof.

[0011] In another preferred example, the antibody or antigen-binding fragment thereof has the following amino acid sequence: a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 2, wherein the mutation is selected from the following group: D99 (W / R), R28S, S30T, S100T, G103S, K104R, E110A, or a combination thereof.

[0012] In another preferred example, the antibody or antigen-binding fragment thereof has a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation site of the mutant sequence includes at least position 53 of SEQ ID NO: 1.

[0013] In another preferred embodiment, the serine (S) at position 53 of SEQ ID NO: 1 is mutated to alanine (A) or arginine (R), preferably alanine (A).

[0014] In another preferred example, the mutation site of the mutant sequence further includes position 54, position 56, position 102 of SEQ ID NO: 1, or a combination thereof.

[0015] In another preferred embodiment, the glycine (G) at position 54 of SEQ ID NO: 1 is mutated to alanine (A) or arginine (R).

[0016] In another preferred embodiment, the threonine (T) at position 56 of SEQ ID NO: 1 is mutated to arginine (R).

[0017] In another preferred embodiment, the aspartic acid (D) at position 102 of SEQ ID NO: 1 is mutated to arginine (R).

[0018] In another preferred embodiment, the mutation of the mutant sequence is that the 53rd serine (S) of SEQ ID NO: 1 is mutated to alanine (A).

[0019] In another preferred embodiment, the mutation of the mutant sequence is that the 53rd serine (S) of SEQ ID NO: 1 is mutated to alanine (A), and the 54th glycine (G) is mutated to alanine (A).

[0020] In another preferred embodiment, the mutation of the mutant sequence is that the 53rd serine (S) of SEQ ID NO: 1 is mutated to arginine (R), the 54th glycine (G) is mutated to arginine (R), and the 102nd aspartic acid (D) is mutated to arginine (R).

[0021] In another preferred embodiment, the mutation of the mutant sequence is that the 53rd serine (S) of SEQ ID NO: 1 is mutated to arginine (R), the 56th threonine (T) is mutated to arginine (R), and the 102nd aspartic acid (D) is mutated to arginine (R).

[0022] In another preferred embodiment, the mutation of the mutant sequence is that the 53rd serine (S) of SEQ ID NO: 1 is mutated to arginine (R), and the 102nd aspartic acid (D) is mutated to arginine (R).

[0023] In another preferred example, the antibody or antigen-binding fragment thereof has a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation sites of the mutant sequence at least include positions 100, 101, 102 and 103 of SEQ ID NO: 1.

[0024] In another preferred embodiment, the serine (S) at position 100 of SEQ ID NO: 1 is mutated to glycine (G), the glycine (G) at position 101 is mutated to serine (S), the aspartic acid (D) at position 102 is mutated to asparagine (N), and the glycine (G) at position 103 is mutated to alanine (A).

[0025] In another preferred example, the mutation site of the mutant sequence further includes position 26, position 31, position 53, position 54, or a combination thereof of SEQ ID NO: 1.

[0026] In another preferred embodiment, the glycine (G) at position 26 of SEQ ID NO: 1 is mutated to alanine (A).

[0027] In another preferred embodiment, the arginine (R) at position 31 of SEQ ID NO: 1 is mutated to serine (S).

[0028] In another preferred embodiment, the serine (S) at position 53 of SEQ ID NO: 1 is mutated to alanine (A).

[0029] In another preferred embodiment, the glycine (G) at position 54 of SEQ ID NO: 1 is mutated to alanine (A).

[0030] In another preferred embodiment, the mutation of the mutant sequence is that the serine (S) at position 100 of SEQ ID NO: 1 is mutated to glycine (G), the glycine (G) at position 101 is mutated to serine (S), the aspartic acid (D) at position 102 is mutated to asparagine (N), and the glycine (G) at position 103 is mutated to alanine (A).

[0031] In another preferred example, the mutation of the mutant sequence is that the 100th serine (S) of SEQ ID NO: 1 is mutated to glycine (G), the 101st glycine (G) is mutated to serine (S), the 102nd aspartic acid (D) is mutated to asparagine (N), the 103rd glycine (G) is mutated to alanine (A), the 26th glycine (G) is mutated to alanine (A), the 31st arginine (R) is mutated to serine (S), and the 53rd serine (S) is mutated to alanine (A).

[0032] In another preferred example, the mutation of the mutant sequence is that the serine (S) at position 100 of SEQ ID NO: 1 is mutated to glycine (G), the glycine (G) at position 101 is mutated to serine (S), the aspartic acid (D) at position 102 is mutated to asparagine (N), the glycine (G) at position 103 is mutated to alanine (A), the glycine (G) at position 26 is mutated to alanine (A), the arginine (R) at position 31 is mutated to serine (S), the serine (S) at position 53 is mutated to alanine (A), and the glycine (G) at position 54 is mutated to alanine (A).

