Monoclonal insulin and its Fc conjugate

Abstract

This specification provides an insulin-Fc fusion polypeptide comprising a single-chain insulin and an Fc region peptide. Further provided is a conjugate of two insulin-Fc fusion polypeptides, wherein the two insulin-Fc fusion polypeptides are linked by at least one covalent bond at the C-terminus of the fusion polypeptide. Also provided is a single-chain insulin with reduced binding affinity for the insulin receptor. Further provided is the use of the insulin-Fc fusion polypeptide, conjugate, or single-chain insulin in medicine.

Description

Technical Field

[0001] This specification provides an insulin-Fc fusion polypeptide comprising a single-chain insulin and an Fc region peptide. Further, a conjugate of two insulin-Fc fusion polypeptides is provided, wherein the two insulin-Fc fusion polypeptides are linked by at least one covalent bond at the C-terminus of the fusion polypeptide. Also provided is a single-chain insulin with reduced binding affinity for the insulin receptor. Further provided is the use of the insulin-Fc fusion polypeptide, conjugate, or single-chain insulin in medicine.

Background Art

[0002] Diabetes mellitus (DM) is a chronic metabolic disease caused by insulin deficiency, insulin resistance, or both. There are mainly two types: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). In T1DM, the pancreatic beta cells that produce insulin are destroyed by the immune system, so T1DM is considered an autoimmune disease. As a result, T1DM patients cannot produce insulin and are completely dependent on insulin administration. T2DM is a complex metabolic disease that takes years to progress. T2DM is mostly associated with other health problems such as obesity, non-alcoholic fatty liver disease, and hypertension. The combination of various pathological conditions is also called metabolic syndrome. In T2DM, the pancreas can still produce insulin, but insulin levels may decrease as the disease progresses (Sapra and Bhandari 2020; Godoy-Matos et al., 2020).

[0003] The main physiological function of insulin is to regulate blood glucose. Blood glucose levels are sensed by pancreatic beta cells, and when a certain threshold is reached, insulin is secreted into the circulation. Circulating insulin mainly causes the uptake of glucose in the blood into the liver, muscle, and adipose tissue. Insulin deficiency or insulin resistance causes an increase in blood glucose levels (hyperglycemia), and if left untreated for a long time, it causes microvascular and microvascular abnormalities, ultimately leading to organ dysfunction. The organs most likely to be affected by microvascular and macrovascular disorders are the kidneys, eyes, nerves, and heart (Kahn et al., 2014; Taylor et al., 2021).

[0004] The insulin receptor (IR) is a heterotetramer consisting of two extracellular alpha subunits and two transmembrane beta subunits. The alpha and beta subunits are linked by disulfide bonds. There is a tyrosine kinase domain in the intracellular part. When bound to insulin, IR dimerizes, bringing the intracellular tyrosine kinase domains closer together, enabling autophosphorylation and the initiation of intracellular signaling cascades. In humans, there are two isoforms of IR called IR-A and IR-B. The difference between these isoforms lies in the presence or absence of an extended region of 12 amino acids at the C-terminus of the A subunit of the IR-B isoform. Both isoforms of IR have different expression patterns and different binding parameters not only for insulin but also for insulin-like growth factors 1 and 2 (IGF1 and IGF2). For example, IR-A has a higher affinity for insulin and IGF2 than IR-B. There are also physiological differences between IR-A and IR-B. IR-B is mainly expressed at high levels in insulin target tissues and mediates metabolic effects and cell proliferation, while IR-A is thought to promote cell growth (Belfiore et al., 2017).

[0005] Insulin-like growth factor (IGF) has a structure similar to insulin. Its biological function is to induce growth or cell differentiation. The signal transduction of IGF is mediated by the IGF-1 receptor, which has a structure similar to the insulin receptor. Since the ligand and the receptor have similar structures, IGF1 and IGF2 can indeed bind to and activate the IR, although at high concentrations. Similarly, insulin can also bind to and activate the IGF-1 receptor. The crosstalk between these signal transduction pathways is further complicated by the possibility that IGF-1R and IR form functional heterodimers (Denley et al., 2007; Hakuno et Takahasi, 2018).

[0006] Insulin is synthesized as a preprohormone in the pancreatic beta cells. The signal sequence induces the secretion of insulin into the lumen of the endoplasmic reticulum (ER). The signal sequence is cleaved during secretion. The resulting proinsulin consists of three parts: the B chain at the N-terminus, the connecting peptide (C peptide), and the A chain at the C-terminus (Dodson and Steiner 1998). Proinsulin is further processed during intracellular transport. The C peptide is cleaved, and two C-terminal arginine residues of the B chain are cleaved (Steiner 2011). Mature insulin is composed of two chains (A chain and B chain) and is linked by two disulfide bonds.

[0007] In contrast to mature insulin, proinsulin is a single-chain protein. Proinsulin can fold into a three-dimensional structure, but the junction of the C peptide with the A and B chains is flexible (Yang et al., 2010). Proinsulin is biologically active in cell assays (Jehle et al., 1996) and is stable in human blood (Bright et al., 2017). There is evidence that proinsulin also plays a physiological role in embryonic development (Hernandez-Sanchez et al., 2006).

[0008] Single-chain insulins (scI) are insulin variants in which the insulin B-chain and the insulin A-chain are linked by a short peptide linker. Such variants can be easily produced by recombinant methods, and depending on the length and sequence of the linker, their activity is still maintained. Alternatively, the insulin a-chain and b-chain can also be linked with a chemical linker. The advantage of scI over native insulin is the improved stability (Hua et al., 1998; Glidden et al., 2018). Due to these characteristics, single-chain insulin is attractive as an insulin replacement drug.

[0009] The aim of insulin replacement therapy is to mimic the natural insulin secretion profile. In the case of human insulin, onset of action is within 30 minutes after administration, and the effect lasts for more than 5 hours. When administered in the afternoon, these characteristics can cause nocturnal hypoglycemia, so patients are recommended to have a snack before bedtime to avoid hypoglycemia. To more precisely control the action of insulin, various insulin formulations and insulin analogs with different pharmacokinetic properties have been developed. Insulin analogs are classified into rapid-acting, short-acting, intermediate-acting, and long-acting types. Insulin analogs (e.g., insulin lispro, insulin aspart, insulin glulisine) can be administered before or after meals. For long-acting or basal insulin analogs (e.g., insulin detemir, insulin degludec, insulin glargine), a more flat and long-lasting insulin profile can be obtained. The action time of long-acting insulin analogs is 24 hours. It has been shown that the risk of nocturnal hypoglycemia can be significantly reduced by using long-acting insulin formulations (Sharma et al., 2019; Mathieu 2021). Insulin icodec, a basal insulin analog formulation that can be administered once a week, has the same blood glucose-lowering effect as daily-administered insulin glargine. (Rosenstock et al., 2020). Such ultra-long-acting insulin analog formulations are useful for improving the QOL of patients because they require fewer injections.

[0010] Based on their structures, long-acting insulin analogs can be classified into two groups: insulin with an altered amino acid sequence (e.g., insulin glargine) and insulin analogs with chemical modifications (e.g., insulin detemir). In long-acting insulin analogs, the mechanism of action may vary. Insulin glargine has a different isoelectric point compared to native insulin. Therefore, insulin glargine precipitates after administration and is slowly released into the blood from this precipitate. Insulin detemir is acetylated with a C14 fatty acid and can bind to albumin. The plasma half-life of human albumin is as long as 20 days. The reason for this long half-life lies in the recycling mechanism. The underlying recycling mechanism uses the same pathway as the recycling mechanism used by antibodies of the immunoglobulin G (IgG) class, which have a plasma half-life of ~20 days. Both albumin and IgG utilize the recycling mechanism based on the neonatal Fc receptor (Sand et al., 2015).

[0011] IgG binds to FcRn via the Fc domain. Fusing or conjugating a peptide or protein to the Fc domain can extend the plasma half-life of the corresponding Fc fusion protein, and several Fc fusion proteins have already been launched on the pharmaceutical market (Rath et al., 2015).

[0012] IgG is a multifunctional protein. The normal function of an antibody is to stimulate the humoral and cellular immune systems upon antigen binding. Antigens are bound by the Fab domain, while the effector functions of the antibody are mediated by the Fc domain. The receptors that mediate the physiological functions of IgG are called Fc-gamma receptors (FcyR). When binding to FcyR, the so-called antibody-dependent cellular cytotoxicity (ADCC) reaction is activated (Bruhns and Joensson, 2015). Another effector function of the antibody is mediated by a protein called C1q. C1q belongs to a defense mechanism known as the complement system. When C1q binds to the antibody, a proteolytic cascade called the complement cascade is induced. This cascade ultimately forms a so-called membrane attack complex that creates holes in the membrane of the target cell. This immune defense mechanism is also known as complement-dependent cytotoxicity (CDC) (West et al., 2018).

[0013] For many therapeutic antibodies, such as those used in cancer treatment, effector functions are required to achieve the therapeutic goal. In fact, to enhance the efficacy of such antibodies, it can be achieved by manipulating the Fc to enhance the binding of the antibody to FcyR or C1q. The Fc domain is glycosylated at position N297. Glycosylation at this site is necessary to fully exert the effector function (Wang et al., 2018). On the other hand, many therapeutic antibodies and Fc fusion proteins are designed to bind to specific targets or capture unwanted and excessive messengers such as inflammatory cytokines. In such cases, especially when administering Fc fusion proteins for a long period of time, effector functions mediated by Fc are not desirable.

[0014] In the case of an Fc fusion protein, it is not usually intended to impair binding to FcRn. In contrast, it may be desirable to reduce the effector function of the Fc domain. Some point mutations within the Fc domain that reduce effector function have been reported. Deglycosylated antibodies or Fc fusion proteins reduce effector function, so glycosylation sites are also included in these sites. In the case of an Fc fusion protein, an aglycosylated protein can be obtained by expressing it in bacteria, such as Escherichia coli (E. coli.). However, aglycosylated variants may have reduced solubility and stability (Schlothauer et al., 2016; Jacobsen et al., 2017; Dumet et al., 2019).

[0015] WO 2016 / 178905 A1 discloses a fusion protein comprising an insulin receptor agonist fused to the human IgG Fc region by using a peptide linker, and the use of such a fusion protein in the treatment of diabetes.

[0016] There remains a need for ultra-long-acting insulin analog formulations that have a low injection frequency and are useful for improving the QOL of diabetic patients.

[0017] In the studies described herein, mutations were introduced at specific positions in the insulin B-chain and A-chain of human insulin to create single-chain insulin analogs with reduced binding affinity to the receptor compared to human insulin. Without being bound by theory, it is speculated that low affinity for the insulin receptor is beneficial for ultra-long-acting insulin because it reduces the clearance of insulin derivatives bound to the insulin receptor. Since activation of the IR-A receptor is thought to be involved in the mitogenic action of insulin, variants were created with reduced activity for IR-A compared to the activity for IR-B, compared to wild-type insulin. Insulin formulations were tested in the form of single-chain insulin Fc fusion polypeptides. Their sequences are shown in Table A in the Examples section. The results of receptor activation are shown in Table 1 in the Examples section. Examples of such insulins with reduced activity for IR-A compared to IR-B and with ultra-long activity are INS009 (SEQ ID NO: 10), INS011 (SEQ ID NO: 12), INS013 (SEQ ID NO: 14) and INS018 (SEQ ID NO: 19). The single-chain insulin analogs present in these fusions have the amino acid sequences shown in SEQ ID NO: 50 (INS009), SEQ ID NO: 52 (INS011), SEQ ID NO: 53 (INS014) and SEQ ID NO: 58 (INS018), respectively.

