New insulin receptor agonists

Abstract

The invention relates to the field of biotechnology, namely to novel insulin receptor agonists, and can be used in medicine. The provided ultra-long-acting insulin receptor agonists are a fusion protein comprising insulin or an analog thereof fused to an Fc fragment polypeptide having a high binding capacity for FcRn and a low binding capacity for FcγRs.

Description

[0001] NEW INSULIN RECEPTOR AGONISTS

[0002] DESCRIPTION OF THE INVENTION FIELD

[0003] The present invention relates to fusion insulin polypeptides useful in treating insulinsensitive diseases or conditions. The invention also relates to methods for preparing such fusion polypeptides, pharmaceutical compositions comprising the same, a method for treating diabetes and hyperglycemia using the fusion insulin polypeptides of the invention, and the use of such fusion insulin polypeptides in the treatment of diabetes and hyperglycemia.

[0004] BACKGROUND

[0005] Natural insulin is a hormone secreted by the pancreas and, when interacting with receptors on target cells, stimulates glucose uptake and inhibition of fat breakdown, thus performing the function of controlling blood glucose levels. Insulin is formed through processing of a precursor that has no biological activity in regulating blood glucose level and is known as proinsulin.

[0006] The amino acid sequences of human insulin are as follows:

[0007] A-chain: GIVEQCCTSICSLYQLENYCN (SEQ ID NO:1)

[0008] B-chain: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2)

[0009] The amino acid sequence of human proinsulin is as follows: MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRE AEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN (SEQ ID NO:3)

[0010] When insulin secretion is below metabolic needs and / or the secreted insulin does not have sufficient activity, a state of hyperglycemia, which is characteristic of type II diabetes mellitus, develops in a body. Type I diabetes mellitus usually occurs due to the damage to the hormone-secreting P-cells of the islets of Langerhans, which results in the pancreas failing to produce insulin required for the regulation of blood glucose levels. There are also other forms of diabetes mellitus where a relative or absolute insulin deficiency is formed.

[0011] For all patients with type I diabetes mellitus, insulin replacement therapy is an integral part of treatment. Drug therapy for type II diabetes mellitus consists of several lines: the treatment usually starts with oral metformin and is then enhanced by other classes of drugs as the disease progresses. Including insulin in the treatment schedule for patients with type II diabetes mellitus is necessary in the later stages of the disease: on average, insulin therapy is received by about 20% of patients with type II diabetes mellitus (Mata-Cases M. et al. Trends in the degree of control and treatment of cardiovascular risk factors in people with type 2 diabetes in a primary care setting in Catalonia during 2007-2018 / / Frontiers in Endocrinology. 2022 Jan 10:12:810757).

[0012] A widespread method of insulin treatment is insulin injections, which a patient performs from one to 4 or more times a day depending on the treatment regimen and individual features. Despite being efficient, lifelong insulin injections cause pain and discomfort to patients, thereby reducing their quality of life. Various attempts were made to overcome this issue. One of them is a method of delivering a peptide drug by oral administration or nasal inhalation due to its improved membrane permeability. Unfortunately, this method exhibited very poor delivery efficiency compared to injectable formulations, and thus the need to reduce the injection burden in insulin therapy still remains a pressing concern.

[0013] The technology of human insulin synthesis using recombinant DNA technology emerged in the 1970s, and its commercialization shortly thereafter, in the early 1980s, represents a significant milestone in the history of this hormone. Recombinant synthesis provided an alternative to animal-derived insulins and, more importantly, allowed for the production of virtually unlimited amounts of human insulin. Recombinant DNA technology has provided cost-effective means of producing modified insulin analogs having improved pharmacological properties.

[0014] Depending on the duration of action, insulin analogs are classified into ultra short- and long-acting insulins.

[0015] Ultra short-acting insulins (fast-acting analogs) are designed to meet the prandial need for insulin. The first of the fast-acting analogs worth mentioning is insulin lispro (Humalog, Eli Lilly), which was first registered in 1996. Insulin lispro is based on the principle of weakened self-association as a result of inversion of the native sequence ProB28-LysB29 (Anderson Jr J. H. et al. Improved mealtime treatment of diabetes mellitus using an insulin analog / / Clinical therapeutics. 1997 Jan-Feb;19(l):62-72). The second insulin analog in this class, insulin aspart (Novolog, Novo Nordisk), first appeared on the market in 2000; it comprises Asp at position B28 (Home P. D. et al. Insulin aspart vs. human insulin in the management of long-term blood glucose control in type 1 diabetes mellitus: a randomized controlled trial / / Diabetic medicine.

[0016] 2000 Nov;17(ll):762-70). Another fast-acting analog of insulin, glulisine (Apidra, Sanofi), was registered in 2004; the structural basis of this analog is the amino acid substitutions of Lys in position B29 with Glu, and Asn in position B3 with Lys42 (Hermansen K. et al. A 26-week, randomized, parallel, treat-to-target trial comparing insulin detemir with NPH insulin as add-on therapy to oral glucose-lowering drugs in insulin-naive people with type 2 diabetes / / Diabetes care. 2006 Jun;29(6): 1269-74). Clinical studies of insulin aspart and insulin glulisine have confirmed that these two insulin analogs provide pharmacokinetic and pharmacodynamic characteristics comparable to those of the first-in-class insulin lispro (Becker R. H. A. et al. Insulin glulisine, a new rapid-acting insulin analog, displays a rapid time-action profile in obese non-diabetic subjects / / Experimental and clinical endocrinology & diabetes. 2005 Sep;113(8):435-43).

[0017] Basal insulin analog is intended to mimic the robust secretion profile of a healthy pancreas (Owens D. R., Matfin G., Monnier L. Basal insulin analogs in the management of diabetes mellitus: what progress have we made? / / Diabetes / metabolism research and reviews.

[0018] 2014 Feb;30(2): 104-19). Historically, a mixture of insulin with neutral protamine Hagedorn (NPH), which is an insulin with the prolonged action achieved by addition of zinc and protamine protein, can be considered the first insulin formulation having increased duration of action, which had been used for decades prior to the advent of the recombinant insulin biosynthesis. The main issues associated with classical NPH compositions were their duration of action insufficient for daily administration and a high degree of variability.

[0019] The primary strategy used in the development of improved basal insulin analogs relies on the formation of a depot at the injection site.

[0020] Insulin glargine (Lantus, Sanofi), which was first registered in 2001, makes use of the isoelectric point shift to drastically reduce solubility at physiological pH values, making insulin much less soluble at the injection site (Hilgenfeld R. Controlling insulin bio availability by crystal contact engineering / / Diabetologia. 1992;35(1): A193). The increase in the protein isoelectric point is achieved by the presence of two additional Arg residues at positions B31 and B32. The Gly residue is introduced at A21 position of the insulin A chain to maintain stability in aqueous solutions at low pH.

[0021] Also, the isoelectric shift method was used to create NovoSol insulin (Novo Nordisk), but the development was discontinued due to inflammatory reaction issues at the injection site (Guthrie R. Is there a need for a better basal insulin? / / Clinical diabetes. 2001;19(2):66-70).

[0022] In the alternative approach for achieving the prolonged hormone effect, insulin was conjugated with the long-chain fatty acids which not only form a local depot due to slow absorption, but also promote prolongation of circulation in blood plasma due to non-covalent albumin binding. This approach was used by Eli Lilly with lipidated insulin comprising palmitic acid attached to LysB29 (W99-S-32), and by Novo Nordisk with LysB29-myristyl-desB30-insulin (Hoeg- Jensen T. Design of insulin variants for improved treatment of diabetes / / Peptide and Protein Design for Biopharmaceutical Applications Editor Jensen KJ. John Wiley & Sons (2009); pp. 249-286.). The latter was registered in 2004 as a basal analog of insulin detemir (Levemir, Novo Nordisk). (Hermansen K. et al. A 26-week, randomized, parallel, treat-to-target trial comparing insulin detemir with NPH insulin as add-on therapy to oral glucose-lowering drugs in insulin-naive people with type 2 diabetes / / Diabetes care. 2006 Jun;29(6): 1269-74).

