Single chain insulin and Fc conjugates thereof

Abstract

Provided herein are insulin-Fc fusion polypeptides comprising a single chain insulin and an Fc region polypeptide. Further provided are conjugates 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 polypeptides. Single chain insulin having reduced binding affinity to insulin receptors is also provided. Further provided is the use of the insulin-Fc fusion polypeptide, the conjugate or the single-chain insulin in a medicament.

Description

Technical Field

[0001] Provided herein is an insulin-Fc fusion polypeptide comprising a single-chain insulin and an Fc region polypeptide. Further provided is a conjugate of two insulin-Fc fusion polypeptides, wherein the two insulin-Fc fusion polypeptides are connected 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 to the insulin receptor. Further provided is the use of the insulin-Fc fusion polypeptide, the conjugate or the 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 two main types of DM, called type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). In T1DM, the pancreatic β cells that produce insulin have been destroyed by the immune system, so T1DM can be considered an autoimmune disease. As a result, T1DM patients cannot produce insulin and are absolutely dependent on the administration of insulin. T2DM is a complex metabolic disease that can take years to develop. T2DM is mainly associated with other health problems, such as obesity, non-alcoholic fatty liver disease, and hypertension. The combination of different conditions is also called metabolic syndrome. In T2DM, the pancreas is still able to 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 sugar. Blood sugar levels are sensed by beta cells in the pancreas, and when a certain threshold is reached, insulin is secreted into the circulation. Circulating insulin causes glucose in the blood to be taken up, primarily into the liver, muscle, and adipose tissue. The absence of insulin or insulin resistance leads to elevated blood sugar levels (hyperglycemia), which, when untreated over a prolonged period of time, can lead to abnormalities in the microvasculature and macrovasculature, ultimately causing organ dysfunction. The organs most susceptible to microvasculature and macrovasculature are the kidneys, eyes, nerves, and heart (Kahn et al., 2014; Taylor et al., 2021).

[0004] Insulin receptor (IR) is a heterotetramer composed of two extracellular α subunits and two transmembrane β subunits. α subunit and β subunit are connected by disulfide bonds. There is a tyrosine kinase domain in the intracellular site. After insulin binding, IR dimerizes and brings the intracellular tyrosine kinase domain into close proximity, which allows autophosphorylation and intracellular signal transduction cascade to start. There are two IR isoforms in humans, called IR-A and IR-B. The difference between these isoforms is the extension of the C-terminal 12 amino acids of the A subunit in the IR-B isoform. The two IR isoforms are different in their expression patterns and in the binding parameters for insulin and 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 primarily expressed at high levels in insulin target tissues and is thought to mediate metabolic effects and cell growth, whereas IR-A is thought to promote cell proliferation (Belfiore et al., 2017).

[0005] Insulin-like growth factors (IGFs) have a similar structure to insulin. Their biological function is to induce growth or cell differentiation. IGF signaling is mediated by the IGF-1 receptor, which also has a similar structure to the insulin receptor. Due to the structural similarities between the ligand and receptor, IGF1 and IGF2 can actually bind to and activate the IR, albeit at higher concentrations. Insulin can bind to and activate the IGF-1 receptor in the same way. The crosstalk between these signaling pathways is further entangled by the possibility that IGF-1R and IR form functional heterodimers (Denley et al., 2007; Hakuno et al., 2018).

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

[0007] Compared to mature insulin, proinsulin is a single-chain protein. Proinsulin can fold into a three-dimensional structure, although the C-peptide and the junctions with the A and B chains are flexible (Yang et al., 2010). Proinsulin has biological activity in cell assays (Jehle et al., 1996) and is stable in human blood (Bright et al., 2017). There is evidence that proinsulin also has physiological effects during embryonic development (Hernández-Sánchez et al., 2006).

[0008] Single-chain insulin (scI) is an insulin variant in which the insulin B chain and the insulin A chain are connected by a short peptide linker. Such variants can be easily produced in a recombinant manner and are still active depending on the length and sequence of the linker. Alternatively, the insulin a chain and the b chain can be connected by a chemical linker. The advantage of scI over natural insulin is that its stability is improved (Hua et al., 1998; Glidden et al., 2018). These features make single-chain insulin attractive as an alternative insulin substitute.

[0009] The goal of insulin replacement therapy is to mimic the natural insulin secretion curve. For human insulin, the onset of action is about 30 minutes after administration, and the action can last for more than 5 hours. If administered in the afternoon, these characteristics may lead to nocturnal hypoglycemia; therefore, patients are urged to eat a small snack at bedtime to avoid hypoglycemia. In order to allow more precise control of insulin action, different insulin formulations and insulin analogs with different pharmacokinetic properties have been developed. Insulin analogs can be classified as rapid-acting, short-acting, intermediate-acting or long-acting. Rapid-acting and short-acting insulin analogs (such as insulin lispro, insulin aspart, insulin glulisine) can be administered shortly before or even after a meal. Using long-acting or basal insulin analogs (such as insulin detemir, insulin degludec, insulin glargine), a flatter and more durable insulin curve can be achieved. Long-acting insulin analogs have an action time of up to 24 hours. The results show that the use of long-acting insulin can significantly reduce the risk of nocturnal hypoglycemia (Sharma et al., 2019; Mathieu 2021). A basal insulin analog, insulin icodec, only needs to be administered once a week to be as effective as daily administration of insulin glargine in lowering blood sugar levels (Rosenstock et al., 2020). Such ultra-long-acting insulin analogs require less frequent injections and can therefore help improve patients' quality of life.

[0010] Based on this structure, long-acting insulin analogs can be divided into two groups: insulin with a changed amino acid sequence (e.g., insulin glargine) and insulin analogs with chemical modifications (e.g., insulin detemir). For long-acting insulin analogs, the mode of action can be different. Compared with natural insulin, the isoelectric point of insulin glargine shifts. As a result, insulin glargine precipitates after administration and is slowly released into the bloodstream from this reservoir. Insulin detemir is acetylated with 14 carbon fatty acids that can bind to albumin. Human albumin has a long plasma half-life of about 20 days. The recovery mechanism is the reason for this long half-life. The potential recovery mechanism uses the same approach as the recovery mechanism used by immunoglobulin G (IgG) class antibodies, and the plasma half-life of these antibodies is also about 20 days. Both albumin and IgG use a recovery mechanism based on neonatal Fc receptors (Sand et al., 2015).

[0011] IgG binds to FcRn via the Fc domain. After a peptide or protein is fused or conjugated to the Fc domain, an extended plasma half-life of the corresponding Fc fusion protein can be achieved, and several Fc fusion proteins have entered the pharmaceutical market (Rath et al., 2015).

[0012] IgG is a multifunctional protein. The normal function of antibodies is to evoke the humoral and cellular immune systems upon antigen binding. While the antigen is bound by the Fab domain, the effector functions of antibodies are mediated by the Fc domain. The receptors that mediate the physiological functions of IgG are called Fc-gamma receptors (FcyR). Binding to FcyR activates the so-called antibody-dependent cellular cytotoxicity (ADCC) response (Bruhns and 2015). Another effector function of antibodies is mediated by the protein C1q. C1q belongs to a defense mechanism called the complement system. When C1q binds to an antibody, a proteolytic cascade is triggered, the complement cascade. This cascade ultimately leads to the assembly of the so-called membrane attack complex, which penetrates 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 to treat cancer, effector functions are necessary to achieve therapeutic goals. In fact, increasing the efficacy of such antibodies can be achieved by Fc engineering to enhance the binding of antibodies to FcyR or C1q. The Fc domain is glycosylated at position N297. Glycosylation at this site is necessary to elicit full effector functions (Wang et al., 2018). On the other hand, many therapeutic antibodies and Fc fusion proteins are designed to bind specific targets or capture unwanted excess messengers (like proinflammatory cytokines). In these cases, Fc-mediated effector functions are undesirable; especially in the case of administering Fc fusion proteins over a long period of time.

[0014] For Fc fusion proteins, it is generally not intended to impair binding to FcRn. In contrast, the reduction of the effector function of the Fc domain is desirable in some cases. Several point mutations in the Fc domain have been described to reduce effector function. Glycosylation sites also belong to these sites, because deglycosylated antibodies or Fc fusion proteins show reduced effector function. For Fc fusion proteins, deglycosylated proteins can be obtained by expressing proteins in bacteria (e.g., Escherichia coli (E. coli)). However, deglycosylated variants may show reduced solubility or 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 a human IgG Fc region by using a peptide linker, and the use of such a fusion protein in treating diabetes.

[0016] There remains a need for ultra long-acting insulin analogs that require less frequent injections and thus can help improve the quality of life of diabetic patients.

[0017] In the research described herein, mutations were introduced into certain positions in the insulin B chain and A chain to generate single-chain insulin analogs with reduced binding affinity to the human insulin receptor compared to human insulin. Without being bound by theory, it is assumed that reduced affinity to the insulin receptor is beneficial for ultra-long-acting insulin because this will reduce the clearance rate of insulin derivatives bound to the insulin receptor. Due to the mitogenic properties of insulin that seek to activate the IR-A receptor, a variant with more reduced activity than IR-B compared to wild-type insulin was produced. Insulin in the form of a single-chain insulin Fc fusion polypeptide was tested. These sequences are provided in Table A of the Examples section. The results of receptor activation are shown in Table 1 of the Examples section. Examples of such insulins with reduced activity to IR-A and ultra-long activity compared to IR-B 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).

[0018] In the research described herein, the generated single-chain insulin analog was fused with the IgG Fc domain. Since the administration of insulin usually occurs over a long period of time, an effector-free Fc domain was selected. In the selected effector-free Fc domain, the hinge domain present at the N-terminal of the Fc region is missing. The missing hinge domain reduces the effector function because the binding site of the FcγR responsible for the antibody effector function and C1q is partially located in the antibody hinge region, while the binding site of FcRn and protein A is located in the more C-terminal part of the Fc region. Therefore, the removal of the hinge region eliminates the antibody effector function without compromising the desired Fc function. In addition, the two C-terminal covalent bonds of the present invention overcome the limitations observed for common hinge-free antibody variants (impaired FcRn binding, reduced stability).

