Anti-serum albumin nanobodies and derivatives thereof

Abstract

The present invention relates to a Nanobody or antigen-binding fragment thereof that specifically binds to serum albumin, a derivative comprising this Nanobody or antigen-binding fragment thereof, a nucleic acid encoding this antibody or antigen-binding fragment thereof, a host cell comprising these, and related uses.Furthermore, the present invention relates to therapeutic uses of the antibody or antigen-binding fragment or derivative thereof.

Description

[Technical field]

[0001] The present application relates to a Nanobody or an antigen-binding fragment thereof capable of specifically binding to serum albumin, a derivative comprising this Nanobody or an antigen-binding fragment thereof, a nucleic acid encoding this Nanobody or an antigen-binding fragment thereof, a host cell containing this nucleic acid, and related uses thereof. In addition, the present application relates to therapeutic uses of the Nanobody or an antigen-binding fragment thereof, or a derivative thereof. [Background technology]

[0002] With the development of technology, antibody drugs have long been important therapeutic agents for diseases that do not depend on chemical products or cell therapy. In the development of antibody drugs, pharmacokinetics (PK) is an important field that describes the actions of antibodies in the body, such as absorption, distribution, and clearance, and half-life is an important parameter of PK. In general, the longer the half-life in vivo, the higher the efficacy of the antibody, but it may also increase toxicity. In addition, when the antibody is tightly bound to a specific target, it may be rapidly internalized and degraded in the cell, resulting in a short half-life in vivo.

[0003] By extending the half-life of macromolecular drugs in vivo, the drug's action time can be extended and the number of administrations can be reduced, thereby reducing the medical burden on patients. Currently, there are several chemical methods to improve the long-acting action of drugs, such as polyethylene glycol (PEG) modification and transferrin fusion, but these methods have some drawbacks. For example, PEG may shield or block the antigen-binding site of antibodies, reducing the efficacy of antibodies and therapeutic proteins, and even causing the loss of antibody activity.

[0004] Human serum albumin (HSA) is the most abundant protein in human plasma, accounting for about half of the serum proteins. It is highly soluble in water and exists mainly in monomeric form. The half-life of serum albumin in the body is about 21 days. It is composed of 585 amino acids and its molecular weight is about 66.5KD. Human serum albumin is stable, safe, non-toxic, and has low immunogenicity in the body, making it an ideal drug carrier. In the case of antibody drugs, modification using human serum albumin can extend the half-life of the drug and improve patient compliance with drug administration.

[0005] The salvage mechanism of HSA is similar to that of IgG and is mediated by FcRn. HSA and IgG are internalized into vascular endothelial cells via pinocytosis. In endosomes, the binding of membrane-bound FcRn to HSA is promoted at endosomal pH of about 6.0, and the bound complex is then recycled to the upper or lower part of the endothelial cell, where it is rapidly dissociated from membrane-bound FcRn and recycled via exocytosis at blood pH of 7.4. Summary of the Invention [Problem to be solved by the invention]

[0006] After extensive research, the inventors of the present application have screened and obtained a series of anti-serum albumin nanobodies. The nanobodies have high binding activity to serum albumin and cross-reactivity with human, monkey and / or mouse serum albumin. In particular, the binding of the nanobodies of the present application to serum albumin is pH-independent and can effectively participate in FcRn-mediated serum albumin circulation, thereby extending the in vivo half-life of the nanobodies. Therefore, the nanobodies of the present application can be conjugated (e.g., fused) to drug molecules (e.g., polypeptide drugs) to extend the in vivo half-life of the drug molecules.

[0007] In addition, nanobodies have the characteristics of small molecular weight and high stability.Compared with conventional antibodies, nanobodies have many advantages in drug development, such as good tissue penetration, flexible administration methods, high degree of humanization, and easy conversion to recombinant proteins.

[0008] On this basis, the application also provides compositions containing this Nanobody or antigen-binding fragment thereof, nucleic acids encoding this Nanobody or antigen-binding fragment thereof, and host cells containing this nucleic acid, as well as related uses thereof. [Means for solving the problem]

[0009] Nanobodies and antigen-binding fragments thereof Thus, in one aspect, the application provides a Nanobody or antigen-binding fragment thereof capable of specifically binding to serum albumin, wherein the Nanobody or antigen-binding fragment thereof comprises: (a) CDR1 having a structure selected from the group consisting of: X1X2X3LX4YYX5 (Formula I, SEQ ID NO:72), SGFTLDYYA (SEQ ID NO:30), GSIWGIYH (SEQ ID NO:37), GFTFSIYS (SEQ ID NO:31), GSIFTFYR (SEQ ID NO:33); (b) CDR2 having a structure selected from the group consisting of: IX6SSGGX7X8 (Formula II, SEQ ID NO: 73), ITVDGST (SEQ ID NO: 46), ISSGGSP (SEQ ID NO: 40), ITTDTST (SEQ ID NO: 42); (c) a CDR3 having a structure selected from the group consisting of: AAAX9LECRTX 10 X 11 X 12 X 13 YX 14 Y (Formula III, SEQ ID NO: 74), AAATX 15 ECRGRSSSYDY (Formula IV, SEQ ID NO:75), AAALLECRVRSWPSDN (SEQ ID NO:49), AAAVLECRAAEYVNS (SEQ ID NO:54), DIRNVGGDY (SEQ ID NO:57), SPITSIFKA (SEQ ID NO:48), NVRNVERDY (SEQ ID NO:50); Where: X1 is selected from the group consisting of (i) amino acid residues G, R, and (ii) amino acid residues that are conservative substitutions for (i); X2 is selected from the group consisting of (i) amino acid residues F, A, S, E, and (ii) an amino acid residue that is a conservative substitution for (i); X3 is selected from the group consisting of (i) amino acid residues T, Q, and (ii) an amino acid residue that is a conservative substitution for (i); X4 is selected from the group consisting of (i) amino acid residues D, E, and (ii) an amino acid residue that is a conservative substitution for (i); X5 is selected from the group consisting of: (i) amino acid residue A, and (ii) an amino acid residue that is a conservative substitution for (i); X6 is selected from the group consisting of (i) amino acid residues A, S, and (ii) an amino acid residue that is a conservative substitution for (i); X7 is selected from the group consisting of (i) amino acid residues A, S, and (ii) an amino acid residue that is a conservative substitution for (i); X8 is selected from the group consisting of: (i) amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X9 is selected from the group consisting of (i) amino acid residues T, V, and (ii) an amino acid residue that is a conservative substitution for (i); X 10 is selected from the group consisting of (i) amino acid residues T, V, and (ii) amino acid residues that are conservative substitutions for (i); X 11 is selected from the group consisting of (i) amino acid residues V, I, L, and (ii) amino acid residues that are conservative substitutions for (i); X 12 is selected from the group consisting of (i) amino acid residues R, V, T, and (ii) amino acid residues that are conservative substitutions for (i); X 13 is selected from the group consisting of (i) amino acid residues G, E, and (ii) amino acid residues that are conservative substitutions for (i); X 14is selected from the group consisting of (i) amino acid residues D, A, and (ii) amino acid residues that are conservative substitutions for (i); X 15 is selected from the group consisting of: (i) amino acid residues L, F, and (ii) amino acid residues that are conservative substitutions for (i).

[0010] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in any one of SEQ ID NOs: 30-38; a CDR2 set forth in any one of SEQ ID NOs: 39-46; and a CDR3 set forth in any one of SEQ ID NOs: 47-59.

[0011] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having a sequence as set forth in X1X2X3LX4YYX5 (Formula I, SEQ ID NO: 72) or SGFTLDYYA (SEQ ID NO: 30); (b) CDR2 having the structure shown in Formula II, SEQ ID NO: 73; (c)AAAX9LECRTX 10 X 11 X 12 X 13 YX 14 Y (Formula III, SEQ ID NO: 74), AAATX 15 CDR3 having a structure selected from the group consisting of ECRGRSSSYDY (Formula IV, SEQ ID NO: 75), AAALLECRVRSWPSDN (SEQ ID NO: 49), AAAVLECRAAEYVNS (SEQ ID NO: 54); Where: X1 is selected from the group consisting of (i) amino acid residues G, R, and (ii) amino acid residues that are conservative substitutions for (i); X2 is selected from the group consisting of (i) amino acid residues F, A, S, E, and (ii) an amino acid residue that is a conservative substitution for (i); X3 is selected from the group consisting of (i) amino acid residues T, Q, and (ii) an amino acid residue that is a conservative substitution for (i); X4 is selected from the group consisting of (i) amino acid residues D, E, and (ii) an amino acid residue that is a conservative substitution for (i); X5 is selected from the group consisting of: (i) amino acid residue A, and (ii) an amino acid residue that is a conservative substitution for (i); X6 is selected from the group consisting of (i) amino acid residues A, S, and (ii) an amino acid residue that is a conservative substitution for (i); X7 is selected from the group consisting of (i) amino acid residues A, S, and (ii) an amino acid residue that is a conservative substitution for (i); X8 is selected from the group consisting of: (i) amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X9 is selected from the group consisting of (i) amino acid residues T, V, and (ii) an amino acid residue that is a conservative substitution for (i); X 10 is selected from the group consisting of (i) amino acid residues T, V, and (ii) amino acid residues that are conservative substitutions for (i); X 11 is selected from the group consisting of (i) amino acid residues V, I, L, and (ii) amino acid residues that are conservative substitutions for (i); X 12 is selected from the group consisting of (i) amino acid residues R, V, T, and (ii) amino acid residues that are conservative substitutions for (i); X 13 is selected from the group consisting of (i) amino acid residues G, E, and (ii) amino acid residues that are conservative substitutions for (i); X 14 is selected from the group consisting of (i) amino acid residues D, A, and (ii) amino acid residues that are conservative substitutions for (i); X 15 is selected from the group consisting of: (i) amino acid residues L, F, and (ii) amino acid residues that are conservative substitutions for (i).