[0023] Conjugation with fatty acids serves as a basis for another basal insulin analog, insulin degludec (Novo Nordisk). Insulin degludec is a des-B30 human insulin with a fatty acid attached at the LysB29 position. The prolonged pharmacodynamic profile of insulin degludec is provided by the additional formation of multihexameric complexes at the injection site (Gough S. C. L. et al. Insulin degludec: overview of a novel ultra long-acting basal insulin / / Diabetes, Obesity and Metabolism. 2013 Apr;15(4):301-9). As long as insulin degludec is in a finished dosage form in the presence of phenol and zinc, it retains a stable dihexameric structure. Upon injection, phenol rapidly dissociates, and the subsequent conformational change mediates formation of a soluble multimeric structure which slowly releases degludec insulin monomers for absorption (Jonassen I. et al. Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin / / Pharmaceutical research. 2012 Aug;29(8):2104-14). The oligomeric structure of insulin degludec is mainly responsible for its prolonged action, while its affinity for plasma albumin is thought to provide a buffering effect and reduce variability after absorption into the bloodstream. Clinical evaluation of insulin degludec therapy showed a sugar-reducing effect lasting up to 42 hours and fewer hypoglycemic episodes, especially at night, compared to insulin glargine therapy (Sorli C. et al. Elderly patients with diabetes experience a lower rate of nocturnal hypoglycemia with insulin degludec than with insulin glargine: a meta-analysis of phase Illa trials / / Drugs & aging. 2013 Dec;30(12): 1009-18).

[0024] Another approach for the manufacture of long-acting insulins is conjugation with polymers. This methodology underlies insulin peglispro (Eli Lilly). Insulin peglispro is made by covalently attaching a 20 kDa linear polyethylene glycol (PEG) polymer to the LysB28 side chain of insulin lispro. Prolonged action is achieved due to the increased hydrodynamic size of the conjugated analog, which is more than four times larger than the unmodified insulin. Larger size leads to a slower subcutaneous absorption, as well as a significant decrease in the renal clearance of the drug, which contributes to a noticeable increase in the blood residence time of the peptide (Caparrotta T. M., Evans M. PEGylated insulin Lispro, (LY2605541) — a new basal insulin analog / / Diabetes, Obesity and Metabolism. 2014 May;16(5):388-95).

[0025] Immunoglobulin Fc domain receptors (FcRs) are divided into two main classes: those involved in effector functions, exemplified by the FcRs for IgG (FcyRI, II and III), IgE (FCERI), IgA (FcaRI), and those responsible for the transport of immunoglobulins through the epithelium - the IgA polyreceptor (plgR) and FcRn, a neonatal IgG transporter.

[0026] The neonatal Fc receptor (FcRn) plays a central role in the regulation of IgG levels and serum albumin (SA) levels in mammals via a recycling mechanism. The mechanism of FcRn- mediated recycling consists in rescuing wild-type IgG from the process of lysosomal degradation, which results in reduced clearance and increased half-life of antibodies. FcRn is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and a 15 kDa p2-microglobulin (P2m). FcRn binds with high affinity to the CH2-CH3 portion of the IgG class antibody Fc region. The interaction between an IgG class antibody and FcRn is pH-dependent and occurs in a stoichiometric ratio of 1:2, i.e., one IgG antibody molecule can interact with two FcRn molecules through its two heavy chain Fc region polypeptides (see, e.g., HUBER A. H. et al., Crystallization and stoichiometry of binding of a complex between a rat intestinal Fc receptor and Fc, Journal of molecular biology, 1993;230:1077-83).

[0027] Thus, the properties / characteristics of binding of IgG molecules to FcRn in vitro are indicative of its in vivo pharmacokinetics in the bloodstream.

[0028] The Fc fragment of an antibody also interacts with various Fc receptors and ligands that mediate the effector functions of immune system cells. This receptor family incorporates FcyRI (CD64), comprising FcyRIa, FcyRIb, and FcyRIc isoforms; FcyRII (CD32), comprising FcyRIIa, FcyRIIb (including FcyRIIb-1 and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD 16), comprising FcyRIIIa and FcyRIIIb isoforms (see NAGELKERKE S. Q., KUIJPERS T. W., Immuno modulation by IVIg and the role of Fc-gamma receptors: classic mechanisms of action after all?, Frontiers in immunology, 2015 Jan 21;5: 674).

[0029] Fc gamma receptors are known to have an extracellular domain mediating interaction with Fc, a transmembrane domain, and an intracellular domain that can mediate some signaling events within the cell. These receptors are expressed by various immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK), and T cells. The formation of the Fc / Fc-gamma-R complex enables interaction of these effector cells with antigens, which typically leads to signal transduction in cells and important subsequent immune responses such as release of inflammatory mediators, B-cell activation, endocytosis, phagocytosis, and antibodydependent cellular cytotoxicity. The ability to mediate phagocytic effector function is a potential mechanism by which antibodies provide protection from infection (see NAGELKERKE S. Q., KUIJPERS T. W., Immunomodulation by IVIg and the role of Fc-gamma receptors: classic mechanisms of action after all?, Frontiers in immunology, 2015 Jan 21;5:674).

[0030] Different amino acid residues of the heavy chain CH2 and CH3 domains are involved in the interaction between FcRn and Fc region of an IgG class antibody. The amino acid residues involved in the interaction with the FcRn are localized from about EU position 243 to about EU position 261, from about EU position 275 to about EU position 293, from about EU position 302 to about EU position 319, from about EU position 336 to about EU position 348, at EU position 408, and from about EU position 424 to about EU position 440. More specifically, the following amino acid residues according to EU Kabat numbering are involved in the interaction between the Fc region and the FcRn: F243, P244, P245 P, K246, P247, K248, D249, T250, L251, M252, 1253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V3O3, S304, V305, L306, T307, V3O8, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, 1336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, 1377, A378, V379, E38O, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439, and S440.

[0031] Studies employing site-specific mutagenesis have confirmed that in the IgG Fc region, histidine 310, histidine 435, and isoleucine 253, and, to a lesser extent, histidine 433 and tyrosine 436 are critical sites for binding to FcRn (see, e.g., KIM J. K. et al., Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn, European journal of immunology, 1999 Sep;29(9):2819-25; RAGHAVAN M. et al., Analysis of the pH dependence of the neonatal Fc receptor / immunoglobulin G interaction using antibody and receptor variants, Biochemistry, 1995 Nov 14;34(45): 14649-57; MEDESAN C. et al., Delineation of the amino acid residues involved in transcytosis and catabolism of mouse IgGl, I Immunol. 1997 Mar 1;158(5):2211-7).

[0032] Methods are known for enhancing the IgG binding to FcRn, which have been accomplished by introducing mutations to various amino acid residues in IgGs: threonine 250, methionine 252, serine 254, threonine 256, threonine 307, glutamic acid 380, methionine 428, histidine 433, and asparagine 434 (see, e.g., KUO T. T. et al., Neonatal Fc receptor: from immunity to therapeutics, lournal of clinical immunology, 2010 Oct 1;30(6):777- 789).