[0019] SEQ ID NO:34 shows the amino acid sequence of the effector-free IgG1 Fc region in which the hinge region is missing. However, the hinge region (SEQ ID NO:41) is fused to the C-terminus of the Fc region via a linker peptide (GGGGSA, SEQ ID NO:42). The covalent connection of two Fc chains is achieved by fusing the hinge sequence to the C-terminus. Advantageously, if the sequence of the antibody hinge region is located at the C-terminus of the Fc portion, an interchain disulfide bond is also formed. Since the sequence of the antibody hinge region can be derived from the human endogenous antibody hinge region, the use of exogenous sequences can be avoided, which reduces the risk of anti-drug antibodies (ADA) formation.

[0020] The linker sequence connecting the single-chain insulin to the Fc domain ( Figure 8 The joint 2 in (2) is derived from C-peptide, which is present in natural proinsulin. C-peptide is a 30 amino acid long peptide (SEQ ID NO:31), mainly composed of small and hydrophilic amino acids. It is stable in blood and may have a chaperone-like effect on insulin (Landreh et al. 2013). The C-peptide in solution is not fully folded, nor does it have a random structure (Munte et al., 2005). It is composed of small and hydrophilic amino acids, stable in blood and structurally flexible, and one or more C-peptides are predetermined, which are derived from the C-peptide used as a joint. In order to further increase the amount of small hydrophilic amino acids in the sequence of the second joint, the amino acid Leu26Ala27Leu28 from SEQ ID NO:31 is deleted and Gly is placed at the N-terminus to obtain the joint sequence of SEQ ID NO:29. Therefore, the joint generated is, for example, used in the constructs designated as INS003 to INS025 (see Table A).

[0021] Surprisingly, the combination of insulin variants with no effector Fc domains resulted in an ultralong mode of action compared to protein constructs in which the insulin variants were fused to a commonly used silent IgG Fc backbone. This is exemplified by a single chain insulin variant having a sequence as shown in SEQ ID NO:50. When fused to a hinge-deficient no effector Fc domain (SEQ ID NO:10, INS009), a longer blood glucose lowering effect was observed compared to INS024 (SEQ ID NO:25) (see Figure 4 and 7 , and Table 2 in the Examples section). INS009 (SEQ ID NO: 10) is The blood glucose lowering activity in the minipig model lasted for at least 216 hr, which is much longer than the case of INS024 (SEQ ID NO 25) which had a blood glucose lowering activity with a duration of action of up to 120 hr in minipigs. INS024 has the hinge in the normal position.

[0022] exist In the minipig model, very long-lasting blood glucose lowering activity was also observed for variants INS013 (SEQ ID NO: 14) and INS011 (SEQ ID NO: 12), with a duration of action of up to 216 hr (INS013, SEQ ID NO: 14) ( Figure 4 ) and 264hr (INS011, SEQ ID NO: 12) (Examples section Figure 5 and Table 2 ).

[0023] Mutations introduced into the insulin sequence, peptides linking the insulin A and B chains ( Figure 8 The linker 2 in the INS011 (SEQ ID NO: 12) and the linker sequence connecting insulin to the Fc domain were accumulated into stable insulin derivatives with ultra-long effects in rat models (up to 168 hr of action time) and minipig models (up to 264 hr of action time [INS011 [SEQ ID NO: 12]). In addition, a plasma half-life of up to 69 hr was measured in cynomolgus monkeys (see Tables 3 and Fig.11 ). Summary of the invention

[0024] Provided herein are insulin analogs with reduced affinity for the insulin receptor, such as those shown in Table B of the Examples section.Further provided are ultra-long-acting insulin analogs comprising single-chain insulin (scI) fused to an Fc domain, advantageously fused to an IgG Fc domain without an N-terminal hinge region.ScI-Fc fusion proteins can be produced recombinantly without the need for further chemical modification.

[0025] First aspect: insulin analogs

[0026] Provided herein are insulin analogs 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).

[0027] Further provided are insulin analogs 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).

[0028] Further provided are insulin analogs 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).

[0029] Further provided are insulin analogs 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 GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75).

[0030] Further provided are insulin analogs comprising an insulin B chain and an insulin A chain, wherein the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 65), and wherein the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0031] Further provided are insulin analogs 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).

[0032] Further provided are insulin analogs 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).

[0033] Typically, insulin analogs are provided as single chain insulins. In single chain insulins, the insulin B chain is typically linked to the insulin A chain via a first linker peptide.

[0034] Second aspect: Insulin-Fc fusion polypeptide and conjugate of two insulin-Fc fusion polypeptides

[0035] Further provided is an insulin-Fc fusion polypeptide comprising, from N-terminus to C-terminus: insulin and an Fc region polypeptide (also referred to herein as "Fc region").

[0036] Typically, the insulin present in the fusion protein is an insulin analog with reduced binding affinity to the human insulin receptor compared to human insulin.In an embodiment, the insulin is a single chain insulin, such as a single chain insulin provided herein (see, e.g., Table B).

[0037] Typically, the single-chain insulin is linked to the Fc region polypeptide via a second linker peptide.

[0038] Thus, the insulin-Fc fusion polypeptide typically comprises, from N-terminus to C-terminus:

[0039] a) Insulin B chain,

[0040] b) a first linker peptide,

[0041] c) Insulin A chain,

[0042] d) a second linker peptide, and

[0043] e) Fc region polypeptide.

[0044] In some embodiments, the insulin-Fc fusion polypeptide further comprises

[0045] f) a third linker peptide,

[0046] g) C-terminal peptide.

[0047] The C-terminal peptide should allow for the formation of at least two covalent bonds between the two insulin-Fc fusion polypeptides, thereby stabilizing the conjugates provided herein.

[0048] Typically, the C-terminal peptide comprises at least two cysteine ​​residues allowing the formation of at least two disulfide bonds between the two insulin-Fc fusion polypeptides present in the conjugate.

[0049] In an embodiment, the C-terminal peptide comprises the amino acid sequence of an antibody hinge region (or a highly similar sequence), wherein the amino acid sequence of the antibody hinge region comprises at least two cysteine ​​residues that allow formation of at least two disulfide bonds between two insulin-Fc fusion polypeptides.

[0050] In some embodiments, the C-terminal peptide comprises or consists of an amino acid sequence as shown in SEQ ID NO:36, 39, 40 or 41.

[0051] In some embodiments, the Fc region polypeptide comprises a constant domain of the constant gene of an IgG, IgM, IgA, IgD or IgE antibody.

[0052] In some embodiments, the Fc region polypeptide comprises the constant domains CH2 and CH3 of the antibody heavy chain of an IgG antibody (eg, an IgG1 antibody or an IgG4 antibody).

[0053] In some embodiments, the Fc region polypeptide comprises or consists of:

[0054] a) the amino acid sequence shown in SEQ ID NO: 34, or

[0055] 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.

[0056] Advantageously, the Fc region polypeptide lacks an antibody hinge region at the N-terminus.Thus, the two insulin-Fc fusion polypeptides are typically not covalently linked via one or more disulfide bonds between the N-termini of each Fc region polypeptide.

[0057] In some embodiments, the insulin-Fc fusion polypeptide comprises a sequence as shown in any one of SEQ ID NOs: 2 to 25 and 77 and 78. These sequences are shown in Table A of the Examples section.

[0058] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO:4 (internal name: "INS003", see Table A).

[0059] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO: 5 (INS004).

[0060] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO: 6 (INS005).

[0061] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO: 7 (INS006).

[0062] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO: 10 (INS009),

[0063] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO: 12 (INS011),

[0064] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO: 14 (INS013),

[0065] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO: 19 (INS018).

[0066] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO:77.

[0067] In some embodiments, the insulin-Fc fusion polypeptide comprises the sequence shown in SEQ ID NO:78.

[0068] Further provided herein is an insulin-Fc conjugate comprising at least two insulin-Fc fusion polypeptides provided herein, wherein each of these insulin-Fc fusion polypeptides comprises, from N-terminus to C-terminus: insulin (such as single-chain insulin provided herein) and an Fc region polypeptide.

[0069] Typically, the conjugate is a homodimer of two insulin-Fc fusion polypeptides provided herein. Thus, the two insulin polypeptides typically have the same sequence, ie, they are identical.

[0070] In one embodiment of the provided insulin-Fc conjugates, the insulin is an insulin analog with reduced binding affinity to the human insulin receptor compared to human insulin. Typically, the insulin is an insulin provided herein (see, e.g., Table B), such as a single-chain insulin provided herein.

[0071] Typically, the conjugate is a conjugate of two insulin-Fc fusion polypeptides, wherein 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.

[0072] In embodiments, at least two covalent bonds are located at the C-termini of the CH2, CH3, and CH4 domains on each fusion polypeptide.

[0073] In alternative embodiments, at least two covalent bonds are located at the C-termini of the CH2 and CH3 domains on each fusion polypeptide.

[0074] Typically, these covalent bonds are disulfide bonds.

[0075] 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.

[0076] Typically, the first insulin-Fc fusion polypeptide and the second insulin Fc fusion polypeptide each comprise a C-terminal peptide at the C-terminus, wherein the C-terminal peptide comprises at least two cysteine ​​residues that allow the formation of two disulfide bonds between the first fusion polypeptide and the second fusion polypeptide. Typically, the C-terminal peptide comprises the amino acid sequence of the hinge region of an antibody (or a highly similar sequence).

[0077] Other aspects

[0078] Further provided are polynucleotides encoding the single-chain insulin provided herein or the insulin-Fc fusion polypeptide provided herein.

[0079] Further 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.

[0080] Further 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 allowing expression of the single-chain insulin or the insulin-Fc fusion polypeptide. The expression of the insulin-Fc fusion polypeptide can result in the formation of an insulin-Fc conjugate as defined herein.

[0081] Further 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.

[0082] Further provided is an insulin-Fc conjugate provided herein, an insulin provided herein (such as a single-chain insulin provided herein), or an insulin-Fc fusion polypeptide provided herein for use as a medicament.