[0012] In certain embodiments, X1 is selected from the group consisting of amino acid residues G and R; X2 is selected from the group consisting of amino acid residues F, A, S and E; X3 is selected from the group consisting of amino acid residues T and Q; X4 is selected from the group consisting of amino acid residues D and E; X5 is amino acid residue A; X6 is selected from the group consisting of amino acid residues A and S; X7 is selected from the group consisting of amino acid residues A and S; X8 is amino acid residue T; X9 is selected from the group consisting of amino acid residues T and V; 10 is selected from the group consisting of amino acid residues T and V; 11 is selected from the group consisting of amino acid residues V, I and L; 12 is selected from the group consisting of amino acid residues R, V and T; 13 is selected from the group consisting of amino acid residues G and E; 14 is selected from the group consisting of amino acid residues D and A; 15 is selected from the group consisting of amino acid residues L and F.

[0013] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in any one of SEQ ID NOs: 30, 32, 34-36, 38; a CDR2 set forth in any one of SEQ ID NOs: 39, 41, 43-45; and a CDR3 set forth in any one of SEQ ID NOs: 47, 49, 51-56, 58-59.

[0014] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) CDR1 having the structure shown in Formula I, SEQ ID NO: 72, or SGFTLDYYA (SEQ ID NO: 30); (b) CDR2 having the structure shown in Formula II, SEQ ID NO: 73; (c) CDR3, 10 X 11 X 12 X 13 YX 14 Y (Formula III, SEQ ID NO: 74), AAATX15 those having a structure selected from the group consisting of ECRGRSSSYDY (Formula IV, SEQ ID NO:75), AAALLECRVRSWPSDN (SEQ ID NO:49), AAAVLECRAAEYVNS (SEQ ID NO:54); Where: X1 is selected from the group consisting of (i) amino acid residues G, R, and (ii) amino acid residues that are conservative substitutions for (i); X2 is selected from the group consisting of (i) amino acid residues F, E, S, and (ii) an amino acid residue that is a conservative substitution for (i); X3 is selected from the group consisting of: (i) amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X4 is selected from the group consisting of: (i) amino acid residue D, and (ii) an amino acid residue that is a conservative substitution for (i); X5 is selected from the group consisting of: (i) amino acid residue A, and (ii) an amino acid residue that is a conservative substitution for (i); X6 is selected from the group consisting of (i) amino acid residues A, S, and (ii) an amino acid residue that is a conservative substitution for (i); X7 is selected from the group consisting of (i) amino acid residues A, S, and (ii) an amino acid residue that is a conservative substitution for (i); X8 is selected from the group consisting of: (i) amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X9 is selected from the group consisting of (i) amino acid residues T, V, and (ii) an amino acid residue that is a conservative substitution for (i); X 10 is selected from the group consisting of: (i) amino acid residue V, and (ii) an amino acid residue that is a conservative substitution for (i); X 11 is selected from the group consisting of (i) amino acid residues V, I, and (ii) amino acid residues that are conservative substitutions for (i); X 12 is selected from the group consisting of (i) amino acid residues R, V, and (ii) amino acid residues that are conservative substitutions for (i); X13 is selected from the group consisting of (i) amino acid residues G, E, and (ii) amino acid residues that are conservative substitutions for (i); X 14 is selected from the group consisting of: (i) amino acid residue D, and (ii) an amino acid residue that is a conservative substitution for (i); X 15 is selected from the group consisting of: (i) amino acid residues L, F, and (ii) amino acid residues that are conservative substitutions for (i).

[0015] In certain embodiments, X1 is selected from the group consisting of amino acid residues G and R; X2 is selected from the group consisting of amino acid residues F, S and E; X3 is amino acid residue T; X4 is amino acid residue D; X5 is amino acid residue A; X6 is selected from the group consisting of amino acid residues A and S; X7 is selected from the group consisting of amino acid residues A and S; X8 is amino acid residue T; X9 is selected from the group consisting of amino acid residues T and V; 10 is an amino acid residue V; 11 is selected from the group consisting of amino acid residues V and I; 12 is selected from the group consisting of amino acid residues R and V; 13 is selected from the group consisting of amino acid residues G and E; 14 is an amino acid residue D; X 15 is selected from the group consisting of amino acid residues L and F.

[0016] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in any one of SEQ ID NOs: 30, 32, 34-36; a CDR2 set forth in any one of SEQ ID NOs: 39, 41, 43-45; and a CDR3 set forth in any one of SEQ ID NOs: 47, 49, 51, 52, 54-56, 58.

[0017] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 32 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 32; (b) CDR2 having the sequence set forth in SEQ ID NO: 41 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 41; and (c) a CDR3 having a sequence as set forth in SEQ ID NO: 49 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 49.

[0018] In certain embodiments, the substitutions are conservative substitutions.

[0019] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO:32, a CDR2 set forth in SEQ ID NO:41, and a CDR3 set forth in SEQ ID NO:49.

[0020] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) CDR1 having a sequence as set forth in SEQ ID NO: 32 or 34, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 32 or 34; (b) CDR2 having the sequence set forth in SEQ ID NO: 43 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 43; and (c) a CDR3 having a sequence as set forth in SEQ ID NO:52 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:52.

[0021] In certain embodiments, the substitutions are conservative substitutions.

[0022] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having a structure shown in Formula I, SEQ ID NO:72, where X1 is amino acid residue G; X2 is selected from the group consisting of (i) amino acid residues F, E, and (ii) an amino acid residue which is a conservative substitution for (i); X3 is amino acid residue T; X4 is amino acid residue D; and X5 is amino acid residue A; (b) CDR2 having the structure shown in IASSGGST (SEQ ID NO: 43); (c) CDR3 having the structure shown in AAAVLECRTVVRGYDY (sequence number 52).

[0023] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 32 or 34; a CDR2 set forth in SEQ ID NO: 43; and a CDR3 set forth in SEQ ID NO: 52.

[0024] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 37 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 37; (b) CDR2 having the sequence set forth in SEQ ID NO: 46 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 46; and (c) a CDR3 having a sequence as set forth in SEQ ID NO:57 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:57.

[0025] In certain embodiments, the substitutions are conservative substitutions.

[0026] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 37, a CDR2 set forth in SEQ ID NO: 46, and a CDR3 set forth in SEQ ID NO: 57.

[0027] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 31 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 31; (b) CDR2 having the sequence set forth in SEQ ID NO: 40 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 40; and (c) a CDR3 having a sequence as set forth in SEQ ID NO: 48 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 48.

[0028] In certain embodiments, the substitutions are conservative substitutions.

[0029] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 31, a CDR2 set forth in SEQ ID NO: 40, and a CDR3 set forth in SEQ ID NO: 48.

[0030] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 33 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 33; (b) CDR2 having the sequence set forth in SEQ ID NO: 42 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 42; and (c) a CDR3 having a sequence as set forth in SEQ ID NO:50 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:50.

[0031] In certain embodiments, the substitutions are conservative substitutions.

[0032] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 33, a CDR2 set forth in SEQ ID NO: 42, and a CDR3 set forth in SEQ ID NO: 50.

[0033] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (1) CDR1 set forth in SEQ ID NO: 30; CDR2 set forth in SEQ ID NO: 39; and CDR3 set forth in SEQ ID NO: 47; (2) CDR1 set forth in SEQ ID NO: 31; CDR2 set forth in SEQ ID NO: 40; and CDR3 set forth in SEQ ID NO: 48; (3) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 49; (4) CDR1 set forth in SEQ ID NO: 33; CDR2 set forth in SEQ ID NO: 42; and CDR3 set forth in SEQ ID NO: 50; (5) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 51; (6) CDR1 set forth in SEQ ID NO: 32 or 34; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 52; (7) CDR1 set forth in SEQ ID NO: 35; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 52; (8) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 53; (9) CDR1 set forth in SEQ ID NO: 36; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 54; (10) CDR1 set forth in SEQ ID NO: 35; CDR2 set forth in SEQ ID NO: 44; and CDR3 set forth in SEQ ID NO: 55; (11) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 45; and CDR3 set forth in SEQ ID NO: 56; (12) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 52; (13) CDR1 set forth in SEQ ID NO: 37; CDR2 set forth in SEQ ID NO: 46; and CDR3 set forth in SEQ ID NO: 57; (14) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 58; or (15) CDR1 set forth in SEQ ID NO: 38; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 59.

[0034] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of: (i) a sequence set forth in any one of SEQ ID NOs: 1 to 3, 7 to 9, 14 to 24, and 28 to 29; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to any one of the sequences set forth in SEQ ID NOs: 1 to 3, 7 to 9, 14 to 24, and 28 to 29; and (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to any one of the sequences set forth in SEQ ID NOs: 1 to 3, 7 to 9, 14 to 24, and 28 to 29.

[0035] In certain embodiments, the substitutions are conservative substitutions.

[0036] In certain embodiments, the Nanobody or antigen-binding fragment thereof is humanized.

[0037] In certain embodiments, the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g. a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), which heavy chain framework region may comprise one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to camelid residues.

