Single-domain antibody targeting serum albumin and fusion protein

Abstract

Provided is a fusion protein formed from an insulin analogue and a single-domain antibody specifically binding to human serum albumin (HSA). By binding to HAS, the half-life of insulin is prolonged by means of an FcRn-mediated recycling effect. Moreover, the single-domain antibody has a shielding effect on the fusion-expressed insulin analogue, whereby the insulin activity can be regulated to achieve a sustained-release effect. Further provided are a single-domain antibody specifically binding to human serum albumin (HSA), or an antigen-binding fragment thereof, and a derivative containing the single-domain antibody or the antigen-binding fragment thereof.

Description

Single-domain antibodies and fusion proteins targeting serum albumin

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application 202410545257.9, filed April 30, 2024, the entire contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application provides fusion proteins of insulin analogs and single-domain antibodies that specifically bind to human serum albumin (HSA) and their therapeutic uses. The present application also provides single-domain antibodies or antigen-binding fragments thereof that specifically bind to human serum albumin (HSA), derivatives containing said single-domain antibodies or antigen-binding fragments thereof, and their uses for extending half-life in vivo. BACKGROUND

[0004] Diabetes is a chronic metabolic disease caused by insufficient insulin secretion or the body's inability to utilize the insulin produced. Currently, diabetes is mainly divided into type I diabetes and type II diabetes, among which type I diabetes is insulin-dependent diabetes, mainly characterized by insufficient insulin secretion, and daily insulin input is required; type II diabetes is non-insulin-dependent diabetes, which is caused by impaired function of pancreatic beta cells and long-term insulin resistance. Type II diabetes patients are the main group.

[0005] Regardless of type I or type II diabetes, the main treatment relies on insulin. However, natural insulin has a short half-life, and patients need to be injected multiple times. Although the current long-acting insulin degludec and glargine achieve a once-a-day dosing frequency, for patients who need to use basal insulin, scientists hope to provide ultra-long-acting insulin with longer action time, lower injection frequency, and better smoothness of drug efficacy to better meet the needs of patients.

[0006] WO2012123519A2 discloses a super long acting insulin Icodec which has been submitted for marketing application, the molecular structure features are that the threonine at position 30 of the B chain of human insulin is removed, and a 20-carbon fatty monoacid is connected to the lysine residue at position 29 of the B chain, and YB16H and FB25H mutations are made to B16 and B25 of the B chain, YA14E mutation is also made to A14 of the A chain, the modified fatty acid chain is combined with serum albumin, and the FcRn-mediated recycling achieves once-a-week super long acting insulin administration. US9855318B2 discloses another super long acting insulin LY3209590 which has completed clinical phase II, the molecule LY3209590 is connected to the B chain and the A chain into a single chain insulin through a linker, and then the insulin is fused and expressed with human Ig2Fc through another linker. WO2021136296A1 discloses another super long acting insulin which has completed clinical phase I, the main principle is the same as Icodec, which is also an acylated derivative of a human insulin analogue obtained by modification of a fatty acid chain. WO2023174370A1 discloses another super long acting insulin in the form of a fusion protein, which is the same as the molecule LY3209590, both of which are single-chain insulin fused and expressed with human Fc through a linker, thereby prolonging the half-life of insulin through Fc and FcRn recycling. CN114174348A discloses another fusion protein, which is a single-chain human insulin fused and expressed with a single-domain antibody VHH of human serum albumin (HSA), the single-domain antibody VHH in the fusion protein binds to HSA, and the half-life of insulin is prolonged through FcRn-mediated recycling.

[0007] Although super long acting insulins have been reported, there is still a need in the art for insulin analogues with longer in vivo half-life and better in vivo efficacy to exhibit longer drug duration and drug smoothness. SUMMARY

[0008] The present application provides a fusion protein of an insulin analogue (INS analogue) and a single-domain antibody (VHH) of human serum albumin (HSA), which prolongs the half-life of insulin through FcRn-mediated recycling, and the VHH has a shielding effect on the fusion-expressed insulin analogue, which can adjust the activity of insulin to achieve a slow-release effect. In addition, the present application also provides a single-domain antibody or antigen-binding fragment thereof specific to human serum albumin (HSA), which can effectively prolong the in vivo half-life of the drug molecule coupled thereto, and has cross-reactivity with human, monkey, mouse, and rat serum albumin, providing convenience for pharmacodynamics, pharmacokinetics, and toxicology research. Thus the following aspects are provided.

[0009] Single-domain antibodies and antigen-binding fragments thereof

[0010] In one aspect, the present application provides a single-domain antibody or an antigen-binding fragment thereof that specifically binds to human serum albumin (HSA), comprising: a CDR1 as set forth in GYSYSSQYMX1 (SEQ ID NO: 10), a CDR2 as set forth in AISATGVYTYYADSVKG (SEQ ID NO: 7), and a CDR3 as set forth in PLPRTSPAX2PLX3VYEYEY (SEQ ID NO: 11); wherein X1 is selected from G or S, X2 is selected from N or F, and X3 is selected from G or M.

[0011] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: a CDR1 comprising a sequence as set forth in SEQ ID NO: 5 or 6, a CDR2 comprising a sequence as set forth in SEQ ID NO: 7, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 8 or 9.

[0012] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises:

[0013] (1) a CDR1 as set forth in SEQ ID NO: 5, a CDR2 as set forth in SEQ ID NO: 7, and a CDR3 as set forth in SEQ ID NO: 8;

[0014] (2) a CDR1 as set forth in SEQ ID NO: 6, a CDR2 as set forth in SEQ ID NO: 7, and a CDR3 as set forth in SEQ ID NO: 8;

[0015] (3) a CDR1 as set forth in SEQ ID NO: 5, a CDR2 as set forth in SEQ ID NO: 7, and a CDR3 as set forth in SEQ ID NO: 9; or

[0016] (4) a CDR1 as set forth in SEQ ID NO: 6, a CDR2 as set forth in SEQ ID NO: 7, and a CDR3 as set forth in SEQ ID NO: 9.

[0017] In certain embodiments, the single-domain antibody comprises a VHH comprising a sequence set forth in SEQ ID NO: 1, or a variant thereof having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or having at least 80% (e.g., 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%) sequence identity. In certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant does not differ from the sequence from which it is derived except in the CDR regions.

[0018] In certain embodiments, the single-domain antibody comprises a VHH comprising a sequence set forth in SEQ ID NO: 1.

[0019] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof is humanized.

[0020] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene), optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from a human-derived residue to a camelid-derived residue.

[0021] In certain embodiments, the single-domain antibody comprises a VHH comprising a sequence set forth in any one of SEQ ID NOs: 2-4, or a variant thereof having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or having at least 80% (e.g., 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%) sequence identity. In certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant does not differ from the sequence from which it is derived except in the CDR regions.

[0022] In certain embodiments, the single-domain antibody comprises a VHH comprising a sequence set forth in any one of SEQ ID NOs: 2-4.

[0023] In certain embodiments, the antigen-binding fragment of the single-domain antibody is truncated at the N- and / or C-terminus compared to the full-length single-domain antibody such that it comprises only part of FR1 and / or FR4.

[0024] In certain embodiments, the single domain antibody or antigen binding fragment thereof described in any of the above embodiments also binds to monkey serum albumin (e.g. cynomolgus serum albumin), mouse serum albumin and / or rat serum albumin.

[0025] Conjugate

[0026] The half-life of a biologically active molecule in vivo can be extended by conjugating a single domain antibody of the present application to the biologically active molecule (e.g. a pharmaceutical agent, a detection agent, etc.).

[0027] In another aspect, the present application provides a conjugate comprising a single domain antibody or antigen binding fragment thereof of the present application and a further biologically active molecule.

[0028] In certain embodiments, the further biologically active molecule is optionally linked to the N- and / or C-terminus of the single domain antibody or antigen binding fragment thereof via a linker.

[0029] In certain embodiments, the further biologically active molecule is a therapeutic agent.

[0030] In certain embodiments, the conjugate is a fusion protein comprising a single domain antibody or antigen binding fragment thereof and a further biologically active polypeptide. In certain embodiments, the further biologically active polypeptide is optionally linked to the N- and / or C-terminus of the single domain antibody or antigen binding fragment thereof via a peptide linker.

[0031] Insulin analogue

[0032] In another aspect, the present application provides a fusion protein comprising an insulin active domain and an antigen binding domain targeting human serum albumin (HSA), wherein,

[0033] the insulin active domain comprises an insulin A chain and an insulin B chain;

[0034] the antigen binding domain targeting human serum albumin (HSA) is selected from a single domain antibody or antigen binding fragment thereof of the present application.

[0035] In certain embodiments, the antigen binding domain comprises a VHH sequence as set forth in SEQ ID NO: 2 or 3.