[0083] Further provided is an insulin-Fc conjugate provided herein, an insulin provided herein (such as a single-chain insulin provided herein), or an insulin-Fc fusion polypeptide provided herein, for use as a medicament for treating 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Analysis of the pharmacodynamic activity (blood glucose lowering activity) of INS007 (SEQ ID NO: 8) in normoglycemic rats. Blood glucose lowering activity was observed to persist for at least 168 hr.

[0085] Figure 2 Analysis of the pharmacodynamic activity (blood glucose lowering activity) of INS009 (SEQ ID NO: 10) and INS013 (SEQ ID NO: 14) in normoglycemic rats. Blood glucose lowering activity was observed to last for at least 144 hr (INS013, SEQ ID NO: 14) and at least 168 hr (INS009, SEQ ID NO: 10).

[0086] Figure 3 Analysis of the pharmacodynamic activity (blood glucose lowering activity) of INS011 (SEQ ID NO: 12), INS018 (SEQ ID NO: 19) and INS019 (SEQ ID NO: 20) in normoglycemic rats. Blood glucose lowering activity was observed to last for at least 144 hr (INS018 [SEQ ID NO: 19], INS019 [SEQ ID NO: 20]) and at least 168 hr (INS011, SEQ ID NO: 12).

[0087] Figure 4 In healthy females Analysis of the pharmacodynamic activity (blood glucose lowering activity) of INS007 (SEQ ID NO: 08), INS009 (SEQ ID NO: 10) and INS013 (SEQ ID NO: 14) in normoglycemic Goettingen minipigs. For all variants, blood glucose lowering activity was observed for at least 216 hr. The single chain insulin of variant INS009 (SEQ ID NO: 10) is the same as that of INS024 (SEQ ID NO: 25) (see Figure 7 ). The difference between these two variants is the architecture of the Fc domain. In INS009 (SEQ ID NO: 10), a single-chain insulin (SEQ ID NO: 50) is fused to an Fc domain (SEQ ID NO: 34) that lacks a hinge at the N-terminus and contains a linker sequence (SEQ ID NO: 42) and a hinge sequence (SEQ ID NO: 41) at the C-terminus.

[0088] Figure 5 Analysis of the pharmacodynamic activity (blood glucose lowering activity) of INS011 (SEQ ID NO: 12) in normoglycemic Goettingen minipigs. Blood glucose lowering activity was observed to persist for at least 264 hr.

[0089] Figure 6 Pharmacodynamic activity (blood glucose lowering activity) analysis of INS019 (SEQ ID NO: 20) and INS020 (SEQ ID NO: 21) in normoglycemic Goettingen minipigs. For both variants, blood glucose lowering activity was observed for at least 192 hr.

[0090] Figure 7 Analysis of pharmacodynamic activity (blood glucose lowering activity) in normoglycemic Goettingen minipigs. The single-chain insulin variant is the same single-chain insulin variant as INS009 (SEQ ID NO: 10) (see Figure 4 ). The only difference is the structure of the Fc domain used. In INS024 (SEQ ID NO: 25), a single-chain insulin (SEQ ID NO: 50) is fused to the hinge domain of an IgG1-Fc domain (SEQ ID NO: 37) via a linker sequence (SEQ ID NO: 29). Blood lowering activity was observed to last for at least 120 hr.

[0091] Figure 8Schematic diagram of insulin-Fc fusion protein provided herein.A) Insulin B chain is connected to insulin A chain with joint 1. This single-chain insulin is connected to the IgG Fc domain lacking hinge region via joint 2. The hinge region is relocated to the C-terminal of the Fc domain. Two Fc chains are connected via the disulfide bridge in the C-terminal hinge.B) Schematic structure of single-chain insulin fused to the hinge region of IgG. Two Fc protein chains are connected via the disulfide bridge in the N-terminal hinge.

[0092] Fig. 9 INS011 (SEQ ID NO: 10) and INS026 (SEQ ID NO: 78) lower blood glucose time course in normoglycemic rats. Blood glucose lowering activity was observed to last for at least 180hr (INS011) and at least 168hr (INS026). In both constructs, the linker sequence of the Fc domain (SEQ ID NO: 29) and the Fc domain (SEQ ID NO: 34) was identical to the C-terminal "hinge" sequence SEQ ID NO: 42 and SEQ ID 41.

[0093] Fig.10 Time course of blood glucose lowering by INS007 (SEQ ID NO: 6) and INS027 (SEQ ID NO: 79) in normoglycemic rats. For both variants, blood glucose lowering activity was observed to last at least 168 hr.

[0094] Fig.11 Mean + SD plasma concentrations of INS009 (SEQ ID NO: 10), INS011 (SEQ ID NO: 12), and INS013 (SEQ ID NO: 14) following subcutaneous administration of a single 1 nmol / kg dose to male cynomolgus monkeys.

[0095] Fig.12 The sequence of the single-chain insulin-Fc fusion polypeptide INS009 (SEQ ID NO: 10). The fusion polypeptide comprises from the N-terminus to the C-terminus:

[0096] a) insulin B chain (SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 73)),

[0097] b) The first linker peptide ( GSYPGGV , SEQ ID NO:26),

[0098] c) Insulin A chain ( GIVEQCCTSICSLEQLENYCG , SEQ ID NO:74),

[0099] d) Second connector ( GEAEDLQVGQVELGGGPGAGSLQPEGSLQ , SEQ ID NO: 29),

[0100] e) an IgG region without the hinge in its normal position (italics, SEQ ID NO: 34),

[0101] f) the third linker peptide ( GGGGSA , SEQ ID NO:42),

[0102] g) C-terminal peptide, the amino acid sequence of the antibody hinge region comprises two cysteine ​​residues (SEQ ID NO: 41, ESKYGPPCPPCPA ).

[0103] Two single-chain insulin-Fc fusion polypeptides form an insulin Fc conjugate via the formation of two disulfide bonds between two cysteine ​​residues present in the C-terminal hinge region (see Figure 8 A).

[0104] definition

[0105] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0106] As used herein, the term "comprising" should be interpreted as specifying the presence of the features, integers, steps or components mentioned, but not excluding the presence or addition of one or more features, integers, steps or components or groups thereof. Thus, for example, a protein comprising a sequence of amino acids may comprise more amino acids than the actually cited protein, i.e. be embedded in a larger protein.

[0107] As used herein, a "polypeptide" is a chain of 10 or more amino acids covalently linked by peptide bonds. Thus, the term "polypeptide" can represent a chain of a multi-chain protein, regardless of the length of such chain. In some embodiments, a polypeptide is a chain of a multi-chain protein.

[0108] As used herein, a "linker" or "linker peptide" is a short and flexible amino acid sequence that connects two regions of a molecule. For example, a linker can connect the B chain of insulin to the A chain. This linker is referred to herein as the "first linker peptide". In addition, a linker can connect a single-chain insulin to the Fc region. This linker is referred to herein as the "second linker peptide". In addition, a linker peptide can connect the Fc region of a molecule comprising at least two interchain covalent bonds to the C-terminal region. This linker is referred to herein as the "third linker peptide".

[0109] As used herein, the expression "insulin analogue" refers to a peptide having a molecular structure that is formally derivable from the structure of naturally occurring insulin (also referred to herein as "parent insulin", such as human insulin).

[0110] As used herein, the expression "parent insulin" refers to naturally occurring insulin, i.e., to wild-type insulin that has not mutated. In some embodiments, the parent insulin is an animal insulin, such as a mammalian insulin. For example, the parent insulin can be human insulin, porcine insulin, or bovine insulin. Typically, the parent insulin is human insulin.

[0111] The human insulin B chain contains the following sequence:

[0112] FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO:32)

[0113] The A chain of human insulin contains the following sequence:

[0114] GIVEQCCTSICSLYQLENYCN(SEQ ID NO:33)

[0115] In an embodiment, the insulin analog provided herein or the insulin analog included in 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 connected to the insulin A chain via a connecting peptide (referred to herein as the first linker peptide).

[0116] Typically, relative to the parent insulin, the insulin analogs provided herein or the insulin analogs included in the insulin-Fc fusion polypeptides provided herein include at least one mutation (amino acid substitution, deletion or addition). As used herein, the term "at least one" means one or more than one, such as "at least two", "at least three", "at least four", "at least five", etc. In some embodiments, the insulin analogs provided herein include at least one mutation in the B chain and at least one mutation in the A chain. In another embodiment, the insulin analogs provided herein include at least two mutations in the B chain and at least one mutation in the A chain.

[0117] Typically, the insulin analog provided herein or the insulin analog included in the insulin-Fc fusion polypeptide provided herein comprises two peptide chains, i.e., chain A and chain B. Typically, the two chains are connected by a disulfide bridge between the cysteine ​​residues. For example, in at least one embodiment, human insulin analogs comprise three disulfide bridges: a disulfide bridge between the cysteine ​​at positions A6 and A11, a disulfide bridge between the cysteine ​​at position A7 of chain A and the cysteine ​​at position B7 of chain B, and a disulfide bridge between the cysteine ​​at position A20 of chain A and the cysteine ​​at position B19 of chain B.

[0118] Typically, the insulin receptor binding affinity of the insulin analogs provided herein or the insulin analogs included in the insulin-Fc fusion polypeptides provided herein is reduced compared to the insulin receptor binding affinity of the corresponding parent insulin (e.g., human insulin). Therefore, the insulin analogs have a very low clearance rate, i.e., a very low insulin receptor-mediated clearance rate.

[0119] The insulin receptor can be any mammalian insulin receptor, such as a bovine, porcine or human insulin receptor. In some embodiments, the insulin receptor is a human insulin receptor, such as a human insulin receptor isoform A or a human insulin receptor isoform B.

[0120] Typically, compared to human insulin, the insulin analogs provided herein or the insulin analogs included in the insulin-Fc fusion polypeptides provided herein have a binding affinity for human insulin receptor isoform B of less than 25%, such as less than 15%. In embodiments, the insulin analogs have a binding affinity for human insulin receptor isoform B of 10%-25% compared to human insulin.

[0121] Typically, compared to human insulin, the insulin analogs provided herein or the insulin-Fc fusion polypeptides provided herein comprise an insulin analog for human insulin receptor isoform a with a binding affinity of less than 15%, such as less than 8%. In an embodiment, compared to human insulin, the insulin analog has a binding affinity of 1%-15%, such as 3%-10% for human insulin receptor isoform A.