[0033] The study on the interactions between immunoglobulins and FcRs revealed that the binding sites of Clq and FcyR are located in the IgG CH2 domain. The IgG2b mAb mutagenesis assay identified glutamic acid, lysine, and lysine residues at positions 318, 320, and 322, respectively, as the key binding motif for Clq. Also, amino acid residues at positions 234-238 are involved in the high-affinity interaction of murine IgG2a with FcyRI. In addition, replacing aspartic acid by alanine at position 265 (D265A) has been shown to abolish interaction between the murine IgGl and low-affinity FcyRIIB and FcyRIII (BAUDINO L. et al., Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions, I Immunol. 2008 Nov 1; 181(9):6664-9). Thus, for some therapeutic applications, it is preferable to avoid activation of complement and FcyRs. For example, it is undesirable to stimulate complement when administering therapeutically active polypeptides fused to antibody Fc fragments (see, e.g. CAPON D. J. et al., Designing CD4 immunoadhesins for AIDS therapy, Nature, 1989 Feb 9;337(6207):525-31). In addition, due to relatively lower affinity of human IgG2 and IgG4 for FcyRs and complement receptors (BRUHNS P. et al., Specificity and affinity of human Fey receptors and their polymorphic variants for human IgG subclasses, Blood, The Journal of the American Society of Hematology, 2009, V. 113, N. 16, p.3716-3725), these two subclasses are used in the development of therapeutic mAbs (e.g., denosumab, natalizumab, panitumumab, and eculizumab) where minimal effector potential is required.

[0034] For these purposes, glucagon like peptide 1 (GLP-1) was fused to human IgG2 to avoid unwanted immunogenicity, and it was shown to have superior therapeutic and pharmacologic properties compared to native GLP-1 in a mouse model of type I diabetes (Wang Q. et al., Novel GLP-1 fusion chimera as potent long acting GLP-1 receptor agonist, PLoS One. 2010 Sep 15;5(9):el2734). However, it is also possible to achieve a decreased effector function of one effector system by the simultaneous activation of another effector system. In particular, it has been observed that FcER-activation of mast cells can be downregulated by the simultaneous activation of inhibitory FcyRIIB (D AERON M. et al., The same tyrosine-based inhibition motif, in the intra-cytoplasmic domain of FcyRIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation, Immunity, 1995, V. 3, N. 5, p.635-646), and this observation stimulated the development of series of Fc-fusion proteins that inhibit inflammation and are useful in treating allergic asthma.

[0035] In summary, it can be assumed that introducing certain modifications to the Fc fragment which a biologically active partner is fused with or conjugated to can enhance the ability of the fusion protein to bind to FcRn, and can reduce its ability to bind to effector FcRs, which can significantly improve pharmacokinetic properties and safety profile of the fusion protein.

[0036] Thus, a modified Fc fragment polypeptide can be a suitable partner for fusing with insulin for producing novel long-acting insulin analogs.

[0037] The present invention is directed to the development of insulin receptor agonists comprising insulin variants and immunoglobulin Fc fragment polypeptide variants having high binding capacity for FcRn and low binding capacity for FcRs, which are useful for the effective treatment of insulin- sensitive conditions or diseases.

[0038] SUMMARY All terms and abbreviations used herein have meanings commonly understood in the art and will be evident to a skilled person.

[0039] The term “insulin receptor agonist” refers to a protein that binds to and activates insulin receptor, resulting in reduced blood glucose levels and / or suppression of the glucose release by the liver, or other features which are consistent with insulin receptor activation and which may be tested and measured using means and methods known to a person skilled in the art.

[0040] The term “insulin analog” means an engineered altered insulin comprising one or more substitutions, deletions, or insertions as compared with natural human insulin, and having insulin activity.

[0041] The term “alteration” means a mutation (substitution), insertion (addition, insert), or deletion of one or more amino acid residues compared to a parent polypeptide.

[0042] The term “amino acid mutation” refers to replacing at least one of the existing amino acid residue with another different amino acid residue (i.e., a substituting amino acid residue). The substituting amino acid residue may be a “naturally occurring amino acid residue”, and may be selected from the group consisting of alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V). The substituting amino acid residue may be a “non-naturally occurring amino acid residue,” including but not limited to a D-stereoisomer of a “naturally occurring amino acid residue”.

[0043] The term “amino acid insertion” means the (additional) incorporation of at least one amino acid residue at a predetermined position in an amino acid sequence. In one embodiment, the “insertion” is the insertion of one, two, three, or more amino acid residues. The inserted amino acid residues may be any naturally occurring or non-naturally occurring amino acid residues, or a combination thereof.

[0044] The term “amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined position of an amino acid sequence.

[0045] A “human antibody” is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human, or produced by a human cell, or derived from a nonhuman source that employs human antibody repertoires or other sequences encoding human antibodies. Such definition of a “human antibody” specifically excludes a humanized antibody that contains antigen-binding residues not found in a human antibody.

[0046] The “class” of an antibody refers to the type of its heavy chain constant domain or constant region. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called a, 5, a, y and p, respectively.

[0047] The term “CH2 domain” means the portion of an antibody heavy chain polypeptide spanning approximately from EU position 231 to EU position 340 (EU numbering system according to Kabat).

[0048] The term “CH3 domain” means a portion of an antibody heavy chain polypeptide spanning approximately from EU position 341 to EU position 446.

[0049] The term “derived from” means that an amino acid sequence is made from a parent amino acid sequence by introducing changes at least at one position. Such altered amino acid sequence differs from the corresponding parent amino acid sequence at least at one corresponding position (Kabat EU index numbering used for numbering antibody Fc regions). In one embodiment, an amino acid sequence derived from a parent amino acid sequence differs in one or more amino acid residues at corresponding positions. Similarly, the altered amino acid sequence has a high amino acid sequence identity to its parent amino acid sequence. In one embodiment, the amino acid sequence derived from a parent amino acid sequence has an amino acid sequence identity of 90% or greater. In one embodiment, the amino acid sequence derived from a parent amino acid sequence has an amino acid sequence identity of 95% or greater.

[0050] The term “human-derived Fc fragment” refers to the C-terminal heavy chain region of a human-derived immunoglobulin, which region comprises at least a portion of the hinge region, and the CH2-domain and the CH3-domain. In one embodiment, the human IgG heavy chain Fc region extends about from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. The C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in KABAT E. A., Sequences of proteins of immunological interest, US Department of Health and Human Services, Public Health Service, National Institutes of Health, 1991, N. 91. The Fc region consists of two heavy chain Fc region polypeptides that can be covalently joined to one another via hinge region cysteine residues involved in the formation of disulfide bonds between the polypeptides.

[0051] The term “FcRn” means a human neonatal Fc receptor. The function of FcRn is to rescue IgG from the process of lysosomal degradation, which results in reduced clearance and increased half-life. FcRn is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and a 15 kDa p2-microglobulin (P2m). FcRn binds with high affinity to the CH2-CH3 portion of the IgG Fc region. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a stoichiometric ratio of 1:2, with one IgG molecule binding through two heavy chains thereof to two FcRn molecules (HUBER A. H. et al., Crystallization and stoichiometry of binding of a complex between a rat intestinal Fc receptor and Fc, Journal of molecular biology, 1993 Apr 5;230(3): 1077-83). FcRn binding occurs in the endosome at acidic pH values (pH<6.5), and IgG is released on the cell surface at neutral pH (pH of about 7.4). The pH-dependence of this interaction contributes to the FcRn-mediated protection of IgG molecules, entering cells through pinocytosis, from intracellular degradation, due to the binding of the receptor within endosomes under acidic conditions. In addition, FcRn promotes IgG return back to the cell surface and subsequent release into the bloodstream after the FcRn-IgG complex is exposed to a neutral pH environment outside the cell.

[0052] The term “FcRn-binding portion of the Fc region” means a portion of an antibody heavy chain polypeptide, which portion extends from about EU position 243 to about EU position 261, and from about EU position 275 to about EU position 293, and from about EU position 302 to about EU position 319, and from about EU position 336 to about EU position 348, and from about EU position 367 to about EU position 393 and about EU position 408, and from about EU position 424 to about EU position 440. In one embodiment, one or more of the following amino acid residues corresponding to EU Kabat numbering are altered: F243, P244, P245 P, K246, P247, K248, D249, T250, L251, M252, 1253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V3O3, S304, V305, L306, T307, V3O8, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, 1336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, 1377, A378, V379, E38O, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 (EU numbering).