[0036] In certain embodiments, the insulin B chain is a native human insulin B chain.

[0037] In certain embodiments, the insulin B chain is a variant of the native human insulin B chain, e.g., the variant can comprise one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions compared to the native human insulin B chain sequence. In certain embodiments, the insulin B chain is a truncation of the native human insulin B chain, e.g., deletion of one or several (e.g., 1, 2, 3, or 4) amino acids at the N- and / or C-terminus compared to the native human insulin B chain sequence.

[0038] In certain embodiments, the insulin B chain comprises the amino acid sequence set forth in FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or a C-terminally truncated version thereof comprising a deletion of 1-4 amino acids at the C-terminus; wherein X1is selected from Y, E, G, A, V, L, I, D, N, K, R, Q, S, or T. In certain embodiments, the C-terminally truncated version comprises a deletion of 1-4 contiguous amino acids in the C-terminal TPKT sequence.

[0039] In certain embodiments, the insulin B chain comprises the amino acid sequence set forth in FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or the amino acid sequence set forth in FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); wherein X1is selected from Y, E, G, A, V, L, I, D, N, K, R, Q, S, or T.

[0040] In certain embodiments, the insulin B chain comprises the amino acid sequence set forth in FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29).

[0041] In certain embodiments, X1is selected from Y or A.

[0042] In certain embodiments, the insulin B chain comprises the sequence set forth in any one of SEQ ID NOs: 12-27.

[0043] In certain embodiments, the insulin B chain comprises the sequence set forth in SEQ ID NO: 12 or 17.

[0044] In certain embodiments, the insulin A chain is the native human insulin A chain.

[0045] In certain embodiments, the insulin A chain is a variant of the native human insulin A chain, e.g., the variant can comprise one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions compared to the native human insulin A chain sequence. In certain embodiments, the insulin A chain is a truncation of the native human insulin A chain, e.g., deletion of one or several (e.g., 1, 2, or 3) amino acids at the N-terminus and / or C-terminus compared to the native human insulin A chain sequence.

[0046] In certain embodiments, the insulin A chain comprises the amino acid sequence set forth in GIVEQCCTSZ1CSLZ2QLENYCZ3(SEQ ID NO: 34), wherein Z1is selected from T or I; Z2is selected from D or Y; and Z3is G or absent.

[0047] In certain embodiments, Z1is T and Z2is D. In certain embodiments, Z1is T and Z2is Y. In certain embodiments, Z1is I and Z2is D.

[0048] In certain embodiments, Z3is G.

[0049] In certain embodiments, the insulin A chain comprises the sequence set forth in any one of SEQ ID NOs: 30-33.

[0050] In certain embodiments, the insulin A chain comprises the sequence set forth in SEQ ID NO: 30.

[0051] In certain embodiments, the insulin B chain comprises the amino acid sequence set forth in FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or a C-terminally truncated version thereof comprising a deletion of 1-4 amino acids at the C-terminus; wherein X1is selected from Y, E, G, A, V, L, I, D, N, K, R, Q, S, or T; and,

[0052] the insulin A chain comprises the amino acid sequence set forth in GIVEQCCTSZ1CSLZ2QLENYCZ3(SEQ ID NO: 34), wherein Z1is selected from T or I; Z2is selected from D or Y; and Z3is selected from G or absent.

[0053] In certain embodiments, the insulin B chain comprises the amino acid sequence set forth in FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); preferably, X1is selected from Y or A; preferably, the insulin B chain comprises the sequence set forth in any one of SEQ ID NOs: 12-27; preferably, the insulin B chain comprises the sequence set forth in SEQ ID NO: 12 or 17.

[0054] In certain embodiments, the insulin A chain comprises the amino acid sequence set forth in GIVEQCCTSZ1CSLZ2QLENYCZ3 (SEQ ID NO: 34), wherein Z3 is G; preferably, Z1 is T and Z2 is D; or, Z1 is T and Z2 is Y; or, Z1 is I and Z2 is D; preferably, the insulin A chain comprises the sequence set forth in any one of SEQ ID NOs: 30-33; preferably, the insulin A chain comprises the sequence set forth in SEQ ID NO: 30.

[0055] In certain embodiments, the insulin B chain and A chain respectively comprise a sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 30, SEQ ID NO: 13 and SEQ ID NO: 30, SEQ ID NO: 14 and SEQ ID NO: 30, SEQ ID NO: 15 and SEQ ID NO: 30, SEQ ID NO: 16 and SEQ ID NO: 30, SEQ ID NO: 17 and SEQ ID NO: 30, SEQ ID NO: 18 and SEQ ID NO: 30, SEQ ID NO: 19 and SEQ ID NO: 30, SEQ ID NO: 20 and SEQ ID NO: 30, SEQ ID NO: 21 and SEQ ID NO: 30, SEQ ID NO: 22 and SEQ ID NO: 30, SEQ ID NO: 23 and SEQ ID NO: 30, SEQ ID NO: 24 and SEQ ID NO: 30, SEQ ID NO: 25 and SEQ ID NO: 30, SEQ ID NO: 26 and SEQ ID NO: 30, SEQ ID NO: 27 and SEQ ID NO: 30, SEQ ID NO: 12 and SEQ ID NO: 32, or SEQ ID NO: 12 and SEQ ID NO: 33.

[0056] In certain embodiments, the insulin B chain and A chain respectively comprise a sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 30, or SEQ ID NO: 17 and SEQ ID NO: 30.

[0057] In another aspect, the present application provides a fusion protein comprising an insulin active domain and a single-domain antibody or an antigen-binding fragment thereof targeting human serum albumin (HSA), wherein:

[0058] The insulin active domain comprises an insulin A chain and an insulin B chain, the insulin B chain comprising an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or a C-terminal truncation thereof comprising a deletion of 1-4 amino acids from the C-terminus; wherein X1 is selected from G, A, V, L, I, D, N, K, Q, S or T.

[0059] In certain embodiments, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); wherein X1 is selected from G, A, V, L, I, D, N, K, Q, S or T.

[0060] In certain embodiments, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29).

[0061] In certain embodiments, X1 is selected from A.

[0062] In certain embodiments, the insulin B chain comprises a sequence represented by any one of SEQ ID NOs: 16-23, 25-27.

[0063] In certain embodiments, the insulin B chain comprises a sequence represented by SEQ ID NO: 17.

[0064] In certain embodiments, the insulin A chain comprises an amino acid sequence represented by GIVEQCCTSZ1CSLZ2QLENYCZ3 (SEQ ID NO: 34), wherein Z1 is selected from T or I; Z2 is selected from D or Y; and Z3 is G or absent.

[0065] In certain embodiments, Z1 is T and Z2 is D; or, Z1 is T and Z2 is Y; or, Z1 is I and Z2 is D.

[0066] In certain embodiments, Z3 is G.

[0067] In certain embodiments, the insulin A chain comprises a sequence represented by any one of SEQ ID NOs: 30-33.

[0068] In certain embodiments, the insulin A chain comprises a sequence represented by SEQ ID NO: 30.

[0069] In certain embodiments, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or a C-terminal truncation thereof comprising a deletion of 1-4 amino acids from the C-terminus; wherein X1is selected from G, A, V, L, I, D, N, K, Q, S, or T; and,

[0070] the insulin A chain comprises an amino acid sequence represented by GIVEQCCTSZ1CSLZ2QLENYCZ3 (SEQ ID NO: 34), wherein Z1is selected from T or I; Z2is selected from D or Y; and Z3is selected from G or absent.

[0071] In certain embodiments, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); preferably, X1is selected from A; preferably, the insulin B chain comprises a sequence represented by any one of SEQ ID NOs: 16-23, 25-27; preferably, the insulin B chain comprises a sequence represented by SEQ ID NO: 17.

[0072] In certain embodiments, the insulin A chain comprises an amino acid sequence represented by GIVEQCCTSZ1CSLZ2QLENYCZ3 (SEQ ID NO: 34), wherein Z3is G; preferably, Z1is T and Z2is D; or, Z1is T and Z2is Y; or, Z1is I and Z2is D; preferably, the insulin A chain comprises a sequence represented by any one of SEQ ID NOs: 30-33; preferably, the insulin A chain comprises a sequence represented by SEQ ID NO: 30.

[0073] In certain embodiments, the insulin B chain and A chain respectively comprise a sequence selected from the group consisting of: SEQ ID NO: 16 and SEQ ID NO: 30, SEQ ID NO: 17 and SEQ ID NO: 30, SEQ ID NO: 18 and SEQ ID NO: 30, SEQ ID NO: 19 and SEQ ID NO: 30, SEQ ID NO: 20 and SEQ ID NO: 30, SEQ ID NO: 21 and SEQ ID NO: 30, SEQ ID NO: 22 and SEQ ID NO: 30, SEQ ID NO: 23 and SEQ ID NO: 30, SEQ ID NO: 25 and SEQ ID NO: 30, SEQ ID NO: 26 and SEQ ID NO: 30, SEQ ID NO: 27 and SEQ ID NO: 30.