[0033] In another preferred example, the antibody or antigen-binding fragment thereof has a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation sites of the mutant sequence include at least positions 26 and 31 of SEQ ID NO: 1.

[0034] In another preferred embodiment, the glycine (G) at position 26 of SEQ ID NO: 1 is mutated to alanine (A), and the arginine (R) at position 31 is mutated to serine (S).

[0035] In another preferred example, the mutation site of the mutant sequence further includes position 53, position 54, position 100, position 101, position 102, position 103, or a combination thereof of SEQ ID NO: 1.

[0036] In another preferred embodiment, the serine (S) at position 53 of SEQ ID NO: 1 is mutated to alanine (A).

[0037] In another preferred embodiment, the glycine (G) at position 54 of SEQ ID NO: 1 is mutated to alanine (A).

[0038] In another preferred embodiment, the serine (S) at position 100 of SEQ ID NO: 1 is mutated to glycine (G).

[0039] In another preferred embodiment, the glycine (G) at position 101 of SEQ ID NO: 1 is mutated to serine (S).

[0040] In another preferred embodiment, the aspartic acid (D) at position 102 of SEQ ID NO: 1 is mutated to asparagine (N).

[0041] In another preferred embodiment, the glycine (G) at position 103 of SEQ ID NO: 1 is mutated to alanine (A).

[0042] In another preferred embodiment, the mutation of the mutant sequence is that glycine (G) at position 26 of SEQ ID NO: 1 is mutated to alanine (A), and arginine (R) at position 31 is mutated to serine (S).

[0043] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) selected from the following group:

[0044] (a1) CDR1 as set forth in SEQ ID NO:22, CDR2 as set forth in SEQ ID NO:23, and CDR3 as set forth in SEQ ID NO:21;

[0045] (a2) CDR1 as set forth in SEQ ID NO:22, CDR2 as set forth in SEQ ID NO:24, and CDR3 as set forth in SEQ ID NO:21;

[0046] (a3) CDR1 as set forth in SEQ ID NO:22, CDR2 as set forth in SEQ ID NO:20, and CDR3 as set forth in SEQ ID NO:25;

[0047] (a4) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20, and CDR3 as set forth in SEQ ID NO: 21;

[0048] (a5) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 23, and CDR3 as set forth in SEQ ID NO: 25;

[0049] (a6) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 24, and CDR3 as set forth in SEQ ID NO: 25;

[0050] (a7) CDR1 as set forth in SEQ ID NO:22, CDR2 as set forth in SEQ ID NO:27, and CDR3 as set forth in SEQ ID NO:28;

[0051] (a8) CDR1 as set forth in SEQ ID NO:22, CDR2 as set forth in SEQ ID NO:29, and CDR3 as set forth in SEQ ID NO:28;

[0052] (a9) CDR1 as set forth in SEQ ID NO:22, CDR2 as set forth in SEQ ID NO:30, and CDR3 as set forth in SEQ ID NO:28; or

[0053] (a10) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO: 26.

[0054] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof further comprises a FR region, and the FR region comprises:

[0055] FR1 as shown in SEQ ID NO: 57, FR2 as shown in SEQ ID NO: 58 or 61, FR3 as shown in SEQ ID NO: 59, 62 or 63, and FR4 as shown in SEQ ID NO: 60.

[0056] In another preferred example, the Nanobody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 3-12, or an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 3-12, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0057] In another preferred example, the Nanobody or antigen-binding fragment thereof has an amino acid sequence as shown in SEQ ID NO: 4, 5 or 6.

[0058] In another preferred example, the antibody or antigen-binding fragment thereof has a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 2, wherein the mutation site of the mutant sequence includes at least position 99 of SEQ ID NO: 2.

[0059] In another preferred embodiment, the aspartic acid (D) at position 99 of SEQ ID NO: 2 is mutated to tryptophan (W) or arginine (R), preferably tryptophan (W).

[0060] In another preferred embodiment, the mutation of the mutant sequence is that aspartic acid (D) at position 99 of SEQ ID NO: 2 is mutated to tryptophan (W).

[0061] In another preferred example, the antibody or antigen-binding fragment thereof has a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 2, wherein the mutation sites of the mutant sequence include at least positions 28 and 30 of SEQ ID NO: 2.

[0062] In another preferred embodiment, the arginine (R) at position 28 of SEQ ID NO: 2 is mutated to serine (S), and the serine (S) at position 30 is mutated to threonine (T).

[0063] In another preferred example, the mutation site of the mutant sequence further includes position 100, position 103, position 104, position 110 of SEQ ID NO: 2, or a combination thereof.

[0064] In another preferred embodiment, the serine (S) at position 100 of SEQ ID NO: 2 is mutated to threonine (T).