[0038] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of: (i) a sequence set forth in any one of SEQ ID NOs: 4 to 6, 10 to 13, 25 to 27, and 78; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to any one of the sequences set forth in SEQ ID NOs: 4 to 6, 10 to 13, 25 to 27, and 78; and (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to any one of the sequences set forth in SEQ ID NOs: 4 to 6, 10 to 13, 25 to 27, and 78.

[0039] In certain embodiments, the substitutions are conservative substitutions.

[0040] In certain embodiments, the serum albumin is selected from the group consisting of human serum albumin (HSA), mouse serum albumin (MSA) and / or cynomolgus serum albumin (CSA).

[0041] In certain embodiments, the binding of the Nanobody or antigen-binding fragment thereof to serum albumin is pH-independent.

[0042] In certain embodiments, the Nanobody or antigen-binding fragment thereof can specifically bind to serum albumin at a pH range of 5 to 8 (eg, 5.5 to 7.4).

[0043] Polypeptide Constructs In another embodiment, the present application also provides a polypeptide construct capable of specifically binding to serum albumin, the construct comprising the above-mentioned Nanobody or an antigen-binding fragment thereof and an immunoglobulin Fc domain.

[0044] As used herein, Fc domain, also referred to as Fc region, refers to a portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc domain includes a hinge, CH2 and CH3. When the Fc domain includes a hinge, the hinge regulates dimerization between two Fc-containing polypeptides. The Fc domain can be any isotype of antibody heavy chain constant region. In some embodiments, the Fc domain is of IgG1, IgG2, IgG3, or IgG4.

[0045] In certain embodiments, the Fc domain contained in the polypeptide construct of the present application is a native Fc region and comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature. For example, the Fc domain can be the Fc region of human IgG1 with native sequence, the Fc region of human IgG2 with native sequence, the Fc region of human IgG3 with native sequence, or the Fc region of human IgG4 with native sequence. The native Fc region can have an effector function. Exemplary "effector functions" include binding to Fc receptors; Clq binding, and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells. Substitution of at least one amino acid residue in a native Fc region with a different residue or chemical modification of at least one amino acid residue can result in a functional change, such as a change in the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby altering (e.g., decreasing or enhancing) effector function.

[0046] Thus, in certain embodiments, the Fc domain contained in the polypeptide construct of the present application may be a variant Fc region and may comprise one or more (e.g., 1-10, e.g., 1-5) amino acid mutations or chemical modifications compared to a native Fc region to alter one or more properties of the antibody of the present application, such as Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0047] In certain embodiments, the Fc domain contained in the polypeptide construct of the present application has ADCC activity. In certain embodiments, the Fc domain contained in the polypeptide construct of the present application does not have ADCC activity.

[0048] In certain embodiments, an immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus (eg, the C-terminus) of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker.

[0049] In certain embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain).

[0050] In certain embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO:68, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions, or additions thereto (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) thereto.

[0051] In certain embodiments, the serum albumin is selected from the group consisting of human serum albumin (HSA), mouse serum albumin (MSA) and / or cynomolgus serum albumin (CSA).

[0052] In certain embodiments, the binding of the polypeptide construct to serum albumin is pH independent.

[0053] In certain embodiments, the polypeptide construct is capable of specifically binding to serum albumin at a pH range of 5 to 8 (eg, 5.5 to 7.4).

[0054] Fusion proteins In another aspect, the application also provides a fusion protein, which comprises the above-mentioned Nanobody or antigen-binding fragment thereof, or the above-mentioned polypeptide construct, and an additional peptide domain.

[0055] In certain embodiments, the additional peptide domain is selected from the group consisting of polypeptide domains with therapeutic effects.

[0056] In certain embodiments, the additional polypeptide domain is linked to the N-terminus and / or C-terminus of the Nanobody or antigen-binding fragment thereof or polypeptide construct, optionally via a peptide linker.

[0057] Isolated Nucleic Acid Molecules In another aspect, the application also provides an isolated nucleic acid molecule, which encodes the above-mentioned Nanobody or antigen-binding fragment thereof, the above-mentioned polypeptide construct, or the above-mentioned fusion protein.

[0058] vector In another aspect, the present application also provides a vector, which comprises the above-mentioned nucleic acid molecule. In certain embodiments, the vector is a cloning vector or an expression vector.

[0059] host cell In another aspect, the present application also provides a host cell, which comprises the above-mentioned nucleic acid molecule or the above-mentioned vector. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E. coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0060] Preparation method The Nanobody or polypeptide construct or fusion protein of the present application can be prepared by various methods known in the art, such as recombinant genetic engineering techniques. For example, a DNA molecule encoding the Nanobody or polypeptide construct or fusion protein of the present application can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under certain conditions to express the Nanobody or polypeptide construct or fusion protein of the present application.

[0061] The antigen-binding fragments of the present application can be obtained by hydrolysis of the intact Nanobody molecule (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr.Opin. Immunol.11:548-557(1999); Little et al., Immunol.Today,21:364-370(2000)). A person of ordinary skill in the art is well aware of other techniques for preparing these antigen-binding fragments.

[0062] In another aspect, the application also provides a method for preparing the above-mentioned Nanobody or antigen-binding fragment thereof, the above-mentioned polypeptide construct, or the above-mentioned fusion protein, the method comprising culturing the above-mentioned host cell under conditions allowing expression of the protein, and recovering the Nanobody or antigen-binding fragment thereof, or the polypeptide construct, or the fusion protein from the culture of the cultured host cell.

[0063] Bispecific or multispecific antibodies In another aspect, the application also provides bispecific or multispecific antibodies, which comprise the above-mentioned Nanobody or antigen-binding fragment thereof, or the above-mentioned polypeptide construct.

[0064] In certain embodiments, a bispecific or multispecific antibody is capable of specifically binding to serum albumin and, in addition, is capable of specifically binding to one or more additional targets.

[0065] In certain embodiments, the bispecific or multispecific antibody further comprises at least one second antibody or antigen-binding fragment thereof that has binding specificity for a second target.

[0066] In certain embodiments, a bispecific or multispecific antibody comprises: (i) as a first antigen-specific binding domain, a Nanobody or antigen-binding fragment thereof as described above, or a polypeptide construct as described above (e.g. a Nanobody or antigen-binding fragment thereof or a polypeptide construct comprising a sequence set forth in any one of SEQ ID NOs: 10, 4, 78); (ii) a second antigen-specific binding domain (e.g., a Fab) comprising a light chain set forth in SEQ ID NO: 79 and a heavy chain set forth in SEQ ID NO: 80; and, (iii) a third antigen-specific binding domain (e.g., a nanobody VHH) set forth in SEQ ID NO: 81.

[0067] In certain embodiments, a bispecific or multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, where the first polypeptide chain comprises (e.g., from N-terminus to C-terminus): a second antigen-specific binding domain, a third antigen-specific binding domain, a light or heavy chain (e.g., VH and CH1) of the first antigen-specific binding domain; and the second polypeptide chain comprises (e.g., from N-terminus to C-terminus): a second antigen-specific binding domain and a heavy chain (e.g., VH and CH1) or light chain of the third antigen-specific binding domain; where the first polypeptide chain pairs with the second polypeptide chain to form a complete second antigen-specific binding domain. In certain embodiments, preferably, each domain of said first polypeptide chain and / or second polypeptide chain is linked by a linker (e.g., a peptide linker comprising one or more glycines and / or one or more serines).

[0068] In certain embodiments, a bispecific or multispecific antibody comprises: (1) a first peptide having the sequence set forth in SEQ ID NO:69, and a second peptide having the sequence set forth in SEQ ID NO:76; (2) a first peptide having the sequence set forth in SEQ ID NO: 70, and a second peptide having the sequence set forth in SEQ ID NO: 76; or (3) A first peptide having the sequence set forth in SEQ ID NO: 71, and a second peptide having the sequence set forth in SEQ ID NO: 76.

[0069] In certain embodiments, the first peptide is present in a first peptide chain and the second peptide is present in a second peptide chain, hi certain embodiments, the first peptide chain and the second peptide chain form a complex.

[0070] Conjugates In another aspect, the present application also provides a conjugate, which comprises the above-mentioned Nanobody or antigen-binding fragment thereof or the above-mentioned polypeptide construct and a therapeutic agent linked to the Nanobody or antigen-binding fragment thereof or the polypeptide construct.

[0071] In certain embodiments, the therapeutic agent is selected from the group consisting of a cytotoxic agent, a hormonal agent, a biological response modifier, an anti-tumor agent such as another antibody or an antigen-binding fragment thereof.

[0072] Pharmaceutical Compositions In another aspect, the present application also provides a pharmaceutical composition, which comprises the nanobody or antigen-binding fragment thereof, polypeptide construct, fusion protein, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific antibody, or conjugate as described above.

[0073] In certain embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient.

[0074] In certain exemplary embodiments, the pharma- ceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., a solution containing 0.01% polysorbate 20), a pH buffer (e.g., a phosphate buffer), Ringer's solution, and any combination thereof.

[0075] In certain embodiments, the pharmaceutical composition comprises the above-mentioned Nanobody or antigen-binding fragment thereof, or polypeptide construct, or a nucleic acid molecule, vector, or host cell encoding the Nanobody or antigen-binding fragment thereof, or polypeptide construct.

[0076] In certain embodiments, the pharmaceutical composition comprises the above-mentioned fusion protein, or a nucleic acid molecule, vector, or host cell encoding the fusion protein.

[0077] In certain embodiments, the pharmaceutical composition comprises the bispecific or multispecific antibody described above, or a nucleic acid molecule, vector, or host cell encoding the bispecific or multispecific antibody.

[0078] In certain embodiments, the pharmaceutical composition comprises a conjugate as described above.