[0074] In certain embodiments, the insulin B chain and A chain respectively comprise a sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 30.

[0075] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof targeting human serum albumin (HSA) is selected from the group consisting of the single-domain antibodies or antigen-binding fragments thereof of the present application.

[0076] In certain embodiments of the fusion protein of any of the above aspects, the insulin active domain is linked to the N-terminus and / or C-terminus of the single-domain antibody or antigen-binding fragment thereof via a peptide linker LI. In certain embodiments, the insulin active domain is linked to the N-terminus of the single-domain antibody or antigen-binding fragment thereof via a peptide linker LI.

[0077] In certain embodiments, the peptide linker LI is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 86, (GGGGQ)n(G)m(SEQ ID NO: 87), (GGGGS)n(G)m(SEQ ID NO: 88), wherein n is an integer selected from 0-5, and m is an integer selected from 0-5.

[0078] In certain embodiments, the peptide linker LI is G„ (SEQ ID NO: 86), and n is 0, 1, 2, 3, or 4. In certain embodiments, the peptide linker LI is selected from the group consisting of the sequences set forth in any one of SEQ ID NOs: 76-78.

[0079] In certain embodiments, the peptide linker LI is (GGGGQ)n(G)m(SEQ ID NO: 87), wherein n is 1, 2, 3, or 4, and m is an integer selected from 1-5, for example m is 2 or 5. In certain embodiments, the peptide linker LI is selected from the group consisting of the sequences set forth in any one of SEQ ID NOs: 75, 80-85.

[0080] In certain embodiments, the peptide linker LI is (GGGGS)n(G)m(SEQ ID NO: 88), wherein n is 1, 2, or 3, and m is an integer selected from 1-5, for example m is 5. In certain embodiments, the peptide linker LI is selected from the group consisting of the sequence set forth in SEQ ID NO: 79.

[0081] In certain embodiments of the fusion protein of any of the above aspects, the insulin active domain is directly linked to the N-terminus and / or C-terminus of the single-domain antibody or antigen-binding fragment thereof. In certain embodiments, the insulin active domain is directly linked to the N-terminus of the single-domain antibody or antigen-binding fragment thereof.

[0082] In certain embodiments of the fusion protein of any of the above aspects, the insulin active domain is a single-chain insulin, wherein the A chain and the B chain are connected to each other by a non-native linker. In addition, the single-chain insulin can include at least one of the native inter-chain and / or intra-chain disulfide bonds to maintain the correct structural folding.

[0083] In certain embodiments of the fusion protein of any of the above aspects, the insulin active domain is a two-chain insulin, wherein the A chain and the B chain are connected to each other by one or more inter-chain and / or intra-chain disulfide bonds, but not by any linker, to maintain the correct structural folding, such as native INS.

[0084] In certain embodiments of the fusion protein of any of the above aspects, the insulin active domain is a single-chain polypeptide formed by an insulin A chain and an insulin B chain connected by a peptide linker L2.

[0085] In certain embodiments, the insulin active domain has a structure of [insulin B chain]-L2-[insulin A chain] or [insulin A chain]-L2-[insulin B chain].

[0086] In certain embodiments, the peptide linker L2 is selected from a peptide linker consisting of one or more glycine (G) and / or serine (S).

[0087] In certain embodiments, the peptide linker L2 consists of 7-11 amino acids.

[0088] In certain embodiments, the peptide linker L2 comprises a sequence of GGSGGGG (SEQ ID NO: 72).

[0089] In certain embodiments, the peptide linker L2 comprises a sequence of (G)nGGSGGGG (SEQ ID NO: 74), n being an integer from 0 to 5.

[0090] In certain embodiments, the peptide linker L2 comprises a sequence of GGGGGSGGGG (SEQ ID NO: 73).

[0091] In certain embodiments of the fusion protein of any of the above aspects, the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 90-126 or a variant thereof having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, or having at least 90% (e.g., 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%) sequence identity.

[0092] In certain embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103-126, or a variant thereof having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to the sequence from which it is derived, or having at least 90% (e.g., 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%) sequence identity.

[0093] In certain embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103-124.

[0094] In certain embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103, 105-124.

[0095] In certain embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103, 114.

[0096] In certain embodiments of any of the above aspects of the fusion protein, the fusion protein can further comprise a tag, e.g., a purification tag, such as a His tag. In certain embodiments, the fusion protein comprises a His tag at its C-terminus. In certain exemplary embodiments, the fusion protein comprising a His tag comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 35-71.

[0097] Preparation of single-domain antibodies and fusion proteins

[0098] The single-domain antibodies and fusion proteins of the present application can be prepared by various methods known in the art, e.g., by genetic engineering recombinant technology. For example, a DNA molecule encoding the single-domain antibodies and fusion proteins is obtained by chemical synthesis or PCR amplification; the resulting DNA molecule is inserted into an expression vector, which is then transfected into host cells; then, the transfected host cells are cultured under specific conditions, and the single-domain antibodies or fusion proteins of the present application are expressed.

[0099] In another aspect, the present application provides an isolated nucleic acid molecule comprising (i) a nucleotide sequence encoding a single-domain antibody or antigen binding fragment thereof of the present application or (ii) a nucleotide sequence encoding a fusion protein of any aspect of the present application.

[0100] In another aspect, the present application provides a vector (e.g., an expression vector) comprising the above-mentioned isolated nucleic acid molecule.

[0101] In another aspect, the present application provides a host cell comprising a nucleic acid molecule or a vector as described above. 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.).

[0102] In another aspect, the present application provides a method of producing a single-domain antibody or an antigen-binding fragment or a fusion protein thereof of the present application, comprising culturing a host cell as described above under conditions permitting protein expression, and recovering said single-domain antibody or an antigen-binding fragment or a fusion protein thereof from the culture of the host cell.

[0103] Therapeutic applications

[0104] In another aspect, the present application provides a pharmaceutical composition comprising: a single-domain antibody or an antigen-binding fragment, conjugate, or fusion protein thereof of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0105] In certain embodiments, the pharmaceutical composition comprises a conjugate of the present application, said conjugate comprising a single-domain antibody or an antigen-binding fragment thereof of the present application and a therapeutic agent.

[0106] In certain embodiments, the pharmaceutical composition comprises a fusion protein of the present application.

[0107] In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0108] In certain embodiments, the additional therapeutic agent is selected from an anti- glycemic or anti-obesity agent.

[0109] In certain embodiments, the additional therapeutic agent is selected from a GLP-1 receptor agonist, a dipeptidyl peptidase 4 (DPP-IV) inhibitor, a native amylin or an analog thereof, a short-acting (prandial) INS analog, a native incretin or an analog thereof, a native insulin-like growth factor (IGF) or an analog thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, a statin, a sulfonylurea (SU), a thiazolidinedione (TZD).

[0110] The pharmaceutical compositions of the present application can be formulated in dosage forms compatible with their intended route of administration. One preferred dosage form is injection. Such injections can be sterile solutions. For example, sterile solutions of the single-domain antibodies or antigen-binding fragments, conjugates, or fusion proteins described herein can be prepared by incorporating the necessary dosage of the single-domain antibodies or antigen-binding fragments, conjugates, or fusion proteins in the appropriate solvent with optional simultaneous incorporation of other desired ingredients, including, but not limited to, pH adjusting agents, surfactants, adjuvants, ion strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof, followed by filtration to remove bacteria. In addition, sterile solutions of the single-domain antibodies or antigen-binding fragments, conjugates, or fusion proteins can be prepared as sterile lyophilized powders for storage and use (e.g., by vacuum or freeze-drying) to facilitate storage and use.

[0111] The pharmaceutical compositions of the present application can be administered by any suitable method known in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration. Parenteral administration refers to modes of administration other than enteral and topical administration, i.e., by injection, rather than by ingestion or topically, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and sternal injection, and infusion. Alternatively, administration can be via a non-parenteral route, such as topical, epidermal, or mucosal administration routes, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

[0112] In another aspect, the present application provides a method for treating a metabolic disease in a subject, comprising: administering to a subject in need thereof an effective amount of a fusion protein of the present application or a pharmaceutical composition comprising the fusion protein. Also provided is the use of a fusion protein of the present application or a pharmaceutical composition comprising the fusion protein in the manufacture of a medicament for treating a metabolic disease.

[0113] In certain embodiments, the metabolic disease is selected from hyperglycemia, diabetes (e.g., Type I diabetes and / or Type II diabetes), obesity, metabolic syndrome.