[0065] In another preferred embodiment, the glycine (G) at position 103 of SEQ ID NO: 2 is mutated to serine (S).

[0066] In another preferred embodiment, the lysine (K) at position 104 of SEQ ID NO: 2 is mutated to arginine (R).

[0067] In another preferred embodiment, the glutamic acid (E) at position 110 of SEQ ID NO: 2 is mutated to alanine (A).

[0068] In another preferred embodiment, the mutation of the mutant sequence is that the arginine (R) at position 28 of SEQ ID NO: 2 is mutated to serine (S), and the serine at position 30 is mutated to threonine (T).

[0069] In another preferred example, the mutation of the mutant sequence is that the arginine (R) at position 28 of SEQ ID NO: 2 is mutated to serine (S), the serine at position 30 is mutated to threonine (T), the serine (S) at position 100 is mutated to threonine (T), the glycine (G) at position 103 is mutated to serine (S), the lysine (K) at position 104 is mutated to arginine (R), and the glutamic acid (E) at position 110 is mutated to alanine (A).

[0070] In another preferred example, the antibody or antigen-binding fragment thereof has a mutant sequence based on the amino acid sequence shown in SEQ ID NO: 2, wherein the mutation sites of the mutant sequence include at least positions 100, 103 and 104 of SEQ ID NO: 2.

[0071] In another preferred embodiment, the serine (S) at position 100 of SEQ ID NO: 2 is mutated to threonine (T), the glycine (G) at position 103 is mutated to serine (S), and the lysine (K) at position 104 is mutated to arginine (R).

[0072] In another preferred embodiment, the mutation site of the mutant sequence further includes position 110, position 28, position 30, or a combination thereof of SEQ ID NO: 2.

[0073] In another preferred embodiment, the glutamic acid (E) at position 110 of SEQ ID NO: 2 is mutated to alanine (A).

[0074] In another preferred embodiment, the arginine (R) at position 28 of SEQ ID NO: 2 is mutated to serine (S).

[0075] In another preferred embodiment, the serine (S) at position 30 of SEQ ID NO: 2 is mutated to threonine (T).

[0076] In another preferred embodiment, the mutation of the mutant sequence is that the serine (S) at position 100 of SEQ ID NO: 2 is mutated to threonine (T), the glycine (G) at position 103 is mutated to serine (S), and the lysine (K) at position 104 is mutated to arginine (R).

[0077] In another preferred embodiment, the mutation of the mutant sequence is that the serine (S) at position 100 of SEQ ID NO: 2 is mutated to threonine (T), the glycine (G) at position 103 is mutated to serine (S), the lysine (K) at position 104 is mutated to arginine (R), and the glutamic acid (E) at position 110 is mutated to alanine (A).

[0078] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) selected from the following group:

[0079] (b1) CDR1 as set forth in SEQ ID NO:34, CDR2 as set forth in SEQ ID NO:32, and CDR3 as set forth in SEQ ID NO:38;

[0080] (b2) CDR1 as set forth in SEQ ID NO:31, CDR2 as set forth in SEQ ID NO:32, and CDR3 as set forth in SEQ ID NO:33;

[0081] (b3) CDR1 as set forth in SEQ ID NO:34, CDR2 as set forth in SEQ ID NO:32, and CDR3 as set forth in SEQ ID NO:35;

[0082] (b4) CDR1 as set forth in SEQ ID NO:34, CDR2 as set forth in SEQ ID NO:32, and CDR3 as set forth in SEQ ID NO:36;

[0083] (b5) CDR1 as set forth in SEQ ID NO:31, CDR2 as set forth in SEQ ID NO:32, and CDR3 as set forth in SEQ ID NO:36; or

[0084] (b6) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 32, and CDR3 shown in SEQ ID NO: 37.

[0085] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof further comprises a FR region, and the FR region comprises:

[0086] FR1 as shown in SEQ ID NO: 57, FR2 as shown in SEQ ID NO: 58 or 61, FR3 as shown in SEQ ID NO: 59, 62 or 63, and FR4 as shown in SEQ ID NO: 60.

[0087] In another preferred example, the Nanobody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 13-18, or an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 13-18, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0088] In another preferred example, the Nanobody or antigen-binding fragment thereof has an amino acid sequence as shown in SEQ ID NO: 18 or 13.

[0089] In another preferred embodiment, the Nanobody is human or humanized.

[0090] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof binds to serum albumin of a human or non-human mammal.

[0091] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof binds to human, cynomolgus monkey, mouse or rat serum albumin.

[0092] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof binds to human serum albumin.

[0093] In another preferred embodiment, the Nanobody or antigen-binding fragment thereof can bind to human serum albumin at pH 3.0-10.0, preferably pH 4.0-9.0, and more preferably pH 5.0-8.0.