[0079] use In another aspect, the application also provides the use of a Nanobody or antigen-binding fragment thereof, a polypeptide construct, a fusion protein, an isolated nucleic acid molecule, a vector, a host cell, a bispecific or multispecific antibody or conjugate, or a pharmaceutical composition as described above in the manufacture of a medicament.

[0080] In certain embodiments, the pharmaceutical agent can directly or indirectly participate in FcRn-mediated serum albumin circulation in a subject.

[0081] In certain embodiments, the medicament exhibits an increased in vivo half-life compared to a corresponding medicament lacking the Nanobody or antigen-binding fragment thereof.

[0082] In certain embodiments, the pharmaceutical agent is a protein pharmaceutical agent.

[0083] In certain embodiments, the subject is a mammal, such as a human, mouse, or cynomolgus monkey.

[0084] Methods for extending the in vivo half-life of drugs In another aspect, the application also provides a method for increasing the in vivo half-life of a drug, the method comprising linking the above-mentioned Nanobody or antigen-binding fragment thereof, or the above-mentioned polypeptide construct to a drug.

[0085] In certain embodiments, the increased in vivo half-life is relative to the in vivo half-life of the agent in the absence of the Nanobody or antigen-binding fragment thereof.

[0086] In certain embodiments, the drug is selected from the group consisting of macromolecular drugs (eg, polypeptide drugs).

[0087] In certain embodiments, the subject is a mammal, such as a human, mouse, or cynomolgus monkey.

[0088] Definition of Terms In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.In addition, the experimental procedures of virology, biochemistry and immunology used herein are all routine processes widely used in the corresponding fields.At the same time, in order to better understand this application, the definitions and explanations of related terms are provided below.

[0089] When the terms "for example," "eg," "such as," "including," "comprising," or variations thereof are used in this specification, these terms are not considered to be limiting terms and are to be interpreted to mean "but not limited to" or "limited to."

[0090] Unless otherwise indicated in this specification or clearly contradicted by context, the terms "a" and "an" and "the" and similar designations should be construed to cover both the singular and the plural in the context of describing this application (particularly in the context of the claims which follow).

[0091] As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), usually derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies are composed of four framework regions and three complementarity determining regions, and have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies may be truncated at the N- or C-terminus to include only a portion of FR1 and / or FR4, or to lack one or two of these framework regions, so long as they substantially retain antigen binding ability and specificity. Nanobodies are also called single domain antibodies (sdAbs), and the two are used interchangeably.

[0092] As used herein, the term "antigen-binding fragment" of a Nanobody refers to a polypeptide comprising a fragment of a Nanobody, which retains the ability to specifically bind to the same antigen as the Nanobody binds and / or competes with the Nanobody for specific binding to an antigen, also referred to as an "antigen-binding portion". See generally Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), which is fully incorporated herein by reference for all purposes. Antigen-binding fragments of the Nanobodies of the present application can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the present application. In some embodiments, an "antigen-binding fragment" of a Nanobody can be truncated at the N-terminus or C-terminus to include only a portion of FR1 and / or FR4, or to lack one or two of these framework regions, as compared to the full-length Nanobody, so long as it substantially retains antigen-binding ability and specificity.

[0093] Antigen-binding fragments of a Nanobody are obtained from a given Nanobody (e.g., a Nanobody provided by the present application) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of a Nanobody are screened for specificity in the same way as the intact Nanobody.

[0094] As used herein, unless the context clearly indicates otherwise, when the term "nanobody" is referred to it includes not only complete nanobodies but also antigen-binding fragments of nanobodies.

[0095] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in nanobodies, which are named CDR1, CDR2 and CDR3. The exact boundaries of these CDRs can be defined according to various numbering systems well known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, those skilled in the art can easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). As used herein, the CDRs of Nanobodies are preferably determined by the IMGT numbering system.

[0096] As used herein, the term "framework region" or "FR" residues refers to amino acid residues of an antibody variable domain other than the CDR residues defined above.

[0097] As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region that contains CH2 and CH3. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding. "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. In some embodiments, the Fc region comprises a hinge, CH2 and CH3. When the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be any isotype of antibody heavy chain constant region, such as, for example, IgG1, IgG2, IgG3, or IgG4.

[0098] The Fc domain may be a native Fc region or a variant Fc region. A native Fc region comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature, for example, a human Fc region having a native sequence includes the Fc region of human IgG1 (both non-A and A subtypes) having a native sequence; the Fc region of human IgG2 having a native sequence; the Fc region of human IgG3 having a native sequence; and the Fc region of human IgG4 having a native sequence, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of an Fc region having a native sequence by at least one amino acid modification. In some embodiments, a variant Fc region may have an altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.

[0099] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and has its amino acid sequence modified to improve homology with the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (receptor antibody). In certain embodiments, the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (receptor antibody). A humanized antibody generally retains the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody may be a camelid-derived antibody having the expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR regions of an antibody derived from an immunized animal may be inserted into a human framework sequence using methods known in the art. In the context of nanobodies, a humanized antibody may refer to a humanized VHH, i.e., a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework regions (FRs) of a human immunoglobulin have been replaced with corresponding non-human residues. In addition, a humanized VHH may contain residues that are not found in either the original VHH or human framework sequence, but are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide.

[0100] The term "identity" is used herein to refer to the sequence match between two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of overlapping identical positions / total number of positions×100%). In certain embodiments, the two sequences are the same length.

[0101] The determination of percent identity between two sequences can also be achieved using mathematical algorithms.A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, which is improved in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877.Such an algorithm is integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0102] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D In this application, "K DThe term "antibody-antigen binding affinity" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the stronger the binding between the antibody and the antigen and the higher the affinity between the antibody and the antigen.

[0103] The specific binding properties between two molecules can be determined using methods known in the art. These include measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. The "association rate constant" (k a or k on ) and "dissociation rate constant" (k dis or k off ) can be calculated from the concentration and the actual binding and dissociation rates (see Malmqvist M, Nature, 1993, 361:186-187). dis / k on The ratio of the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59:439-473). D , k on and k dis The value of can be measured by any effective method. In certain embodiments, the dissociation constant can be measured by surface plasmon resonance (SPR) using a Biacore instrument. In addition, the dissociation constant can be measured by bioluminescence interferometry or Kinexa technology.

[0104] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector is introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda and M13 phages, animal viruses, and the like. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papoviruses (e.g., SV40), and the like. The vector may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication.

[0105] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus spp., insect cells such as S2 Drosophila cells or Sf9, fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or animal cells such as human cells.

[0106] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacement of an amino acid residue with an amino acid residue having a similar side chain, e.g., with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417(1997), which are incorporated herein by reference).

[0107] The compilation of the 20 conventional amino acids involved herein follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S.Golub and D.R.Gren, Eds., Sinauer Associates, Sunderland, Mass.(1991)), which is incorporated herein by reference. In this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And, in this application, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0108] As used herein, the term "pharmaceutical acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, osmotic pressure maintaining agents, absorption retarding agents, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, etc. Absorption retardants include, but are not limited to, monostearate salts, gelatin, etc. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and can stabilize the desired activity of active ingredients in the drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or their degradation products (e.g., lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharma- ceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), a sodium chloride solution (e.g., 0.9% (w / v) NaCl), a glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., a solution containing 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0109] As used herein, the term "in vivo half-life" has the meaning known to those of skill in the art and can generally be defined as the time it takes for the serum concentration of a molecule in vivo to decrease by 50% (e.g., due to degradation of the ligand and / or clearance or chelation of the ligand by natural mechanisms). Methods for determining in vivo half-life are well known to those of skill in the art, for example, by pharmacokinetic analysis.

[0110] Beneficial Effects of the Present Application The present application provides Nanobodies with high binding activity to serum albumin, and cross-reactivity with human, monkey and / or mouse serum albumin. In particular, the binding of the Nanobodies of the present application to serum albumin is pH independent and can effectively participate in FcRn-mediated serum albumin circulation, thereby extending the in vivo half-life of the Nanobodies. Thus, the Nanobodies of the present application can be linked (e.g. fused) to drug molecules (e.g. polypeptide drugs) to extend the half-life of the drug molecules in vivo.

[0111] In addition, nanobodies have the characteristics of small molecular weight and high stability.Compared with conventional antibodies, they have many advantages in drug development, such as good tissue penetration, flexible administration, high degree of humanization, and easy conversion to recombinant proteins.

[0112] Hereinafter, the embodiments of the present application will be described in detail with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to explain the present application and do not limit the scope of the present application. From the following drawings and detailed description of the preferred embodiments, various objects and advantages of the present application will become apparent to those skilled in the art. [Brief description of the drawings]

[0113] [Figure 1] FIG. 1 shows the time course of the concentrations of Bi-340-352, Bi-340-353, Bi-340-354 and Bi-340-356 in mouse serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0114] Specific Model for Implementing the Invention The invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the invention.