[0053] The term “human IgG Fc fragment polypeptide” refers to an amino acid sequence derived from a human IgG native Fc region polypeptide or a human IgG wild-type Fc region polypeptide as a result of at least one amino acid alteration.

[0054] The term “heterodimer”, or “heterodimeric” means a molecule comprising two polypeptide chains (e.g., of comparable length), wherein the two polypeptide chains have amino acid sequences differing at least in one amino acid residue at corresponding positions.

[0055] The terms “homodimer” and “homodimeric” mean a molecule comprising two polypeptide chains of comparable length, wherein the two polypeptide chains have the identical amino acid sequences at corresponding positions. The term “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule comprised in cells that normally comprise a nucleic acid molecule, but the nucleic acid molecule is present either extrachromosomally or at a location on a chromosome that is different from its natural location on the chromosome.

[0056] According to the present invention, a “nucleic acid” or “nucleic acid molecule” is intended to mean a polynucleotide molecule which may refer to a DNA or RNA type, preferably a DNA type, and, in particular, may be double- stranded. It may be of a natural or synthetic origin. Synthetic nucleic acids are generated in vitro. Examples of such synthetic nucleic acids are acids wherein the codons encoding the polypeptide(s) have been optimized for the host organism intended for expression of the polypeptide(s) (e.g., by substituting codons with the codons which are more preferred or most preferred according to the codon usage tables for the host organism or the group to which the host organism belongs, compared to the original host). Methods for codon optimization are well known to a skilled person.

[0057] For the purposes of the present disclosure, the terms “nucleic acid” and “polynucleotide” are used in the same sense and are synonymous. For example, the phrase "encoding nucleic acid" is equivalent to the phrase "encoding polynucleotide".

[0058] As used herein, the terms “including” or “comprising” a particular ‘X’ sequence, in relation to a DNA, polypeptide, or protein, refers to a DNA or protein including or comprising at least the X sequence in such a way that the other nucleotide or amino acid sequences, e.g., a selectable marker, and / or a 5 ’-leader sequence, or a 3 ’-trailer sequence can be included at the 5 ’(or N-terminal) and / or 3’ (or C-terminal) end.

[0059] The terms “comprise”, “comprising”, or “comprises”, as used throughout this description and appended claims, is intended to include a certain number of structural elements, substances, or steps, or a certain number of groups of structural elements, substances, or steps, but not to exclude any other number of structural elements, substances, or steps, or groups of structural elements, substances, or steps.

[0060] “Percent (%) 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 with the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. As used herein, “percent identity” between two amino acid sequences means the percentage of identical amino acids in the two sequences to be compared, obtained by optimal alignment of said sequences, wherein the percentage is purely statistical, and the differences between the two sequences are randomly distributed over the amino acid sequences. As used herein, the “best alignment” or “optimal alignment” means an alignment characterized by a specified maximum percentage of identity (see below). Comparison of two amino acid sequences is typically carried out by comparing data for the pre-aligned sequences according to the most optimal alignment; this comparison is performed within comparison windows to identify and compare the local regions of similarity. Along with the manual method, the most optimal sequence alignment for the purpose of comparison may be carried out using the local homology and general homology search algorithm, using software that implements such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, T FAST A), using MUSCLE multiple sequence alignment algorithms (EDGAR R. C., MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic acids research, 2004 Mar 19;32(5): 1792-7). In order to obtain the best alignment, it may be preferable to use BLAST software with BLOSUM 62 matrix. The percent identity between two amino acid sequences is determined by comparing the two optimally aligned sequences, wherein said amino acid sequences can incorporate additions and deletions relative to the reference sequence to obtain the optimal alignment between the two sequences. The percent identity is calculated by determining the number of identical positions in the two sequences, dividing this number by the total positions compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0061] The term “vector” as used herein refers to a nucleic acid molecule having the ability to reproduce another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure including a “cloning vector”, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0062] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transfected cells," which include the primary transfected cell and progeny derived therefrom without regard to the number of passages. Progeny may be not completely identical in nucleic acid content to a parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity used for screening or selecting the originally transfected cell are included into the present invention.

[0063] The term “peptide linker” as used herein means a peptide with various amino acid sequences, which, in one embodiment, has a synthetic origin.

[0064] The term “individual” or “subject” means a mammal. Mammals include, but are not limited to, domesticated animals e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as apes and monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, a human is the individual or subject.

[0065] For the purposes of the present invention, the term “medicament” refers to a substance or a mixture of substances of synthetic or natural origin formulated in a dosage form, including, but not limited to the form of a tablet, capsule, solution, ointment, aerosol, etc., used for the prevention, diagnosis and treatment of diseases.

[0066] The term “pharmaceutical composition” refers to a preparation in such a form that contributes to the effective biological activity of the active ingredient comprised therein, and which does not contain any additional components that are unacceptably toxic to a subject who will be receiving the composition.

[0067] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition other than the active ingredient and being non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to a buffer, excipient, stabilizer, or preservative.

[0068] As used herein, the term “treatment” (as well as “therapy”) refers to clinical measures aiming to alter the natural course of disease in the individual being treated, and it may be carried out either for preventing, or in the course of treatment of a clinical pathological condition. Desirable treatment effects include, but are not limited to, preventing the disease onset or recurrence, ameliorating symptoms, minimizing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, reducing the intensity of symptoms or temporary alleviating the disease state, and remission or improved prognosis.

[0069] As used herein, the term “instructions for use” refers to instructions commonly included in commercial packages of therapeutic products that contain information on indications, use, dosage, administration, combination therapy, contraindications and / or warnings regarding the use of such therapeutic products.

[0070] EMBODIMENTS

[0071] The present invention provides ultra-long-acting insulin receptor agonists, which are fusion proteins comprising an insulin or analog thereof fused to an Fc fragment polypeptide that has modified binding properties to human Fc receptors as compared to the corresponding wildtype Fc fragment.

[0072] In one embodiment of the invention, the human Fc receptor is selected from a human neonatal Fc receptor and a human Fey receptor. In one embodiment, the human Fey receptor is selected from a human FcyRI receptor, a human FcyRII receptor, and a human FcyRIII receptor.

[0073] In one embodiment, the insulin receptor agonist is a fusion protein comprising consecutively fused insulin B chain or insulin analog B chain, insulin A chain or insulin analog A chain, and human IgG Fc fragment polypeptide.

[0074] In one embodiment, the insulin A chain is the human insulin A chain of SEQ ID NO: 1. In one embodiment, the insulin analog A chain has an amino acid sequence having at least 90% identity to the amino acid sequence of the human insulin A chain.

[0075] In one embodiment, the insulin analog A chain has an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence of SEQ ID NO:1.

[0076] In one embodiment, the insulin analog A chain differs from the human insulin A chain in 1 amino acid residue, or 2, or 3, or 4, or 5, or 6 amino acid residues at the corresponding position(s).

[0077] In one embodiment, the insulin analog A chain comprises one or more of the following mutations or combinations of mutations: T8H, Y14E, Y14D, and N21G.

[0078] In one embodiment, the insulin analog A chain comprises T8H mutation.

[0079] In one embodiment of the invention, the insulin analog A chain comprises Y14E mutation.

[0080] In one embodiment, the insulin analog A chain comprises T8H and Y14E mutations. In one embodiment, the insulin analog A chain comprises Y14D and N21G mutations. In one embodiment, the insulin analog A chain comprises the amino acid sequence of GIVEQCCX8SICSLXi4QLENYCX2ias set forth in SEQ ID NO: 15, wherein

[0081] X8is T or H,

[0082] X14 is D, E, or Y, and

[0083] X2iis G or N.

[0084] In one embodiment, the insulin analog A chain comprises the amino acid sequence of GIVEQCCX8SICSLXI4QLENYCX2I as set forth in SEQ ID NO: 15, wherein X8is T, Xi4is Y, and X2iis N.