[0114] In certain embodiments, the subject is a mammal, e.g., a human.

[0115] In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent.

[0116] In certain embodiments, the additional therapeutic agent is selected from an antihyperglycemic agent or an antiobesity agent.

[0117] In certain embodiments, the additional therapeutic agent is selected from the group consisting of a GLP-1 receptor agonist, a dipeptidyl peptidase 4 (DPP-IV) inhibitor, a native amylin or an analog thereof, a short acting (prandial) INS analog, a native incretin or an analog thereof, a native insulin-like growth factor (IGF) or an analog thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, a statin, a sulfonylurea (SU), a thiazolidinedione (TZD).

[0118] The fusion protein or pharmaceutical composition of the present application can be formulated into any dosage forms known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended administration mode and therapeutic use. The fusion protein or pharmaceutical composition of the present application should be sterile and stable under the conditions of production and storage. A preferred dosage form is an injection.

[0119] The fusion protein or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, endocytic reticulum, inguinal, intravesical, local (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred administration route / way is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled person will understand that the administration route and / or way will vary depending on the intended purpose. In certain embodiments, the fusion protein or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0120] The fusion protein or pharmaceutical composition of the present application can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0121] Prolonged half-life applications

[0122] In another aspect, the present application provides the use of a single-domain antibody or antigen binding fragment thereof or conjugate of the present application for the manufacture of a medicament, wherein the medicament exhibits an extended in vivo half-life relative to a corresponding medicament lacking the single-domain antibody or antigen binding fragment thereof.

[0123] In certain embodiments, the medicament is a polypeptide or protein drug.

[0124] In certain embodiments, the subject is a mammal, e.g., a human.

[0125] In another aspect, the present application provides a method of extending the in vivo half-life of a drug, comprising: linking a single domain antibody or antigen binding fragment thereof of the present application to the drug.

[0126] In certain embodiments, the method comprises conjugating a single domain antibody or antigen binding fragment thereof of the present application to the drug to provide a conjugate.

[0127] In certain embodiments, the method comprises fusing a single domain antibody or antigen binding fragment thereof of the present application to the drug to provide a fusion protein.

[0128] In certain embodiments, the extension of the in vivo half-life is relative to the in vivo half-life of the drug in the absence of the single domain antibody or antigen binding fragment thereof.

[0129] In certain embodiments, the drug is a polypeptide or protein drug.

[0130] In certain embodiments, the subject is a mammal, e.g., a human.

[0131] Definitions of terms

[0132] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the viral, biochemical, immunological laboratory procedures described herein are in accordance with conventional techniques of the corresponding field. In addition, the following terms are defined and explained as follows for better understanding of the present application.

[0133] When the terms "for example," "for instance," "such as," "including," "containing," or "comprising" or variations thereof are used herein, these terms are not to be interpreted in an exclusionary or exhaustive sense, but rather are to be interpreted in an illustrative sense, meaning that the named item(s) is / are being exemplified.

[0134] Unless otherwise indicated, the terms "a" and "an" and "the" and similar referents are to be construed to cover both the singular and the plural unless otherwise indicated by context.

[0135] As used herein, the term "insulin" or "INS" refers to insulin obtained or derived from any species, such as a mammalian species, especially a human, wherein the native form is a heterodimeric peptide with two peptide chains (e.g., A chain and B chain) connected by two disulfide bonds, and wherein the A chain further has a single intramolecular disulfide bond. In humans, INS processing starts from preproinsulin, which is processed into proinsulin (comprising A chain, B chain and C peptide, with the structure B-C-A). Proinsulin is further processed, wherein the C peptide is cleaved to give INS. The sequences of the A chain and B chain of native human INS are both known to those skilled in the art, see various public databases, e.g., UniProt P01308-1.

[0136] As used herein, the term "insulin analogue" or "INS analogue" refers to a compound, e.g., a polypeptide, which activates the insulin receptor (IR) and elicits at least one in vivo or in vitro effect elicited by native INS, but which varies in some way in the amino acid sequence compared to native INS. In some embodiments, the INS analogue herein has one or more additions, deletions, insertions and / or substitutions compared to native INS, preferably binds to the IR with lower affinity.

[0137] As used herein, the term "ultra-long acting insulin" refers to an insulin which still has a significant glucose lowering effect 24 hours after subcutaneous injection in a diabetic patient.

[0138] As used herein, the term "in vivo half-life" has the meaning commonly known to those skilled in the art and can generally be defined as the time required for the serum concentration of a molecule to decrease by 50% in vivo, e.g., due to degradation of the ligand and / or clearance or sequestration of the ligand by natural mechanisms. Methods for determining in vivo half-life are well known to those skilled in the art, e.g., can be determined by pharmacokinetic analysis.

[0139] As used herein, the term "single-domain antibody" (sdAb) has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or shark antibody). A single-domain antibody is typically composed of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, so long as it substantially retains antigen binding and specificity. A single-domain antibody is also known as a nanobody, and the two are used interchangeably.

[0140] As used herein, the term "antigen binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind the same antigen to which the nanobody binds, and / or competes with the single-domain antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". In some embodiments, the "antigen binding fragment" of a single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, as compared to a full-length single-domain antibody, so long as it substantially retains antigen binding and specificity.

[0141] Antigen binding fragments of a single-domain antibody can be obtained from a given single-domain antibody (e.g., a single-domain antibody provided herein) using routine techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and screened for specificity in the same manner as for the intact single-domain antibody.

[0142] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in a Nanobody, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g. according to the definition in 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), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a skilled person will readily identify the CDRs as defined by the various numbering systems. Moreover, the correspondence between the different numbering systems is well known to the skilled person (see e.g. Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In the present text, the CDRs of a Nanobody are preferably determined by the Kabat numbering system.

[0143] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody that are not CDR residues as defined above.

[0144] As used herein, the term "humanized antibody" refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) from a human-derived immunoglobulin (acceptor antibody). In certain embodiments, a humanized antibody has CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) from a human-derived immunoglobulin (acceptor antibody). A humanized antibody typically retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a camelid antibody having the desired properties (e.g., antigen specificity, affinity, reactivity, etc.). To make a humanized antibody, the CDR regions of the immunized animal can be inserted into a human framework sequence using methods known in the art. In the context of a nanobody, a humanized antibody can refer to a humanized VHH, i.e., a VHH in which one or more of the framework regions have been substantially replaced by human framework regions. In some cases, certain framework regions (FRs) of a human immunoglobulin are replaced by the corresponding non-human residues. In addition, a humanized VHH can comprise residues that are found neither in the original VHH nor in the human framework sequences but are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide.

[0145] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between 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 purposes (e.g., introduction of gaps to achieve the best comparison). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions x 100%). In certain embodiments, the two sequences are the same length.

[0146] The determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0147] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (K D ) of the interaction. In the present application, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0148] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. Both the "association rate constant" (kaor kon) and the "dissociation rate constant" (kdisor koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187). The ratio of kdis / kon is equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). K D , konand kdisvalues can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore. In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0149] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector that is capable of mediating the expression of an inserted polynucleotide is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell takes up and expresses the genetic material elements carried by the vector. 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) or P1 -derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0150] 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 E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0151] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising 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 substitutions of an amino acid residue for an amino acid residue with similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution is one in which the replacement amino acid residue has an atom or a group of atoms that is similar in size, shape, charge, chemical properties, etc. to the atom or group of atoms of the original amino acid residue. Methods for identifying amino acid conservative 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).

[0152] The nomenclature used herein to refer to the twenty conventional amino acids follows conventional usage. See, e.g., 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 the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the single and three letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0153] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in pharmacology and / or physiology, with the subject and the active ingredient, which are well known in the art, and include but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include but are not limited to phosphate buffer. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Agents to maintain osmotic pressure include but are not limited to sugars, NaCl and the like. Agents to delay absorption include but are not limited to monostearate and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol) and the like. Preservatives include but are not limited to various antibacterial agents and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid and the like.

[0154] Advantages of the invention

[0155] The present invention provides a fusion protein formed by an insulin analogue and a single-domain antibody that specifically binds to human serum albumin (HSA). The insulin analogue in the fusion protein can be formed by recombinant synthesis into a single-chain polypeptide, is biologically active without endoprotease hydrolysis processing, and has reduced affinity to insulin receptor (IR) compared to native human INS, while the affinity (or function) to insulin-like growth factor (IGF-1R) is also significantly reduced, increasing the selectivity to IR and reducing the receptor-mediated clearance rate. The single-domain antibody in the fusion protein not only binds to albumin in human serum, but also extends the half-life of the drug through FcRn-mediated recycling, and also masks the insulin analogue to varying degrees, thereby having adjustable pharmacodynamics of the INS analogue. Thus, the present invention provides an ultra-long-acting insulin that has significantly prolonged duration of action, longer in vivo half-life, and better in vivo efficacy compared to the marketed degludec or other insulin derivatives. Meanwhile, compared with the Icodec that has been applied for marketing, the fusion protein of the present invention also exhibits longer drug duration and drug stability at the same dosage.