[0115] Unless otherwise specified, the molecular biological experimental methods and immunoassays used in the present invention were essentially performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compendium of Molecular Biology Experimental Guide, 3rd edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used according to the conditions recommended by the product manufacturers. Those skilled in the art will be familiar with the following examples, which are illustrative of the present invention and are not intended to limit the scope of the invention as claimed. EXAMPLES

[0116] [Example 1]

[0117] Screening of anti-HSA nanobodies 1.1 Construction of nanobody libraries Animal immunization 1 mg of human serum albumin (HSA) antigen (purchased from AcroBiosystems) was mixed with an equal amount of Freund's adjuvant, and two alpacas were immunized once a week for a total of four times to stimulate B cells to express antigen-specific nanobodies. After four immunizations, 50 ml of alpaca peripheral blood was extracted, and lymphocytes were isolated using lymphocyte separation fluid. Total RNA was extracted using RNA extraction reagent Trizol (purchased from Invitrogen). Total cDNA of alpaca lymphocytes was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0118] Amplification of nanobody genes The first PCR was performed to amplify the IgG2 and IgG3 sequences from the cDNA obtained in the previous step. The primers used for amplification are shown in Table 1:

[0119] [Table 1]

[0120] The first round PCR products were subjected to agarose gel electrophoresis and the 750 bp band of interest was excised from the gel and collected for the second round amplification of the VHH sequence. The second round PCR amplification primers are shown in Table 2:

[0121] [Table 2]

[0122] The second PCR product was used as a template to perform a third PCR to add homology arm sequences homologous to the display vector to the VHH genes. The third PCR amplification primers are shown in Table 3:

[0123] [Table 3]

[0124] Building a library The linearized yeast display vector (artificially synthesized) and the third PCR product were mixed and electrotransformed into Saccharomyces cerevisiae (purchased from ATCC) to construct anti-HSA nanobody libraries from two animals. The library sizes were measured to be 2.1 × 10 7 and 3.2 x 10 7 It was.

[0125] 1.2 Screening of HSA nanobodies Biotinylation of HSA protein To dissolve HSA protein (purchased from AcroBiosystems, catalog number: HSA-H5220), an appropriate amount of double-distilled water was taken, and biotin was dissolved according to the product instructions of the biotin labeling kit (purchased from Thermo), mixed with the protein solution, and incubated for 2 hours at 4°C. Excess biotin was removed using a desalting column (purchased from Thermo), and the pretreatment of the desalting column and the sample recovery operation were all performed according to the product instructions.

[0126] Enrichment of yeast specifically capable of binding to HSA by MACS The VHH library constructed in step 1.1 was inoculated into SD-CAA development medium (1L of SD-CAA development medium containing 6.7g YNB, 5g tyrosine, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4, and 2% glucose) and cultured overnight at 30℃ and 225 rpm. An appropriate amount of yeast cells was collected, centrifuged at 3000 rpm for 5 minutes to remove the medium (parameters for the following centrifugation operation are the same as here), and the yeast cells were resuspended in SD-CAA induction medium and induced overnight. The library concentration after induction was measured, and an appropriate amount of yeast cells was collected, centrifuged to remove the medium. The yeast cells were suspended in 50ml of PBS and centrifuged to remove the supernatant. The yeast cells were suspended in 10ml of PBS.

[0127] The biotin-labeled HSA protein (final concentration 100 nM) obtained in the above step was added and incubated at room temperature for 30 minutes, after which the yeast cells were collected by centrifugation and washed three times with 50 ml of PBS. The yeast cells were resuspended in 5 ml of washing solution, 200 μl of SA magnetic beads (purchased from Miltenyi Biotec) were added, and incubated for 10 minutes. The mixture of yeast cells and magnetic beads was washed three times with PBS and added to an LS purification column (purchased from Miltenyi Biotec). The LS purification column was placed on a magnetic grid and washed with PBS to remove nonspecifically bound yeast cells. The column was removed from the magnetic grid, and PBS was added to elute the yeast cells. After centrifugation, the eluted yeast cells were transferred to SD-CAA development medium and developed.

[0128] Flow cytometric sorting to obtain high affinity yeast cells Yeast cells enriched by MACS were inoculated into SD-CAA development medium and cultured overnight at 30°C with shaking at 225 rpm. Yeast cells were resuspended in SD-CAA induction medium and induced overnight. Anti-c-Myc mouse antibody (purchased from Thermo, catalog number: MA1-980) and 100 nM biotin-labeled HSA antigen were added and incubated for 10 min. Yeast cells were washed three times with PBS, and then goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen, catalog number: A32723) and streptavidin-APC conjugate fluorescent antibody (purchased from Invitrogen, catalog number: SA1005) were added and incubated for 15 min. Cells were resuspended in PBS and sorted using a BD FACSAriaII instrument to obtain yeast cells with high binding ability to HSA antigen.

[0129] Sequencing of vHH candidates The yeast products with high binding ability to HSA antigen obtained by MACS and FACS enrichment were cultured in SD-CAA development medium at 30°C and 225 rpm overnight, and yeast plasmids were extracted according to the operation of the yeast plasmid extraction kit (purchased from Tiangen). The plasmids were transformed into Top10 competent cells (purchased from Tiangen) by electroporation, plated on plates containing ampicillin, and cultured at 37°C overnight. The monoclonal cells were harvested for sequencing, VHH sequences were obtained, and CDR sequences were determined according to the IMGT numbering system. The sequence information of the obtained monoclonal nanobodies is shown in Table 4.

[0130] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Example 2]

[0131] Construction, expression and purification of heavy chain antibodies 2.1. Construction of pCDNA3.1 expression vector with antibody gene inserted The VHH gene sequence was ligated to the coding sequence of the Fc fragment of human IgG1 (LALA mutation) (amino acid sequence shown in SEQ ID NO: 68) and inserted into the pCDNA3.1 vector linearized by EcoRI / NotI double enzyme digestion using a recombinase that catalyzes homologous recombination (purchased from Vazyme, catalog number: c115-02). The treatment was performed according to the product manual. The homologous recombination product was transferred to Top10 competent cells, spread on a plate containing ampicillin, and cultured overnight at 37°C. Single clones were picked for sequencing, and plasmids were extracted.

[0132] The sequence of SEQ ID NO:68 was as follows: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0133] 2.2. Cell transfection and protein purification The extracted plasmid obtained in the above-mentioned process was introduced into Expi-CHO cells using the ExpiCHO (registered trademark) Expression System Kit (purchased from Thermo). The transfection method was in accordance with the product manual. After 5 days of cell culture, the supernatant was collected, and the protein was purified by the sorting method using protein A magnetic beads (purchased from GenScript). The magnetic beads were resuspended in an appropriate amount (1 to 4 times the amount of magnetic beads) of binding buffer (PBS + 0.1% Tween 20, pH 7.4), added to the sample to be purified, and incubated at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic grid (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. An elution buffer (0.1 M sodium citrate, pH 3.2) was added in an amount 3 to 5 times the amount of magnetic beads, and the mixture was shaken at room temperature for 5 to 10 minutes. After returning the mixture to the magnetic grid, the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1 M Tris, pH 8.54), and mixed thoroughly to obtain the target protein. [Example 3]

[0134] Affinity determination ForteBio affinity measurements were performed according to existing methods (Estep, P et al., Determination of antibody-antigen affinity and epitope binding based on high-throughput methods. MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated offline in assay buffer for 30 min, then tested online for 60 s to establish a baseline, and purified antibodies obtained as described above were loaded online onto the AHQ sensor. The sensor was then exposed to 100 nM HSA antigen (purchased from AcroBiosystems, catalog number: HSA-H5220) for 5 min, and the sensor was transferred to PBS for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model. The affinity data were shown in Table 5.

[0135] [Table 5]

[0136] In this example, we further detected the cross-reaction of the candidate molecules with serum albumins from different species, including mouse serum albumin (MSA, purchased from AcroBiosystems, catalog number: MSA-M52H8) and cynomolgus serum albumin (CSA, purchased from AcroBiosystems, catalog number: CSA-C52H4). The affinity data is shown in Table 6.

[0137] [Table 6] [Example 4]

[0138] Construction of humanized anti-HSA antibody To reduce the immunogenicity of monoclonal antibodies in humans, three antibodies, HSA-59, HSA-M-27, and HSA-PH-8, were humanized. The humanization method employed was the VHH humanized universal framework grafting method, and several amino acids in the antibody framework 2 were mutated according to a method reported in the literature (Vincke, C., et al., General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem 284(5): 3273-3284). The sequence information of the humanized antibodies is shown in Table 7.

[0139] [Table 7-1] [Table 7-2] [Table 7-3]

[0140] In this study, IMGT (http: / / www.imgt.org) was used to evaluate the humanization levels of HSA-59, HSA-M-27, HSA-PH-8 and humanized sequences. The results are shown in Table 8. The humanization levels of all humanized samples were improved and met the requirements for late-stage drug development.

[0141] [Table 8]

[0142] The construction, expression and purification methods of the protein were the same as those in Example 2. The purity of the protein was determined by HPLC detection, and the purity detection results are shown in Table 9. HPLC was performed as follows, mobile phase: 150 mM Na2HPO4·12H2O, pH 7.0; chromatographic conditions: detection wavelength: 280 nm; column temperature: 25°C; flow rate: 0.35 ml / min; detection time: 20 min; Zenix-C SEC-300 column (SEPAX, 4.6×300 mm, 3 μm).

[0143] [Table 9] [Example 5]

[0144] Affinity determination of humanized anti-HSA antibodies 5.1. Affinity Determination of Humanized Antibodies In this example, the binding affinity of the purified humanized anti-HSA antibody to HSA was detected. The experimental method was the same as in Example 3. The results are shown in Table 10. As a result, the humanized anti-HSA antibody had good binding activity to HSA protein.

[0145] [Table 10]

[0146] In this example, the binding affinity of the purified humanized anti-HSA antibody to MSA / CSA was also detected. The experimental method was the same as in Example 3. The experimental results are shown in Table 11.