[0018] In the studies described herein, the single-chain insulin analogs created were fused to the IgGFc domain. Since insulin administration is typically carried out over a long period of time, an Fc domain without effectors was selected. In the selected Fc domain without effectors, the hinge domain present at the N-terminus of the Fc region was removed. This is because the binding sites for FcγR and C1q, which are responsible for the effector functions of the antibody, are partially in the antibody hinge region, whereas the binding sites for FcRn and protein A are more C-terminal in the Fc region. Thus, removal of the hinge region results in loss of the effector functions of the antibody without impairing the desired Fc functions. Furthermore, the two C-terminal covalent linkages of the present invention overcome the limitations (reduced FcRn binding, reduced stability) seen in normal antibody variants without a hinge.

[0019] Sequence number 34 shows the amino acid sequence of an effector-free IgG1 Fc region lacking the hinge region. However, the hinge region (sequence number 41) was fused to the C-terminus of the Fc region via a linker peptide (GGGGSA, sequence number 42). The covalent bond of the two Fc chains was achieved by fusing the hinge sequence to the C-terminus. Advantageously, the sequence of the antibody hinge region forms an interchain disulfide bond even when located at the C-terminus relative to the Fc portion. Since the sequence of the antibody hinge region can be derived from the human endogenous antibody hinge region, the use of heterologous-derived sequences can be avoided, and the risk of anti-drug antibody (ADA) formation can be reduced.

[0020] The linker sequence (linker 2 in Figure 8) connecting the single-chain inulin and the Fc domain is derived from the C-peptide present in native proinsulin. The C-peptide is a 30-amino acid-long peptide (sequence number 31) mainly composed of small hydrophilic amino acids. This peptide is stable in the blood and probably has a chaperone-like effect on insulin (Landreh et al. 2013). The C-peptide in solution is not fully folded and does not adopt a random structure (Munte et al., 2005). Since it consists of small and hydrophilic amino acids, is stable in the blood, and is structurally flexible, the C-peptide or a peptide derived from the C-peptide is used as the linker. To further increase the amount of small hydrophilic amino acids in the sequence of the second linker, amino acids Leu26Ala27Leu28 of sequence number 31 were removed and Gly was placed at the N-terminus, resulting in the linker sequence of sequence number 29. The linker thus created was used in constructs named, for example, INS003 to INS025 (see Table A).

[0021] Surprisingly, the combination of an insulin variant and an effector-free Fc domain results in a mechanism of action with an extremely long duration, compared to a protein construct in which the insulin variant is fused to a commonly used silencer IgG Fc backbone. This is exemplified by a single-chain insulin variant having a sequence as shown in SEQ ID NO: 50. When these are fused to an effector-free Fc domain (SEQ ID NO: 10, INS009) with the hinge removed, a much longer hypoglycemic effect was observed compared to INS024 (SEQ ID NO: 25) (see Figures 4 and 7 and Table 2 in the Examples section). The hypoglycemic activity of INS009 (SEQ ID NO: 10) lasted at least 216 hours in the Göttingen minipig model and was much longer than that of INS024 (SEQ ID NO: 25) with a hypoglycemic activity duration of up to 120 hours in minipigs. The hinge of INS024 was in its normal position.

[0022] Also, for the variants INS013 (SEQ ID NO: 14) and INS011 (SEQ ID NO: 12), very long hypoglycemic activities were observed in the Göttingen minipig model, with action times of up to 216 hours (INS013 [SEQ ID NO: 14] (Figure 4)) and 264 hours (INS011, SEQ ID NO: 12 (Figure 5 and Table 2 in the Examples section)).

[0023] Mutations introduced into the insulin sequence, the peptide connecting the insulin A and B chains (linker 2 in Figure 8), and the linker sequence connecting insulin and the Fc domain accumulated in stable insulin derivatives with an extremely long duration of action in the rat model (maximum action time of 168 hours) and the minipig model (maximum action time of 264 hours [INS011 [SEQ ID NO: 12]]). Furthermore, in cynomolgus monkeys, a plasma half-life of up to 69 hours was measured (see Table 3 and Figure 11). SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0024] This specification provides insulin analogs with reduced affinity for the insulin receptor, such as the insulin analogs shown in Table B of the Examples section. Further provided are ultra-long-acting insulin analogs comprising single chain insulins (scI) fused to an Fc domain, preferably an IgG Fc domain without an N-terminal hinge region. The scI-Fc fusion proteins do not require further chemical modification and can be produced by recombinant methods.

[0025] First aspect: Insulin analog Provided herein is an insulin analog comprising an insulin B chain and an insulin A chain, wherein the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 73), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0026] Further provided is an insulin analog comprising an insulin B chain and an insulin A chain, wherein the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTPKT (SEQ ID NO: 70), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0027] Further provided is an insulin analog comprising an insulin B chain and an insulin A chain, wherein the insulin B chain comprises or consists of the amino acid sequence FVNQHLCGSHLVEALHLVCGERGFHYTPKT (SEQ ID NO: 71), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75).

[0028] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 73), and the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75).

[0029] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 65), and the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0030] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 66), and wherein the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0031] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 67), and wherein the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0032] Typically, the insulin analog is provided as single-chain insulin. In single-chain insulin, the insulin B chain is typically linked to the insulin A chain via a first linker peptide.

[0033] Second Aspect: Insulin-Fc Fusion Polypeptide and Conjugate of Two Insulin-Fc Fusion Polypeptides Furthermore, provided is an insulin-Fc fusion polypeptide comprising, from the N-terminus to the C-terminus: insulin and an Fc region polypeptide (also referred to herein as the "Fc region").

[0034] Typically, the insulin contained in the fusion protein is an insulin analog with a lower binding affinity to the human insulin receptor compared to human insulin. In one embodiment, the insulin is a single-chain insulin such as the single-chain insulin provided herein (see, for example, Table B).

[0035] Typically, the single-chain insulin is linked to the Fc region polypeptide via a second linker peptide.

[0036] Thus, the insulin-Fc fusion polypeptide typically comprises, from the N-terminus to the C-terminus: a) insulin B chain, and b) a first linker peptide, and c) insulin A chain, and d) a second linker peptide, and e) an Fc region polypeptide. and includes.

[0037] In some embodiments, the insulin-Fc fusion polypeptide further comprises f) a third linker peptide, and g) a C-terminal peptide. and further includes.

[0038] The C-terminal peptide enables the formation of at least two covalent bonds between two insulin-Fc fusion polypeptides, thereby stabilizing the conjugate provided herein.

[0039] Typically, the C-terminal peptide contains at least two cysteine residues, enabling the formation of at least two disulfide bonds between two insulin-Fc fusion polypeptides present in the conjugate.

[0040] In one embodiment, the C-terminal peptide contains the amino acid sequence (or a highly similar sequence) of the antibody hinge region that enables the formation of at least two disulfide bonds between two insulin-Fc fusion polypeptides.

[0041] In some embodiments, the C-terminal peptide contains or consists of an amino acid sequence as set forth in SEQ ID NO: 36, 39, 40 or 41.

[0042] In some embodiments, the Fc region polypeptide contains the constant document of the constant region of an IgG, IgM, IgA, IgD or IgE antibody.

[0043] In some embodiments, the Fc region polypeptide contains the constant domains CH2 and CH3 of the heavy chain of an IgG antibody such as an IgG1 antibody or an IgG4 antibody.

[0044] In some embodiments, the Fc region polypeptide a) the amino acid sequence set forth in SEQ ID NO: 34, or b) an amino acid sequence that is 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% or at least 99% identical to SEQ ID NO: 34, contains or consists of.

[0045] Advantageously, the Fc region polypeptide does not have an antibody hinge region at the N-terminus. Thus, the two insulin-Fc fusion polypeptides are not usually covalently linked by one or more disulfide bonds between the N-termini of each Fc region polypeptide.

[0046] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in any one of SEQ ID NOs: 2-25, 77, and 78. The sequence is shown in Table A in the Examples section.

[0047] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 4 (internal name: "INS003", see Table A).

[0048] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 5 (INS004).

[0049] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 6 (INS005).

[0050] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 7 (INS006).

[0051] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 10 (INS009), In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 12 (INS011). In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 14 (INS013). In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 19 (INS018).

[0052] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 77.

[0053] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 78.

[0054] Also provided herein is an insulin-Fc conjugate comprising at least two insulin-Fc fusion polypeptides provided herein, wherein each of the insulin-Fc fusion polypeptides comprises, from the N-terminus to the C-terminus: insulin, such as a single-chain insulin provided herein, and an Fc region polypeptide.

[0055] Typically, the conjugate is a homodimer of two insulin-Fc fusion polypeptides provided herein. Thus, both fusion polypeptides typically have the same sequence, i.e., are identical.

[0056] In one embodiment of the insulin-Fc conjugate provided, the insulin is an insulin analog with a reduced binding affinity for the human insulin receptor as compared to human insulin. Typically, the insulin is an insulin provided herein (e.g., see Table B), such as a single-chain insulin provided herein.

[0057] Typically, the conjugate is a conjugate of two insulin-Fc fusion polypeptides, and the two insulin-Fc fusion polypeptides are linked by at least two covalent bonds at the C-terminus of the fusion polypeptide. Thus, the two fusion polypeptides are linked by at least two covalent bonds located at the C-terminus of the Fc region polypeptide.

[0058] In one embodiment, at least two covalent bonds are located at the C-terminus of the CH2, CH3, and CH4 domains on each fusion polypeptide.

[0059] In another embodiment, at least two covalent bonds are located at the C-terminus of the CH2 and CH3 domains on each fusion polypeptide.

[0060] Typically, the covalent bond is a disulfide bond.

[0061] Typically, the two insulin-Fc fusion polypeptides are not covalently linked by one or more disulfide bonds between the N-termini of each Fc region polypeptide.

[0062] Typically, each of the first insulin-Fc fusion polypeptide and the second insulin-Fc fusion polypeptide includes a C-terminal peptide at the C-terminus, and the C-terminal peptide includes at least two cysteine residues that enable the formation of two disulfide bonds between the first and second fusion polypeptides. Typically, the C-terminal peptide includes the amino acid sequence of an antibody hinge region (or a highly similar sequence).

[0063] Further aspects Furthermore, provided is a polynucleotide encoding a single-chain insulin provided herein, or an insulin-Fc fusion polypeptide provided herein.

[0064] Furthermore, provided is a host cell comprising an insulin-Fc conjugate provided herein, a single-chain insulin provided herein, or an insulin-Fc fusion polypeptide provided herein, and / or a polynucleotide provided herein.

[0065] Furthermore, provided is a method for producing an insulin-Fc conjugate provided herein, a single-chain insulin provided herein, or an insulin-Fc fusion polypeptide provided herein, the method comprising incubating a host cell provided herein under conditions capable of expressing a single-chain insulin or an insulin-Fc fusion polypeptide. Expression of the insulin-Fc fusion polypeptide may result in the formation of an insulin-Fc conjugate as defined herein.