[0156] In addition, the present invention also provides a single-domain antibody that specifically binds to human serum albumin and has cross-binding properties to monkey, mouse and rat serum albumin, providing convenience for pharmacodynamics, pharmacokinetics and toxicology research, and making it easier to translate from these species to humans or other species listed above.

[0157] Embodiments of the present application will be described in detail below with reference to the attached drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not a limitation on the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0158] Figure 1 shows a structural schematic diagram of an INS analogue.

[0159] Figure 2 shows the affinity detection results of camel and humanized single domain antibodies.

[0160] Figures 3A-3C show the in vitro binding activity detection results of INS analogue 1-37 to IR-B.

[0161] Figure 4 shows the in vitro binding activity detection results of INS analogue 3 / 6 / 14 / 25 to IGF1R.

[0162] Figure 5 shows the hypoglycemic ability test results of INS analogue 3 / 6 in streptozotocin (STZ) treated rats.

[0163] Figures 6A-6C show the hypoglycemic ability test results of INS analogue 6 / 14 in streptozotocin (STZ) treated rats.

[0164] Figure 7 shows the hypoglycemic ability test results of INS analogue 14 / 25 / 36 / 37 in streptozotocin (STZ) treated rats.

[0165] Figure 8 shows the hypoglycemic ability test results of INS analogue 14 / 15 / 19 / 22 / 25 in streptozotocin (STZ) treated rats.

[0166] Figures 9A-9B show the hypoglycemic ability test results of INS analogue 25 and control molecules in streptozotocin (STZ) treated rats.

[0167] Figures 10A-10B show the pharmacokinetic test results of INS analogue 14 / 25 in wt rats.

[0168] SEQUENCE INFORMATION

[0169] The information of part of the sequences involved in the present application is provided in the following table.

[0170] Table 1 DETAILED DESCRIPTION

[0171] The present application will now be described with reference to the following examples, which are intended to be illustrative only and not limiting of the present application.

[0172] It is to be understood that the examples are described herein in terms of specific embodiments thereof and that no limitation is intended to the scope of protection sought for this application. Experimental methods in the examples are carried out in accordance with conventional methods unless otherwise specified. Where specific conditions are not specified, conditions are carried out under conventional conditions or as suggested by the manufacturer. Where the manufacturer of reagents or instruments is not indicated, conventional products available from commercial vendors are used.

[0173] Example 1: Obtaining of HSA single domain antibody

[0174] 1.1 Preparation of camelid single domain antibody

[0175] A llama was immunized with human serum albumin (HSA) antigen (Acro, HSA-H522a) (the immunization was completed by Shanxi Namibiosci Biotech Development Co., Ltd.). The peripheral blood of the llama was extracted, and lymphocytes were obtained by using lymphocyte separation medium. Total RNA was extracted, and total cDNA of llama lymphocytes was obtained by reverse transcription. Then, a nanobody phage library was constructed. Monoclonal antibodies specifically binding to HSA were obtained by affinity screening, and the VHH amino acid sequence was obtained by sequencing. The CDR region sequence was determined according to the Kabat numbering system. The VHH sequence of the finally obtained single domain antibody is shown as SEQ ID NO: 1, and the sequences of CDR1-CDR3 are shown as SEQ ID NO: 5, 7, and 8, respectively.

[0176] 1.2 Preparation of humanized single domain antibody

[0177] The camelid single domain antibody was humanized by using a general CDR grafting method, and three humanized single domain antibodies were obtained, the sequences of which are summarized in the following table.

[0178] Table 2: Sequences of humanized single domain antibodies

[0179] 1.3 Activity detection of camelid and humanized single domain antibodies

[0180] The affinity detection results of the camel and humanized single-domain antibodies to human serum albumin (HAS), cynomolgus serum albumin (CSA), rat serum albumin (RSA) and mouse serum albumin (MSA) are shown in the following table. The results show that the camel and humanized single-domain antibodies have good binding activity to human serum albumin, and also have good cross-binding activity to cynomolgus serum albumin, rat serum albumin and mouse serum albumin.

[0181] Table 3-1: Affinity detection results

[0182] Table 3-2: Affinity detection results

[0183] Table 3-3: Affinity detection results

[0184] Example 2: Preparation of INS analogs

[0185] 2.1 Sequence design of INS analogs

[0186] The structural schematic diagram of the INS analogs is shown in Figure 1, which comprises B-L2-A-L1-VHH from N-terminal to C-terminal, B is an insulin B chain, A is an insulin A chain, L1 and L2 are both peptide linkers, and VHH is the HSA single-domain antibody prepared in Example 1. The specific structures of each INS analog are shown in the following table.

[0187] Table 4: Structures and sequences of INS analogs

[0188] 2.2 CHO expression of INS analogs

[0189] The above INS analogs were produced in a mammalian cell expression system by using Expi-CHO. The cDNA sequence encoding the amino acid sequence of each of the above INS analogs was subcloned into a pCDNA3.4 expression plasmid. Each of the above INS analogs was further linked to a His tag at its C-terminal, and the amino acid sequence of each of the INS analogs containing the His tag is shown in SEQ ID NOs: 35-71, respectively. In the following examples, the INS analogs all contain the His tag unless otherwise specified. The cDNA sequence was in-frame fused with the coding sequence of the signal peptide sequence MHSSALLCCLVLLTGVRA (SEQ ID NO: 90) to enhance the secretion of the INS analog into the tissue culture medium.

[0190] CHO cells were transfected by electroporation with appropriate amount of recombinant expression plasmid. After electroporation, cells in cuvettes were aliquoted into previously prepared flasks containing culture medium. After 40 minutes incubation, flasks were put into 37°C, 120rpm, 8% CO2 incubator. After 24 hours, feed / butyric acid sodium / dual antibody were added and cells were cultured for another 4 days.

[0191] The supernatant of INS analogues (INS-VHH-his) secreted by cell culture was purified by Ni affinity chromatography column. The column was equilibrated with appropriate volume of 1xPBS. After equilibration, the sample was loaded at a flow rate of 1ml / min. After loading, the column was washed with appropriate volume at a flow rate of 1ml / min. Then the column was eluted with 5mM imidazole at a flow rate of 1ml / min at pH 8.0. Subsequently, the sample was eluted with 150mM imidazole at a flow rate of 1ml / min at pH 8.0. The collected sample was determined for protein concentration by NanoDrop. Finally, the high concentration protein was absorbed into dialysis bag and dialyzed in a beaker containing 1xPBS. The final sample was subjected to a series of quality control tests including SDS-PAGE, SEC and endotoxin assay.

[0192] 2.3 Pichia pastoris expression of INS analogues

[0193] The above INS analogues were produced by using Pichia pastoris in eukaryotic expression system. The cDNA sequences encoding the amino acid sequences of INS analogues 3, 6, 14, 25 were subcloned into pZaA yeast expression plasmid.

[0194] Pichia pastoris competent cells were transfected by electroporation with appropriate amount of recombinant expression plasmid. After electroporation, 0.6ml of 1M sorbitol was added and transferred to 1.5ml centrifuge tube. The tube was incubated at 30°C for 1 hour. Then 0.5ml of YPD medium was added to the incubated tube. After shaking culture at 30°C, 225rpm for 1.5h, the sample was diluted and plated on YPDS, 600ug / ml zeocin plate for selective culture for 48h. After two days, single colonies were picked from the plate and cultured in YPD medium for 48h. Appropriate amount of bacteria solution was transferred to BMGY medium and continued to shake culture for 24h. After BMGY culture, appropriate amount of bacteria solution was centrifuged, resuspended and transferred to BMMY medium for induction expression. 1% methanol was added every day for continuous shaking culture (1-4 days).

[0195] The supernatant of the INS analogues secreted by the yeast culture was purified by Ni affinity chromatography column. The chromatography column was equilibrated with an appropriate volume of 1xPBS, and after equilibration, the sample was loaded at a flow rate of 1 ml / min. After loading, an appropriate volume was used for the initial wash, and then the impurities were eluted at a pH of 7.4, a flow rate of 1 ml / min, and 5 mM imidazole. Subsequently, the sample was eluted at a flow rate of 1 ml / min with 250 mM and 500 mM imidazole, pH 7.4. The collected sample was determined for protein concentration using a NanoDrop. Finally, the target protein was concentrated by a 10K ultrafiltration tube and exchanged into 1xPBS. The final sample was subjected to SDS-PAGE, SEC, and a series of quality control tests.