[0122] Methods for determining the binding affinity of insulin analogs to insulin receptors are well known in the art. For example, insulin receptor binding affinity can be determined by scintillation proximity assays, which are based on the evaluation of competitive binding between [125I]-labeled parent insulin (such as [125I]-labeled human insulin) and (unlabeled) insulin analogs and insulin receptors. The insulin receptor may be present in the membrane of a cell (e.g., a CHO (Chinese hamster ovary) cell overexpressing a recombinant insulin receptor). In an embodiment, insulin receptor binding affinity is determined as described in the example section of WO2020 / 120463 A1 (incorporated herein with respect to all disclosures).

[0123] The binding of naturally occurring insulin or insulin analogs to the insulin receptor activates the insulin signaling pathway. The insulin receptor has tyrosine kinase activity. The binding of insulin to its receptor induces conformational changes that stimulate the autophosphorylation of the receptor on tyrosine residues. The autophosphorylation of the insulin receptor stimulates the activity of the receptor's tyrosine kinase to intracellular substrates involved in signal transduction. Therefore, the autophosphorylation of the insulin receptor by the insulin analog is considered to be a measure of the signal transduction caused by the conjugate. In some embodiments, relative to human insulin, the insulin analogs provided herein can induce 10%-25% of the human insulin receptor isoform B autophosphorylation, and / or induce 1%-15% of the human insulin receptor isoform A autophosphorylation relative to human insulin. In addition, in some embodiments, the insulin analogs provided herein can induce 3%-7% (such as 5%-7%) of insulin receptor autophosphorylation relative to the parent insulin (such as human insulin). The insulin receptor autophosphorylation relative to the parent insulin can be determined as described in the example section.

[0124] In the insulin-Fc fusion polypeptide provided herein, insulin (such as single-chain insulin) should be operably linked to the Fc region polypeptide. In antibodies, the Fc (fragment crystallizable) region is a region that interacts with cell surface receptors known as Fc receptors to activate the immune system. It is known that the Fc region of IgG carries a highly conserved N-glycosylation site that is essential for Fc receptor-mediated activity.

[0125] The two insulin-Fc fusion polypeptides present in the conjugates provided herein form an antibody Fc region.

[0126] As used herein, "Fc region" is a fragment of an immunoglobulin molecule formed by several constant heavy chain (CH) immunoglobulin domains of two polypeptides. In natural antibodies, the Fc region mediates the binding to Fc receptors and complement system components. For IgG, IgA and IgD, the Fc region is formed by the CH2 and CH3 constant domains of two heavy chains. For IgM and IgE, the Fc region is formed by the CH2, CH3 and CH4 constant domains of two 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., CH2 and CH3 domains or CH2, CH3 and CH4 domains. The Fc region of the present invention comprises CH domains, which are identical to or derived from the CH domains of natural immunoglobulins, such as by inserting one or more point mutations.

[0127] The term "Fc region polypeptide" is a fragment of an antibody heavy chain, and the fragment comprises the constant domain of an antibody heavy chain. Therefore, the Fc region polypeptide typically comprises 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 comprises (the antibody heavy chain) CH2 and CH3 domains. In an embodiment, the Fc region polypeptide comprises or is composed of the following: a) an amino acid sequence as shown in SEQ ID NO: 34, or b) 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 sequence to SEQ ID NO: 34. Typically, two Fc region polypeptides form an antibody Fc region in a conjugate provided herein.

[0128] The Fc region polypeptide may comprise additional elements, such as a third linker peptide, a C-terminal peptide allowing formation of the at least two covalent bonds between the two insulin Fc fusion polypeptides. Typically, however, the Fc region polypeptide lacks an antibody hinge region at the N-terminus.

[0129] As used herein, the term "hinge region" refers to a portion of an antibody sequence located between the Fc region and the Fab region. It provides segment flexibility and can stabilize antibody molecules. The hinge region can be clearly defined based on structural data, such as the IgG1 hinge region comprising residues 221-237 (R. Nezlin, "The Immunoglobulins [immunoglobulins] ", Academic Press, 1998, pp. 23-26). In IgG antibodies, the hinge region comprises a disulfide bond connecting the two heavy chains of the antibody. IgG1 and IgG4 comprise 2 such disulfide bonds, while IgG2 comprises 4, and IgG3 comprises 11 (Liu and May, 2012 MAbs [monoclonal antibodies]. 2012 January-February; 4 (1): 17-23).

[0130] In this article, if a region is located at the N-terminus of the Fc region, it is typically designated as a "hinge region" only, as is the case with all natural antibodies. If an amino acid sequence identical or highly similar to the hinge region is located at the C-terminus of the Fc region (as is the case in some embodiments of the molecules of the invention), it is designated as an "amino acid sequence comprising an (antibody) hinge region amino acid sequence" etc. High similarity means having at least 70% identity with the sequence of the antibody hinge region and / or comprising at least 7 consecutive amino acids of the antibody hinge region sequence.

[0131] As used herein, a "covalent bond" is a chemical bond involving the sharing of electron pairs between atoms. Covalent bonds are distinct from non-covalent interactions, such as electrostatic interactions or hydrophobic interactions.

[0132] The indication that a covalent bond (e.g., a disulfide bond) is "located at the C-terminus (of the Fc region)" means that, on the two polypeptides forming the Fc region, the amino acid residue (e.g., a cysteine ​​residue) involved in forming the covalent bond is located at the C-terminus of a part of the polypeptides forming the Fc region.

[0133] The indication that no covalent bond (e.g., disulfide bond) is "located at the N-terminus (of the Fc region)" means that, on the two polypeptides forming the Fc region, no amino acid residue participating in the formation of such a covalent bond (e.g., cysteine ​​residue) is located at the N-terminus of a part of the polypeptides forming the Fc region.

[0134] "Percentage (%) of amino acid sequence identity" with respect 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 in a reference polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity. Typically, standard parameters are applied to determine the degree of sequence 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 optimally aligned sequences over a comparison window, wherein a fragment of the amino acid sequence in the comparison window may contain additions or deletions (e.g., gaps or overhangs) for optimal alignment when compared to a reference sequence (which does not contain additions or deletions). The percentage can be calculated by determining the number of positions at which the same amino acid residue appears in the two sequences to produce 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 obtain the percentage of sequence identity. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Add. APL. Math. [Advances in Applied Mathematics] 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. [Journal of Molecular Biology] 48:443 (1970), by the similarity search method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) [Proceedings of the National Academy of Sciences of the United States of America] 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are typically employed to determine their optimal alignment, and therefore the degree of identity. Typically, default values ​​of a gap weight of 5.00 and a gap weight length of 0.30 are used.In an 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, which has been incorporated into 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), the BLOSUM62 scoring matrix, and a gap opening penalty of 10 and a gap extension penalty of 0.5. A preferred non-limiting example of the parameters used to align two amino acid sequences using the Needle program is the default parameters, including the EBLOSUM62 scoring matrix, a gap opening penalty of 10 and a gap extension penalty of 0.5.

[0135] The term "at least 80% identical" with respect to two sequences means that the two sequences have 80% or higher sequence identity. 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.

[0136] As used herein, the term "cell" or "host cell" refers to an intact cell, i.e., a cell with an intact membrane that has not released its normal intracellular components (such as enzymes, organelles, or genetic material). In certain exemplary embodiments, an intact cell is a living cell, i.e., a living cell that is able to perform its normal metabolic functions. In certain exemplary embodiments, a cell or host cell is any cell that can be transfected or transformed with an exogenous nucleic acid. In certain exemplary embodiments, when transfected or transformed with an exogenous nucleic acid and transferred to a recipient, the cell can express the nucleic acid in the recipient.

[0137] The term "cell" includes prokaryotic cells (such as bacterial cells) and eukaryotic cells (such as yeast cells, fungal cells or mammalian cells). Suitable bacterial cells include, but are not limited to, cells from gram-negative bacterial strains (such as strains of Escherichia coli, Proteus and Pseudomonas) and gram-positive bacterial strains (such as strains of Bacillus, Streptomyces, Staphylococcus and Lactococcus). Suitable fungal cells include, but are not limited to, cells from Trichoderma, Neurospora and Aspergillus species. Suitable yeast cells include, but are not limited to, cells from species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula. Suitable mammalian cells include, but are not limited to, for example, CHO (Chinese hamster ovary) cells, BHK cells, HeLa cells, COS cells, HEK-293, etc. In one embodiment, HEK-293 cells are used. In another embodiment, CHO cells are used. However, amphibian cells, insect cells, plant cells, and any other cells in the art for expressing heterologous proteins may also be used. In certain exemplary embodiments, adoptive transfer is performed using mammalian cells (e.g., cells from humans, mice, hamsters, pigs, goats, or primates).

[0138] In some embodiments, the host cell comprises a polynucleotide encoding an insulin, fusion protein or insulin-Fc conjugate provided herein, and / or a vector comprising the polynucleotide. Typically, the vector is an expression vector.

[0139] The cell or host cell can be isolated or part of a tissue or organism (e.g., a "non-human organism"). As used herein, the term "non-human organism" is meant to include non-human primates or other animals, such as mammals, such as 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.

[0140] The pharmaceutical compositions as described herein typically comprise insulin or insulin-Fc conjugates provided herein and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable" refers to materials that are non-toxic and do not interact with the effects of the active agent (i.e., insulin or insulin-Fc conjugate) of the pharmaceutical composition in certain exemplary embodiments.

[0141] As used herein, the term "carrier" refers to an organic or inorganic composition of natural or synthetic nature, in which the active ingredients are combined to facilitate, enhance or enable application. Typically, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject.

[0142] Suitable carrier materials for parenteral administration include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, bacteriostatic saline (e.g., saline containing 0.9% benzyl alcohol), phosphate buffered saline (PBS), Hank's solution, polyalkylene glycols, hydrogenated naphthalenes, and particularly biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0143] As used herein, the term "excipient" is intended to include all substances that may be present in a pharmaceutical composition and are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffer substances, flavoring agents or coloring agents.