[0085] In one embodiment, the insulin analog A chain comprises the amino acid sequence of GIVEQCCX8SICSLXI4QLENYCX2I as set forth in SEQ ID NO: 15, wherein X8is H, Xi4is Y, and X2iis N. In one embodiment, the insulin analog A chain comprises the amino acid sequence of GIVEQCCX8SICSLXi4QLENYCX2ias set forth in SEQ ID NO: 15, wherein X8is T, Xi4is E, and X2iis N.

[0086] In one embodiment, the insulin analog A chain comprises the amino acid sequence of GIVEQCCX8SICSLXi4QLENYCX2ias set forth in SEQ ID NO: 15, wherein X8is H, Xi4is E, and X2iis N.

[0087] In one embodiment, the insulin analog A chain comprises the amino acid sequence of GIVEQCCX8SICSLXI4QLENYCX2I as set forth in SEQ ID NO: 15, wherein X8is T, Xi4is D, and X2iis G.

[0088] In one preferred embodiment, the insulin analog A chain has the amino acid sequence of SEQ ID NO: 11.

[0089] In one preferred embodiment, the insulin analog A chain has the amino acid sequence of SEQ ID NO: 12.

[0090] In one preferred embodiment, the insulin analog A chain has the amino acid sequence of SEQ ID NO: 13.

[0091] In one preferred embodiment, the insulin analog A chain has the amino acid sequence of SEQ ID NO: 14.

[0092] In one embodiment, the insulin B chain is a human insulin B chain of SEQ ID NO:2. In one embodiment, the insulin analog B chain has an amino acid sequence having at least 90% identity to the amino acid sequence of the human insulin B chain.

[0093] In one embodiment, the insulin analog B chain has an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:2.

[0094] In one embodiment, the insulin analog B chain differs from the human insulin B chain in 1 amino acid residue, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13 amino acid residues at the corresponding position(s).

[0095] In one embodiment, the insulin analog B chain comprises one or more of the following mutations, or combinations of mutations: F1H, Y16H, Y16E, Y16D, F24H, F25H, Y26H, T27del, P28del, K29del, T30del.

[0096] In one embodiment, the insulin analog B chain comprises F25H, Y26H, and T30del mutations. In one embodiment, the insulin analog B chain comprises F24H, Y26H, T27del, P28del, K29del, and T30del mutations.

[0097] In one embodiment, the insulin analog B chain comprises Y16H, F25H, and T30del mutations.

[0098] In one embodiment, the insulin analog B chain comprises F1H, F25H, and T30del mutations.

[0099] In one embodiment, the insulin analog B chain comprises Y16E, F25H, and T30del mutations.

[0100] In one embodiment, the insulin analog B chain comprises Y16H, F24H, T27del, P28del, K29del, and T30del mutations.

[0101] In one embodiment, the insulin analog B chain comprises Y16D, F25H, and T30del mutations.

[0102] In one embodiment, the insulin analog B chain comprises F25H, T27del, P28del, K29del, and T30del mutations.

[0103] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein

[0104] Xi is F or H,

[0105] Xi6 is H, Y, E, or D,

[0106] X24 is H or F,

[0107] X25 is F or H,

[0108] X26is Y or H,

[0109] X27 is T or absent,

[0110] X28is P or absent,

[0111] X29 is K or absent.

[0112] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is F, X16 is Y, X24 is F, X25 is H, X26 is H, X27 is T, X28is P, X29 is K.

[0113] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is F, X16 is Y, X24 is H, X25 is F, X26 is H, X27 is absent, X28is absent, X29 is absent. In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is F, Xi6 is H, X24 is F, X25 is H, X26 is Y, X27 is T, X28 is P, X29 is K.

[0114] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is H, X16 is Y, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K.

[0115] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is F, X16 is E, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K.

[0116] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is F, X16 is H, X24 is H, X25 is F, X26 is Y, X27 is absent, X28is absent, X29 is absent.

[0117] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is F, X16 is D, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K.

[0118] In one embodiment, the insulin analog B chain comprises the amino acid sequence of XiVNQHECGSHEVEAEXi6EVCGERGX24X25X26X27X28X29 as set forth in SEQ ID NO:24, wherein Xi is F, X16 is Y, X24 is F, X25 is H, X26 is Y, X27 is absent, X28is absent, X29 is absent.

[0119] In one preferred embodiment, the insulin analog B chain has the amino acid sequence of SEQ ID NO: 16.

[0120] In one preferred embodiment, the insulin analog B chain has the amino acid sequence of SEQ ID NO: 17.

[0121] In one preferred embodiment, the B -chain of the insulin analog is the amino acid sequence of SEQ ID NO: 18.

[0122] In one preferred embodiment, the insulin analog B chain has the amino acid sequence of SEQ ID NO: 19.

[0123] In one preferred embodiment, the insulin analog B chain has the amino acid sequence of SEQ ID NO:20.

[0124] In one preferred embodiment, the insulin analog B chain has the amino acid sequence of SEQ ID NO:21. In one preferred embodiment, the insulin analog B chain has the amino acid sequence of SEQ ID NO:22.

[0125] In one preferred embodiment, the insulin analog B chain has the amino acid sequence of SEQ ID NO:23.

[0126] In one embodiment of the present invention, the Fc fragment polypeptide is a human IgG class Fc fragment.

[0127] In one embodiment, the Fc fragment polypeptide is a human IgGl class Fc fragment. In one embodiment, the Fc fragment polypeptide is a human IgG2 class Fc fragment. In one embodiment, the Fc fragment polypeptide is a human IgG3 class Fc fragment. In one embodiment, the Fc fragment polypeptide is a human IgG4 class Fc fragment. In one embodiment, a pair of two Fc fragment polypeptides may form a functional dimer. In one embodiment of the invention, the human Fc receptor is selected from a human neonatal Fc receptor and a human Fey receptor.

[0128] In one embodiment, the human Fey receptor is selected from a human FcyRI receptor, a human FcyRII receptor, and a human FcyRIII receptor.

[0129] In one embodiment, the Fc fragment polypeptide differs from the wild-type Fc fragment polypeptide in 1 amino acid residue, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12 amino acid residues at the corresponding position(s) according to the Kabat EU index numbering system.

[0130] In one embodiment, the Fc fragment polypeptide comprises one or more mutations at positions corresponding to positions 222, 223, 224, 225, 226-238, 226, 234, 235, 236, 237, 239, 250, 252, 252, 254, 256, 262, 264, 264, 264, 268, 272, 274, 283, 285, 286, 288, 294, 296, 297, 300, 302, 303, 308, 308, 309, 309, 312, 315, 327, 328, 330, 330, 331, 339, 355, 356, 358, 378, 397, 409, 419, 427, 428, 434, 434, 445, 444-446 according to the Kabat EU index numbering system.

[0131] In one embodiment, the Fc fragment polypeptide comprises one or more of the following mutations or combinations of mutations:

[0132] K222D, C223K, C224T, V225H, E226-P238del, E226W, P234E, 234insF235, V235E, A236G, G237M, S239E, T250Q, M252Y, M252W, S254T, T256E, V262W, V264del, V264D, V264E, H268Q, E272Q, Q274K, E283Q, H285E, N286E, K288M, E294Q, F296Y, N297D, F300Y, V302W, V3O3del, V3O8W, V3O8F, V309del, V309E, D312N, N315D, G327A, E328G, A33OS, A33OF, P331S, T339A, R355Q, D356E, E358M, S378A, M397V, K409R, Q419E, V427L, M428L, N434Y, N434W, P445L, S444-G446del according to the Kabat EU index numbering system.

[0133] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to V262W, V264del, V302W, V3O3del, V3O8W, V309del, and S378A mutations according to the Kabat EU index numbering system.

[0134] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to M252Y, S254T, T256E, and S378A mutations according to the Kabat EU index numbering system.

[0135] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to T250Q, M428L and S378A mutations according to the Kabat EU index numbering system.