[0094] In another preferred example, the EC50 value of the Nanobody or antigen-binding fragment thereof binding to human serum albumin is less than the EC50 value of the Nanobody of SEQ ID NO: 1 binding to human serum albumin.

[0095] In another preferred example, the EC50 value of the Nanobody or its antigen-binding fragment binding to human serum albumin is in the range of 2nM to 8nM, preferably, in the range of 2nM to 5nM.

[0096] In another preferred example, the EC50 value of the Nanobody or antigen-binding fragment thereof binding to human serum albumin is less than the EC50 value of the Nanobody of SEQ ID NO: 2 binding to human serum albumin.

[0097] In another preferred example, the EC50 value of the Nanobody or its antigen-binding fragment binding to human serum albumin is in the range of 1.5 nM to 5 nM, preferably, in the range of 1.5 nM to 3 nM.

[0098] The second aspect of the present invention provides an anti-serum albumin antibody, which comprises one or more serum albumin-binding Nanobodies or antigen-binding fragments thereof as described in the first aspect of the present invention.

[0099] In another preferred embodiment, the anti-serum albumin antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0100] The third aspect of the present invention provides a multispecific antibody, wherein at least one antigen-binding domain of the antibody comprises a serum albumin-binding Nanobody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0101] In another preferred embodiment, the multispecific antibody further comprises an antigen binding domain that binds to an antigen selected from the group consisting of IL-4R, IL-4Rα, TNF-α, VEGF, PD-1, PD-L1, 4-1BB, CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD38, IL-5, TSLP, BCMA, GLP-1, Trop2, TIGIT, or a combination thereof.

[0102] In another preferred embodiment, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

[0103] In a fourth aspect of the present invention, an isolated polynucleotide is provided, which encodes a Nanobody or an antigen-binding fragment thereof that binds to serum albumin as described in the first aspect of the present invention, an anti-serum albumin antibody as described in the second aspect of the present invention, or a multispecific antibody as described in the third aspect of the present invention.

[0104] In another preferred embodiment, the polynucleotide includes RNA, DNA, and cDNA.

[0105] In another preferred embodiment, the sequence of the polynucleotide is as shown in any one of SEQ ID NOs: 40-49.

[0106] In another preferred embodiment, the sequence of the polynucleotide is as shown in any one of SEQ ID NOs: 51-56.

[0107] The fifth aspect of the present invention provides an expression vector, which comprises the isolated polynucleotide as described in the fourth aspect of the present invention.

[0108] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0109] In another preferred embodiment, the viral vector includes a lentiviral vector, an adenoviral vector, an AAV viral vector, a retroviral vector, or a combination thereof.

[0110] In the sixth aspect of the present invention, a host cell is provided, which contains the expression vector as described in the fifth aspect of the present invention, or expresses the serum albumin-binding Nanobody or its antigen-binding fragment as described in the first aspect of the present invention, the anti-serum albumin antibody as described in the second aspect of the present invention, or the multispecific antibody as described in the third aspect of the present invention.

[0111] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0112] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0113] The seventh aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0114] (a) a serum albumin-binding Nanobody or antigen-binding fragment thereof as described in the first aspect of the invention, an anti-serum albumin antibody as described in the second aspect of the invention, or a multispecific antibody as described in the third aspect of the invention; and

[0115] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle or nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0116] In another preferred embodiment, the components (a) and (b) are operably linked.

[0117] In an eighth aspect of the present invention, a recombinant protein is provided, comprising:

[0118] (i) a serum albumin-binding Nanobody or antigen-binding fragment thereof as described in the first aspect of the invention, an anti-serum albumin antibody as described in the second aspect of the invention, or a multispecific antibody as described in the third aspect of the invention; and

[0119] (ii) Optional polypeptide molecules or fragments having therapeutic functions.

[0120] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are not limited to: polypeptide molecules or fragments targeting IL-4R, IL-4Rα, TNF-α, VEGF, PD-1, PD-L1, 4-1BB, CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD38, IL-5, TSLP, BCMA, GLP-1, Trop2 or TIGIT.

[0121] In another preferred embodiment, the polypeptide molecules or fragments having therapeutic functions include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, antibody fragments, and cytokines.

[0122] In another preferred embodiment, the polypeptide molecule or fragment with therapeutic function includes a single-chain antibody (scFv), a double-chain antibody, a monoclonal antibody, or a chimeric antibody.

[0123] In another preferred embodiment, the recombinant protein further comprises a tag sequence to assist expression and / or purification.

[0124] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0125] The ninth aspect of the present invention provides a pharmaceutical composition comprising:

[0126] The serum albumin-binding Nanobody or antigen-binding fragment thereof as described in the first aspect of the invention, the anti-serum albumin antibody as described in the second aspect of the invention, or the multispecific antibody as described in the third aspect of the invention, the immunoconjugate as described in the seventh aspect of the invention, or the recombinant protein as described in the eighth aspect of the invention; and a pharmaceutically acceptable carrier.