[0147] [Table 11]

[0148] 5.2. Determining the affinity of humanized antibodies at different pH values The recycling mechanism of HSA is similar to that of IgG. HSA is internalized into vascular endothelial cells via pinocytosis. In endosomes, the binding of membrane-bound FcRn to HSA is promoted at endosomal pH of about 6.0; the complex in the endosome is then recycled into the blood; and at pH 7.4, membrane-bound FcRn and HSA rapidly dissociate, allowing the recycling of HSA.

[0149] In this example, the affinity of the purified humanized anti-HSA antibody to HSA was detected under different pH conditions. The experimental method was the same as in Example 3. The experimental results are shown in Table 12. As a result, the affinity of the humanized anti-HSA antibody to HSA protein was good under different pH conditions.

[0150] [Table 12] [Example 6]

[0151] Detection of the half-life extension effect of anti-HSA nanobody 6.1. Molecular construction and protein preparation In order to confirm the half-life extension effect of anti-HSA nanobody, in this example, multispecific antibodies were constructed. Multispecific antibodies Bi-340-352, Bi-340-353 and Bi-340-354 were each independently composed of peptide chain #1 and peptide chain #2, and contained three antigen-binding domains, i.e., Fab targeting the first antigen, nanobody (VHH1) targeting the second antigen, and nanobody (VHH2) targeting the third antigen, where VHH2 was Hu-HSA-M-27-1, Hu-HSA-59-1(.), Hu-HSA-PH-8-5 or a non-HSA-related VHH as a control. The structures and sequences of the multispecific antibodies are shown in Table 13-1 and Table 13-2, and the domains of peptide chain #1 and peptide chain #2 were linked by a linker containing GGGGS.

[0152] [Table 13]

[0153] [Table 14-1] [Table 14-2] [Table 14-3]

[0154] Gene sequences encoding peptide chain #1 and peptide chain #2 of Bi-340-352, Bi-340-353, Bi-340-354, and Bi-340-356 were synthesized by GENEWIZ. Using a recombinase that catalyzes homologous recombination (purchased from Vazyme), the encoding gene sequences were constructed in pCDNA3.1 vectors linearized by EcoRI / NotI double enzyme digestion. The construction process was performed according to the product's instructions. The homologous recombination products of peptide chain #1 and peptide chain #2 of each multispecific antibody were co-transformed into Top10 competent cells, spread on plates containing ampicillin, and cultured at 37°C overnight, and single clones were picked for sequencing.

[0155] The plasmid was transfected into Expi-CHO cells using the ExpiCHO® Expression System Kit (purchased from Thermo). The transfection process was performed according to the manufacturer's instructions. After 5 days of cell culture, the supernatant was collected and purified using a KappaSelect (GE) affinity chromatography column. The specific method was as follows: the sample was filtered through a 0.2 um sterile syringe filter PES with a syringe. The column was equilibrated with 5 column volumes of equilibration buffer (20 mM PB + 0.15 M NaCl, pH 7.4) until the conductivity and pH of the effluent did not change. The sample was loaded at a flow rate of 0.5 ml / min. After loading, the column was continuously washed with equilibration buffer until the permeation was complete and the UV value did not decrease. Elution was performed using elution buffer (0.1 M glycine-HCl, pH 3.0), and the effluent was collected. After elution, the recovered antibody solution must be immediately neutralized with an alkaline buffer (eg, 1 M Tris / HCl, pH 8.0) to a pH at which the antibody is stable.

[0156] The purity of the obtained protein was detected by HPLC. HPLC was performed as follows: mobile phase: 150 mM Na2HPO4·12H2O, pH 7.0; chromatographic conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.35 ml / min, detection time: 20 min, Zenix-C SEC-300 column (SEPAX, 4.6 × 300 mm, 3 μm).

[0157] 6.2. Protein Half-Life Detection The injection dose for mice was 10 mg / kg. Blood sample collection time points: 3 min, 30 min, 2 h, 6 h, 24 h, 48 h, 96 h, and 168 h after administration. The whole blood sample was left to stand at 2-8 °C for 30 min, centrifuged at 12000 rpm for 5 min to collect serum, and the obtained serum was centrifuged at 2-8 °C, 12000 rpm for 5 min, and stored at -80 °C. The free antibody concentration in the serum was detected by ELISA. The results are shown in Figure 1. As a result, the half-life of the molecules with anti-HSA nanobody in mice was about 23.7 h (Bi-340-352), 24.2 h (Bi-340-353), and 10.9 h (Bi-340-354), respectively, and the half-life of the molecule with non-HSA-related nanobody (Bi-340-356) was about 1.4 h. The results showed that anti-HSA nanobodies were able to significantly extend the in vivo half-life of this molecule.

[0158] Although the specific embodiments of the present application have been described in detail, it will be understood that those skilled in the art can make various modifications and changes to the details based on all the teachings disclosed, and all of these modifications are within the scope of protection of the present application. The entirety of the present application is defined by the appended claims and their equivalents.

Claims

1. 1. A Nanobody or antigen-binding fragment thereof capable of specifically binding to serum albumin, wherein said Nanobody or antigen-binding fragment thereof comprises: (a) a CDR1:X having a structure selected from the group consisting of: １ X ２ X ３ LX ４ YYX ５ (Formula I, SEQ ID NO:72), SGFTLDYYA (SEQ ID NO:30), GSIWGIYH (SEQ ID NO:37), GFTFSIYS (SEQ ID NO:31), GSIFTFYR (SEQ ID NO:33); (b) a CDR2 having a structure selected from the group consisting of: ６ SSGGX ７ X ８ (Formula II, SEQ ID NO:73), ITVDGST (SEQ ID NO:46), ISSGGSP (SEQ ID NO:40), ITTDTST (SEQ ID NO:42); (c) a CDR3 having a structure selected from the group consisting of: AAAX ９ LECRTX １０ X １１ X １２ X １３ YX １４ Y (Formula III, SEQ ID NO:74), AAATX １５ ECRGRSSSYDY (Formula IV, SEQ ID NO:75), AAALLECRVRSWPSDN (SEQ ID NO:49), AAAVLECRAAEYVNS (SEQ ID NO:54), DIRNVGGDY (SEQ ID NO:57), SPITSIFKA (SEQ ID NO:48), NVRNVERDY (SEQ ID NO:50); Where: X １ is selected from the group consisting of: (i) amino acid residues G, R, and (ii) amino acid residues that are conservative substitutions for (i); X ２ is selected from the group consisting of: (i) amino acid residues F, A, S, E, and (ii) amino acid residues that are conservative substitutions for (i); X ３ is selected from the group consisting of: (i) amino acid residues T, Q, and (ii) amino acid residues that are conservative substitutions for (i); X ４ is selected from the group consisting of (i) amino acid residues D, E, and (ii) amino acid residues that are conservative substitutions for (i); X ５ is selected from the group consisting of: (i) amino acid residue A, and (ii) an amino acid residue that is a conservative substitution for (i); X ６ is selected from the group consisting of (i) amino acid residues A, S, and (ii) amino acid residues that are conservative substitutions for (i); X ７ is selected from the group consisting of (i) amino acid residues A, S, and (ii) amino acid residues that are conservative substitutions for (i); X ８ is selected from the group consisting of: (i) the amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X ９ is selected from the group consisting of: (i) amino acid residues T, V, and (ii) amino acid residues that are conservative substitutions for (i); X １０ is selected from the group consisting of: (i) amino acid residues T, V, and (ii) amino acid residues that are conservative substitutions for (i); X １１ is selected from the group consisting of (i) amino acid residues V, I, L, and (ii) amino acid residues that are conservative substitutions for (i); X １２ is selected from the group consisting of (i) amino acid residues R, V, T, and (ii) amino acid residues that are conservative substitutions for (i); X １３ is selected from the group consisting of: (i) amino acid residues G, E, and (ii) amino acid residues that are conservative substitutions for (i); X １４ is selected from the group consisting of: (i) amino acid residues D, A, and (ii) amino acid residues that are conservative substitutions for (i); X １５ is selected from the group consisting of: (i) amino acid residues L, F, and (ii) amino acid residues that are conservative substitutions for (i); Preferably, said Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in any one of SEQ ID NOs: 30-38; a CDR2 set forth in any one of SEQ ID NOs: 39-46; and a CDR3 set forth in any one of SEQ ID NOs: 47-59.