[0136] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to M252Y, T256E, V264D, N297D, V3O8F, S378A, and N434Y mutations according to the Kabat EU index numbering system.

[0137] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to P234E, 234insF235, V235L, A236G, T256E, V264D, H268Q, H285E, N286E, N297D, F300Y, V309L, A33OS, P331S, R355Q, S378A, M397V, K409R, Q419E, M428L, P445L mutations according to the Kabat EU index numbering system.

[0138] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to M252W, T256E, S378A, M428L, and N434Y mutations according to the Kabat EU index numbering system.

[0139] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to S239E, T256E, V264E, A33OF, S378A, and N434Y mutations according to the Kabat EU index numbering system.

[0140] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to ins221D, V222K, ins223T, E224H, ins225W, G237M, T250Q, M252Y, V264D mutations according to the Kabat EU index numbering system.

[0141] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to M252Y, T256E, V264E, N297D, V3O8F, A33OF, S378A, and N434W mutations according to the Kabat EU index numbering system.

[0142] In one preferred embodiment, the Fc fragment polypeptide comprises a combination of mutations corresponding to mutations T250Q, M252Y, T256E, V264E, V3O8F, A33OF, S378A, and N434W according to the Kabat EU index numbering system. In one embodiment, the Fc fragment polypeptide has an amino acid sequence having at least 90% identity to the amino acid sequence of a human IgG2 Fc fragment.

[0143] In one preferred embodiment, the Fc fragment polypeptide comprises the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX2O8HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein

[0144] Xi is E or D,

[0145] X2 is K or absent,

[0146] X3is T or absent,

[0147] X4 is H or absent,

[0148] X5is W or absent,

[0149] X13 is P or E,

[0150] X14 is F or absent,

[0151] X15 is V or L,

[0152] Xi6 is A or G,

[0153] X17 is G or M,

[0154] X19 is S or E,

[0155] X30is T or Q,

[0156] X32is M, Y, or W,

[0157] X34 is S or T,

[0158] X36 is T or E,

[0159] X44 is V, E, or D,

[0160] X48is H or Q,

[0161] Xes is H or E, X66 is N or E,

[0162] X77 is N or D,

[0163] X80is F or Y,

[0164] X88is V or F,

[0165] X89is V or L,

[0166] X110is A, F, or S,

[0167] X111is P or S,

[0168] X119 is T or A,

[0169] X135 is R or Q,

[0170] X177 is M or V,

[0171] X189is K or R,

[0172] X199 is Q or E,

[0173] X208is M or L,

[0174] X214 is N, Y, or W, and

[0175] X225 is P or L.

[0176] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX208HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X 5 is absent, Xi3is P, Xi4is absent, Xi5is V, X16 is A, Xi7is G, X19 is S, X30 is T, X32 is Y, X34 is T, X36 is E, X44 is V, X48 is H, Xes is H, X66 is N, X77 is N, X8o is F, X88is V, X89 is V, Xno is A, Xm is P, X119 is T, X135 is R, X177 is M, Xi89 is K, X199 is Q, X2o8 is M, X214 is N, and X225 is P.

[0177] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPX177LDSDGSFFLYSX189LTVDKSRWQX199GNV FSCSVX208HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X5is absent, Xi3is P, Xi4is absent, Xi5is V, X16 is A, Xi7is G, X19 is S, X30is Q, X32is M, X34is S, X36 is T, Xu is V, X^ is H, X63is H, X66 is N, X77 is N, X§o is F, X88is V, X8g is V, Xno is A, Xm is P, X119 is T, Xi33is R, X177 is M, Xi8g is K, X199 is Q, X208 is L, X2i4is N, and X225 is P.

[0178] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of XiX2X3X4X5CPPCPAPXi3Xi4Xi5Xi6Xi7PXi9VFLFPPKPKDX3oLX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX1 I9KGQPREPQVYTLPPSXI35EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPX177LDSDGSFFLYSX189LTVDKSRWQX199GNV FSCSVX2O8HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X5is absent, Xi3is P, Xi4is absent, Xi5is V, X16 is A, Xi7is G, X19 is S, X30is T, X32is Y, X34is S, X36 is E, X44is D, X48is H, X63is H, X66 is N, X77 is D, X8o is F, X88is F, X89 is V, Xno is A, Xm is P, X119 is T, Xi33is R, X177 is M, Xi89 is K, X199 is Q, X208 is M, X2i4is Y, and X225 is P.

[0179] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of XiX2X3X4X5CPPCPAPXi3Xi4Xi5Xi6Xi7PXi9VFLFPPKPKDX3oLX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX1 I9KGQPREPQVYTLPPSXI35EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPX177LDSDGSFFLYSX189LTVDKSRWQX199GNV FSCSVX208HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X5 is absent, Xi3is E, Xi4is F, X15 is L, X16 is G, X17 is G, X19 is S, X30is T, X32is M, X34is S, X36 is E, Xu is D, Xu is Q, X63is E, X66 is E, X77 is D, X80is Y, X88is V, X89 is L, Xno is S, Xm is S, X119 is A, Xi33is Q, X177 is V, Xi89 is R, X199 is E, X208 is L, X2i4is N, and X225 is L.

[0180] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of XiX2X3X4X5CPPCPAPXi3Xi4Xi5Xi6Xi7PXi9VFLFPPKPKDX3oLX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX1 I9KGQPREPQVYTLPPSXI35EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPX177LDSDGSFFLYSX189LTVDKSRWQX199GNV FSCSVX208HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X5is absent, Xi3is P, Xi4is absent, Xi5is V, X16 is A, Xi7is G, X19 is S, X30is T, X32is W, X34is S, X36 is E, Xu is V, Xu is H, X63is H, X66 is N, X77 is N, X8o is F, X88is V, X89 is V, Xno is A, Xm is P, X119 is T, X135 is R, X177 is M, Xi89is K, X199 is Q, X2Q8is L, X214 is Y, and X225 is P.

[0181] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX2O8HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X5is absent, Xi3is P, Xi4is absent, Xi5is V, X16 is A, Xi7is G, X19 is E, X30is T, X32is M, X34is S, X36 is E, X44is E, X48is H, X65 is H, X66 is N, X77 is N, X8o is F, X88is V, X89 is V, Xno is F, Xm is P, X119 is T, X135 is R, X177 is M, Xi89is K, X199 is Q, X2O8is M, X214 is Y, and X225 is P.

[0182] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX2O8HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is D, X2is K, X3 is T, X4 is H, X5 is W, X13 is P, X14 is absent, X15 is V, X16 is A, X17 is M, X19 is S, X30 is Q, X32is Y, X34 is S, X36 is T, X44is D, X48is H, X65 is H, X66 is N, X77 is N, X80is F, X88is V, X89 is V, Xno is A, Xm is P, X119 is T, X135 is R, X177 is M, Xi89is K, X199 is Q, X2os is M, X214 is N, and X225 is P.

[0183] In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX2O8HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X5is absent, Xi3is P, Xi4is absent, Xi5is V, X16 is A, Xi7is G, X19 is S, X30is T, X32is Y, X34is S, X36 is E, X44is E, X48is H, X65 is H, X66 is N, X77 is D, X8o is F, X88is F, X89is V, Xno is F, Xm is P, X119 is T, X135 is R, X177 is M, Xi89is K, X199 is Q, X2Q8is M, X214 is W, and X225 is P. In one embodiment, the Fc fragment polypeptide comprises the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX2O8HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, wherein Xi is E, X2is absent, X3is absent, X4is absent, X5is absent, Xi3is P, Xi4is absent, Xi5is V, X16 is A, Xi7is G, X19 is S, X30is Q, X32is Y, X34is S, X36 is E, X44is E, X48is H, X65 is H, X66 is N, X77 is N, X8o is F, X88is F, X89is V, Xno is F, Xm is P, X119 is T, X135 is R, X177 is M, Xi89is K, X199 is Q, X2O8is M, X214 is W, and X225 is P.

[0184] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:25.