[0144] The form of the pharmaceutical composition, route of administration, dosage and regimen will naturally depend on the condition to be treated, the severity of the disease, age, weight and sex of the patient, etc.

[0145] The pharmaceutical composition can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for subcutaneous administration.

[0146] In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for a formulation that can be administered parenterally, such as intravenously or subcutaneously. These may be in particular isotonic sterile saline solutions (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dry, in particular freeze-dried compositions, which, after addition of sterile water or physiological saline, as the case may be, allow the constitution of injectable solutions.

[0147] The insulin or insulin-Fc conjugates described herein can be administered via any conventional route, such as oral, pulmonary administration (by inhalation), or parenteral (including by injection or infusion). In some embodiments, parenteral administration is used, such as intravenous, intraarterial, subcutaneous, intradermal, or intramuscular administration. In some embodiments, the insulin or insulin-Fc conjugates provided herein are administered subcutaneously.

[0148] Insulin or insulin-Fc conjugates, or pharmaceutical compositions described herein, are generally administered in a therapeutically effective amount. The term "therapeutically effective amount" is understood by the skilled artisan. In some embodiments, the term refers to an amount that alone or in combination with another dose achieves a desired therapeutic response or desired therapeutic effect, optionally without causing or only minimally causing unacceptable or unwanted side effects.

[0149] Typically, 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 currently defines one 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 [World Health Organization's proposal to initiate a project to evaluate the international standard for human recombinant insulin candidates], WHO / BS / 10.2143–working document QAS / 10.381, 2010). Typically, one unit of insulin analogs is biologically equivalent to one unit of human insulin.

[0150] In embodiments, an insulin analog provided herein or an 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.

[0151] In an embodiment, the insulin-Fc conjugate provided herein is administered once a week.

[0152] For example, the insulin analog provided herein or the insulin-Fc conjugate provided herein is administered once a week in an amount of 0.01-100 U / kg body weight, such as 1-20 U / kg body weight, such as 1-10 U / kg body weight.

[0153] The terms "subject" and "patient" are used interchangeably herein. A "subject" or "patient" can be a vertebrate. The term includes humans and other animals, particularly mammals and other organisms. Thus, herein, a subject can be an animal, such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel, or primate. 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.

[0154] In some embodiments, the subject suffers from a disease or disorder as mentioned herein. For example, the subject can be an obese subject. In some embodiments, the patient is at risk of suffering from a disease or disorder as mentioned herein.

[0155] Typically, the term "disease or disorder" refers to any pathological or unhealthy state that can be treated by administering insulin or an insulin-Fc conjugate or a pharmaceutical composition provided herein, in particular diabetes (such as gestational diabetes, type 1 diabetes or type 2 diabetes) and / or hyperglycemia.

[0156] As used herein, "diabetes mellitus" (also referred to as "diabetes") refers to a group of metabolic diseases characterized by high levels of blood sugar caused by 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 as follows: fasting plasma glucose ≥ 7.0 mmol / l (126 mg / dL) or 2-hour plasma glucose ≥ 11.1 mmol / l (200 mg / dL).

[0157] In some embodiments, diabetes is type 1 diabetes. As used herein, "type 1 diabetes" is a condition characterized by high blood sugar levels caused by a complete lack of insulin. This occurs when the body's immune system attacks the insulin-producing beta cells in the pancreas and destroys them. The pancreas then produces almost no insulin. Removal or disease of the pancreas may also result in the loss of insulin-producing beta cells. Type 1 diabetes accounts for 5%-10% of diabetes cases.

[0158] In some embodiments, diabetes is type 2 diabetes. As used herein, "type 2 diabetes" is a condition characterized by excess glucose production despite the availability of insulin, and because of inadequate glucose clearance (insulin action), circulating glucose levels remain too high.

[0159] In some embodiments, the diabetes is gestational diabetes. As used herein, "gestational diabetes" is a condition in which a woman who has not previously been diagnosed with diabetes exhibits high blood sugar levels during pregnancy, particularly during the third trimester. Gestational diabetes affects 3%-10% of pregnancies, depending on the population studied.

[0160] As used herein, the term "hyperglycemia" refers to an excess of sugar (glucose) in the blood.

[0161] As used herein, the term "treating" or "treatment" refers to administering a compound or composition or a combination of compounds or compositions to a subject to: prevent, improve or eliminate the subject's herein mentioned diseases and / or disorders, such as diabetes. Therefore, the term encompasses the treatment of existing diseases or disorders mentioned herein, or the prevention (prevention) of diseases or disorders, i.e., prophylaxis. Therefore, it will be appreciated that in some embodiments, the treatments mentioned herein may be preventive. In some embodiments, the term refers to the treatment of existing diseases or disorders mentioned herein. Therefore, the subject suffers from the disease or disorder. DETAILED DESCRIPTION

[0162] First aspect: Insulin

[0163] Provided herein are insulin analogs comprising an insulin B chain and a corresponding insulin A chain, as shown in Table B in the Examples section. Insulin analogs are typically provided as single chain insulins.

[0164] Provided herein are insulin analogs 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: 69) and the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0165] Further provided are insulin analogs 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).

[0166] Further provided are insulin analogs 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).

[0167] Further provided are insulin analogs comprising an insulin B chain comprising or consisting of the amino acid sequence SFVNQHLCGSHLVEALELVCGERGFHYTPKT (SEQ ID NO: 73) and an insulin A chain comprising or consisting of the amino acid sequence GIVEQCCTSICSLSQLEDYCG (SEQ ID NO: 75).

[0168] Further provided are insulin analogs comprising an insulin B chain and an insulin A chain, wherein the insulin B chain comprises or consists of the amino acid sequence SFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 65), and wherein the insulin A chain comprises or consists of the amino acid sequence GIVEQCCTSICSLEQLENYCG (SEQ ID NO: 74).

[0169] Further provided are insulin analogs 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).

[0170] Further provided are insulin analogs 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).

[0171] Typically, the insulin analogs provided herein are provided as single chain insulins. In single chain insulins, the insulin B chain is typically linked to the insulin A chain via a first linker peptide.

[0172] Therefore, the single-chain insulin comprises from N-terminus to C-terminus:

[0173] a) Insulin B chain,

[0174] b) a first linker peptide, and

[0175] c) Insulin A chain.

[0176] In an embodiment, the length of the first joint peptide is 1 to 50 amino acids. In another embodiment, the length of the first joint peptide is 5 to 15 amino acids. In another embodiment, the length of the first joint peptide is 5 to 10 amino acids. In another embodiment, the length of the first joint peptide is 7 amino acids.

[0177] 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).

[0178] In an embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0179] SFVNQHLCGSHLVEALELVCGERGFHYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 50).

[0180] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0181] SFVNQHLCGSHLVEALHLVCGERGFAYTPKTGSYPGGVGIVEQCCT SICSLEQLENYCG (SEQ ID NO: 52).

[0182] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0183] FVNQHLCGSHLVEALHLVCGERGFHYTPKTGSYPGGVGIVEQCCTSI CSLSQLEDYCG (SEQ ID NO: 53).

[0184] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0185] FVNQHLCGSHLVEALELVCGERGFHYTPKT GSYPGGV GIVEQCCTSI CSLSQLEDYCG(SEQ IDNO:58).

[0186] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0187] SFVNQHLCGSHLVEALYLVCGERGFFYTPKTEEYPGDVGIVEQCCTSICSLEQLENYCG(SEQ IDNO:45).

[0188] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0189] SFVNQHLCGSHLVEALYLVCGERGFFYTPKTGSYPGGVGIVEQCCTSICSLEQLENYCG (SEQ ID NO: 81).

[0190] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0191] SFVNQHLCGSHLVEALHLVCGERGFAYTDKTEEYPGDVGIVEQCCT SICSLEQLENYCG (SEQ ID NO: 82).

[0192] In another embodiment, the single-chain insulin comprises or consists of the following amino acid sequence:

[0193] SFVNQHLCGSHLVEALELVCGERGFHYTDKTEEYPGDVGIVEQCCT SICSLEQLENYCG (SEQ ID NO: 47).

[0194] Second aspect: Insulin Fc fusion polypeptide and conjugate thereof

[0195] Further provided herein is an insulin-Fc fusion polypeptide comprising, from N-terminus to C-terminus, insulin and an Fc region polypeptide.

[0196] Also provided is an insulin-Fc conjugate of two insulin-Fc fusion polypeptides provided herein. The two insulin-Fc fusion polypeptides should form an antibody Fc region (via the Fc region).

[0197] 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.

[0198] Typically, the insulin comprised by the insulin-Fc fusion polypeptides provided herein is an insulin analog that has reduced binding affinity for the human insulin receptor compared to human insulin.

[0199] Typically, an insulin-Fc fusion polypeptide comprises, from N-terminus to C-terminus:

[0200] a) Insulin B chain,

[0201] b) a first linker peptide,

[0202] c) Insulin A chain,

[0203] d) a second linker peptide,

[0204] e) Fc region (also referred to herein as Fc region polypeptide).

[0205] In the embodiment of insulin-Fc fusion polypeptide, the insulin is a single-chain insulin, such as any of the single-chain insulins shown in Table B in the Examples section. Typically, the insulin is a single-chain insulin comprising, from N-terminus to C-terminus, an insulin B chain, a first linker peptide and an insulin A chain.

[0206] In an embodiment of the insulin-Fc fusion polypeptide, the single-chain insulin comprises or consists of the following amino acid sequence: SFVNQHLCGSHLVEALELVCGERGFHYTPKTGSYPGGVG IVEQCCTSICSLEQLENYCG (SEQ ID NO: 50),

[0207] SFVNQHLCGSHLVEALHLVCGERGFAYTPKTGSYPGGVGIVEQCCT SICSLEQLENYCG(SEQ IDNO:52),

[0208] FVNQHLCGSHLVEALHLVCGERGFHYTPKTGSYPGGVGIVEQCCTSI CSLSQLEDYCG(SEQ IDNO:53),

[0209] FVNQHLCGSHLVEALELVCGERGFHYTPKTGSYPGGVGIVEQCCTSI CSLSQLEDYCG(SEQ IDNO:58),

[0210] SFVNQHLCGSHLVEALYLVCGERGFFYTPKTEEYPGDVGIVEQCCTS ICSLEQLENYCG(SEQ IDNO:45)

[0211] SFVNQHLCGSHLVEALYLVCGERGFFYTPKTGSYPGGVGIVEQCCTS ICSLEQLENYCG(SEQ IDNO:81)

[0212] SFVNQHLCGSHLVEALHLVCGERGFAYTDKTEEYPGDVGIVEQCCT SICSLEQLENYCG(SEQ IDNO:82), or

[0213] SFVNQHLCGSHLVEALELVCGERGFHYTDKTEEYPGDVGIVEQCCT SICSLEQLENYCG(SEQ IDNO:47)

[0214] Typically, the single-chain insulin is connected to the Fc region polypeptide via a second linker peptide. The second linker peptide is typically 1 to 100 amino acids in length, such as 5 to 40 amino acids. In an embodiment of an insulin-Fc fusion polypeptide, the second linker is human insulin C-peptide, or a fragment thereof.