2. The nanobody or antigen-binding fragment thereof comprises: (a) CDR1, １ X ２ X ３ LX ４ YYX ５ (Formula I, SEQ ID NO:72) or SGFTLDYYA (SEQ ID NO:30); (b) CDR2, comprising: IX ６ SSGGX ７ X ８ (Formula II, SEQ ID NO:73); (c) CDR3, ９ LECRTX １０ X １１ X １２ X １３ YX １４ Y (Formula III, SEQ ID NO:74), AAATX １５ those having a structure selected from the group consisting of ECRGRSSSYDY (Formula IV, SEQ ID NO:75), AAALLECRVRSWPSDN (SEQ ID NO:49), AAAVLECRAAEYVNS (SEQ ID NO:54); Where: X １ is selected from the group consisting of: (i) amino acid residues G, R, and (ii) amino acid residues that are conservative substitutions for (i); X ２ is selected from the group consisting of: (i) amino acid residues F, A, S, E, and (ii) amino acid residues that are conservative substitutions for (i); X ３ is selected from the group consisting of: (i) amino acid residues T, Q, and (ii) amino acid residues that are conservative substitutions for (i); X ４ is selected from the group consisting of (i) amino acid residues D, E, and (ii) amino acid residues that are conservative substitutions for (i); X ５ is selected from the group consisting of: (i) amino acid residue A, and (ii) an amino acid residue that is a conservative substitution for (i); X ６ is selected from the group consisting of (i) amino acid residues A, S, and (ii) an amino acid residue that is a conservative substitution for (i) (.Base); X ７ is selected from the group consisting of (i) amino acid residues A, S, and (ii) amino acid residues that are conservative substitutions for (i); X ８ is selected from the group consisting of: (i) the amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X ９ is selected from the group consisting of: (i) amino acid residues T, V, and (ii) amino acid residues that are conservative substitutions for (i); X １０ is selected from the group consisting of: (i) amino acid residues T, V, and (ii) amino acid residues that are conservative substitutions for (i); X １１ is selected from the group consisting of (i) amino acid residues V, I, L, and (ii) amino acid residues that are conservative substitutions for (i); X １２ is selected from the group consisting of (i) amino acid residues R, V, T, and (ii) amino acid residues that are conservative substitutions for (i); X １３ is selected from the group consisting of: (i) amino acid residues G, E, and (ii) amino acid residues that are conservative substitutions for (i); X １４ is selected from the group consisting of: (i) amino acid residues D, A, and (ii) amino acid residues that are conservative substitutions for (i); X １５ is selected from the group consisting of: (i) amino acid residues L, F, and (ii) amino acid residues that are conservative substitutions for (i); Preferably, X １ is selected from the group consisting of amino acid residues G and R; ２ is selected from the group consisting of amino acid residues F, A, S and E; ３ is selected from the group consisting of amino acid residues T and Q; ４ is selected from the group consisting of amino acid residues D and E; ５ is an amino acid residue A; ６ is selected from the group consisting of amino acid residues A and S; ７ is selected from amino acid residues A and S; ８ is the amino acid residue T; ９ is selected from the group consisting of amino acid residues T and V; １０ is selected from the group consisting of amino acid residues T and V; １１ is selected from the group consisting of amino acid residues V, I and L; １２ is selected from the group consisting of amino acid residues R, V and T; １３ is selected from the group consisting of amino acid residues G and E; １４ is selected from the group consisting of amino acid residues D and A; １５ is selected from the group consisting of amino acid residues L and F; 2. The nanobody or antigen-binding fragment thereof of claim 1, wherein the nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in any one of SEQ ID NOs: 30, 32, 34-36, 38; a CDR2 set forth in any one of SEQ ID NOs: 39, 41, 43-45; and a CDR3 set forth in any one of SEQ ID NOs: 47, 49, 51-56, 58-59.

3. The nanobody or antigen-binding fragment thereof comprises: (a) CDR1, １ X ２ X ３ LX ４ YYX ５ (Formula I, SEQ ID NO:72) or SGFTLDYYA (SEQ ID NO:30); (b) CDR2, comprising: IX ６ SSGGX ７ X ８ (Formula II, SEQ ID NO:73); (c) CDR3, ９ LECRTX １０ X １１ X １２ X １３ YX １４ Y (Formula III, SEQ ID NO:74), AAATX １５ those having a structure selected from the group consisting of ECRGRSSSYDY (Formula IV, SEQ ID NO:75), AAALLECRVRSWPSDN (SEQ ID NO:49), AAAVLECRAAEYVNS (SEQ ID NO:54); Where: X １ is selected from the group consisting of: (i) amino acid residues G, R, and (ii) amino acid residues that are conservative substitutions for (i); X ２ is selected from the group consisting of: (i) amino acid residues F, E, S, and (ii) amino acid residues that are conservative substitutions for (i); X ３ is selected from the group consisting of: (i) the amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X ４ is selected from the group consisting of: (i) amino acid residue D, and (ii) an amino acid residue that is a conservative substitution for (i); X ５ is selected from the group consisting of: (i) amino acid residue A, and (ii) an amino acid residue that is a conservative substitution for (i); X ６ is selected from the group consisting of (i) amino acid residues A, S, and (ii) amino acid residues that are conservative substitutions for (i); X ７ is selected from the group consisting of (i) amino acid residues A, S, and (ii) amino acid residues that are conservative substitutions for (i); X ８ is selected from the group consisting of: (i) the amino acid residue T, and (ii) an amino acid residue that is a conservative substitution for (i); X ９ is selected from the group consisting of: (i) amino acid residues T, V, and (ii) amino acid residues that are conservative substitutions for (i); X １０ is selected from the group consisting of: (i) amino acid residue V, and (ii) an amino acid residue that is a conservative substitution for (i); X １１ is selected from the group consisting of (i) amino acid residues V, I, and (ii) amino acid residues that are conservative substitutions for (i); X １２ is selected from the group consisting of (i) amino acid residues R, V, and (ii) amino acid residues that are conservative substitutions for (i); X １３ is selected from the group consisting of: (i) amino acid residues G, E, and (ii) amino acid residues that are conservative substitutions for (i); X １４ is selected from the group consisting of: (i) amino acid residue D, and (ii) an amino acid residue that is a conservative substitution for (i); X １５ is selected from the group consisting of: (i) amino acid residues L, F, and (ii) amino acid residues that are conservative substitutions for (i); Preferably, X １ is selected from the group consisting of amino acid residues G and R; ２ is selected from the group consisting of amino acid residues F, S and E; ３ is the amino acid residue T; ４ is an amino acid residue D; X ５ is an amino acid residue A; ６ is selected from the group consisting of amino acid residues A and S; ７ is selected from the group consisting of amino acid residues A and S; ８ is the amino acid residue T; ９ is selected from the group consisting of amino acid residues T and V; １０ is an amino acid residue V; １１ is selected from the group consisting of amino acid residues V and I; １２ is selected from the group consisting of amino acid residues R and V; １３ is selected from the group consisting of amino acid residues G and E; １４ is an amino acid residue D; X １５ is selected from the group consisting of amino acid residues L and F; 3. The nanobody or antigen-binding fragment thereof of claim 1 or 2, wherein the nanobody or antigen-binding fragment thereof comprises a CDR1 as set forth in any one of SEQ ID NOs: 30, 32, 34-36; a CDR2 as set forth in any one of SEQ ID NOs: 39, 41, 43-45; and a CDR3 as set forth in any one of SEQ ID NOs: 47, 49, 51, 52, 54-56, 58.

4. The nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having a sequence as set forth in SEQ ID NO:32 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:32; (b) CDR2 having a sequence as set forth in SEQ ID NO: 41 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 41; and (c) a CDR3 having a sequence as set forth in SEQ ID NO:49 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:49; Preferably, the substitutions are conservative substitutions; 2. The nanobody or antigen-binding fragment thereof of claim 1, wherein the nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 32, a CDR2 set forth in SEQ ID NO: 41, and a CDR3 set forth in SEQ ID NO:

49.

5. The nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having a sequence as set forth in SEQ ID NO: 32 or 34, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 32 or 34; (b) CDR2 having a sequence as set forth in SEQ ID NO: 43 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 43; and (c) a CDR3 having a sequence as set forth in SEQ ID NO:52 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:52; Preferably, the substitutions are conservative substitutions; Preferably, said Nanobody or antigen-binding fragment thereof comprises: (a) CDR1, １ X ２ X ３ LX ４ YYX ５ (Formula I, SEQ ID NO:72), wherein X １ is an amino acid residue G; ２ is selected from the group consisting of (i) amino acid residues F, E, and (ii) amino acid residues that are conservative substitutions for (i); ３ is the amino acid residue T; ４ is an amino acid residue D; X ５ is amino acid residue A; (b) CDR2 having the structure set forth in IASSGGGST (SEQ ID NO: 43); (c) a CDR3 having the structure AAAVLECRTVVRGYDY (SEQ ID NO:52); 2. The nanobody or antigen-binding fragment thereof of claim 1, wherein the nanobody or antigen-binding fragment thereof comprises a CDR1 as set forth in SEQ ID NO: 32 or 34; a CDR2 as set forth in SEQ ID NO: 43; and a CDR3 as set forth in SEQ ID NO:

52.

6. The nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having a sequence as set forth in SEQ ID NO:37 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:37; (b) CDR2 having a sequence as set forth in SEQ ID NO: 46 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 46; and (c) a CDR3 having a sequence as set forth in SEQ ID NO:57 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:57; Preferably, the substitutions are conservative substitutions; 2. The nanobody or antigen-binding fragment thereof of claim 1, wherein the nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 37, a CDR2 set forth in SEQ ID NO: 46, and a CDR3 set forth in SEQ ID NO:

57.

7. The nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having a sequence as set forth in SEQ ID NO:31 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:31; (b) CDR2 having a sequence as set forth in SEQ ID NO: 40 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 40; and (c) a CDR3 having a sequence as set forth in SEQ ID NO: 48 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 48; Preferably, the substitutions are conservative substitutions; Preferably, the nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 31, a CDR2 set forth in SEQ ID NO: 40, and a CDR3 set forth in SEQ ID NO:

48.

8. The nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having a sequence as set forth in SEQ ID NO: 33 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 33; (b) CDR2 having a sequence as set forth in SEQ ID NO: 42 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO: 42; and (c) a CDR3 having a sequence as set forth in SEQ ID NO:50 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence as set forth in SEQ ID NO:50; Preferably, the substitutions are conservative substitutions; 2. The nanobody or antigen-binding fragment thereof of claim 1, wherein the nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 33, a CDR2 set forth in SEQ ID NO: 42, and a CDR3 set forth in SEQ ID NO:

50.