[0185] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:26.

[0186] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:27.

[0187] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:28.

[0188] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:29.

[0189] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:30.

[0190] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:31.

[0191] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:32.

[0192] In one preferred embodiment, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:33.

[0193] Binding to Fc receptors can be determined, for example, by ELISA, by the surface plasmon resonance (SPR) using standard equipment such as the BIAcore device (GE Healthcare), and using such Fc receptors that can be obtained by recombinant expression method. Alternatively, the binding affinity of Fc domains or antibodies comprising Fc domain to Fc receptors can be assessed using cell lines known to express specific Fc receptors, such as human NK cells expressing Fcyllla receptor, and the like.

[0194] Fc receptor binding can also be determined, e.g., by the label- free bio layer interferometry (BLI) technique using ForteBio Octet system (Sartorius) (see, e.g., TOBIAS R., MA W., Analysis of FcRn-Antibody Interactions on the Octet Platform, ForteBio Appl Note 17, 2019, p.1-13; BAJARDI-TACCIOLI A. et al., Biolayer Interferometry-based FcyRIIa binding assay for a therapeutic antibody with strong effector function, Analytical biochemistry, 2020 Dec 15:611:113842).

[0195] The effector function of an Fc domain or antibody comprising an Fc domain can be assessed by methods known in the art, for example, by evaluating the ADCC activity of the molecule of interest (CLYNES R. et al., Fc receptors are required in passive and active immunity to melanoma, Proceedings of the National Academy of Sciences, 1998 Jan 20;95(2):652-6). Alternatively, methods based on non-radioactive analysis may be used. Acceptable effector cells for such assays are peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0196] Methods for testing FcRn binding and measuring in vivo clearance or half-life are also known to a skilled person (PETKOVA S. B. et al., Enhanced half-life of genetically engineered human IgGl antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease, International immunology, 2006 Dec;18(12): 1759-69).

[0197] In the insulin receptor agonist provided by the invention, an insulin or analog thereof can be genetically fused to an Fc fragment polypeptide. The genetic fusion of the insulin or analog thereof to the Fc fragment polypeptide may be designed so that to fuse the sequence of the insulin or analog thereof to the polypeptide directly or indirectly through a linker sequence. The linker composition and length can be determined using methods well known in the art, and their efficacy can be evaluated. Specific linker peptides are provided above herein. Where appropriate, additional sequences, e.g., an endopeptidase-recognizable sequence, may also be included at the cleavage site to separate individual fusion components. In addition, the provided fusion polypeptide can be chemically synthesized using polypeptide synthesis techniques well known in the art (e.g., Merrifield solid phase synthesis).

[0198] In one embodiment, the A chain and B chain of the insulin or insulin analog can be linked together by a peptide linker.

[0199] In one embodiment, the A chain and the B chain of the insulin or insulin analog are linked via the peptide linker having SEQ ID NO:4. In one embodiment, the A chain and the B chain of the insulin or insulin analog are linked via the peptide linker having SEQ ID NO:5.

[0200] In one embodiment, the A chain and the B chain of the insulin or insulin analog are linked via the peptide linker having SEQ ID NO:6.

[0201] The insulin or analog thereof comprised in the insulin receptor agonist of the present disclosure may be fused to an Fc fragment polypeptide directly or through a linker peptide comprising one or more amino acids, typically about 2-20 amino acids, to form a fusion construct. Linker peptides are known in the art and are described herein. Acceptable non-immunogenic linker peptides include, for example, (G4S)n, (SG4)n, (G4S)n, or G4(SG4)n, where “n” typically denotes an integer ranging from 1 to 10, typically from 2 to 4. In one embodiment, the linker peptide consists of at least 5 amino acids, in one embodiment, of 5-100 amino acids, in a further embodiment, of 10-50 amino acids. In a particular embodiment, the linker peptide is comprised of 15 amino acids. In one embodiment, the linker peptide is (GxS)nor (GxS)nGm, wherein G denotes glycine, S denotes serine, and (x=3, n=3, 4, 5 or 6 and m=0, 1, 2, or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2, or 3), in one embodiment, x=4 and n=2 or 3, in another embodiment, x=4 and n=3. In particular embodiments, the linker peptide is (648)3 or (046)365.

[0202] In one embodiment, the linker peptide has (or consists of) the amino acid sequence of SEQ ID NO:8.

[0203] In one embodiment, the linker peptide has (or consists of) the amino acid sequence of SEQ ID NO:9.

[0204] The insulin or analog thereof in the insulin receptor agonist of the present invention may be conjugated to the Fc fragment polypeptide via a non-peptide linker.

[0205] The non-peptide linker used in the present invention may be selected from the group consisting of, but not limited to polyethylene glycol, polypropylene glycol, ethylene glycolpropylene glycol copolymer, polyoxy ethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymers such as polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, their derivatives, and / or combinations thereof. Preferably, it is a polyethylene glycol.

[0206] In addition, in the present invention, a non-peptide linker may be comprised not only of one kind of polymer, but also a combination of different kinds of polymers.

[0207] The non-peptide linker used in the present invention has reactive groups capable of binding to the insulin or analog thereof, and to the Fc fragment polypeptide. The reactive groups at both ends of the non-peptide polymer are preferably selected from the group consisting of a reactive aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. In particular, when the non-peptide polymer has reactive aldehyde groups at both of its ends, the insulin or analog thereof and the Fc fragment polypeptide effectively bind to the both ends of the non-peptide linker, respectively, while minimizing nonspecific reactions. The final product formed by reductive alkylation via aldehyde bond is significantly more stable than that linked via the amide bond. The aldehyde reactive group can selectively bind to the N-terminus at low pH and can form a covalent bond with a lysine residue at high pH, e.g., pH 9.0.

[0208] The reactive groups at both ends of the non-peptide linker can be the same or different. For example, the non-peptide linker may have a maleimide group at one end, and an aldehyde group, a propionaldehyde group, or a butylaldehyde at another end. When a polyethylene glycol having hydroxyl reactive groups at both ends is used as the non-peptide linker, the hydroxyl groups can be activated to various reactive groups by known chemical reactions. Alternatively, a commercially available polyethylene glycol having a modified reactive group can be used to produce the conjugate of the present invention.

[0209] In one embodiment, the insulin receptor agonist comprises the amino acid sequence XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO:83, wherein

[0210] Xi is F or H,

[0211] Xi6 is Y, H, D, or E,

[0212] X24 is H or F,

[0213] X25 is F or H,

[0214] X26is H or Y,

[0215] X27 is T or absent,

[0216] X28is P or absent, X29 is K or absent,

[0217] X35 is S or Q,

[0218] X4o is G or absent,

[0219] X48 is T or H,

[0220] X54 is Y, E, or D,

[0221] Xei is N or G,

[0222] X§2 is D or E,

[0223] X83is K or absent,

[0224] X84is T or absent,

[0225] X85is H or absent,

[0226] X86 is W or absent,

[0227] X97 is M or G,

[0228] X99 is S or E,

[0229] X110is Q or T,

[0230] X112 is Y, M, or W,

[0231] X114 is S or T,

[0232] X116is T or E,

[0233] X124 is D, E, or V,

[0234] X190 is A or F,

[0235] X288is M or L,

[0236] X294 is N or Y.

[0237] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, X112 is Y, Xn4is T, Xue is E, Xi24 is V, Xi90is A, X288is M, X294 is N.

[0238] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24 is H, X25 is F, X26 is H, X27 is absent, X38is absent, X29is absent, X35is S, X40is absent, X48is T, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xn2is Y, Xn4is T, Xue is E, X124 is V, X190 is A, X288is M, X294 is N.

[0239] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, X112 is Y, Xn4is T, Xn6 is E, Xi24 is V, Xi90is A, X288is M, X294 is N.

[0240] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is H, Xi6is Y, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xn2is Y, Xn4is T, Xue is E, Xi24is V, Xi90is A, X288is M, X294is N.