[0127] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as therapeutic drugs, for example, drugs for treating tumors, autoimmune diseases, infectious diseases, metabolic diseases, inflammatory diseases, proliferative diseases, viral diseases, immune diseases, allergic reactions or parasitic reactions.

[0128] In the tenth aspect of the present invention, there is provided a use of a serum albumin-binding Nanobody or an antigen-binding fragment thereof as described in the first aspect of the present invention, an anti-serum albumin antibody as described in the second aspect of the present invention, or a multispecific antibody as described in the third aspect of the present invention, an immunoconjugate as described in the seventh aspect of the present invention, or a recombinant protein as described in the eighth aspect of the present invention for preparing a long-acting protein drug.

[0129] In another preferred embodiment, the long-acting protein drug is used to treat diseases including (but not limited to) the following: tumor diseases, autoimmune diseases, infectious diseases, metabolic diseases, inflammatory diseases, proliferative diseases, viral diseases, immune diseases, allergic reactions, and parasitic reactions.

[0130] In the eleventh aspect of the present invention, there is provided a use of a Nanobody or antigen-binding fragment thereof that binds to serum albumin as described in the first aspect of the present invention, an anti-serum albumin antibody as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention for preparing a reagent, a detection plate or a kit for detecting serum albumin.

[0131] In another preferred embodiment, the reagent is a diagnostic reagent.

[0132] In another preferred embodiment, the reagent is used to detect serum albumin or a fragment thereof in a sample.

[0133] The twelfth aspect of the present invention provides an immunoadsorbent material for purifying serum albumin, wherein the immunoadsorbent material comprises the serum albumin-binding Nanobody or its antigen-binding fragment as described in the first aspect of the present invention, or the anti-serum albumin antibody as described in the second aspect of the present invention.

[0134] In another preferred embodiment, the immunoadsorption material further comprises a carrier.

[0135] In another preferred embodiment, the carrier includes but is not limited to: magnetic beads, agarose gel, silica microspheres, and porous materials.

[0136] The thirteenth aspect of the present invention provides a method for producing an anti-serum albumin nanobody or an antigen-binding fragment thereof, comprising the steps of:

[0137] (1) culturing the host cell as described in the sixth aspect of the present invention under conditions suitable for producing Nanobodies, thereby obtaining a culture containing anti-serum albumin Nanobodies or antigen-binding fragments thereof; and

[0138] (2) separating or recovering the anti-serum albumin Nanobody from the culture; and optionally

[0139] (3) purifying and / or modifying the anti-serum albumin nanobody obtained in step (b).

[0140] In another preferred example, the anti-serum albumin Nanobody or its antigen-binding fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 3-12; or an amino acid sequence as shown in any one of SEQ ID NOs: 13-18.

[0141] A fourteenth aspect of the present invention provides a method for detecting serum albumin or a fragment thereof in a sample in vitro, the method comprising the steps of:

[0142] (S1) contacting the sample in vitro with the serum albumin-binding Nanobody or antigen-binding fragment thereof as described in the first aspect of the invention, the anti-serum albumin antibody as described in the second aspect of the invention, or the immunoconjugate as described in the seventh aspect of the invention;

[0143] (S2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of serum albumin or a fragment thereof in the sample.

[0144] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0145] In the fifteenth aspect of the present invention, a method for treating a disease is provided, comprising the step of administering to a subject in need thereof a Nanobody or an antigen-binding fragment thereof that binds to serum albumin as described in the first aspect of the present invention, an anti-serum albumin antibody as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, an immunoconjugate as described in the seventh aspect of the present invention, a recombinant protein as described in the eighth aspect of the present invention, or a drug combination as described in the ninth aspect of the present invention.

[0146] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0147] In another preferred embodiment, the diseases include, but are not limited to, the following diseases: tumor diseases, autoimmune diseases, infectious diseases, metabolic diseases, inflammatory diseases, proliferative diseases, viral diseases, immune diseases, allergic reactions, and parasitic reactions.

[0148] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] FIG1 shows a diagram of the binding pattern of Nb1 to human albumin.

[0150] FIG2 shows the binding pattern of Nb2 to human albumin.

[0151] FIG3 shows the results of ELISA assay for the binding activity of Nb1 mutants to human albumin at pH 7.4.

[0152] FIG4 shows the results of ELISA assay for the binding activity of Nb1 mutants to human albumin at pH 5.0.

[0153] FIG5 shows the results of ELISA detection of the binding activity of Nb2 mutants to human albumin at pH 7.4.