[0066] Furthermore, provided is a pharmaceutical composition comprising a pharmaceutically effective amount of an insulin-Fc conjugate provided herein, a single-chain insulin provided herein, or an insulin-Fc fusion polypeptide provided herein.

[0067] Furthermore, provided herein are insulin-Fc conjugates provided herein, insulin provided herein (such as single-chain insulin provided herein), or insulin-Fc fusion polypeptides provided herein for use as a medicament.

[0068] Furthermore, provided herein are insulin-Fc conjugates provided herein, insulin provided herein (such as single-chain insulin provided herein), or insulin-Fc fusion polypeptides provided herein for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes, and hyperglycemia and / or for use as a medicament for reducing blood glucose levels.

Brief Description of the Drawings

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[0070] Definitions As used herein in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0071] As used herein, the term "comprising" is to be interpreted as specifying the presence of the stated feature, integer, step, or component referred to, but not precluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, for example, a protein comprising an amino acid sequence may contain more amino acids than those actually recited, i.e., it may be incorporated into a larger protein.

[0072] As used herein, "polypeptide" refers to a chain of 10 or more amino acids covalently linked by peptide bonds. Thus, the term "polypeptide" can mean a chain of a multi-chain protein, regardless of the length of such a chain. In some embodiments, the polypeptide is a chain of a multi-chain protein.

[0073] As used herein, "linker" or "linker peptide" refers to a short, flexible amino acid sequence that connects two regions of a molecule. For example, a linker can connect the B-chain and A-chain of insulin. This linker is referred to herein as the "first linker peptide". Furthermore, a linker can connect single-chain insulin and the Fc region. This linker is referred to herein as the "second linker peptide". Furthermore, a linker peptide can connect the Fc region and the C-terminal region of a molecule containing at least two inter-chain covalent bonds. This linker is referred to herein as the "third linker peptide".

[0074] The expression "insulin analog" as used herein refers to a peptide having a molecular structure formally derived from the structure of naturally occurring insulin (also referred to herein as "parent insulin", e.g., human insulin).

[0075] The expression "parent insulin" as used herein refers to naturally occurring insulin, i.e., wild-type insulin that has not mutated. In some embodiments, the parent insulin is insulin derived from an animal, e.g., insulin derived from a mammal. For example, the parent insulin may be human insulin, porcine insulin, bovine insulin. Usually, the parent insulin is human insulin.

[0076] The B-chain of human insulin contains the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 32) The A-chain of human insulin contains the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 33)

[0077] In one embodiment, the insulin analog provided herein or the insulin analog comprising the insulin-Fc fusion polypeptide provided herein is a single-chain insulin. A single-chain insulin is a single polypeptide chain in which the insulin B chain is continuously linked to the insulin A chain via a linking peptide herein referred to as the first linker peptide.

[0078] The insulin analog provided herein or the insulin analog comprising the insulin-Fc fusion polypeptide provided herein typically contains at least one mutation (amino acid substitution, deletion or addition) relative to the parent insulin. As used herein, the term "at least one" means one or more, such as "at least two", "at least three", "at least four", "at least five", etc. In some embodiments, the insulin analog provided herein contains at least one mutation in the B chain and at least one mutation in the A chain. In a further embodiment, the insulin analog provided herein contains at least two mutations in the B chain and at least one mutation in the A chain.

[0079] Typically, the insulin analog provided herein or the insulin analog comprising the insulin-Fc fusion polypeptide provided herein contains two peptide chains, an A chain and a B chain. Usually, the two chains are linked by disulfide bonds between cysteine residues. For example, in at least one embodiment, the human insulin analog contains three disulfide bonds: one disulfide bond between the cysteines at positions A6 and A11 of the A chain, one disulfide bond between the cysteine at position A7 of the A chain and the cysteine at position B7 of the B chain, and one disulfide bond between the cysteine at position A20 of the A chain and the cysteine at position B19 of the B chain.

[0080] Typically, an insulin analog provided herein or an insulin analog composed of an insulin-Fc fusion polypeptide provided herein has a reduced insulin receptor binding affinity compared to the corresponding parent insulin, such as human insulin. Thus, the insulin analog has a very slow clearance rate, i.e., a very slow clearance rate via the insulin receptor.

[0081] The insulin receptor can be a mammalian insulin receptor such as the bovine, porcine, or human insulin receptor. In some embodiments, the insulin receptor is a human insulin receptor, such as human insulin receptor isoform A or human insulin receptor isoform B.

[0082] Typically, an insulin analog provided herein or an insulin analog composed of an insulin-Fc fusion polypeptide provided herein has a binding affinity of less than 25%, such as less than 15%, for human insulin receptor isoform B compared to human insulin, i.e., exhibits. In one embodiment, the insulin analog has a binding affinity for human insulin receptor isoform B of 10 - 25% compared to human insulin.

[0083] Typically, an insulin analog provided herein or an insulin analog composed of an insulin-Fc fusion polypeptide provided herein has a binding affinity of less than 15%, such as less than 8%, for human insulin receptor isoform a compared to human insulin, i.e., exhibits. In one embodiment, the insulin analog has a binding affinity for human insulin receptor isoform A of 1 - 15%, such as 3 - 10%, compared to human insulin.

[0084] Methods for measuring the binding affinity of insulin analogs to the insulin receptor are known in the art. For example, insulin receptor binding affinity can be measured by a scintillation proximity assay based on the evaluation of competitive binding of [125I]-labeled parent insulin, such as [125I]-labeled human insulin, and (unlabeled) insulin analogs to the insulin receptor. The insulin receptor can be present in the membrane of cells that overexpress the recombinant insulin receptor, such as CHO (Chinese hamster ovary) cells. In one embodiment, the insulin receptor binding affinity is measured as described in the Examples section of International Publication No. WO 2020 / 120463 A1, which is incorporated herein by reference in its entirety.

[0085] When a naturally occurring insulin or insulin analog binds to the insulin receptor, the insulin signaling pathway is activated. The insulin receptor has tyrosine kinase activity. Binding of insulin to the receptor causes a structural change that stimulates autophosphorylation of tyrosine residues of the receptor. Autophosphorylation of the insulin receptor stimulates the tyrosine kinase activity of the receptor towards intracellular substrates involved in signal transduction. Thus, autophosphorylation of the insulin receptor by an insulin analog is considered an indicator of signal transduction by said analog. In some embodiments, the insulin analogs provided herein can induce 10 - 25% autophosphorylation of human insulin receptor isoform B and / or 1 - 15% autophosphorylation of human insulin receptor isoform A relative to human insulin. Further, in some embodiments, the insulin analogs provided herein can induce 3 - 7%, such as 5 - 7%, autophosphorylation of the insulin receptor relative to parent insulin (such as human insulin). Autophosphorylation of the insulin receptor relative to parent insulin can be measured as described in the Examples section.

[0086] In the insulin-Fc fusion polypeptide provided herein, insulin, such as single-chain insulin, is to be linked in an operable state to the Fc region polypeptide. In an antibody, the Fc (fragment crystallizable) region is a region that interacts with cell surface receptors called Fc receptors, thereby activating the immune system. The Fc region of IgG is known to have a highly conserved N-glycosylation site, which is essential for activity via Fc receptors.

[0087] The two insulin-Fc fusion polypeptides present in the conjugate provided herein form the Fc region of an antibody.

[0088] As used herein, the "Fc region" is a fragment of an immunoglobulin molecule formed by multiple constant heavy chain (CH) immunoglobulin domains of two polypeptides. In a natural antibody, the Fc region is involved in binding to Fc receptors and components of the complement system. In the case of IgG, IgA, and IgD, the Fc region is formed by the CH2 and CH3 constant domains of both heavy chains. In the case of IgM and IgE, the Fc region is formed by the CH2, CH3, and CH4 constant domains of both heavy chains. The Fc region of the present invention can be formed by the same CH domains as the Fc region of a natural antibody, i.e., the CH2 and CH3 domains or the CH2, CH3, and CH4 domains. The Fc region of the present invention is identical to the CH domain of a natural immunoglobulin or includes a CH domain derived from the CH domain of a natural immunoglobulin by, for example, inserting one or several point mutations.

[0089] The term "Fc region polypeptide" is a fragment of an antibody heavy chain, and the fragment includes the constant domain of the antibody heavy chain. Thus, an Fc region polypeptide typically includes the constant domain of an IgG, IgM, IgA, IgD, or IgE antibody heavy chain. For example, if the antibody is an IgG antibody, the Fc region polypeptide includes the CH2 domain and the CH3 domain (of the antibody heavy chain). In one embodiment, the Fc region polypeptide is a) the amino acid sequence shown in SEQ ID NO: 34, or b) an array having 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% or at least 99% identity to SEQ ID NO: 34, or consisting of or comprising the same. Typically, the two Fc region polypeptides form the antibody Fc region in the conjugates provided herein.

[0090] The Fc region polypeptide may comprise additional elements such as a third linker peptide C-terminal peptide that allows for the formation of the at least two covalent bonds between the two insulin-Fc fusion polypeptides. However, typically, the Fc region polypeptide lacks the N-terminal antibody hinge region.

[0091] As used herein, the term "hinge region" refers to a portion of the antibody sequence located between the Fc region and the Fab region. This provides flexibility between segments and can stabilize the antibody molecule. The hinge region can be clearly defined based on structural data. For example, the IgG1 hinge region includes residues 221-237 (R. Nezlin, "The Immunoglobulins", Academic Press, 1998, pages 23-26). In IgG antibodies, the hinge region includes disulfide bonds that connect the two heavy chains of the antibody. IgG1 and IgG4 contain two such disulfide bonds, IgG2 contains four, and IgG3 contains eleven (Liu and May, 2012 MAbs. 2012 Jan-Feb;4(1):17-23).

[0092] Here, a region is usually called a "hinge region" only when it is located at the N-terminus of the Fc region, as is the case with all natural antibodies. When an amino acid sequence identical or highly similar to the hinge region (such as in some embodiments of the molecules of the present invention) is arranged at the C-terminus of the Fc region, it is designated as "an amino acid sequence containing the amino acid sequence of the (antibody) hinge region" and the like. Highly similar means having at least 70% identity with the sequence of the antibody hinge region and / or containing at least 7 consecutive amino acids of the sequence of the antibody hinge region.

[0093] As used herein, "covalent bond" refers to a chemical bond in which electron pairs are shared between atoms. A covalent bond is different from non-covalent interactions such as electrostatic interactions and hydrophobic effects.

[0094] The statement that a covalent bond, such as a disulfide bond, is "located at the C-terminus" of the Fc region means that in both polypeptides forming the Fc region, the amino acid residues involved in the formation of such a covalent bond, such as cysteine residues, are located at the C-terminus of the portion of the polypeptide forming the Fc region.

[0095] The statement that there is no covalent bond, such as a disulfide bond, "located at the N-terminus" of the Fc region means that in both polypeptides forming the Fc region, there are no amino acid residues involved in the formation of such a covalent bond, such as cysteine residues, located at the N-terminus of the portion of the polypeptide forming the Fc region.