[0196] The results of the yeast expression test are shown in Figure 2. It can be found that INS analogues 3 / 6 / 14 can be expressed in Pichia pastoris and the target protein with good purity can be obtained, which can greatly reduce the production cost.

[0197] Example 3: Albumin binding assay of INS analogues

[0198] The in vitro binding of INS analogues to human, cynomolgus monkey, mouse and rat serum albumin was determined by Octet. The binding of INS analogues 3, 6, 14, 25 to serum albumin of each species was detected on a ForteBio Octet HTX instrument. Bio-HSA / bio-CSA / bio-RSA / bio-MSA was configured in running buffer at a concentration of 5 ug / ml, and the ligand was immobilized on the SA probe according to the process of 60s baseline / 200s loading / 60s baseline / 120s association / 180s disscociation / 5s regeneration. The obtained sensorgram was analyzed by 1:1 binding kinetics or steady-state affinity model fitting analysis to calculate the equilibrium dissociation constant (KD).

[0199] The results are shown in Table 5. The tested INS analogues have good affinity to human, cynomolgus monkey, mouse and rat serum albumin.

[0200] Table 5: Binding kinetics of INS analogues 3, 6, 14, 25 to human, cynomolgus monkey, rat and mouse serum albumin at 25°C

[0201] Example 4: In vitro activity detection of INS analogues on IR-B report cell line

[0202] Preparation of IR-B reporter gene cell line: The insulin report cell line (Genomeditech, GM-C26180, which is a reporter gene cell line constructed by Genomeditech. When INS or INS analogs bind to INR-B, it can activate the downstream signaling pathway, thereby activating the expression of luciferase. The luminescence intensity is detected by luciferase substrate, so as to judge the strength of the action of INS or INS analogs with INR-B) was resuscitated and passaged. The cells were digested the night before the functional experiment, and then plated in a 96-well white transparent bottom microplate (Thermo, 165306) at a density of 100ul, 4x10^4 / well. The plate was incubated in a 37℃, 5% CO2 incubator overnight.

[0203] Receptor binding function assay scheme: INS analogs 1-37 were added at concentrations of 10000nM, 4000nM, 1600nM, 640nM, 256nM, 102.4nM, 40.96nM and 16.384nM. The cells after adding sample were placed in a 37℃, 5% CO2 incubator for continued incubation for 6 hours. After 6 hours, 100ul of firefly luciferase detection reagent kit (ONE-Glo TM Luciferase Assay System, Promega, E6120) was added to each well and incubated at 25℃ for 5 minutes. The detection was performed on a multifunctional enzyme label instrument (Perkin Elmer, Envision), and the values were recorded. The control sample insulin was used for activity determination at concentrations of 100nM, 40nM, 16nM, 6.4nM, 2.56nM, 1.024nM, 0.41nM and 0.164nM. For the masking test experiment, different concentrations of HSA (final concentration 5uM / 50uM or 250uM) were mixed with the INS analog sample and added to the test cells for determination. All tests were performed in duplicate. The obtained data were analyzed and curve fitted by Prism9.0 software.

[0204] The results are shown in Figures 3A-3C. Compared with native INS, the affinity of INS analogs for insulin receptor is reduced, thereby reducing the rate of receptor-mediated clearance.

[0205] Example 5: In vitro functional detection of INS analogs on IGF1R

[0206] Preparation of IGF1R overexpression cell line: The expanded HEK293T cells (obtained from the Chinese Academy of Sciences Typical Culture Collection Committee Cell Bank) were trypsinized (Thermo, TrypLE TMDigestion was performed in 96-well plates using Express Enzyme (1X), phenol red, 12605010) and the digested cells were adjusted to a density of 6x10^5 / mL, and the treated cells were added to the pre-coated 96-well plates (100ul / well 0.01mg / ml Poly-D-lysine hydrobromide incubated at 37℃ for 2 hours) at an amount of 100ul per well, and incubated overnight at 37℃, 5% CO2.

[0207] Binding function assay protocol: INS analogs 3 / 6 / 14 / 25 and control Insulin (MCE, HY-P0035) and control Icodec (WO2012123519A2, control compound synthesized by Pharming) were added at concentrations of 10000nM, 4000nM, 1600nM, 640nM, 256nM, 102.4nM, 40.96nM and 16.384nM, and control IGF1 was added at concentrations of 100nM, 40nM, 16nM, 6.4nM, 2.56nM, 1.024nM, 0.41nM and 0.164nM, and the added cells were incubated at 37℃, 5% CO2 for 20 minutes, and then removed for subsequent experiments according to the operating instructions of the p-AKT HTRF assay kit (Cisbio, pan-Akt (Phospho-Ser473) TR-FRET Assay Kit, 64AKSPEH). The obtained data were analyzed and curve-fitted by Prism 9.0 software.

[0208] The results are shown in Figure 4. Compared with native INS, the affinity of the INS analogs for insulin-like growth factor 1 receptor (IGF-1R) was significantly reduced, thereby not only increasing the selectivity for IR but also reducing the receptor-mediated clearance rate.

[0209] Example 6: Test of the hypoglycemic ability of INS analogs in streptozotocin (STZ)-treated rats

[0210] STZ rats: SPF level male rats of Sprague Dawley (SD) (obtained from Sibeifu (Beijing) Biotechnology Co., Ltd.) were selected for this experiment, with a body weight of 280-320g, and all animals were purchased for at least 5 days of adaptive feeding. The rats were raised in polycarbonate boxes, with a maximum of 4 animals of the same sex in the same group per cage. After grouping and dosing, a maximum of 3 animals of the same sex in the same group were raised per cage. The temperature was controlled at 20-26℃, the humidity was controlled at 40-70%, and the light was alternated for 12 hours.

[0211] STZ modeling: SPF male SD rats were fasted without water restriction at 17:00 the day before injection, and injection was started at 08:30 the next day. The normal control group was not operated, and the rest of the animals were used for the model group and the test group, and STZ was injected. The STZ solution needs to be prepared immediately, and it needs to be transported and stored in low temperature and light after being weighed and dried. The injection should be completed within 15 minutes after dissolution (1g / bottle of STZ is weighed as 6 tubes, and it is dissolved and injected in batches. Each tube is prepared as a 10mg / mL solution according to the corresponding STZ weight, and the whole process is stored in ice under light protection). Each rat was injected with 10mg / mL STZ solution in the abdominal cavity, the injection dose was 65mg / kg, and the injection volume was 0.65mL / 100g. After injection, the rats were allowed to eat freely until the end of the experiment, and 5% glucose was given at the same time. The next morning from 9:00 to 10:00, normal drinking water was changed.

[0212] Non-fasting blood glucose was detected at 9:00-11:00 on the third day after injection of STZ, and animals with blood glucose of 300-550mg / dL (16.7mmol / L-30.6mmol / L) were selected for grouping. According to the blood glucose and body weight, the animals were divided into groups and administered drugs according to the grouping. The administration day was D0. All rats were given subcutaneous injection on the nape of the neck, and the administration volume of each group was 0.5mL / animal. The administration amount of each animal was determined according to the latest body weight measured before administration, and the administration time was recorded as 0h.

[0213] Blood glucose monitoring: Before administration, non-fasting blood glucose was detected at 4h, 8h, 12h, 18h, D1, D2, D3, D4, D5, D6, D7, D8, D9 after administration. Except for 4h, 8h, 12h, 18h, non-fasting blood glucose was detected at 9:00-11:00 on the same day. When detecting blood glucose, the rat tail was disinfected, and after the alcohol evaporated, a disposable blood collection needle was used to prick the tail tip, a cotton ball was used to wipe off the first drop of blood, and the second drop of blood was used for blood glucose monitoring. After measurement, the rats were returned to the cage. On D9, the blood glucose curve was drawn using GraphPad Prism, and the area under the curve AUC was calculated.

[0214] Body weight monitoring: All rats were weighed (non-fasting body weight) on D0 (fasting body weight), D4, D7 during the administration grouping. Each time, the blood glucose was measured first, and then the weight was measured.

[0215] Data processing and statistical analysis: The effective digits of the data in this experiment were rounded off, and the statistical and plotting software was GraphPad Prism 8. The measurement data was represented by mean ± standard deviation (Mean ± SD), and the statistical analysis was performed by one-way ANOVA.

[0216] As shown in Figure 5, after a single injection, both INS analogue 3 and INS analogue 6 showed hypoglycemic effect compared with the model group, in which INS analogue 3 reached the lowest hypoglycemic effect at D2 and maintained a stable period for one day, and then the blood glucose began to rise from D3 and returned to the initial level at D6. At the same dosage (250 nmol / rat), INS analogue 6 showed stronger hypoglycemic effect than INS analogue 3, and the blood glucose was stable during D1-D5, and then rapidly rose after D5 and returned to the pre-administration level at D9. Compared with the control group (control 1 and control 2 have the same structure as the INS analogue, but the VHH sequence is different from that in the INS analogue), both INS analogue 3 and INS analogue 6 showed good hypoglycemic effect.