[0154] FIG6 shows the results of ELISA detection of the binding activity of Nb2 mutants to human albumin at pH 5.0. DETAILED DESCRIPTION

[0155] Through extensive and intensive research and extensive screening, the present inventors have successfully obtained a group of nanobodies with enhanced binding to serum albumin. Based on the interaction sites between albumin nanobodies Nb1 (SEQ ID NO: 1) and Nb2 (SEQ ID NO: 2) and human serum albumin, the present inventors modified the CDR1, CDR2, and CDR3 of these two antibodies, thereby obtaining albumin nanobodies with enhanced binding activity to serum albumin. This work, based on the results of the present invention, was completed.

[0156] the term

[0157] As used herein, the term "serum albumin" refers to the most abundant blood protein and acts as a carrier protein for steroids, fatty acids, and thyroid hormones in the blood, and plays a major role in stabilizing the volume of extracellular fluid. For nomenclature purposes only and not limitation, an exemplary sequence of human serum albumin is shown in NCBI GenBank Accession No. AEE60908. It should be understood that reference to "serum albumin" or "albumin" includes preproalbumin, which includes an N-terminal peptide, proalbumin, and secretory albumin. Albumin comprises three homologous domains, each of which is the product of two subdomains with a common structural motif. Domains I, II, and III can be defined with reference to human serum albumin.

[0158] As used herein, the terms "anti-serum albumin Nanobody of the invention," "serum albumin-binding Nanobody of the invention," "serum albumin-binding molecule of the invention," and "albumin Nanobody mutant of the invention" are used interchangeably and all refer to Nanobodies that specifically recognize and bind to serum albumin (including human serum albumin). Particularly preferred are Nanobodies whose VHH chain amino acid sequences are as shown in any one of SEQ ID NOs: 3-12; or Nanobodies whose VHH chain amino acid sequences are as shown in any one of SEQ ID NOs: 13-18.

[0159] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0160] As used herein, the terms "single domain antibody (sdAb, or VHH)" and "nanobody" have synonymous meanings and refer to the cloning of the variable region of an antibody heavy chain to construct a nanobody consisting solely of a single heavy chain variable region. This is the smallest fully functional antigen-binding fragment. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody consisting solely of a single heavy chain variable region.

[0161] Nanobodies / single-domain antibodies (Nanobodies) are a new type of small-molecule antibody fragment, cloned from the variable heavy chain region (VHH) of natural camel heavy-chain antibodies. Nanobodies (Nb) possess excellent biological properties, with a molecular weight of 12-15 kDa, one-tenth the size of a full-length antibody. They exhibit excellent tissue penetration, high specificity, and good water solubility. Due to their unique structural properties, they combine the advantages of traditional antibodies and small-molecule drugs, almost perfectly overcoming the drawbacks of traditional antibodies, such as their long development cycles, low stability, and demanding storage conditions. They are gradually becoming an emerging force in the next generation of antibody therapies, showing broad application prospects in immune diagnosis and treatment.

[0162] As used herein, the term "variable" refers to certain portions of the variable region in an antibody that differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, which in some cases may form a partial β-pleated structure. The CDRs in each chain are closely together through the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0163] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0164] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0165] The antibodies of the present invention refer to polypeptides that have serum albumin binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides that include the aforementioned CDR regions and have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion, insertion, and / or substitution of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or similar properties generally does not alter the function of the protein. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0166] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0167] The invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the invention also encompasses fragments of the Nanobodies of the invention. Typically, the fragment will have at least about 50 consecutive amino acids of an antibody of the invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0168] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0169] Table A

[0170] Based on the interaction sites between albumin nanoantibodies Nb1 (SEQ ID NO: 1) and Nb2 (SEQ ID NO: 2) and human serum albumin analyzed by crystal structure, the CDR1, CDR2, and CDR3 of the two antibodies were modified to obtain albumin nanoantibody mutants with better binding activity to serum albumin.

[0171] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0172] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0173] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0174] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0175] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0176] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0177] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0178] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0179] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0180] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0181] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0182] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0183] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0184] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0185] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. drug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0186] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof or fusion protein thereof, as well as a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0187] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0188] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0189] The present invention also relates to a method for detecting serum albumin. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of serum albumin in the dissolved sample using the antibody or antigen-binding fragment thereof described herein.

[0190] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage medium.

[0191] The present invention also provides a test plate or kit containing the antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0192] The present invention also provides a test kit for detecting serum albumin levels, comprising an antibody that recognizes serum albumin, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, a detection marker, and a detection substrate. The test kit can be an in vitro diagnostic device.

[0193] The nanobodies of the present invention have broad biological and clinical applications. Their applications include using anti-serum albumin nanobodies to prepare long-acting protein drugs, providing a research and development foundation for the development of long-acting protein drugs. They also involve using anti-serum albumin nanobodies to prepare detection reagents, test plates, or kits, which can be used for clinical diagnosis or scientific research.