[0096] The "percent amino acid sequence identity (%)" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to maximize the percent sequence identity. Typically, standard parameters are applied to determine the degree of identity between two sequences. In some embodiments, the degree of sequence identity is calculated over the entire length of the two sequences. In some embodiments, the degree of identity is determined by comparing two sequences optimally aligned over a comparison window. Here, an amino acid sequence fragment within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to a reference sequence (without additions or deletions) for optimal alignment. This percentage is calculated by determining the number of positions where identical amino acid residues occur in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percent sequence identity. Optimal alignment of sequences for comparison may be performed by the local homology algorithm by Smith and Waterman Add.APL.Math.2:482 (1981), the homology alignment algorithm by Needleman and Wunsch J.Mol.Biol.48:443 (1970), the similarity search method by Pearson and Lipman Proc.Natl.Acad.Sci.(USA) 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, TFASTA within the Wisconsin Genetics Software Package by Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. When two sequences are identified for comparison, GAP and BESTFIT are typically used to determine their optimal alignment and thus the degree of identity. Usually, default values of 5.00 for the gap weight and 0.30 for the length of the gap weight are used.In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman 1970, J. Mol. Biol. (48):444-453) incorporated in the needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice, P., Longden, I., and Bleasby, A., Trends in Genetics 16(6), 276-277, 2000), with a BLOSUM62 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5. Preferred non-limiting examples of the parameters used to align two amino acid sequences using the needle program are the default parameters including the EBLOSUM62 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5.

[0097] The term "at least 80% identical" with respect to two sequences means that the two sequences have a sequence identity of 80% or more. In some embodiments, the sequences are at least 85% or 87% identical. In some embodiments, the sequences are at least 90% identical. In some embodiments, the sequences are at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the sequences are identical, i.e., 100% identical.

[0098] As used herein, the terms "cell" or "host cell" refer to intact cells, i.e., cells with an intact membrane that do not release normal intracellular components such as enzymes, organelles, genetic material, etc. In certain exemplary embodiments, an intact cell is a viable cell, i.e., a living cell capable of performing normal metabolic functions. In certain exemplary embodiments, a cell or host cell is any cell that can be transfected or transformed with exogenous nucleic acid. In certain specific exemplary embodiments, a cell is transfected or transformed with exogenous nucleic acid and can express the nucleic acid in a recipient when introduced into the recipient.

[0099] The term "cell" includes prokaryotic cells such as bacterial cells, and eukaryotic cells such as yeast cells, fungal cells, and mammalian cells. Suitable bacterial cells include Gram-negative bacterial strains such as Escherichia coli, Proteus genus, and Pseudomonas genus, and cells of Gram-positive bacterial strains such as Bacillus genus, Streptomyces genus, Staphylococcus genus, and Lactococcus genus, but are not limited thereto. Suitable fungal cells include cells derived from species of the Trichoderma genus, Neurospora genus, and Aspergillus genus, but are not limited thereto. Suitable yeast cells include cells derived from species of the Saccharomyces genus (e.g., Saccharomyces cerevisiae), Schizosaccharomyces genus (e.g., Schizosaccharomyces pombe), Pichia genus (e.g., Pichia pastoris and Pichia methanolica), and Hansenula genus, but are not limited thereto. Suitable mammalian cells include, for example, Chinese Hamster Ovary (CHO) cells, BHK cells, HeLa cells, COS cells, HEK-293, etc., but are not limited thereto. In one embodiment, HEK-293 cells are used. In another embodiment, CHO cells are used. However, amphibian cells, insect cells, plant cells, and other cells used in the art for the expression of heterologous proteins can equally be used. In certain exemplary embodiments, mammalian cells (e.g., human, mouse, hamster, pig, goat, or primate cells) are used for transplantation.

[0100] In some embodiments, the host cell comprises a polynucleotide encoding an insulin, a fusion protein, or an insulin-Fc conjugate provided herein, and / or a vector comprising the polynucleotide. Typically, the vector is an expression vector.

[0101] The cell or host cell may be isolated or may be a tissue or part of an organism such as a "non-human organism". As used herein, the term "non-human organism" means mammals other than humans, such as non-human primates or other animals, for example, cows, horses, pigs, sheep, goats, dogs, cats, rabbits, or rodents (e.g., mice, rats, guinea pigs, and hamsters). In some embodiments, the non-human organism is a cynomolgus monkey.

[0102] The pharmaceutical compositions defined herein typically comprise an insulin or insulin-Fc conjugate provided herein together with a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable" means non-toxic substances that do not interact with the action of the active agent of the pharmaceutical composition, i.e., insulin or insulin-Fc conjugate, in certain exemplary embodiments.

[0103] As used herein, the term "carrier" refers to natural or synthetic organic or inorganic components into which the active ingredient is formulated to facilitate, enhance, or enable administration. Generally, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject.

[0104] Carrier substances suitable for parenteral administration include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, bacteriostatic physiological saline (e.g., physiological saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), Hank's solution, polyalkylene glycol, hydrogenated naphthalene, and especially biocompatible polylactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0105] As used herein, the term "excipient" is intended to include all substances that may be present in a pharmaceutical composition and that are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffering substances, flavoring agents, or coloring agents.

[0106] Of course, the form, route of administration, dosage, and regimen of a pharmaceutical composition depend on the condition to be treated, the severity of the disease, the age, weight, gender, etc. of the patient.

[0107] A pharmaceutical composition can be formulated for topical, oral, parenteral, nasal, intravenous, intramuscular, subcutaneous, intraocular, etc. administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for subcutaneous administration.

[0108] In some embodiments, a pharmaceutical composition contains a pharmaceutically acceptable solvent as a formulation that enables parenteral administration such as intravenous or subcutaneous. These may in particular be isotonic, sterile, physiological saline (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or a mixture of these salts), or a dried, especially lyophilized, composition, which in some cases can form an injectable solution by adding sterile water or physiological saline.

[0109] The insulin or insulin-Fc conjugate described herein may be administered by any conventional route including injection or infusion, e.g., orally, by pulmonary administration, inhalation or parenterally. In some embodiments, parenteral administration such as intravenous, intraarterial, subcutaneous, intradermal or intramuscular administration may be used. In some embodiments, the insulin or insulin-Fc conjugate provided herein is administered subcutaneously.

[0110] The insulin or insulin-Fc conjugate, or pharmaceutical composition, described herein is typically administered in a therapeutically effective amount. The term "therapeutically effective amount" is understood by those skilled in the art. In some embodiments, the term refers to an amount that achieves the desired therapeutic response or desired therapeutic effect, alone or in combination with further dosages, optionally, without causing unacceptable or undesirable side effects, or with minimal side effects.

[0111] Generally, the activity of insulin analogs is provided in units of human insulin, which is the current standard for insulin administration to patients. The World Health Organization (WHO) currently defines 1 unit of human insulin (EP / US / IU) as 0.0347 mg of human insulin (Burns C, Morris T, Jones B, et al. World Health Organization. Proposal to Initiate a Project to Evaluate a Candidate International Standard for Human Recombinant Insulin. WHO / BS / 10.2143 - Working document QAS / 10.381, 2010). Usually, 1 unit of an insulin analog is biologically equivalent to 1 unit of human insulin.

[0112] In one embodiment, the insulin analog provided herein or the insulin-Fc conjugate provided herein is administered in an amount corresponding to 200 to 1500 U of human insulin, such as 400 U to 1000 U, such as 400 U, 450 U, 500 U, 600 U, 700 U, 800 U, 900 U, or 1000 U.

[0113] In one embodiment, the insulin-Fc conjugate provided herein is administered once a week.

[0114] For example, the insulin analog provided herein or the insulin-Fc conjugate provided herein is administered once a week in an amount of 0.01 to 100 U / kg body weight, such as 1 to 20 U / kg body weight, such as 1 to 10 U / kg body weight.

[0115] As used herein, the terms "subject" and "patient" are used interchangeably. A "subject" or "patient" may be a vertebrate. The term includes both humans and other animals, particularly mammals, and other organisms. Thus, as used herein, a subject may be an animal such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, cow, horse, camel, primate, etc. In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human. In some embodiments, the subject is 16 years of age or older.

[0116] In some embodiments, the subject has a disease or disorder referred to herein. For example, the subject may be an obese subject. In some embodiments, the patient is at risk of having a disease or disorder referred to herein.

[0117] Typically, the term "disease or disorder" refers to any pathological or unhealthy condition that can be treated by administering the insulin or insulin-Fc conjugate, or pharmaceutical composition, provided herein, particularly diabetes (such as gestational diabetes, type 1 diabetes or type 2 diabetes) and / or hyperglycemia.

[0118] "Diabetes mellitus" (also simply referred to as "diabetes") as used herein refers to a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin production, insulin action, or both. In one embodiment, diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, and gestational diabetes. The current WHO diagnostic criteria for diabetes are a fasting plasma glucose level of 7.0 mmol / l or higher (126 mg / dL) or a 2-hour plasma glucose level of 11.1 mmol / l or higher (200 mg / dL).

[0119] In some embodiments, diabetes is type 1 diabetes. As used herein, "type 1 diabetes" refers to a condition characterized by hyperglycemia caused by a complete lack of insulin. This occurs when the body's immune system attacks and destroys the insulin-producing beta cells in the pancreas. Subsequently, the pancreas produces little or no insulin. The removal of the pancreas or a disease may also cause the loss of beta cells that produce insulin. Type 1 diabetes accounts for 5% to 10% of diabetes patients.

[0120] In some embodiments, diabetes is type 2 diabetes. As used herein, "type 2 diabetes" refers to a condition characterized in that, despite the availability of insulin, glucose is overproduced and the circulating glucose level remains excessively high as a result of insufficient glucose clearance (insulin action).

[0121] In certain embodiments, the diabetes is gestational diabetes. As used herein, "gestational diabetes" refers to a condition in which women who have not previously been diagnosed with diabetes exhibit elevated blood glucose levels during pregnancy (particularly in the third trimester). Gestational diabetes affects 3-10% of pregnancies, depending on the population studied.

[0122] As used herein, the term "hyperglycemia" means an excess of sugar (glucose) in the blood.

[0123] As used herein, the terms "administering" or "administration" when referring to a subject, means administering to the subject a compound or composition, or a combination of compounds or compositions, for preventing, ameliorating, or eliminating a disease and / or disorder referred to herein, such as diabetes. Thus, the term encompasses both treatment of an existing disease or disorder referred to herein, or prevention of a disease or disorder, i.e., prophylaxis. Thus, it will be appreciated that treatment as referred to herein may, in some embodiments, be prophylactic. In some embodiments, the term refers to treatment of an existing disease or disorder referred to herein. Thus, the subject is suffering from said disease or disorder.

[0124] First aspect: Insulin Provided herein is an insulin analog that includes an insulin B-chain corresponding to an insulin A-chain, as shown in Table B of the Examples section. Insulin analogs are typically provided as single-chain insulin.

[0125] Provided herein is an insulin analog that includes an insulin B-chain and an insulin A-chain, where the insulin B-chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 69), and the insulin A-chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0126] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTPKT (SEQ ID NO: 70), and the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0127] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence FVNQHLCGSHLVEALHLVCGERGFHYTPKT (SEQ ID NO: 71), and the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75).