[0217] Example 7: Test of hypoglycemic ability of humanized INS analogue in streptozotocin (STZ)-treated rats

[0218] The steps used were the same as in Example 6.

[0219] As shown in Figures 6A-6C, after a single injection, there was no significant difference in the effect of INS analogue 6 and INS analogue 14 at different dosages (120 nmol / rat, 200 nmol / rat and 280 nmol / rat), but at high dosages (200 nmol / rat and 280 nmol / rat), the duration of stable blood glucose was extended from D2-D3 at low dosage (120 nmol / rat) to D2-D5. Regardless of INS analogue 6 or INS analogue 14, the duration of stable blood glucose was significantly longer than that of Icodec at the same dosage (120 nmol / rat), and the blood glucose of the Icodec administration group returned to the pre-administration level at D4, while the blood glucose of INS analogue 6 and INS analogue 14 returned to the pre-administration level at D6 after administration.

[0220] Example 8: Test of hypoglycemic ability of optimized INS analogue in streptozotocin (STZ)-treated rats

[0221] The steps used were the same as in Example 6, in which the blood glucose was monitored at D0, 4h, 8h after administration, D1, D2, D3, D4, D5, D6, D7, D8, D9, and the non-fasting blood glucose was detected at 9:00-11:00 at all time points except 4h and 8h.

[0222] Results are shown in Figure 7. All samples showed good blood glucose lowering effect, and the high dose group showed more obvious blood glucose lowering effect. INS analogues 14 and 25 showed better blood glucose lowering intensity than the other samples at the same dose, but INS analogue 25 showed the best effect in terms of stability and effective duration at a dose of 120 nmol / rat.

[0223] Example 9: Test of blood glucose lowering ability of INS analogues in streptozotocin (STZ)-treated rats

[0224] The steps used were the same as in Example 6, and blood glucose was monitored at D0, 4h, 8h after administration, D1, D2, D3, D4, D5, D6, D7, and non-fasted blood glucose was measured at 9:00-11:00 at all time points except 4h and 8h.

[0225] Results are shown in Figure 8. All samples showed good blood glucose lowering effect at the same dose (120 nmol / rat), and all samples showed good stability at D1-D4 except INS analogue 15. INS analogue 25 showed the best blood glucose recovery rate at D4-D7.

[0226] Example 10: Test of blood glucose lowering ability of INS analogues and control samples in streptozotocin (STZ)-treated rats

[0227] The steps used were the same as in Example 6, and blood glucose was monitored at D0, 2h, 4h, 6h, 8h, 10h, 12h after administration, D1, D2, D3, D4, D5, D6, D7, and non-fasted blood glucose was measured at 9:00-11:00 at all time points except 4h and 8h.

[0228] Results are shown in Figures 9A and 9B. The control molecule (INS analogue 38, SEQ ID NO: 127, see CN114174348A) significantly lowered blood glucose in rats at 2h after administration at a low dose (80 nmol / rat), and significantly lowered blood glucose in rats at 4h after administration at a high dose (120 nmol / rat), and the dose-effect relationship was not obvious. The candidate molecule INS analogue 25 (no his-tag, SEQ ID NO: 114) significantly lowered blood glucose in rats at 2h after administration, and showed a certain dose-effect relationship. The effect of the high dose group (120 nm / rat) was comparable to that of the control molecule, and blood glucose gradually recovered from the fourth day.

[0229] Example 11: Test of pharmacokinetics of INS analogues in wt rats

[0230] Experimental animal SD rats dosing: 7-8 weeks old male wild type SD rats were provided by Vantianlihua, all animals were given normal diet and water before dosing, rats with similar body weight and age were randomly divided into groups, weighed 0.5-1 h before dosing, and the dosing amount was calculated according to the body weight. Subcutaneous injection, dosing according to the dose of 1.05 mg / kg, 2.1 mg / kg and 4.2 mg / kg respectively. After dosing, samples were taken at 0 h (30 min before dosing); 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240 h after dosing.

[0231] PK detection was performed on samples of different dosing amounts. The anti-his antibody was coated at 4 degrees overnight, and the next day the plate was washed after blocking with blocking solution. After blocking, 3200 / 1280 / 640 / 320 / 160 / 80 / 32 ng / ml standard and samples taken at different time points were added, and incubated at room temperature for 2 h, then secondary antibody was added, incubated at room temperature for 0.5 h, then the plate was washed, developed and read. According to the PK detection results, the PK parameters including (if data allows) but not limited to peak concentration (Cmax), peak time (Tmax), elimination half-life (T1 / 2), serum concentration-time area under the curve (AUC), mean residence time (MRT) and the like were calculated and analyzed.

[0232] Table 6: PK detection results of INS analogue 14

[0233] Table 7: PK detection results of INS analogue 25

[0234] The results are shown in Tables 6-7, Figures 10A and 10B. According to the PK detection results, both molecules INS analogue-14 (SEQ ID NO: 48) and INS analogue-25 (SEQ ID NO: 59) showed good dose linearity, among which molecule INS analogue-14 reached Cmax at 24 h, while INS analogue-25 showed that most animals reached peak at 48 h, and then showed slow elimination. The half-life of INS analogue-14 molecule was about 30 h, while the half-life of INS analogue-25 was in the range of 40-50 h.

[0235] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present application. The entire disclosure of the present application is given by the appended claims and any equivalents thereof

Claims

1. A single-domain antibody or antigen-binding fragment thereof that specifically binds to human serum albumin (HSA), comprising: a CDR1 set forth in GYSYSSQYMX1 (SEQ ID NO: 10), a CDR2 set forth in AISATGVYTYYADSVKG (SEQ ID NO: 7), and a CDR3 set forth in PLPRTSPAX2PLX3VYEYEY (SEQ ID NO: 11); wherein, X1 is selected from G or S, X2 is selected from N or F, and X3 is selected from G or M.

2. The single-domain antibody or antigen-binding fragment thereof of claim 1, comprising: a CDR1 comprising the sequence set forth in SEQ ID NO: 5 or 6, a CDR2 comprising the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the sequence set forth in SEQ ID NO: 8 or 9.

3. The single-domain antibody or antigen-binding fragment thereof of claim 1 or 2, comprising: (1) a CDR1 set forth in SEQ ID NO: 5, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO: 8; (2) a CDR1 set forth in SEQ ID NO: 6, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO: 8; (3) a CDR1 set forth in SEQ ID NO: 5, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO: 9; or (4) a CDR1 set forth in SEQ ID NO: 6, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO:

9.

4. The single-domain antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein, the single-domain antibody comprises a VHH comprising the sequence set forth in SEQ ID NO: 1 or a variant thereof having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to the sequence from which it is derived, or having at least 80% sequence identity; Preferably, the single-domain antibody comprises a VHH comprising the sequence set forth in SEQ ID NO:

1.

5. The single-domain antibody or antigen-binding fragment thereof of any one of claims 1-3, which is humanized; Preferably, the single-domain antibody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin, which heavy chain framework region optionally comprises a back-mutation from a human-derived residue to a camelid-derived residue; Preferably, the single-domain antibody comprises a VHH comprising the sequence set forth in any one of SEQ ID NOs: 2-4 or a variant thereof having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to the sequence from which it is derived, or having at least 80% sequence identity; Preferably, the single-domain antibody comprises a VHH comprising the sequence set forth in any one of SEQ ID NOs: 2-4.

6. The single-domain antibody or antigen-binding fragment thereof of any one of claims 1-5, which further binds to monkey serum albumin, mouse serum albumin, and / or rat serum albumin.

7. A conjugate comprising the single-domain antibody or antigen-binding fragment thereof of any one of claims 1-6 and a further biologically active molecule; Preferably, the further biologically active molecule is optionally linked to the N- and / or C-terminus of the single-domain antibody or antigen-binding fragment thereof via a linker; Preferably, the further biologically active molecule is a therapeutic agent.

8. A fusion protein comprising an insulin active domain and an antigen binding domain targeting human serum albumin (HSA), wherein, the insulin active domain comprises an insulin A chain and an insulin B chain; the antigen binding domain targeting human serum albumin (HSA) is selected from the single domain antibody or antigen binding fragment thereof of any one of claims 1-6.

9. The fusion protein of claim 8, wherein: the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or a C-terminal truncation thereof comprising a deletion of 1-4 amino acids at the C-terminus; wherein X1 is selected from Y, E, G, A, V, L, I, D, N, K, R, Q, S or T.