[0194] The main advantages of the present invention include:

[0195] (1) The anti-serum albumin nanobody of the present invention has a high degree of humanization, with a homology of more than 90% with human immunoglobulin.

[0196] (2) The anti-serum albumin nanobody of the present invention can bind to human serum albumin under different pH conditions.

[0197] (3) The anti-serum albumin nanobody of the present invention has better serum albumin binding ability than its parent antibody (i.e., the antibody before mutation / transformation).

[0198] (4) The anti-serum albumin nanoantibody of the present invention can significantly prolong the half-life of protein drugs, providing a research and development basis for the development of long-acting protein drugs.

[0199] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0200] Example 1: Interaction between albumin nanobodies and human albumin

[0201] High-purity human blood-derived albumin was mixed with nanoantibodies to form a complex, which was then purified by molecular sieves. The complex protein was concentrated to a high concentration and screened on a floor to obtain optimal crystallization conditions. After further optimization on a hanging drop plate, crystals with good morphology were obtained. X-ray diffraction revealed that the albumin nanoantibody Nb1 Diffraction data of albumin nanobody Nb2 was obtained The diffraction data of the two antibodies were obtained. Schematic diagrams of the simulated structures are shown in Figures 1 and 2. The CDR1, CDR2, and CDR3 regions of the two antibodies are all involved in the interaction with albumin.

[0202] Example 2: Mutant design and expression based on structural analysis

[0203] Based on the interaction sites analyzed by the crystal structure, the CDR1, CDR2, and CDR3 regions of the nanoantibody were modified, and multiple mutants were designed to obtain albumin nanoantibodies with better binding characteristics.

[0204] Single or combined point mutations were designed for amino acids 26, 31, 53, 54, 100, 101, 102, 103, 107, and 112 of Nb1; single or combined point mutations were designed for amino acids 28, 30, 99, 100, 103, 104, and 110 of Nb2. The design methods are shown in Table 1. The designed mutant amino acids were codon-optimized for E. coli, cloned into the pMECS vector, and expressed in E. coli. High-purity mutant proteins were obtained by nickel column purification.

[0205] Full-length amino acid sequence of Nb1 (SEQ ID NO: 1):

[0206] Among them, the underlined parts are CDR1, CDR2 and CDR3 respectively.

[0207] The full-length amino acid sequence of Nb2 (SEQ ID NO: 2):

[0208] Among them, the underlined parts are CDR1, CDR2 and CDR3 respectively.

[0209] Table 1 Design of Nb1 albumin nanobody mutants

[0210] Table 2 Design of Nb2 albumin nanobody mutants

[0211] Table 3 Sequence information of Nanobody mutants

[0212] Example 3: Analysis of binding activity of mutant antibodies under different pH conditions

[0213] The binding activity of the above mutant proteins with human albumin was detected by ELISA. Human serum albumin was diluted to 10 μg / mL with pH 8.2 NaHCO3 and coated onto the ELISA plate, which was then placed at 4°C overnight. The plate was washed with PBST and blocked with skim milk for 2 hours at room temperature. The plate was washed with PBST and the test antibody was diluted in series (each antibody was diluted with pH 5.0 and pH 7.4 PBS solutions, starting at 1000 nM and then diluted 6-fold). The plate was incubated at 37°C for 1 hour. The plate was washed with PBST and the diluted anti-his (HRP) antibody was added and incubated for 1 hour. The plate was washed with PBST and TMB colorimetric solution was added. The color development was then stopped by adding 50 μL of 2 M sulfuric acid. The absorbance was read at a wavelength of 450 nm using a microplate reader.

[0214] The results are shown in Figures 3 to 6.

[0215] The results showed that Nb1-Mut2 had the best human serum albumin binding activity and was significantly better than Nb1. The binding activities of Nb1-Mut3 and Nb1-Mut4 were comparable to that of Nb1 ( Figures 3 and 4 ).

[0216] Nb2-Mut6 has the best human serum albumin binding activity and is significantly better than Nb2. The binding activity of Nb2-Mut1 is comparable to that of Nb2 (Figures 5 and 6).

[0217] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A nanobody or its antigen-binding fragment that binds to serum albumin, characterized in that, The antibody or its antigen-binding fragment has an amino acid sequence selected from the group consisting of: (Z1) A mutant sequence based on the amino acid sequence shown in SEQ ID NO:1, wherein the mutation occurs at a site corresponding to SEQ ID NO:1 selected from the group consisting of: positions 53, 54, 100, 101, 102, 103, 26, 31, 107, 112, or a combination thereof; or (Z2) A mutant sequence based on the amino acid sequence shown in SEQ ID NO:2, wherein the mutation occurs at a site corresponding to SEQ ID NO:2 selected from the group consisting of: positions 99, 28, 30, 100, 103, 104, 110, or a combination thereof.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has a mutant sequence based on the amino acid sequence shown in SEQ ID NO:1, wherein the mutation site of the mutant sequence at least includes position 53 of SEQ ID NO:

1.

3. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has a mutant sequence based on the amino acid sequence shown in SEQ ID NO:1, wherein the mutation site of the mutant sequence at least includes positions 100, 101, 102, and 103 of SEQ ID NO:

1.

4. The nanobody or antigen-binding fragment thereof according to claim 1, wherein The antibody or its antigen-binding fragment has a mutant sequence based on the amino acid sequence shown in SEQ ID NO:1, wherein the mutation site of the mutant sequence at least includes positions 26 and 31 of SEQ ID NO:

1.

5. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has a mutant sequence based on the amino acid sequence shown in SEQ ID NO:2, wherein the mutation site of the mutant sequence at least includes position 99 of SEQ ID NO:

2.

6. The nanobody or its antigen-binding fragment according to claim 1, wherein The antibody or its antigen-binding fragment has a mutant sequence based on the amino acid sequence shown in SEQ ID NO:2, wherein the mutation site of the mutant sequence at least includes positions 28 and 30 of SEQ ID NO:

2.

7. The nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, The antibody or its antigen-binding fragment has a mutant sequence based on the amino acid sequence shown in SEQ ID NO:2, wherein the mutation site of the mutant sequence at least includes positions 100, 103, and 104 of SEQ ID NO:

2.

8. An anti-serum albumin antibody, the antibody comprising one or more of the nanobodies or their antigen-binding fragments that bind to serum albumin as claimed in claim 1.

9. A multispecific antibody, characterized in that, At least one antigen-binding domain of the antibody comprises the nanobody or its antigen-binding fragment that binds to serum albumin as claimed in claim 1.

10. An isolated polynucleotide, characterized in that, The polynucleotide encodes the nanobody or its antigen-binding fragment that binds to serum albumin as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, or the multispecific antibody as claimed in claim 9.

11. An expression vector, characterized in that, The expression vector comprises the isolated polynucleotide as claimed in claim 10.

12. A host cell, characterized in that, The host cell contains the expression vector as claimed in claim 11, or expresses the nanobody or its antigen-binding fragment that binds to serum albumin as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, or the multispecific antibody as claimed in claim 9.

13. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) The nanobody that binds to serum albumin or its antigen-binding fragment as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, or the multispecific antibody as claimed in claim 9; and (b) A conjugate moiety selected from the group consisting of: a detectable label, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle or nanorod, a magnetic nanoparticle, a viral capsid protein or VLP, or a combination thereof.

14. A recombinant protein, characterized in that, The recombinant protein comprises: (i) The nanobody that binds to serum albumin or its antigen-binding fragment as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, or the multispecific antibody as claimed in claim 9; and (ii) Optionally, a polypeptide molecule or fragment having a therapeutic function.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: The nanobody that binds to serum albumin or its antigen-binding fragment as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, the multispecific antibody as claimed in claim 9, the immunoconjugate as claimed in claim 13, or the recombinant protein as claimed in claim 14; and a pharmaceutically acceptable carrier.

16. Use of a nanobody that binds to serum albumin or its antigen-binding fragment as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, the multispecific antibody as claimed in claim 9, the immunoconjugate as claimed in claim 13, or the recombinant protein as claimed in claim 14 for the preparation of a long-acting protein drug.

17. Use of a nanobody that binds to serum albumin or its antigen-binding fragment as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, or the immunoconjugate as claimed in claim 13 for the preparation of a reagent, test strip or kit for detecting serum albumin.

18. An immunosorbent material for purifying serum albumin, wherein, The immunosorbent material contains the nanobody that binds to serum albumin or its antigen-binding fragment as claimed in claim 1, or the anti-serum albumin antibody as claimed in claim 8.

19. A method for generating an anti-serum albumin nanobody or its antigen-binding fragment, comprising the steps of: (1) Culturing the host cell as claimed in claim 12 under conditions suitable for generating nanobodies, thereby obtaining a culture containing the anti-serum albumin nanobody or its antigen-binding fragment; and (2) Separating or recovering the anti-serum albumin nanobody from the culture; and optionally (3) Purifying and / or modifying the anti-serum albumin nanobody obtained in step (b).

20. A method for in vitro detecting serum albumin or its fragment in a sample, the method comprising the steps of: (S1) In vitro, contacting the sample with the nanobody that binds to serum albumin or its antigen-binding fragment as claimed in claim 1, the anti-serum albumin antibody as claimed in claim 8, or the immunoconjugate as claimed in claim 13; (S2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of serum albumin or its fragment in the sample.