[0128] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 73), and the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75).

[0129] Furthermore, an insulin analog comprising an insulin B chain and an insulin A chain is provided, wherein the insulin B chain comprises, or consists of, the amino acid sequence the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 65), and the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0130] Furthermore, there is provided an insulin analog comprising an insulin B chain and an insulin A chain, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 66), and wherein the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0131] Furthermore, there is provided an insulin analog comprising an insulin B chain and an insulin A chain, wherein the insulin B chain comprises, or consists of, the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 67), and wherein the insulin A chain comprises, or consists of, the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0132] Typically, the insulin analogs provided herein are provided as single-chain insulin. In single-chain insulin, the insulin B chain is typically linked to the insulin A chain via a first linker peptide.

[0133] Accordingly, said single-chain insulin has, from the N-terminus to the C-terminus: a) an insulin B chain; b) a first linker peptide; c) an insulin A chain and comprises.

[0134] In one embodiment, the first linker peptide has a length of 1 to 50 amino acids. In another embodiment, the first linker peptide has a length of 5 to 15 amino acids. In another embodiment, the first linker peptide has a length of 5 to 10 amino acids. In another embodiment, the first linker peptide has a length of 7 amino acids.

[0135] Typically, the first linker peptide comprises or consists of the amino acid sequence EEYPGDV (SEQ ID NO: 27). Also typically, the first linker peptide comprises or consists of the amino acid sequence GSYPGGV (SEQ ID NO: 26).

[0136] In one embodiment, the single-chain insulin comprises or consists of the following amino acid sequence: SFVNQHLCGSHLVEALELVCGERGFHYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 50).

[0137] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence: SFVNQHLCGSHLVEALHLVCGERGFAYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 52).

[0138] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence: FVNQHLCGSHLVEALHLVCGERGFHYTPKTGSYPGGVGIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 53).

[0139] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

Chemical formula

[0140] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence: SFVNQHLCGSHLVEALYLVCGERGFFYTPKTEEYPGDVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 45).

[0141] In another embodiment, the single-chain insulin comprises, or consists of, the following amino acid sequence: SFVNQHLCGSHLVEALYLVCGERGFFYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 81).

[0142] In another embodiment, the single-chain insulin comprises, or consists of, the following amino acid sequence: SFVNQHLCGSHLVEALHLVCGERGFAYTDKTEEYPGDVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 82).

[0143] In another embodiment, the single-chain insulin comprises, or consists of, the following amino acid sequence: SFVNQHLCGSHLVEALELVCGERGFHYTDKTEEYPGDVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 47).

[0144] Second aspect: Insulin Fc fusion polypeptides and their conjugates Further provided herein is an insulin-Fc fusion polypeptide comprising an insulin and an Fc region polypeptide from the N-terminus to the C-terminus.

[0145] Also provided herein are insulin-Fc conjugates of two insulin-Fc fusion polypeptides provided herein. The two insulin-Fc fusion polypeptides are assumed to form an antibody Fc region (via the Fc region).

[0146] Typically, the conjugate is a homodimer of two insulin-Fc fusion polypeptides provided herein. Thus, the two insulin-Fc fusion polypeptides typically have the same amino acid sequence.

[0147] Typically, the insulin composed of the insulin-Fc fusion polypeptide provided herein is an insulin analog with a reduced binding affinity to the human insulin receptor compared to human insulin.

[0148] Typically, the insulin-Fc fusion polypeptide comprises, from the N-terminus to the C-terminus: a) an insulin B chain, b) a first linker peptide, c) an insulin A chain, d) a second linker peptide, e) an Fc region (also referred to herein as the Fc region polypeptide), and.

[0149] In one embodiment of the insulin-Fc fusion polypeptide, the insulin is single-chain insulin, for example, any one of the single-chain insulins shown in Table B of the Examples section. Typically, the insulin comprises a single-chain insulin consisting of an insulin B chain, a first linker peptide, and an insulin A chain from the N-terminus to the C-terminus.

[0150] In one embodiment of the insulin-Fc fusion polypeptide, the single-chain insulin comprises or consists of the following amino acid sequence: SFVNQHLCGSHLVEALELVCGERGFHYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 50), SFVNQHLCGSHLVEALHLVCGERGFAYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 52), FVNQHLCGSHLVEALHLVCGERGFHYTPKTGSYPGGVGIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 53), FVNQHLCGSHLVEALELVCGERGFHYTPKTGSYPGGVGIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 58), SFVNQHLCGSHLVEALYLVCGERGFFYTPKTEEYPGDVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 45) SFVNQHLCGSHLVEALYLVCGERGFFYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 81) SFVNQHLCGSHLVEALHLVCGERGFAYTDKTEEYPGDVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 82), or SFVNQHLCGSHLVEALELVCGERGFHYTDKTEEYPGDVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 47)

[0151] Typically, single-chain insulin is linked to the Fc region polypeptide via a second linker peptide. The second linker peptide typically has a length of 1 to 100, such as 5 to 40 amino acids. In one embodiment of the insulin-Fc fusion polypeptide, the second linker is the human insulin C peptide, or a fragment thereof.

[0152] The human C peptide has the following amino acid sequence:

Chemical formula

[0153] A fragment of the human C peptide is, for example, a fragment having a length of at least 5, 10, 15, 20, or 22 amino acids of the human C peptide having the sequence as shown in SEQ ID NO: 31.

[0154] In one embodiment, the amino acids Leu26Ala27Leu28 in the C peptide (shown in bold in SEQ ID NO: 31 above) are deleted in the linker. Thus, the second linker comprises, or consists of, the amino acid sequence EAEDLQVGQVELGGGPGAGSLQPEGSLQ (SEQ ID NO: 8).

[0155] To increase the distance from the second linker to insulin, one or more amino acids may be added to the second linker, such as an N-terminal glycine residue. Thus, the second linker may comprise or consist of the amino acid sequence GEAEDLQVGQVELGGGPGAGSLQPEGSLQ (SEQ ID NO: 29).

[0156] It is also contemplated that the second linker comprises or consists of the amino acid sequence EAEDLQVGQVELGG (SEQ ID NO: 28).

[0157] In one embodiment, the Fc region present in the Fc region polypeptide of the invention is the Fc region of an IgG, IgM, IgA, IgD or IgE antibody. Thus, the Fc region polypeptide typically comprises heavy chain constant domains such as CH2 and CH3 of an IgG antibody.

[0158] In one embodiment, the Fc region is the Fc region of an IgG antibody. Thus, these two polypeptides form the Fc region of an IgG antibody. For example, the Fc region may be the Fc region of an IgG1 antibody. Alternatively, the Fc region may be the Fc region of an IgG4 antibody.

[0159] In one embodiment, the Fc region present in the fusion polypeptide provided herein does not include, i.e., lacks, an antibody hinge region located at the N-terminus of the Fc region. Thus, the two fusion polypeptides forming the conjugate are not linked by one or more disulfide bonds located at the N-terminus of the portion forming the Fc region of each fusion polypeptide.

[0160] Typically, the insulin Fc fusion polypeptide is covalently bound by at least two covalent bonds at the C-terminus of the fusion polypeptide. For example, it is linked by two covalent bonds at the C-terminus of the fusion polypeptide.

[0161] The expression "at the C-terminus of the fusion polypeptide" encompasses, for example, the 20 C-terminal amino acids of the fusion polypeptide.

[0162] In some embodiments, at least two of the covalent bonds are located at the C-terminus of the CH2, CH3, and / or CH4 domains on each fusion polypeptide.

[0163] In some embodiments, at least two of the covalent bonds are located at the C-terminus of the CH2 and / or CH3 domains on each fusion polypeptide.

[0164] In some embodiments, at least two of the covalent bonds are located at the C-terminus of the CH2 and CH3 domains on each polypeptide.

[0165] Typically, the two or more covalent bonds described above are located at the C-terminus of the portion forming the Fc region of each fusion polypeptide.

[0166] In one embodiment of the provided insulin-Fc conjugate, the covalent bond is a disulfide bond. Such a disulfide bond may be formed between the thiol groups of two cysteine residues. Accordingly, the insulin Fc fusion polypeptides referred to herein typically contain at least one cysteine residue, for example two cysteine residues. In one embodiment, the cysteine residue or cysteine residues are present within the C-terminal region of the fusion polypeptide, for example within the 20 C-terminal amino acids of the fusion polypeptide.

[0167] Typically, the two fusion polypeptides are not covalently linked by one or more disulfide bonds between the N-termini of each IgG Fc region polypeptide.

[0168] Typically, each of the first insulin-Fc fusion polypeptide and the second insulin-Fc fusion polypeptide includes an amino acid sequence (also referred to herein as the "C-terminal peptide") containing at least two cysteine residues that enable the formation of two disulfide bonds between the first fusion polypeptide and the second fusion polypeptide at the C-terminus. In this case, at least two C-terminal covalent bonds are at least two disulfide bonds within this sequence.

[0169] The C-terminal peptide may have a length of, for example, 7 to 20 amino acids, for example, 10 to 20 amino acids.

[0170] The amino acid sequence at the C-terminus of the fusion polypeptide (i.e., the C-terminal peptide) provided herein can be the amino acid sequence of a naturally occurring antibody hinge region, or a sequence highly similar to that sequence, for example, the sequence of the hinge region of an IgG1 antibody or an IgG4 antibody (or a sequence highly similar to that sequence).

[0171] The amino acid sequence of the constant region of the IgG1 heavy chain is shown in SEQ ID NO: 35. The hinge region of this antibody is at positions 99 to 110 of this sequence and has the following amino acid sequence: EPKSCDKTHTCP (SEQ ID NO: 36).

[0172] The amino acid sequence of the constant region of the IgG4 heavy chain is shown in SEQ ID NO: 38. The hinge region is at positions 99 to 110 of this sequence and has the following amino acid sequence: ESKYGPPCPSCP (SEQ ID NO: 39). Further, the hinge region is at positions 99 to 111 of this sequence and has the following amino acid sequence: ESKYGPPCPSCPA (SEQ ID NO: 40).

[0173] Therefore, the C-terminal peptide present at the C-terminus can include or be composed of an amino acid sequence as shown in SEQ ID NO: 36, 39, or 40.

[0174] However, in some embodiments, the C-terminal peptide present at the C-terminus has at least 70% identity (i.e., is highly similar) to the antibody hinge region, i.e., the sequence of the naturally occurring antibody hinge region. In some embodiments, the sequence has at least 75% identity to the sequence of the antibody hinge region. In some embodiments, the sequence has at least 80% identity to the sequence of the antibody hinge region. In some embodiments, the sequence has at least 90% identity to the sequence of the antibody hinge region. In some embodiments, the sequence comprises at least 7 contiguous amino acids of the sequence of the antibody hinge region. In some embodiments, the sequence comprises at least 10 contiguous amino acids of the sequence of the antibody hinge region. In some embodiments, the sequence comprises at least 15 contiguous amino acids of the sequence of the antibody hinge region. Typically, the antibody hinge region (to form disulfide bonds between two fusion polypeptides) contains two or more cysteine residues.

[0175] Since the sequence of the antibody hinge region can be derived from the human endogenous antibody hinge region, the use of heterologous-derived sequences can be avoided, and the risk of anti-drug antibody (ADA) formation can be reduced.