[0215] Human C-peptide has the following amino acid sequence:

[0216] EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 31).

[0217] The fragment of human C-peptide is, for example, a fragment of human C-peptide having a sequence as shown in SEQ ID NO: 31 having a length of at least 5, 10, 15, 20 or 22 amino acids.

[0218] In an embodiment, amino acids Leu26Ala27Leu28 in the C-peptide (indicated in bold in SEQ ID NO:31 above) are missing in the linker. Thus, the second linker comprises or consists of the amino acid sequence EAEDLQVGQVELGGGPGAGSLQPEGSLQ (SEQ ID NO:8).

[0219] One or more amino acids may be added to the second linker, such as a glycine residue at the N-terminus, in order to increase the distance from the second linker to the insulin. Thus, the second linker may comprise or consist of the amino acid sequence GEAEDLQVGQVELGGGPGAGSLQPEGSLQ (SEQ ID NO: 29).

[0220] Additionally, it is envisioned that the second linker comprises or consists of the amino acid sequence EAEDLQVGQVELGG (SEQ ID NO: 28).

[0221] In an embodiment, the Fc region present in the Fc region polypeptide of the present invention is the Fc region of an IgG, IgM, IgA, IgD or IgE antibody. Thus, the Fc region polypeptide typically comprises a heavy chain constant domain, such as CH2 and CH3 of an IgG antibody.

[0222] In an embodiment, the Fc region is the Fc region of an IgG antibody. Thus, the two polypeptides form the Fc region of an IgG antibody. For example, the Fc region can be the Fc region of an IgG1 antibody. Alternatively, the Fc region can be the Fc region of an IgG4 antibody.

[0223] In an 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. Therefore, the two pancreatic polypeptides forming the conjugate are not connected to a portion of each fusion polypeptide forming the Fc region by one or more disulfide bonds located at the N-terminus.

[0224] Typically, the insulin Fc fusion polypeptide is covalently linked by at least two covalent bonds at the C-terminus of the fusion polypeptide. For example, they are covalently linked by two covalent bonds at the C-terminus of the fusion polypeptide.

[0225] The expression "at the C-terminus of the fusion polypeptide" encompasses, for example, the 20 C-terminal amino acids of the fusion polypeptide.

[0226] In some embodiments, at least two covalent bonds are located at the C-terminus of the CH2, CH3, and / or CH4 domains on each fusion polypeptide.

[0227] In some embodiments, at least two covalent bonds are located at the C-terminus of the CH2 and / or CH3 domains on each fusion polypeptide.

[0228] In some embodiments, at least two covalent bonds are located at the C-termini of the CH2 and CH3 domains on each polypeptide.

[0229] Typically, the two or more covalent bonds are located at the C-terminus of a portion of each fusion polypeptide that forms the Fc region.

[0230] In the embodiment of the provided insulin-Fc conjugate, the covalent bond is a disulfide bond. Such a disulfide bond can be formed between the thiol groups of two cysteine ​​residues. Therefore, the insulin Fc fusion polypeptide as mentioned herein typically comprises at least one cysteine ​​residue, such as two cysteine ​​residues. In an embodiment, one or more cysteine ​​residues are present in the C-terminal region of the fusion polypeptide, for example, within the 20 C-terminal amino acids of the fusion polypeptide.

[0231] Typically, the two fusion polypeptides are not covalently linked via one or more disulfide bonds between the N-termini of each IgG Fc region polypeptide.

[0232] Typically, the first insulin-Fc fusion polypeptide and the second insulin Fc fusion polypeptide each comprise an amino acid sequence at the C-terminus comprising at least two cysteine ​​residues that allow the formation of two disulfide bonds between the first fusion polypeptide and the second fusion polypeptide (also referred to herein as "C-terminal peptides"). In this case, the at least two C-terminal covalent bonds are at least two disulfide bonds within this sequence.

[0233] The length of the C-terminal peptide may be, for example, 7 to 20 amino acids, such as 10 to 20 amino acids.

[0234] The amino acid sequence at the C-terminus of the fusion polypeptide provided herein (i.e., the C-terminal peptide) can be the amino acid sequence of a naturally occurring antibody hinge region or a sequence highly similar to the sequence, such as the sequence of the hinge region of an IgG1 or IgG4 antibody (or a sequence highly similar to the sequence).

[0235] The amino acid sequence of the IgG1 heavy chain constant region is shown in SEQ ID NO: 35. The hinge region of this antibody can be found at positions 99 to 110 of this sequence and has the following amino acid sequence: EPKSCDKTHTCP (SEQ ID NO: 36).

[0236] The amino acid sequence of the IgG4 heavy chain constant region is shown in SEQ ID NO: 38. The hinge region can be found at positions 99 to 110 of this sequence and has the following amino acid sequence: ESKYGPPCPSCP (SEQ ID NO: 39). In addition, the hinge region can be found at positions 99 to 111 of this sequence and has the following amino acid sequence: ESKYGPPCPSCPA (SEQ ID NO: 40).

[0237] Therefore, the C-terminal peptide present at the C-terminus may comprise or consist of the amino acid sequence as shown in SEQ ID NO: 36, 39 or 40.

[0238] However, in some embodiments, the C-terminal peptide present in the C-terminus has at least 70% identity (i.e., highly similar) to the sequence of the antibody hinge region (i.e., 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 consecutive amino acids of the sequence of the antibody hinge region. In some embodiments, the sequence comprises at least 10 consecutive amino acids of the sequence of the antibody hinge region. In some embodiments, the sequence comprises at least 15 consecutive amino acids of the sequence of the antibody hinge region. Typically, the antibody hinge region comprises two or more cysteine ​​residues (to form a disulfide bond between two fusion polypeptides).

[0239] Since the sequence of the antibody hinge region can be derived from the endogenous human antibody hinge region, the use of foreign sequences can be avoided, which reduces the risk of anti-drug antibody (ADA) formation.

[0240] 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 highly similar to the naturally occurring antibody hinge region. Therefore, the C-terminal peptide present at the C-terminus may comprise or consist of the amino acid sequence shown in SEQ ID NO: 41.

[0241] In one embodiment, the Fc region and the C-terminal region are connected by a third linker peptide. The third linker is typically 1 to 10 amino acids in length, such as 6 amino acids in length. In an embodiment, the third linker peptide comprises or consists of a sequence as shown in GGGGSA (SEQ ID NO: 42).

[0242] Therefore, in some embodiments, the insulin-Fc fusion polypeptide provided herein comprises, from N-terminus to C-terminus:

[0243] a) Insulin B chain,

[0244] b) a first linker peptide,

[0245] c) Insulin A chain,

[0246] d) a second linker peptide,

[0247] e) Fc region,

[0248] d) a third linker peptide, and

[0249] e) C-terminal peptide.

[0250] The C-terminal peptide should allow for the formation of at least two covalent bonds, i.e., interchain covalent bonds, with another insulin-Fc fusion polypeptide (typically a fusion polypeptide with the same sequence). Thus, due to the at least two interchain covalent bonds at the C-terminus, a stable dimer is formed. 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.

[0251] Typically, the C-terminal peptide comprises the amino acid sequence of the hinge region of an antibody (or a sequence highly similar thereto). In some embodiments, the C-terminal peptide has an amino acid sequence as shown in SEQ ID NO: 36, 39, 40 or 41.

[0252] As described above, in some embodiments, the Fc region lacks an antibody hinge region at the N-terminus. Thus, the fusion polypeptide (within the conjugates provided herein) is not linked to a portion of each polypeptide of the Fc region forming the conjugate via a disulfide bond located at the N-terminus.

[0253] Thus, the two fusion polypeptides form the antibody Fc region.

[0254] wherein the two polypeptides are linked to a portion of each polypeptide forming the Fc region via at least two covalent bonds located at the C-terminus,

[0255] wherein the two polypeptides are not linked to a portion of each polypeptide forming the Fc region via a disulfide bond located at the N-terminus.

[0256] In an embodiment, the Fc region lacking the antibody hinge region comprises or consists of:

[0257] a) For example, SEQ ID NO:34 (GGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG), or

[0258] 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:34.

[0259] SEQ ID NO: 34 is the Fc region of a human IgG1 antibody (without hinge and C-terminal Lys) and is used in many of the constructs described in Table A. It includes the region from positions 119 to 329 of human IgG1. The hinge at position 330 and the C-terminal Lys are not included.

[0260] In another embodiment, the Fc region lacking the antibody hinge region comprises or consists of:

[0261] a) the sequence shown in SEQ ID NO: 37, or

[0262] 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.

[0263] In some embodiments, the Fc region present in the fusion polypeptide provided herein comprises the following amino acid sequence:

[0264]

[0265] In the example part, a sequence including a third joint peptide and a C-terminal hinge region is used. The sequence of the third joint peptide is underlined. The C-terminal hinge region is highlighted in bold.

[0266] In some embodiments, the Fc region present in the fusion polypeptide provided herein comprises the following amino acid sequence:

[0267] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAESKYGPPCPPCPA(SEQ ID NO:84).

[0268] In some embodiments, the Fc region present in the fusion polypeptide provided herein comprises the following amino acid sequence:

[0269] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPCPPCPA(SEQ ID NO:85).