9. (1) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 49; (2) CDR1 set forth in SEQ ID NO: 32 or 34; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 52; (3) CDR1 set forth in SEQ ID NO: 37; CDR2 set forth in SEQ ID NO: 46; and CDR3 set forth in SEQ ID NO: 57; (4) CDR1 set forth in SEQ ID NO: 30; CDR2 set forth in SEQ ID NO: 39; and CDR3 set forth in SEQ ID NO: 47; (5) CDR1 set forth in SEQ ID NO: 31; CDR2 set forth in SEQ ID NO: 40; and CDR3 set forth in SEQ ID NO: 48; (6) CDR1 set forth in SEQ ID NO: 33; CDR2 set forth in SEQ ID NO: 42; and CDR3 set forth in SEQ ID NO: 50; (7) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 51; (8) CDR1 set forth in SEQ ID NO: 35; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 52; (9) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 43; and CDR3 set forth in SEQ ID NO: 53; (10) CDR1 set forth in SEQ ID NO: 36; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 54; (11) CDR1 set forth in SEQ ID NO: 35; CDR2 set forth in SEQ ID NO: 44; and CDR3 set forth in SEQ ID NO: 55; (12) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 45; and CDR3 set forth in SEQ ID NO: 56; (13) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 52; (14) CDR1 set forth in SEQ ID NO: 32; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO: 58; or (15) The nanobody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising: CDR1 set forth in SEQ ID NO: 38; CDR2 set forth in SEQ ID NO: 41; and CDR3 set forth in SEQ ID NO:

59.

10. The amino acid sequence is selected from the group consisting of: (i) a sequence set forth in any one of SEQ ID NOs: 3, 9, 24, 1-2, 7-8, 14-23, 28-29; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to any one of the sequences set forth in SEQ ID NOs: 3, 9, 24, 1-2, 7-8, 14-23, 28-29; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 3, 9, 24, 1-2, 7-8, 14-23, and 28-29; Preferably, the substitutions are conservative substitutions. A nanobody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. the Nanobody or antigen-binding fragment thereof is humanized; 10. The Nanobody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g. a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), wherein the heavy chain framework region optionally comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to camelid residues.

12. The Nanobody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of: (i) a sequence set forth in any one of SEQ ID NOs: 4 to 6, 10 to 13, 25 to 27, and 78; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to any one of the sequences set forth in SEQ ID NOs: 4 to 6, 10 to 13, 25 to 27, and 78; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-6, 10-13, 25-27, and 78; 12. The nanobody or antigen-binding fragment thereof of claim 11 , wherein the substitutions are conservative substitutions.

13. the serum albumin is selected from the group consisting of human serum albumin (HSA), mouse serum albumin (MSA) and / or cynomolgus serum albumin (CSA); Preferably, the binding of said Nanobody or antigen-binding fragment thereof to serum albumin is pH-independent; 13. The Nanobody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the Nanobody or antigen-binding fragment thereof is capable of specifically binding to serum albumin at a pH range of 5 to 8 (e.g., 5.5 to 7.4).

14. 14. A polypeptide construct capable of specifically binding to serum albumin, comprising a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 13 and an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus (e.g. the C-terminus) of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker; Preferably, the immunoglobulin Fc domain is an IgG Fc domain (e.g., an IgG1 Fc domain); Preferably, the immunoglobulin Fc domain comprises a polypeptide construct comprising the sequence set forth in SEQ ID NO: 68, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions thereto (e.g. one, two, three, four or five amino acid substitutions, deletions or additions thereto).

15. the serum albumin is selected from the group consisting of human serum albumin (HSA), mouse serum albumin (MSA) and / or cynomolgus serum albumin (CSA); Preferably, the binding of the polypeptide construct to serum albumin is pH independent; Preferably, the polypeptide construct is capable of specifically binding to serum albumin at a pH range of 5 to 8 (eg, 5.5 to 7.4), according to claim 14 .

16. Comprising a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 13, or a polypeptide construct according to claim 13 or 14, and an additional peptide domain; Preferably, said additional peptide domain is selected from polypeptide domains having a therapeutic effect; Preferably, a fusion protein, wherein said additional peptide domain is linked to the N-terminus and / or C-terminus of the Nanobody or antigen-binding fragment thereof or polypeptide construct, optionally via a peptide linker.

17. 17. An isolated nucleic acid molecule encoding a nanobody or antigen-binding fragment thereof described in any one of claims 1 to 13, a polypeptide construct described in claim 14 or 15, or a fusion protein described in claim 16.

18. A vector comprising the nucleic acid molecule of claim 17, preferably said vector being a cloning vector or an expression vector.

19. 19. A host cell comprising the nucleic acid molecule of claim 17 or the vector of claim 18.

20. 20. A method for preparing a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 13, a polypeptide construct according to claims 14 or 15, or a fusion protein according to claim 16, comprising culturing a host cell according to claim 19 under conditions allowing expression of the protein, and recovering the Nanobody or antigen-binding fragment thereof, or the polypeptide construct, or the fusion protein from a culture of the cultured host cells.

21. A bispecific or multispecific antibody, comprising a nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 13, or a polypeptide construct according to claim 14 or 15; Preferably, the bispecific or multispecific antibody is capable of specifically binding to serum albumin and, in addition, is capable of specifically binding to one or more additional targets; Preferably, the bispecific or multispecific antibody further comprises at least one second antibody having binding specificity for a second target; Preferably, said bispecific or multispecific antibody comprises: (i) as a first antigen-specific binding domain, a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 13, or a polypeptide construct according to claim 14 or 15 (e.g. a Nanobody or antigen-binding fragment thereof or a polypeptide construct comprising a sequence according to any one of SEQ ID NOs: 10, 4, 78); (ii) a second antigen-specific binding domain (e.g., a Fab) comprising a light chain set forth in SEQ ID NO:79 and a heavy chain set forth in SEQ ID NO:80; and, (iii) a third antigen-specific binding domain set forth in SEQ ID NO: 81 (e.g. Nanobody VHH); Preferably, a bispecific or multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein said first polypeptide chain comprises (e.g., from N-terminus to C-terminus): said second antigen-specific binding domain, said third antigen-specific binding domain, a light or heavy chain (e.g., VH and CH1) of said first antigen-specific binding domain; and said second polypeptide chain comprises (e.g., from N-terminus to C-terminus): a second antigen-specific binding domain and a heavy chain (e.g., VH and CH1) or light chain of the third antigen-specific binding domain; wherein said first polypeptide chain pairs with said second polypeptide chain to form a complete second antigen-specific binding domain; Preferably, each domain of the first polypeptide chain and / or the second polypeptide chain is linked by a linker (e.g., a peptide linker comprising one or more glycines and / or one or more serines); Preferably, the bispecific or multispecific antibody comprises: (1) a first peptide having a sequence set forth in SEQ ID NO:69, and a second peptide having a sequence set forth in SEQ ID NO:76; (2) a first peptide having a sequence set forth in SEQ ID NO: 70, and a second peptide having a sequence set forth in SEQ ID NO: 76; or (3) a first peptide having a sequence set forth in SEQ ID NO: 71, and a second peptide having a sequence set forth in SEQ ID NO: 76; Preferably, said first peptide is present in said first peptide chain and said second peptide is present in said second peptide chain; preferably, said first peptide chain and said second peptide chain form a complex. A bispecific or multispecific antibody.

22. 16. A conjugate comprising a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 13 or a polypeptide construct according to claim 14 or 15, and a therapeutic agent linked to said nanobody or antigen-binding fragment thereof or said polypeptide construct; Preferably, the therapeutic agent is selected from the group consisting of a cytotoxic agent, a hormonal agent, a biological response modifier, an anti-tumor agent such as another antibody or an antigen-binding fragment thereof, conjugate.

23. 20. A pharmaceutical composition comprising a nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 13, a polypeptide construct according to claim 14 or 15, a fusion protein according to claim 16, an isolated nucleic acid molecule according to claim 17, a vector according to claim 18, a host cell according to claim 19, a bispecific or multispecific antibody according to claim 21, or a conjugate according to claim 22; Preferably, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition comprises a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 13, a polypeptide construct according to claim 14 or 15, or a nucleic acid molecule, vector or host cell encoding said Nanobody or antigen-binding fragment or polypeptide construct; Preferably, the pharmaceutical composition comprises a fusion protein according to claim 16, or a nucleic acid molecule, vector or host cell encoding said fusion protein; Preferably, said pharmaceutical composition comprises a bispecific or multispecific antibody according to claim 21 or a nucleic acid molecule, vector or host cell encoding said bispecific or multispecific antibody; Preferably, said pharmaceutical composition comprises a conjugate according to claim 22.

24. Use of a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 13, a polypeptide construct according to claim 14 or 15, a fusion protein according to claim 16, an isolated nucleic acid molecule according to claim 17, a vector according to claim 18, a host cell according to claim 19, a bispecific or multispecific antibody according to claim 21, or a conjugate according to claim 22, or a pharmaceutical composition according to claim 23 in the manufacture of a medicament; Preferably, the medicament is capable of directly or indirectly influencing FcRn-mediated serum albumin circulation in a subject; Preferably, the medicament exhibits an increased in vivo half-life compared to the corresponding agent lacking the Nanobody or antigen-binding fragment thereof; Preferably, the pharmaceutical agent is a protein drug; Preferably, the subject is a mammal, such as a human, mouse, or cynomolgus monkey.

25. A method for increasing the in vivo half-life of a drug, comprising linking to said drug a nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 13 or a polypeptide construct according to claim 14 or 15; Preferably, said increased in vivo half-life is relative to the in vivo half-life of said agent in the absence of said Nanobody or antigen-binding fragment thereof; Preferably, the drug is selected from the group consisting of macromolecular drugs (e.g., polypeptide drugs); Preferably, the subject is a mammal, such as a human, mouse, or cynomolgus monkey.

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