[0176] In the studies described herein, the following peptide was used as the C-terminal peptide: ESKYGPPCPPCPA (SEQ ID NO: 41). The sequence of this peptide is very similar to the hinge region of a naturally occurring antibody. Thus, the C-terminal peptide present at the C-terminus can comprise or consist of an amino acid sequence as set forth in SEQ ID NO: 41.

[0177] In one embodiment, the Fc region and the C-terminal region are linked by a third linker peptide. The third linker typically has a length of 1 to 10 amino acids, for example, a length of 6 amino acids. In an embodiment, the third linker peptide comprises or consists of a sequence as set forth in GGGGSA (SEQ ID NO: 42).

[0178] Thus, in some embodiments, the insulin-Fc fusion polypeptide provided herein comprises, from N-terminus to C-terminus: a) an insulin B chain, and b) a first linker peptide, and c) an insulin A chain, and d) a second linker peptide, and e) an Fc region, and d) a third linker peptide, and e) a C-terminal peptide. It is included.

[0179] The C-terminal peptide shall enable the formation of at least two covalent bonds, i.e., interchain covalent bonds, with at least two additional insulin-Fc fusion polypeptides (typically fusion polypeptides having the same sequence). As a result, a stable dimer is formed by at least two interchain covalent bonds at the C-terminus. In some embodiments, the at least two covalent bonds are at least two disulfide bonds. In some embodiments, the at least two covalent bonds are two disulfide bonds.

[0180] Typically, the C-terminal peptide comprises (or has a sequence highly similar to) the amino acid sequence of the antibody hinge region. In some embodiments, the C-terminal peptide has an amino acid sequence as set forth in SEQ ID NO: 36, 39, 40, or 41.

[0181] As described above, in some embodiments, the Fc region lacks the N-terminal antibody hinge region. Thus, the fusion polypeptides in the conjugate provided herein are not linked by disulfide bonds located at the N-terminus of the portion of each polypeptide forming the Fc region of the conjugate.

[0182] Thus, the two fusion polypeptides form the antibody Fc region, wherein the two polypeptides are linked by at least two covalent bonds located at the C-terminus of the portion of each polypeptide forming the Fc region, Here, the two polypeptides are not linked by a disulfide bond located at the N-terminus with respect to the portion of each polypeptide that forms the Fc region.

[0183] In one embodiment, the Fc region lacking the antibody hinge region comprises or consists of: a) The amino acid sequence shown in SEQ ID NO: 34:

Chemical formula

[0184] SEQ ID NO: 34 is the Fc region of a human IgG1 antibody (excluding the hinge and C-terminal Lys) and is used in many of the constructs described in Table A. It includes the region from position 119 to position 329 of human IgG1. The hinge and the C-terminal Lys at position 330 are not included.

[0185] In another embodiment, the Fc region lacking the antibody hinge region comprises or consists of: a) A sequence as shown in SEQ ID NO: 37, or b) A sequence that is 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%, or at least 99% identical to SEQ ID NO: 37.

[0186] In some embodiments, the Fc region present in the fusion polypeptide provided herein comprises the following amino acid sequence:

Chemical formula

[0187] The sequence containing the third linker peptide and the C-terminal hinge region was used in the Examples section. The sequence of the third linker peptide is underlined. The C-terminal hinge region is highlighted in bold.

[0188] In some embodiments, the Fc region present in the fusion polypeptides provided herein comprises the following amino acid sequence:

Chemical Formula

[0189] In some embodiments, the Fc region present in the fusion polypeptides provided herein comprises the following amino acid sequence:

Chemical Formula

[0190] Further aspects (host cells, polynucleotides, medical uses) Furthermore, provided is a polynucleotide encoding the single-chain insulin provided herein, or the insulin-Fc fusion polypeptide provided herein.

[0191] Furthermore, provided is a host cell comprising the insulin-Fc conjugate provided herein, the single-chain insulin provided herein, or the insulin-Fc fusion polypeptide provided herein, and / or the polynucleotide provided herein.

[0192] Furthermore, provided is a method for producing the insulin-Fc conjugate provided herein, the single-chain insulin provided herein, or the insulin-Fc fusion polypeptide provided herein, the method comprising incubating the host cell provided herein under conditions capable of expressing the insulin-Fc conjugate, single-chain insulin, or insulin-Fc fusion polypeptide.

[0193] Furthermore, there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of the insulin-Fc conjugate provided herein, the single-chain insulin provided herein, or the insulin-Fc fusion polypeptide provided herein.

[0194] Furthermore, there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of the insulin-Fc conjugate provided herein, the single-chain insulin provided herein, or the insulin-Fc fusion polypeptide provided herein.

[0195] Furthermore, there is provided the insulin-Fc conjugate provided herein, the insulin (such as the single-chain insulin provided herein) provided herein, or the insulin-Fc fusion polypeptide provided herein for use as a medicament.

[0196] Furthermore, there is provided the insulin-Fc conjugate provided herein, the insulin (such as the single-chain insulin provided herein) provided herein, or the insulin-Fc fusion polypeptide provided herein for use as a medicament for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes, and hyperglycemia and / or for reducing blood glucose levels.

[0197] Embodiments In the following embodiments of the conjugate provided, the fusion polypeptide and insulin are disclosed. The above definitions and descriptions herein apply mutatis mutandis to the following as well.

[0198] 1. An insulin-Fc conjugate of two insulin-Fc fusion polypeptides, wherein each of the two insulin-Fc fusion polypeptides comprises, from N-terminus to C-terminus: insulin and an Fc region.

[0199] 2. The insulin-Fc conjugate according to embodiment 1, wherein the two insulin-Fc fusion polypeptides are linked by at least two covalent bonds at the C-terminus of the fusion polypeptide, for example, wherein the at least two covalent bonds are disulfide bonds.

[0200] 3. The insulin-Fc conjugate according to embodiment 1 or 2, wherein the conjugate is a homodimer of the two insulin-Fc fusion polypeptides and / or the two insulin-Fc fusion polypeptides form an antibody Fc region.

[0201] 4. The insulin-Fc conjugate according to any one of embodiments 1 to 3, wherein the insulin is single-chain insulin.

[0202] 5. Each of the two insulin-Fc fusion polypeptides comprises, from the N-terminus to the C-terminus: a) an insulin B chain, and b) a first linker peptide, and c) an insulin A chain, and d) a second linker peptide. The insulin-Fc conjugate according to any one of embodiments 1 to 4.

[0203] 6. Each of the two insulin-Fc fusion polypeptides further comprises: f) a third linker peptide, and g) a C-terminal peptide that enables the formation of the at least two covalent bonds between the two insulin-Fc fusion polypeptides. The insulin-Fc conjugate according to embodiment 5.

[0204] 7. The C-terminal peptide of g) contains at least two cysteine residues (e.g., two cysteine residues) that enable the formation of at least two disulfide bonds between the two insulin-Fc fusion polypeptides. For example, the C-terminal peptide contains the amino acid sequence (or a very similar sequence) of an antibody hinge region that contains at least two cysteine residues enabling the formation of at least two disulfide bonds between the two insulin-Fc fusion polypeptides. The insulin-Fc conjugate according to embodiment 6.

[0205] 8. The amino acid sequence of the C-terminal peptide contains or consists of the amino acid sequence shown in SEQ ID NO: 36, 39, 40, or 41. The insulin-Fc conjugate according to embodiment 7.

[0206] 9. The two insulin-Fc fusion polypeptides are not covalently linked by one or more disulfide bonds between the N-termini of each Fc region polypeptide. The insulin-Fc conjugate according to any one of embodiments 1 to 8.

[0207] 10. The Fc region polypeptide lacks the N-terminal antibody hinge region. The insulin-Fc conjugate according to any one of embodiments 1 to 9.

[0208] 11. The Fc region polypeptide contains the constant region of an IgG, IgM, IgA, IgD, or IgE antibody heavy chain. The insulin-Fc conjugate according to any one of embodiments 1 to 10.

[0209] 12. The Fc region polypeptide contains the CH2 and CH3 of the constant region of an IgG1 antibody or IgG4 antibody heavy chain. The insulin-Fc conjugate according to embodiment 12.

[0210] 13. The first linker peptide has a length of 1 to 50 amino acids, such as 5 to 15 amino acids, such as 5 to 10 amino acids, such as 7 amino acids. The insulin-Fc conjugate according to any one of embodiments 1 to 12.

[0211] 14. The insulin-Fc conjugate according to embodiment 13, wherein the first linker peptide comprises or consists of the amino acid sequence GSYPGGV (SEQ ID NO: 26) or EEYPGDV (SEQ ID NO: 27).

[0212] 15. The insulin-Fc conjugate according to any one of embodiments 1 to 14, wherein the second linker peptide has a length of 1 to 100 amino acids, for example 5 to 40 amino acids.

[0213] 16. The insulin-Fc conjugate according to embodiment 15, wherein the second linker peptide is the human insulin C peptide or a fragment thereof.

[0214] 17. The insulin-Fc conjugate according to embodiment 16, wherein the second linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 28 or 29.

[0215] 18. The insulin-Fc conjugate according to any one of embodiments 1 to 17, wherein the third linker has a length of 1 to 10 amino acids, for example 6 amino acids.

[0216] 19. The insulin-Fc conjugate according to embodiment 18, wherein the third linker peptide comprises or consists of the amino acid sequence shown in GGGGSA (SEQ ID NO: 42).

[0217] 20. The insulin-Fc conjugate according to any one of embodiments 1 to 19, wherein the insulin has a reduced binding affinity for the human insulin receptor as compared to the binding affinity of human insulin for the human insulin receptor.

[0218] 21. The insulin-Fc conjugate according to any one of embodiments 1 to 20, wherein the insulin is the insulin shown in Table B, for example, the single-chain insulin shown in Table B.

[0219] 22. An insulin-Fc conjugate, wherein i) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 69), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), ii) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTPKT (SEQ ID NO: 70), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), iii) the insulin B chain comprises or consists of the amino acid sequence FVNQHLCGSHLVEALHLVCGERGFHYTPKT (SEQ ID NO: 71), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75), iv) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 73), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75), v) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 65), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), vi) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 66), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), or vii) The insulin-Fc conjugate according to embodiment 21, wherein the insulin B chain contains or consists of the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 67), and the insulin A chain contains or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0220] 23. The insulin-Fc conjugate according to embodiment 21 or 22, wherein the insulin is a single-chain insulin containing the amino acid sequence shown in SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 81, or SEQ ID NO: 82.

[0221] 24. The insulin-Fc conjugate according to any one of embodiments 1 to 23, wherein each of the two insulin-Fc fusion polypeptides contains the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0222] 25. Insulin according to the definition of the insulin in embodiment 22 or 23, for example, the single-chain insulin according to the definition of the insulin in embodiment 23.

[0223] 26. An insulin-Fc fusion polypeptide according to the definition of the insulin-Fc fusion polypeptide according to any one of embodiments 1 to 24.

[0224] 27. A pharmaceutical composition comprising the insulin-Fc conjugate according to any one of embodiments 1 to 24, the insulin according to embodiment 25, or the insulin-Fc fusion polypeptide according to embodiment 26 in a pharmaceutically effective amount.