[0270] Other aspects (host cells, polynucleotides, medical uses)

[0271] Further provided are polynucleotides encoding the single-chain insulin provided herein or the insulin-Fc fusion polypeptide provided herein.

[0272] Further 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.

[0273] Further 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 allowing expression of the insulin-Fc conjugate, the single-chain insulin, or the insulin-Fc fusion polypeptide.

[0274] Further 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.

[0275] Further 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.

[0276] Further provided is an insulin-Fc conjugate provided herein, an insulin provided herein (such as a single-chain insulin provided herein), or an insulin-Fc fusion polypeptide provided herein for use as a medicament.

[0277] Further provided is an insulin-Fc conjugate provided herein, an insulin provided herein (such as a single-chain insulin provided herein), or an insulin-Fc fusion polypeptide provided herein, for use as a medicament for treating 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.

[0278] Example

[0279] In the following examples of the provided conjugates, fusion polypeptides and insulin are disclosed. The definitions and explanations given above apply mutatis mutandis to the following.

[0280] 1. An insulin-Fc conjugate having 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 polypeptide.

[0281] 2. The insulin-Fc conjugate of embodiment 1, wherein the two insulin-Fc fusion polypeptides are linked by at least two covalent bonds at the C-termini of the fusion polypeptides, for example, wherein the at least two covalent bonds are disulfide bonds.

[0282] 3. The insulin-Fc conjugate of embodiment 1 or 2, wherein the conjugate is a homodimer of the two insulin-Fc fusion polypeptides, and / or wherein the two insulin-Fc fusion polypeptides form an antibody Fc region.

[0283] 4. The insulin-Fc conjugate according to any one of the preceding embodiments, wherein the insulin is a single-chain insulin.

[0284] 5. The insulin-Fc conjugate of any one of the preceding embodiments, wherein each of the two insulin-Fc fusion polypeptides comprises, from N-terminus to C-terminus:

[0285] a) Insulin B chain,

[0286] b) a first linker peptide,

[0287] c) Insulin A chain,

[0288] d) a second linker peptide,

[0289] e) Fc region polypeptide.

[0290] 6. The insulin-Fc conjugate of embodiment 5, wherein each of the two insulin-Fc fusion polypeptides further comprises

[0291] f) a third linker peptide,

[0292] g) a C-terminal peptide which allows the formation of the at least two covalent bonds between the two insulin-Fc fusion polypeptides.

[0293] 7. The insulin-Fc conjugate as described in Example 6, wherein the C-terminal peptide under g) comprises at least two cysteine ​​residues (such as two cysteine ​​residues) that allow formation of at least two disulfide bonds between the two insulin-Fc fusion polypeptides, for example, wherein the C-terminal peptide comprises the amino acid sequence of the antibody hinge region (or a highly similar sequence), and the amino acid sequence of the antibody hinge region comprises at least two cysteine ​​residues that allow formation of at least two disulfide bonds between the two insulin-Fc fusion polypeptides.

[0294] 8. The insulin-Fc conjugate as described in Example 7, wherein the amino acid sequence of the C-terminal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 36, 39, 40 or 41.

[0295] 9. The insulin-Fc conjugate of any one of the preceding embodiments, wherein the two insulin-Fc fusion polypeptides are not covalently linked via one or more disulfide bonds between the N-termini of each Fc region polypeptide.

[0296] 10. The insulin-Fc conjugate of any one of the preceding embodiments, wherein the Fc region polypeptide lacks an antibody hinge region at the N-terminus.

[0297] 11. The insulin-Fc conjugate of any one of the preceding embodiments, wherein the Fc region polypeptide comprises the constant domain of an IgG, IgM, IgA, IgD or IgE antibody heavy chain.

[0298] 12. The insulin-Fc conjugate as described in Example 12, wherein the Fc region polypeptide comprises the constant domains CH2 and CH3 of the antibody heavy chain of an IgG1 antibody or an IgG4 antibody.

[0299] 13. The insulin-Fc conjugate according to any one of the preceding embodiments, 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.

[0300] 14. The insulin-Fc conjugate of 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).

[0301] 15. The insulin-Fc conjugate according to any one of the preceding embodiments, wherein the second linker peptide has a length of 1 to 100 amino acids, such as 5 to 40 amino acids.

[0302] 16. The insulin-Fc conjugate as described in Example 15, wherein the second linker peptide is human insulin C-peptide or a fragment thereof.

[0303] 17. The insulin-Fc conjugate of embodiment 16, wherein the second linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 28 or 29.

[0304] 18. The insulin-Fc conjugate according to any one of the preceding embodiments, wherein the third linker is 1 to 10 amino acids in length, such as 6 amino acids in length.

[0305] 19. The insulin-Fc conjugate as described in embodiment 18, wherein the third linker peptide comprises or consists of the amino acid sequence as shown in GGGGSA (SEQ ID NO: 42).

[0306] 20. The insulin-Fc conjugate of any one of the preceding embodiments, wherein the insulin is an insulin having reduced binding affinity to the human insulin receptor compared to the binding affinity of human insulin to the human insulin receptor.

[0307] 21. The insulin-Fc conjugate of any one of the preceding embodiments, wherein the insulin is an insulin as shown in Table B, such as a single-chain insulin as shown in Table B.

[0308] 22. The insulin-Fc conjugate of embodiment 21, wherein

[0309] 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);

[0310] 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);

[0311] 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);

[0312] 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);

[0313] 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);

[0314] 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

[0315] vii) 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).

[0316] 23. The insulin-Fc conjugate of embodiment 21 or 22, 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.

[0317] 24. The insulin-Fc conjugate of any one of the preceding embodiments, 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.

[0318] 25. An insulin according to the definition of insulin in embodiment 22 or 23, such as a single-chain insulin according to the definition of insulin in embodiment 23.

[0319] 26. An insulin-Fc fusion polypeptide, which is defined as an insulin-Fc fusion polypeptide according to any one of embodiments 1 to 24.

[0320] 27. A pharmaceutical composition comprising a pharmaceutically effective amount of the insulin-Fc conjugate of any one of Embodiments 1 to 24, the insulin of Embodiment 25, or the insulin-Fc fusion polypeptide of Embodiment 26.

[0321] 28. The insulin-Fc conjugate of any one of embodiments 1 to 24, the insulin of embodiment 25, or the insulin-Fc fusion polypeptide of embodiment 26, for use as a medicament.

[0322] 29. The insulin-Fc conjugate of any one of embodiments 1 to 24, the insulin of embodiment 25, or the insulin-Fc fusion polypeptide of embodiment 26, for use as a medicament for treating 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.

[0323] 30. A polynucleotide encoding the insulin of embodiment 25 or the insulin-Fc fusion polypeptide of embodiment 26.

[0324] 31. A host cell comprising the insulin-Fc conjugate of any one of embodiments 1 to 24, the insulin of embodiment 25, or the insulin-Fc fusion polypeptide of embodiment 26, and / or the polynucleotide of embodiment 20.

[0325] 32. A method for producing the insulin-Fc conjugate of any one of embodiments 1 to 24, the insulin of embodiment 25, or the insulin-Fc fusion polypeptide of embodiment 26, the method comprising incubating the host cell of embodiment 21 under conditions that allow expression of the insulin-Fc conjugate of any one of embodiments 1 to 24, the insulin of embodiment 25, or the insulin-Fc fusion polypeptide of embodiment 26.

[0326] Examples

[0327] method:

[0328] Protein expression and purification

[0329] The scI-Fc proteins INS001-INS027 (SEQ ID NO: 2 to 25, 77 to 79, also see Table A) are fused to a leader sequence (SEQ ID NO: 1) that directs protein expression into the culture supernatant. The scI-Fc proteins are produced in HEK293 or CHO cells by transient transfection. Proteins are purified from the culture supernatant using protein A (mab select sure, GE Healthcare) affinity chromatography. After protein A purification, the scI-Fc proteins are further purified using a gel filtration column (Superdex 200, GE Healthcare) balanced in phosphate buffered saline (PBS, Gibco). The fractions containing the desired protein are collected, concentrated and stored at -80°C until further use.

[0330] In vitro insulin receptor phosphorylation assay

[0331] The scI-Fc protein produced is subjected to insulin receptor phosphorylation assay. Receptor phosphorylation (also referred to herein as "autophosphorylation") is measured substantially according to the protocol described by Sommerfeld et al. (Sommerfeld et al., 2010). In the cell Wester technique, CHO cells expressing the corresponding insulin receptor are used. CHO-IR cells are cultured in a Hart F12-nutrient mixture / GlutaMax medium (Gibco / Thermo Fisher Scientific) containing the following: 10% (v / v) fetal bovine serum (PAN-Biotech), 1x non-essential amino acids (Gibco / Thermo Fisher Scientific) and enough antibiotics for selection. In order to determine the receptor phosphorylation after insulin stimulation, cells are inoculated into 96-well plates and grown for 44h. Cells are serum starved for 2h with serum-free Hart nutrient mixture F12 medium, and then treated with increasing concentrations of insulin or scI-Fc variants at 37°C for 20min. After incubation, the culture medium was discarded and the cells were fixed in 3.75% freshly prepared paraformaldehyde for 20 min. The cells were permeabilized with 0.1% Triton X-100 in PBS (Gibco) for 20 min, followed by blocking with Odyssey blocking buffer (LI-COR Biosciences) at ambient temperature for 1 hr. Anti-phosphotyrosine antibody 4G10 (Millipore) was used as a primary antibody. After incubation for 2 h at ambient temperature, the cells were washed 3 times with PS+0.1% Twen 20. The secondary anti-mouse IgG G-800-CW and DNA stain DRAQ5 (Cell Signaling) required for cell number normalization were added and incubated for 1 h. The cells were washed 3 times with PBS+0.1% Tween 20, and the fluorescence signal at 700 and 800 nm was determined with a LI-COR infrared imaging system (Odyssey, LI-COR Biosciences). Data were obtained in relative fluorescence units (RFU).

[0332] Single chain insulins were tested in the constructs shown in Table A. The sequences of the single chain insulins are also shown in Table B, using human insulin (B chain: SEQ ID NO: 32, A chain: SEQ ID NO: 33) as a control.