10. The fusion protein of claim 9, wherein, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); wherein X1 is selected from Y, E, G, A, V, L, I, D, N, K, R, Q, S or T; preferably, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); preferably, X1 is selected from Y or A; preferably, the insulin B chain comprises a sequence represented by any one of SEQ ID NOs: 12-27; preferably, the insulin B chain comprises a sequence represented by SEQ ID NO: 12 or 17.

11. A fusion protein comprising an insulin active domain and a single-domain antibody or antigen-binding fragment thereof targeting human serum albumin (HSA), wherein: the insulin active domain comprises an insulin A chain and an insulin B chain, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or a C-terminal truncation thereof comprising a deletion of 1-4 amino acids at the C-terminus; wherein X1 is selected from G, A, V, L, I, D, N, K, Q, S or T.

12. The fusion protein of claim 11, wherein, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHYTPKT (SEQ ID NO: 28) or an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); wherein X1 is selected from G, A, V, L, I, D, N, K, Q, S or T; preferably, the insulin B chain comprises an amino acid sequence represented by FVNQHLCGSHLVEALX1LVCGERGFHY (SEQ ID NO: 29); preferably, X1 is selected from A; preferably, the insulin B chain comprises a sequence represented by any one of SEQ ID NOs: 16-23, 25-27; preferably, the insulin B chain comprises a sequence represented by SEQ ID NO:

17.

13. The fusion protein of any one of claims 8-12, wherein, the insulin A chain comprises an amino acid sequence represented by GIVEQCCTSZ1CSLZ2QLENYCZ3 (SEQ ID NO: 34), wherein Z1 is selected from T or I; Z2 is selected from D or Y; and Z3 is G or null; Preferably, Z1 is T and Z2 is D; or, Z1 is T and Z2 is Y; or, Z1 is I and Z2 is D; Preferably, Z3 is G. Preferably, the insulin A chain comprises a sequence represented by any one of SEQ ID NOs: 30-33. Preferably, the insulin A chain comprises a sequence represented by SEQ ID NO:

30.

14. The fusion protein of any one of claims 8-13, wherein, Preferably, the insulin active domain is directly connected to the N-terminus and / or C-terminus of the single-domain antibody or antigen-binding fragment thereof, such as the N-terminus. Preferably, the peptide linker L1 is selected from an amino acid sequence represented by Gn (SEQ ID NO: 86), (GGGGQ)n(G)m (SEQ ID NO: 87), (GGGGS)n(G)m (SEQ ID NO: 88), wherein n is an integer selected from 0-5, and m is an integer selected from 0-5. Preferably, the peptide linker L1 is Gn (SEQ ID NO: 86), and n is 0, 1, 2, 3, or 4; preferably, the peptide linker L1 is selected from a sequence represented by any one of SEQ ID NOs: 76-78. Preferably, the peptide linker L1 is (GGGGQ)n(G)m (SEQ ID NO: 87), wherein n is 1, 2, 3, or 4, and m is an integer selected from 1-5, such as m is 2 or 5; preferably, the peptide linker L1 is selected from a sequence represented by any one of SEQ ID NOs: 75, 80-85. Preferably, the peptide linker L1 is (GGGGS)n(G)m (SEQ ID NO: 88), wherein n is 1, 2, or 3, and m is an integer selected from 1-5, such as m is 5; preferably, the peptide linker L1 is selected from a sequence represented by SEQ ID NO:

79.

15. The fusion protein of claim 14, wherein, Preferably, the insulin active domain is directly connected to the N-terminus and / or C-terminus of the single-domain antibody or antigen-binding fragment thereof, such as the N-terminus.

16. The fusion protein of any one of claims 8-15, wherein, Preferably, the insulin active domain is a single-chain polypeptide formed by an insulin A chain and an insulin B chain connected by a peptide linker L2. Preferably, the insulin active domain has a structure represented by [insulin B chain]-L2-[insulin A chain] or [insulin A chain]-L2-[insulin B chain]. Preferably, the peptide linker L2 is selected from a peptide linker consisting of one or more glycine (G) and / or serine (S). Preferably, the peptide linker L2 consists of 7-11 amino acids. Preferably, the peptide linker L2 comprises a sequence represented by GGSGGGG (SEQ ID NO: 72). Preferably, the peptide linker L2 comprises a sequence represented by (G)nGGSGGGG (SEQ ID NO: 74), wherein n is an integer selected from 0-5. Preferably, the peptide linker L2 comprises a sequence represented by GGGGGSGGGG (SEQ ID NO: 73).

17. The fusion protein of any one of claims 8-16, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 90-126 or a variant thereof having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to the sequence from which it is derived, or having at least 90% sequence identity; Preferably, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103-126 or a variant thereof having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to the sequence from which it is derived, or having at least 90% sequence identity; Preferably, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103-124; Preferably, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103, 105-124; Preferably, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 95, 103, 114.

18. An isolated nucleic acid molecule encoding (i) the single-domain antibody or antigen binding fragment thereof of any one of claims 1-6; or (ii) the fusion protein of any one of claims 8-17.

19. A vector comprising the nucleic acid molecule of claim 18.

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

21. A method of making the single-domain antibody or antigen binding fragment thereof of any one of claims 1-6 or the fusion protein of any one of claims 8-17, comprising culturing the host cell of claim 20 under conditions permitting protein expression, and recovering the single-domain antibody or antigen binding fragment thereof or fusion protein from the cultured host cell culture.

22. A pharmaceutical composition comprising: the single-domain antibody or antigen binding fragment thereof of any one of claims 1-6, the conjugate of claim 7, or the fusion protein of any one of claims 8-17, and a pharmaceutically acceptable carrier and / or excipient.

23. The pharmaceutical composition of claim 22, further comprising an additional therapeutic agent; Preferably, the additional therapeutic agent is selected from an anti-glycemic agent or an anti-obesity agent; Preferably, the additional therapeutic agent is selected from a GLP-1 receptor agonist, a dipeptidyl peptidase 4 (DPP-IV) inhibitor, a native amylin or an analog thereof, a short-acting (prandial) INS analog, a native incretin or an analog thereof, a native insulin-like growth factor (IGF) or an analog thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, a statin, a sulfonylurea (SU), a thiazolidinedione (TZD).

24. Use of the fusion protein of any one of claims 8-17 or a pharmaceutical composition comprising the fusion protein in the manufacture of a medicament for the treatment of a metabolic disease. Preferably, the metabolic disease is selected from hyperglycemia, diabetes, obesity, metabolic syndrome; Preferably, the fusion protein is administered in combination with an additional therapeutic agent; Preferably, the additional therapeutic agent is selected from an anti-hyperglycemic agent or an anti-obesity agent; Preferably, the additional therapeutic agent is selected from a GLP-1 receptor agonist, a dipeptidyl peptidase 4 (DPP-IV) inhibitor, a native amylin or an analog thereof, a short acting (prandial) INS analog, a native incretin or an analog thereof, a native insulin-like growth factor (IGF) or an analog thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, a statin, a sulfonylurea (SU), a thiazolidinedione (TZD).

25. A method for treating a metabolic disease in a subject comprising: administering to a subject in need thereof an effective amount of the fusion protein or a pharmaceutical composition comprising the fusion protein of any one of claims 8-17; Preferably, the metabolic disease is selected from hyperglycemia, diabetes, obesity, metabolic syndrome; Preferably, the fusion protein is administered in combination with an additional therapeutic agent; Preferably, the additional therapeutic agent is selected from an anti-hyperglycemic agent or an anti-obesity agent; Preferably, the additional therapeutic agent is selected from a GLP-1 receptor agonist, a dipeptidyl peptidase 4 (DPP-IV) inhibitor, a native amylin or an analog thereof, a short acting (prandial) INS analog, a native incretin or an analog thereof, a native insulin-like growth factor (IGF) or an analog thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, a statin, a sulfonylurea (SU), a thiazolidinedione (TZD).

26. Use of a single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or a conjugate according to claim 7 or a fusion protein according to any one of claims 8 to 17 for the manufacture of a medicament, wherein, The drug exhibits an extended in vivo half-life relative to a corresponding drug lacking the single-domain antibody or antigen-binding fragment thereof; Preferably, the drug is a polypeptide or a protein drug; Preferably, the subject is a mammal, e.g., a human.

27. A method of extending the half-life of a drug in vivo comprising: The single-domain antibody or antigen-binding fragment thereof of any one of claims 1-6 is linked to the drug; Preferably, the extension of the in vivo half-life is relative to the in vivo half-life of the drug lacking the single-domain antibody or antigen-binding fragment thereof; Preferably, the drug is a polypeptide or a protein drug; Preferably, the subject is a mammal, e.g., a human.

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