[0225] 28. The insulin-Fc conjugate according to any one of embodiments 1 to 24, the insulin according to embodiment 25, or the insulin-Fc fusion polypeptide according to embodiment 26 for use as a medicament.

[0226] Use as a medicament for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes, and hyperglycemia, and / or for lowering blood glucose levels, of an insulin-Fc conjugate according to any one of Embodiments 1 to 24, the insulin according to Embodiment 25, or the insulin-Fc fusion polypeptide according to Embodiment 26.

[0227] 30. A polynucleotide encoding the insulin of Embodiment 25 or the insulin-Fc fusion polypeptide of Embodiment 26.

[0228] 31. A host cell comprising an insulin-Fc conjugate according to any one of Embodiments 1 to 24, the insulin according to Embodiment 25, or the insulin-Fc fusion polypeptide according to Embodiment 26, and / or the polynucleotide according to Embodiment 20.

[0229] 32. A method for producing an insulin-Fc conjugate according to any one of Embodiments 1 to 24, the insulin according to Embodiment 25, or the insulin-Fc fusion polypeptide according to Embodiment 26, the method comprising incubating a host cell according to Embodiment 21 under conditions capable of expressing the insulin-Fc conjugate according to any one of Embodiments 1 to 24, the insulin according to Embodiment 25, or the insulin-Fc fusion polypeptide according to Embodiment 26.

Example

[0230] Method: Expression and purification of protein The scI-Fc proteins INS001 to INS027 (SEQ ID NOs: 2 to 25, 77 to 79, see also Table A) were fused to a leader sequence (SEQ ID NO: 1) that directs protein expression into the culture supernatant. The scI-Fc proteins were produced by transient transfection into HEK293 cells or CHO cells. The proteins were purified from the culture supernatant using Protein A (mab select sure, GE Healthcare) affinity chromatography. After Protein A purification, the scI-Fc proteins were further purified using a gel filtration column (Superdex 200, GE healthcare) equilibrated with phosphate buffered saline (PBS, Gibco). Fractions containing the protein of interest were collected, concentrated, pooled, and stored at -80 °C until further use.

[0231] In vitro assay of insulin receptor phosphorylation The generated scI-Fc protein was subjected to an insulin receptor phosphorylation assay. Receptor phosphorylation (also referred to herein as "autophosphorylation") was measured basically according to the protocol described by Sommerfeld et al. (Sommerfeld et al., 2010). Using intracellular Western technology, CHO cells expressing the corresponding insulin receptor were used. The CHO-IR cells were cultured in Ham’s F12-Nutrient Mix / GlutaMax medium (Gibco / Thermo Fisher scientific) containing 10% (v / v) fetal calf serum (PAN-Biotech), 1× non-essential amino acids (Gibco / Thermo Fisher scientific), and appropriate antibiotics for selection. To measure receptor phosphorylation after insulin stimulation, the cells were seeded in 96-well plates and cultured for 44 hours. The cells were serum-starved in serum-free Ham’s Nutrient Mixture F12 medium for 2 hours, and then the concentration of insulin or scI-Fc variant was increased and the cells were treated at 37 °C for 20 minutes. After incubation, the medium was discarded and the cells were fixed with 3.75% fresh paraformaldehyde for 20 minutes. The cells were permeabilized with 0.1% Triton X-100 in PBS (Gibco) for 20 minutes and then blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 hour at ambient temperature. Anti-phosphotyrosine antibody 4G10 (Millipore) was used as the primary antibody. After incubation for 2 hours at ambient temperature, the cells were washed 3 times with PS + 0.1% Tween 20. Secondary anti-mouse IgG G-800-CW and the DNA staining agent DRAQ5 (Cell Signaling) necessary for normalization of the cell number were added and incubated for 1 hour. The cells were washed 3 times with PBS + 0.1% Tween 20, and the fluorescence signals at 700 nm and 800 nm were measured with a LI-COR infrared imaging system (Odyssey, LI-COR Biosciences). The data were obtained in relative fluorescence units (RFU).

[0232] The single-chain insulin contained in the construct shown in Table A was used as the test subject. Since human insulin (B chain: SEQ ID NO: 32, A chain: SEQ ID NO: 33) was used as a control, the sequence of the single-chain insulin is also shown in Table B.

[0233]

Table 1

[0234]

Table 2

[0235]

Table 3

[0236]

Table 4

[0237]

Table 5

[0238]

Table 6

[0239]

Table 7

[0240]

Table 8

[0241]

Table 9

[0242]

Table 10

[0243]

Table 11

[0244]

Table 12

[0245]

Table 13

[0246] PK / PD of Rats In Vivo Pharmacodynamic data were collected basically as described by Faust et al. (Faust et al., 2020). Eight male Sprague-Dawley rats (Charles River) with a body weight of 320 - 400 g at the start of the test were randomly assigned to each treatment group and allowed free access to tap water and food. The protein was dissolved in PBS (Gibco) and administered subcutaneously. Only PBS was used as the placebo control. Blood glucose levels were measured using whole blood collected from the tip of the tail at the specified time points. The blood was lysed with hemolysate (Hemolysis Reagent H, Glucose / Hexokinase Solution 5 + 1, Hengler analytic), and quantified using the Gluco-quant Glucose / Hexokinase kit (Roche Dagnostics) on a Beckman Coulter AU640 chemical analyzer.

[0247] PK / PD of Miniature Pigs In Vivo Healthy female Göttingen Miniature Pigs (Ellegaard Goettingen Minipigs) (15 - 18 months old, body weight ~20 - 25 kg) were used to evaluate the pharmacodynamic and pharmacokinetic effects of the long-acting scI-Fc protein. The animals were housed in a standard breeding environment and fed once a day, with free access to tap water.

[0248] Animals that had been fasted overnight were injected subcutaneously once with placebo (PBS) or the corresponding scI-Fc variant. Blood sampling was performed using a previously indwelling central venous catheter. The first blood sample was collected before the first administration and used as the baseline. Blood was collected at predetermined time points (1 - 4 times per day during the test period, using K-EDTA-containing sample tubes), immediately stored on ice, and after centrifugation, the plasma was stored at -80 °C until further use. Blood glucose was quantified from lysed samples (250 μl of hemolysis reagent (Hengler Analytik) added to 5 μl of capillary blood) using an Olympus AU680 autoanalyzer device (Beckman Coulter) with an enzymatic UV test (hexokinase method) (Beckman Coulter). All animals were observed regularly, and clinical signs were recorded at least twice a day (on treatment days) and once a day (for the remaining test time). The animals were carefully monitored for clinical signs of hypoglycemia, such as behavior, coat, urine and feces excretion, the condition of body orifices, and signs of illness. In cases of severe hypoglycemia, food was given or glucose solution was administered intravenously as needed.

[0249] PK / PD of Cynomolgus Monkeys In Vivo In the monkey experiment, male cynomolgus monkeys (Macaca fascicularis) (Nveprim Ltd, Mauritius) were acclimatized for at least 4 weeks after arrival. The monkeys were fed 200 g of NHP pellets (Ssniff, Germany) per day and were allowed to freely consume food and tap water using an automatic waterer. The animals were group-housed in cages suitable for their size and species, with a space of 260×200×220 cm. The monkeys were housed under environmental conditions (temperature 20 - 24°C, relative humidity 40 - 75%) on a 12-hour / 12-hour light / dark cycle. The monkeys were not fasted on the night before compound administration. Intravenous administration was performed on the saphenous vein using a Venoflux microinfuser (23G). After injection, the solution remaining in the microinfuser was flushed with 0.9% NaCl. Administration of the Sc compound was performed directly at the site within the scapula using a 26G needle. Blood was collected from the saphenous vein using a 2-ml syringe equipped with a 21G needle. The animals were observed at each blood sampling time point at least once a day. No side effects were observed during the experimental period. Glucose concentration was measured from whole blood immediately after blood sampling using an Accu-Check Active blood glucose meter (Roche Diagnostic). Hypoglycemia was not observed.

[0250] Detection of scI-Fc in plasma using LC-MS / MS 50 μL of plasma sample was diluted with 250 μL of phosphate-buffered saline (PBS) containing IS (500 ng / mL) and immunoconcentrated on a Thermo MSIA (trademark) platform using an MSIA Streptavidin D.A.R.T.S. chip. The analyte and insulin standard (IS) were eluted with 65 μL of water / acetonitrile / TFA (66 / 33 / 0.4 v / v / v). Then, 100 μL of digestion buffer (100 mM ammonium bicarbonate, pH 8.5), 2 μL of 0.1 N sodium hydroxide, and 3 μL of dithiothreitol (DDT, 500 mM, in digestion buffer) were added to the tube and incubated on a thermomixer at 60 °C and 500 rpm for 0.5 h. Then, 5 μL of iodoacetamide (IAA, 500 mM, in digestion buffer) was added to the tube, mixed, and left standing in the dark at room temperature and 500 rpm for 45 min. Then, 2 μL of DDT (dithiothreitol, 500 mM, in digestion buffer) was added to the tube and incubated at room temperature and 500 rpm for 0.5 h. 10 μL of 100 μg / mL of TCPK-trypsin (Thermo) in digestion buffer was added to the tube and digested on a ThermoMixer at 500 rpm and 37 °C for 1.5 h. 10 μL of 10% formic acid dissolved in water was added to stop the reaction, mixed, and analyzed by LC-MS / MS.

[0251] Detection of scI-Fc in plasma by ELISA Plates of MaxiSorp flat-bottom plates (Nunc) were coated with anti-His-Tag monoclonal antibody (Novagen) at 1 μg / mL overnight at +4°C. After washing three times with PBS / 0.05% Tween-20, they were blocked with 150 μL of 1% powdered milk for 1 hour at room temperature (RT), and then washed three times with PBS / 0.05% Tween-20. 50 μL of recombinant insulin receptor (R&D Systems), which is a capture tool, was applied at 1.0 μg / mL, 50 μL / well in PBS / 0.05% Tween-20, and incubated at RT for 1 hour with shaking at 600 rpm. The plates were first washed three times with PBS / 0.05% Tween-20. Then, specimens diluted with MRD (including standards, QC, and PK specimens) were loaded onto the plates, and the plates were incubated at room temperature for 1.5 hours. Then, the plates were washed three times with PBS / 0.05% Tween-20, and 100 μL of a 1:20,000 dilution of HRP-conjugated detection antibody (mouse anti-human IgG-Fc, Southern Biotech) in PBS / 0.05% Tween-20 was applied at 100 μL / well for 1 hour with shaking at 600 rpm to form a complex of the detection tool and scI. The plates were washed three times with PBS / 0.05% Tween-20, incubated with 100 μL of TMB for 20 minutes, and then 100 μL of stop solution (0.3 M HCl) was added. The OD signal was obtained at 450 nm using the Infinity 1000 series of the Tecan plate reader.