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347] In vivo rat PK / PD

[0348] Pharmacodynamic data were collected essentially as described by Faust et al. (Faust et al., 2020). Male Sprague-Dawly rats (Charles River) weighing 320-400g at the start of the study were randomly assigned to 8 treatment groups, allowing them to freely access tap water and food. The protein was dissolved in PBS (Gibco) and administered subcutaneously. Only PBS was used as a placebo control. Blood glucose was determined by whole blood collected from the tip of the tail at the indicated time point. The blood was hemolyzed with hemolysate (hemolysis reagent H, glucose hexokinase fluid 5+1, Hengler analytical company (Hengleranalytic)), and quantified using Gluco-quant glucose / hexokinase kit (Roche Diagnostics (RocheDagnostics)) with Beckman Coulter AU640 chemical analyzer.

[0349] In vivo minipig PK / PD

[0350] Use healthy female Miniature Pig (Ellegaard Minipigs (15-18 months old, weighing approximately 20-25 kg) were used to evaluate the pharmacodynamic and pharmacokinetic effects of long-acting scI-Fc proteins. The animals were maintained under standard animal housing conditions and fed once daily with ad libitum access to tap water.

[0351] Animals fasted overnight are given a single subcutaneous injection containing placebo (PBS) or corresponding scI-Fc variants. Blood is collected via the central venous catheter before implantation. The first blood sample is collected before the first application and used as a baseline. Blood is collected at a given time point (1-4 times a day in the study time, sample tubes containing K-EDTA), immediately stored on ice, and after centrifugation, plasma is stored at -80 ° C until further use. Enzyme UV test (hexokinase method) (Beckman Coulter) is used, and on Olympus AU680 automatic analyzer device (Beckman Coulter), by hemolyzed sample (5 μl capillary blood is added to 250 μl hemolysis reagent (Hengler Analytik)) quantitative blood sugar. All animals are regularly treated, and clinical signs are recorded at least twice a day (treatment day) and once a day (remaining study time). Animals were carefully monitored for any clinical signs of hypoglycemia, including behavior, fur, urinary and fecal excretions, condition of body orifices, and any signs of illness. In cases of severe hypoglycemia, food was provided or glucose solution was administered intravenously (iv), as needed.

[0352] In vivo cyno PK / PD

[0353] For monkey experiments, male cynomolgus monkeys (cynomolgusmonkey / Macaca fascicularis) were adapted for at least 4 weeks after arrival (Nveprim, Mauritius). Animals were fed 200gr of NHP-Pellet (Ssniff, Germany) every day, and food and controlled tap water were freely available through an automatic waterer. Animals were grouped and housed in cages suitable for size and species, with a space of 260x 200x220cm. Under controlled environmental conditions (temperature 20°C to 24°C, relative humidity 40% to 75%), monkeys were housed under a 12h / 12 light / dark cycle. Before applying the compound, the animals were not fasted overnight. Venoflux micro-injector (23G) was used to iv apply to the saphenous vein. After injection, the remaining solution in the micro-injector was rinsed with 0.9% NaCl. 26G needle was used to directly apply sc compounds in the intrascapular region. Blood was extracted from the saphenous vein using a 2ml syringe with a 21G needle. Animals were observed at least once daily and at each blood collection time point. During the experiment, no adverse effects were observed. Glucose concentrations were determined from whole blood immediately after collection using an Accu-Check Active blood glucose meter (Roche Diagnostics). No hypoglycemia was observed.

[0354] Detection of scI-Fc in plasma using LC-MS / MS

[0355] 50 μL of plasma sample was diluted with 250 μL of PBS (phosphate buffered saline) containing IS (500 ng / mL) and quantified using MSIA Streptavidin DART'S tips on a Thermo MSIA TM Immunoenrichment on platform.With 65 μ L water / acetonitrile / TFA (66 / 33 / 0.4v / v / v) elution analyte and insulin standard (IS).After this, 100 μ L digestion buffer (100mM ammonium bicarbonate, pH 8.5), 2 μ L 0.1N sodium hydroxide and 3 μ L DDT (dithiothreitol 500mM in digestion buffer) are added in pipe, to hatch 0.5h at 60 ℃ on ThermoMixer with 500rpm.5 μ L IAA (iodoacetamide 500mM in digestion buffer) is added in pipe, mixes, and at room temperature in the dark under 500rpm, places 45min.After this, 2 μ L DDT (dithiothreitol 500mM in digestion buffer) are added in pipe to hatch 0.5h at room temperature with 500rpm. 10 μL of 100 μg / mL TCPK-trypsin (Thermo) in digestion buffer was added to the tube for digestion for 1.5 h on a ThermoMixer at 500 rpm and 37° C. The reaction was stopped by adding 10 μL of 10% aqueous formic acid, mixed and analyzed by LC-MS / MS.

[0356] Detection of scI-Fc in plasma using ELISA

[0357] MaxiSorp flat-bottom plates (Nunc) were coated with 1 μg / mL of anti-His-Tag monoclonal antibody (Novagen) at +4°C overnight, washed three times with PBS / 0.05% Tween-20, then blocked with 150 μL of 1% milk powder for 1 hour at room temperature (RT), and washed three times with PBS / 0.05% Tween-20. The capture tool, i.e., 50 μL of recombinant insulin receptor (R&D Systems), was applied at 1.0 μg / mL in PBS / 0.05% Tween-20, 50 μL / well, and incubated at room temperature for 1 hour while shaking at 600 rpm. The plate was washed three times with PBS / 0.05% Tween-20. The diluted samples of MRD (including standards, QC and PK samples) were then loaded onto the plate, and the plate was incubated at room temperature for 1.5 hours. The plate was then washed three times with PBS / 0.05% Tween-20, and a 1:20000 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 while shaking at 600 rpm to allow complexation between the detection means and scI. The plate was washed three times with PBS / 0.05% Tween-20, and then the plate was incubated with 100 μL TMB for 20 minutes, followed by the addition of 100 μL stop solution (0.3 M HCl). 1000 series Tecan microplate reader to obtain 450nm The OD signal below.

[0358] Table 1: Insulin receptor phosphorylation.

[0359]

[0360]

[0361] Table 2 In female Pharmacokinetic parameters of selected scI-Fc variants in minipigs.

[0362] female PK Data in Minipigs

[0363] Mean plasma pharmacokinetic parameters (n=3)

[0364] For calculation of mean concentrations, values ​​below the lower limit of quantitation were set to zero. Pharmacokinetic parameters were calculated by the program Phoenix WinNonlin 6.4 (Certara USA, Inc., Princeton, NJ) using a non-compartmental model and linear trapezoidal interpolation.

[0365] Lower limit of quantitation (LLOQ) as measured in plasma.

[0366]

[0367]

[0368] Tmax: time of maximum observed drug plasma concentration

[0369] Cmax: maximum observed plasma drug concentration

[0370] C0: initial plasma drug concentration

[0371] Tlast: The time of the last measurable concentration

[0372] AUClast: The area under the plasma drug concentration-time curve from the time of administration to the last measurable concentration

[0373] AUCinf: AUC from the time of administration to the last measurable concentration and extrapolated to infinity

[0374] AUCextr: Extrapolated AUC estimated as (AUCinf–AUClast / AUCinf)

[0375] T1 / 2: Elimination time

[0376] CL: plasma clearance

[0377] Vss: apparent steady-state volume of distribution

[0378] F (%): Bioavailability estimated as the ratio of AUCinf between sc and iv administration

[0379] MRT: Mean dwell time

[0380] Table 3 Pharmacokinetic parameters of selected scI-Fc variants in male cynomolgus monkeys

[0381]

[0382]

[0383] LLOQ = 50 ng / mL in plasma

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Claims

1. An insulin-Fc conjugate having 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 polypeptide, and wherein the two insulin-Fc fusion polypeptides are linked by at least two covalent bonds at the C-termini of the fusion polypeptides.

2. The insulin-Fc conjugate of 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 wherein the two insulin-Fc fusion polypeptides form an antibody Fc region.

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

5. The insulin-Fc conjugate according to any one of the preceding claims, wherein each of the two insulin-Fc fusion polypeptides comprises, from N-terminus to C-terminus: a) Insulin B chain, b) a first linker peptide, c) Insulin A chain, d) a second linker peptide, e) Fc region polypeptide.

6. The insulin-Fc conjugate of claim 5, wherein each of the two insulin-Fc fusion polypeptides further comprises f) a third linker peptide, g) a C-terminal peptide which allows the formation of the at least two covalent bonds between the two insulin-Fc fusion polypeptides.

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

8. The insulin-Fc conjugate of any one of the preceding claims, wherein 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 wherein the Fc region polypeptide lacks an antibody hinge region at the N-terminus.

9. The insulin-Fc conjugate of any of the preceding claims, wherein the Fc region polypeptide comprises the constant domains of the heavy chain of an IgG, IgM, IgA, IgD or IgE antibody, for example wherein the Fc region polypeptide comprises the constant domains CH2 and CH3 of the antibody heavy chain of an IgG1 antibody or an IgG4 antibody.

10. The insulin-Fc conjugate according to any one of the preceding claims, 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 wherein the first linker peptide comprises or consists of the amino acid sequence GSYPGGV (SEQ ID NO: 26) or EEYPGDV (SEQ ID NO: 27), and / or wherein the second linker peptide has a length of 1 to 100 amino acids, such as 5 to 40 amino acids, for example, wherein the second linker peptide is human insulin C-peptide or a fragment thereof, for example, wherein the second linker comprises 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 a length of 6 amino acids, for example, wherein the third linker peptide comprises or consists of an amino acid sequence as shown in GGGGSA (SEQ ID NO: 42).

11. The insulin-Fc conjugate of any one of the preceding claims, wherein the insulin is an insulin having a reduced binding affinity to the human insulin receptor compared to the binding affinity of human insulin to the human insulin receptor, and / or wherein the insulin is an insulin as shown in Table B, such as a single-chain insulin as shown in Table B, for example 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) 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).

12. The insulin-Fc conjugate of 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 of any one of the preceding claims, 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. An insulin as defined in claim 11 or 12.

15. An insulin-Fc fusion polypeptide, which is as defined in any one of claims 1 to 14.

Images