[0252]

Table 14

[0253]

Table 15

[0254]

Table 16

[0255]

Table 17

[0256] References Belfiore A, Malaguarnera R, Vella V, et al. Insulin Receptor Isoforms in Physiology and Disease: An Updated View. Endocr Rev. 2017;38(5):379 - 431. doi:10.1210 / er.2017 - 00073 Bright DJ, Dunseath GJ, Peter R, Luzio S. Stability of proinsulin in whole blood. Clin Biochem. 2018;52:153 - 155. doi:10.1016 / j.clinbiochem.2017.10.005 Bruhns P, Joensson F. Mouse and human FcR effector functions. Immunol Rev. 2015;268(1):25 - 51. doi:10.1111 / imr.12350 Denley A, Carroll JM, Brierley GV, et al. Differential activation of insulin receptor substrates 1 and 2 by insulin - like growth factor - activated insulin receptors [published correction appears in Mol Cell Biol. 2007 Sep;27(17):6264]. Mol Cell Biol. 2007;27(10):3569 - 3577. doi:10.1128 / MCB.01447 - 06 Dodson G, Steiner D. The role of assembly in insulin’s biosynthesis. Curr Opin Struct Biol. 1998;8(2):189 - 194. doi:10.1016 / s0959 - 440x(98)80037 - 7 Dumet C, Pottier J, Gouilleux-Gruart V, Watier H. Insights into the IgG heavy chain engineering patent landscape as applied to IgG4 antibody development [published correction appears in MAbs. 2019 Nov-Dec;11(8):1]. MAbs. 2019;11(8):1341-1350. doi:10.1080 / 19420862.2019.1664365 Faust C, Ochs C, Korn M, et al. Production of a novel heterodimeric two-chain insulin-Fc fusion protein. Protein Eng Des Sel. 2020;33:gzaa026. doi:10.1093 / protein / gzaa026 Glidden MD, Aldabbagh K, Phillips NB, et al. An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein. J Biol Chem. 2018;293(1):47-68. doi:10.1074 / jbc.M117.808626 Godoy-Matos AF, Silva Junior WS, Valerio CM. NAFLD as a continuum: from obesity to metabolic syndrome and diabetes. Diabetol Metab Syndr. 2020;12:60. Published 2020 Jul 14. doi:10.1186 / s13098-020-00570-y Hakuno F, Takahashi SI. IGF1 receptor signaling pathways. J Mol Endocrinol. 2018;61(1):T69-T86. doi:10.1530 / JME-17-0311 Hernandez-Sanchez C, Mansilla A, de la Rosa EJ, de Pablo F. Proinsulin in development: New roles for an ancient prohormone. Diabetologia. 2006;49(6):1142-1150. doi:10.1007 / s00125-006-0232-5 Hua QX, Hu SQ, Jia W, et al. Mini-proinsulin and mini-IGF-I: homologous protein sequences encoding non-homologous structures. J Mol Biol. 1998;277(1):103-118. doi:10.1006 / jmbi.1997.1574 Jacobsen FW, Stevenson R, Li C, et al. Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability. J Biol Chem. 2017;292(5):1865-1875. doi:10.1074 / jbc.M116.748525 Jehle PM, Lutz MP, Fussgaenger RD. High affinity binding sites for proinsulin in human IM-9 lymphoblasts. Diabetologia. 1996;39(4):421-432. doi:10.1007 / BF00400673 Kahn SE, Cooper ME, Del Prato S. Pathophysiology and treatment of type 2 diabetes: perspectives on the past, present, and future. Lancet. 2014;383(9922):1068-1083. doi:10.1016 / S0140-6736(13)62154-6 Landreh M, Johansson J, Joernvall H. C-peptide: a molecule balancing insulin states in secretion and diabetes-associated depository conditions. Horm Metab Res. 2013;45(11):769-773. doi:10.1055 / s-0033-1347208 Mathieu C. Minimising hypoglycaemia in the real world: the challenge of insulin [published online ahead of print, 2021 Jan 16]. Diabetologia. 2021;10.1007 / s00125-020-05354-7. doi:10.1007 / s00125-020-05354-7 Munte CE, Vilela L, Kalbitzer HR, Garratt RC. Solution structure of human proinsulin C-peptide. FEBS J. 2005;272(16):4284-4293. doi:10.1111 / j.1742-4658.2005.04843.x Rath T, Baker K, Dumont JA, et al. Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics. Crit Rev Biotechnol. 2015;35(2):235-254. doi:10.3109 / 07388551.2013.834293 Rosenstock J, Bajaj HS, Janez A, et al. Once-Weekly Insulin for Type 2 Diabetes without Previous Insulin Treatment. N Engl J Med. 2020;383(22):2107-2116. doi:10.1056 / NEJMoa2022474 Saltiel AR. Insulin signaling in health and disease. J Clin Invest. 2021;131(1):e142241. doi:10.1172 / JCI142241 Sand KM, Bern M, Nilsen J, Noordzij HT, Sandlie I, Andersen JT. Unraveling the Interaction between FcRn and Albumin: Opportunities for Design of Albumin-Based Therapeutics. Front Immunol. 2015;5:682. Published 2015 Jan 26. doi:10.3389 / fimmu.2014.00682 Sapra A, Bhandari P. Diabetes Mellitus. In: StatPearls. Treasure Island(FL): StatPearls Publishing; November 19, 2020. Schlothauer T, Herter S, Koller CF, et al. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng Des Sel. 2016;29(10):457-466. doi:10.1093 / protein / gzw040 Sharma AK, Taneja G, Kumar A, et al. Insulin analogs: Glimpse on contemporary facts and future prospective. Life Sci. 2019;219:90 - 99. doi:10.1016 / j.lfs.2019.01.011 Sommerfeld MR, Mueller G, Tschank G, et al. In vitro metabolic and mitogenic signaling of insulin glargine and its metabolites. PLoS One. 2010;5(3):e9540. Published 2010 Mar 4. doi:10.1371 / journal.pone.0009540 Steiner DF. On the discovery of precursor processing. Methods Mol Biol. 2011;768:3 - 11. doi:10.1007 / 978 - 1 - 61779 - 204 - 5_1 Taylor SI, Yazdi ZS, Beitelshees AL. Pharmacological treatment of hyperglycemia in type 2 diabetes. J Clin Invest. 2021;131(2):142243. doi:10.1172 / JCI142243 Wang X, Mathieu M, Brezski RJ. IgG Fc engineering to modulate antibody effector functions. Protein Cell. 2018;9(1):63 - 73. doi:10.1007 / s13238 - 017 - 0473 - 8 West EE, Kolev M, Kemper C. Complement and the Regulation of T Cell Responses. Annu Rev Immunol. 2018;36:309 - 338. doi:10.1146 / annurev - immunol - 042617 - 053245 Yang Y, Hua QX, Liu J, et al. Solution structure of proinsulin: connecting domain flexibility and prohormone processing. J Biol Chem. 2010;285(11):7847-7851. doi:10.1074 / jbc.C109.084921

Claims

1. An insulin-Fc conjugate that is a conjugate of two insulin-Fc fusion polypeptides, wherein each of the two insulin-Fc fusion polypeptides comprises, from the N-terminus to the C-terminus: insulin and an Fc region polypeptide, and wherein the two insulin-Fc fusion polypeptides are linked by at least two covalent bonds at the C-terminus of the fusion polypeptide, an insulin-Fc conjugate.

2. The insulin-Fc conjugate according to claim 1, wherein the at least two covalent bonds are disulfide bonds.

3. The insulin-Fc conjugate according to claim 1 or 2, wherein the conjugate is a homodimer of the two insulin-Fc fusion polypeptides, and / or the two insulin-Fc fusion polypeptides form an antibody Fc region.

4. The insulin-Fc conjugate according to any one of claims 1 to 3, wherein the insulin is single-chain insulin.

5. Each of the two insulin-Fc fusion polypeptides comprises, from the N-terminus to the C-terminus: a) an insulin B chain, b) a first linker peptide, c) an insulin A chain, and d) a second linker peptide, the insulin-Fc conjugate according to any one of claims 1 to 4.

6. Each of the two insulin-Fc fusion polypeptides further comprises: f) a third linker peptide, and g) a C-terminal peptide that enables the formation of the at least two covalent bonds between the two insulin-Fc fusion polypeptides, the insulin-Fc conjugate according to claim 5.

7. The C-terminal peptide of g) comprises a sequence of an antibody hinge region comprising at least two cysteine residues that enable the formation of at least two disulfide bonds between the two insulin-Fc fusion polypeptides. For example, the sequence of the antibody hinge region comprises or consists of the amino acid sequence shown in SEQ ID NO: 36, 39, 40 or 41, the insulin-Fc conjugate according to claim 6.

8. The two insulin-Fc fusion polypeptides are not covalently linked by one or more disulfide bonds between the N-termini of each Fc region polypeptide, and / or the Fc region polypeptide lacks an antibody hinge region at the N-terminus, the insulin-Fc conjugate according to any one of claims 1 to 7.

9. The Fc region polypeptide includes the constant region of the heavy chain of an IgG, IgM, IgA, IgD, or IgE antibody. For example, the Fc region polypeptide includes the constant domains CH2 and CH3 of the heavy chain of an IgG1 antibody or an IgG4 antibody. The insulin-Fc conjugate according to any one of claims 1 to 8

10. An insulin-Fc conjugate according to any one of claims 1 to 9, wherein the first linker peptide has a length of 1 to 50 amino acids, such as 5 to 15 amino acids, such as 5 to 10 amino acids, such as 7 amino acids. For example, the first linker peptide includes or consists of the amino acid sequence GSYPGGV (SEQ ID NO: 26) or EEYPEGDV (SEQ ID NO: 27), and / or the second linker peptide has a length of 1 to 100 amino acids, such as 5 to 40 amino acids. For example, the second linker peptide is the human insulin C peptide or a fragment thereof. For example, the second linker includes or consists of the amino acid sequence shown in SEQ ID NO: 28 or 29, and / or the third linker has a length of 1 to 10 amino acids, such as 6 amino acids. For example, the third linker peptide includes or consists of the amino acid sequence shown in GGGGSAA (SEQ ID NO: 42). An insulin-Fc conjugate

11. The insulin-Fc conjugate according to any one of claims 1 to 10, wherein the insulin has a reduced binding affinity to the human insulin receptor as compared to the binding affinity of to insulin to the human insulin receptor, and / or the insulin is an insulin shown in Table B, for example, a single-chain insulin shown in Table B. For example, here i) the insulin B chain includes or consists of the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 69), and the insulin A chain includes or consists of the amino acid sequence GIVEQCCTSICSLENYCG (SEQ ID NO: 74), or ii) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTPKT (SEQ ID NO: 70), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), iii) the insulin B chain comprises or consists of the amino acid sequence FVNQHLCGSHLVEALHLVCGERGFHYTPKT (SEQ ID NO: 71), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75), iv) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALEELVCGERGFHYTPKT (SEQ ID NO: 73), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75), v) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 65), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), vi) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 66), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), or vii) the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALHLVCGERGFAYTDKT (SEQ ID NO: 67), and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74), an insulin-Fc conjugate.

12. The insulin-Fc conjugate according to claim 11, wherein the insulin is a single-chain insulin comprising the amino acid sequence shown in SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 81, or SEQ ID NO:

82.

13. The insulin-Fc conjugate according to any one of claims 1 to 12, wherein each of the two insulin-Fc fusion polypeptides comprises the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

14. The insulin according to the definition of insulin recited in claim 11 or 12.

15. The insulin-Fc fusion polypeptide according to the definition of insulin recited in any one of claims 1 to 14.

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