[0241] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX1 I6PEVTCVVXI24DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35 is Q, X40is absent, X48is H, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xn2is Y, Xn4is T, Xue is E, Xi24is V, Xi90is A, X288is M, X294is N.

[0242] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX1 I6PEVTCVVXI24DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40is absent, X48is H, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xn2is Y, Xn4 is T, Xn6 is E, Xi24is V, Xi90is A, X288is M, X294is N.

[0243] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is Q, X4o is absent, X48is T, X54is E, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xn2is Y, Xn4is T, Xue is E, Xi24is V, Xi90is A, X288is M, X294is N.

[0244] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX1 I6PEVTCVVXI24DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35is S, X40is absent, X48is H, X54is E, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xn2is Y, Xn4is T, Xue is E, Xi24is V, Xi90is A, X288is M, X294is N.

[0245] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX1 I6PEVTCVVXI24DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is H, X24is H, X23is F, X26 is Y, X27 is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is T, X54is E, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xn2is Y, Xn4 is T, Xn6 is E, Xi24is V, X190 is A, X288is M, X294is N.

[0246] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24is F, X23is H, X26 is Y, X27 is T, X28is P, X29 is K, X35is S, X40is absent, X48is H, X54is E, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99 is S, Xno is T, X112 is Y, Xn4is T, Xue is E, Xi24is V, Xi90is A, X288 is M, X294 is N.

[0247] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is D, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is T, X54is D, Xei is G, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99 is S, Xno is T, Xii2is Y, Xn4is T, Xue is E, Xi24is V, Xi90is A, X288 is M, X294 is N.

[0248] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24is F, X25is H, X26 is Y, X27is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is H, X54is E, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99 is S, Xno is T, X112 is Y, Xn4 is T, Xn6 is E, Xi24 is V, X190 is A, X28s is M, X294 is N.

[0249] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0250] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0251] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0252] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24 is H, X25 is F, X26 is H, X27 is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is T, X54is Y, Xei is N, X82is E, X83 is absent, X84is absent, X85is absent, X86 is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0253] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24is H, X25 is F, X26 is H, X27 is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is T, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0254] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24is H, X25 is F, X26 is H, X27 is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is T, X54is Y, Xei is N, X82is D, X83is K, X84is T, X85 is H, X86 is W, X97is M, X99is S, X110is Q, X112is Y, X114is S, X116is T, X124is D, X190is A, X288is M, X294is N.

[0255] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0256] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG, set forth in SEQ ID NO:83, wherein Xi is F, X16 is H, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99 is E, Xno is T, Xii2is M, Xn4is S, Xue is E, Xi24is E, Xi90is F, X288 is M, X294is Y.

[0257] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0258] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is H, Xi6is Y, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99 is S, Xno is T, X112 is W, Xn4is S, Xue is E, Xi24is V, X190 is A, X288is L, X294 is Y.

[0259] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is H, Xi6is Y, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99 is E, Xno is T, Xii2is M, Xn4is S, Xue is E, Xi24is E, Xi90is F, X288 is M, X294is Y.

[0260] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is H, Xi6is Y, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is T, X54is Y, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0261] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35 is Q, X40 is absent, X48is H, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0262] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35 is Q, X40 is absent, X48is H, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0263] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is Y, X24is F, X25is H, X26is H, X27is T, X28is P, X29is K, X35 is Q, X40 is absent, X48is H, X54is Y, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0264] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is H, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0265] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is H, X54is Y, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0266] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is H, X54is Y, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0267] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is Q, X40 is absent, X48is T, X54is E, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0268] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is Q, X40 is absent, X48is T, X54is E, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0269] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is Q, X40 is absent, X48is T, X54is E, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0270] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is H, X54is E, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0271] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is H, X54is E, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0272] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, Xi6is H, X24is F, X25is H, X26is Y, X27is T, X28is P, X29is K, X35 is S, X40 is absent, X48is H, X54is E, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0273] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is H, X24 is H, X25 is F, X26 is Y, X27 is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is T, X54is E, Xei is N, X82is E, X83 is absent, X84is absent, X85is absent, X86 is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0274] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is H, X24is H, X25 is F, X26 is Y, X27 is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is T, X54is E, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0275] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is H, X24is H, X25 is F, X26 is Y, X27 is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is T, X54is E, Xei is N, X82is D, X83is K, X84is T, X85 is H, X86 is W, X97is M, X99is S, X110is Q, X112is Y, X114is S, X116is T, X124is D, X190is A, X288is M, X294is N.

[0276] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is H, X54is E, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0277] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is H, X54is E, Xei is N, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0278] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is E, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is H, X54is E, Xei is N, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0279] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is D, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is T, X54is D, Xei is G, X82is E, X83 is absent, X84 is absent, X85 is absent, X86is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0280] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is D, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is T, X54is D, Xei is G, X82is E, X83 is absent, X84 is absent, X85 is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0281] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is D, X24 is F, X25 is H, X26 is Y, X27 is T, X28is P, X29 is K, X35 is S, X40 is absent, X48is T, X54is D, Xei is G, X82is D, X83 is K, X84 is T, X85 is H, X86 is W, X97 is M, X99 is S, Xno is Q, X112 is Y, X114 is S, Xn6 is T, Xi24 is D, X190 is A, X28s is M, X294 is N.

[0282] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX6IGGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24is F, X25is H, X26 is Y, X27is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is H, X54is E, Xei is N, X82is E, X83 is absent, X84is absent, X85is absent, X86 is absent, X97is G, X99is S, X110is T, X112is W, X114is S, X116is E, X124is V, X190is A, X288is L, X294is Y.

[0283] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110EX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24is F, X25is H, X26 is Y, X27is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is H, X54is E, Xei is N, X82is E, X83is absent, X84is absent, X85is absent, X86 is absent, X97 is G, X99is E, X110is T, X112is M, X114is S, X116is E, X124is E, X190is F, X288is M, X294is Y.

[0284] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of XIVNQHLCGSHLVEALXI6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4OGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO: 83 wherein Xi is F, X16 is Y, X24is F, X25is H, X26 is Y, X27is absent, X28is absent, X29is absent, X35is S, X40is absent, X48is H, X54is E, Xei is N, X82is D, X83is K, X84is T, X85 is H, X86 is W, X97is M, X99is S, X110is Q, X112is Y, X114is S, X116is T, X124is D, X190is A, X288is M, X294is N.

[0285] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:35.

[0286] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:36.

[0287] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:37.

[0288] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:38. In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:39.

[0289] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:40.

[0290] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:41.

[0291] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:42.

[0292] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:43.

[0293] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:44.

[0294] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:45.

[0295] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:46.

[0296] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:47.

[0297] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:48.

[0298] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:49.

[0299] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:50.

[0300] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:51.

[0301] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:52. In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:53.

[0302] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:54.

[0303] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:55.

[0304] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:56.

[0305] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:57.

[0306] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:58.

[0307] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:59.

[0308] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:60.

[0309] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:61.

[0310] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:62.

[0311] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:63.

[0312] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:64.

[0313] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:65.

[0314] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:66. In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:67.

[0315] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:68.

[0316] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:69.

[0317] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:70.

[0318] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:71.

[0319] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:72.

[0320] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:73.

[0321] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:74.

[0322] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:75.

[0323] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:76.

[0324] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:77.

[0325] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:78.

[0326] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:79.

[0327] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO: 80. In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:81.

[0328] In one embodiment, the insulin receptor agonist comprises the amino acid sequence of SEQ ID NO:82.

[0329] In one embodiment, the insulin receptor agonist may further comprise a leader peptide at the N-terminus.

[0330] In one embodiment, the leader peptide is a SUMO peptide of SEQ ID NO:7.

[0331] In one embodiment, the insulin receptor agonist is a monomer comprising a single polypeptide chain.

[0332] In one embodiment of the invention, a pair of two insulin receptor agonists may form a functional dimer.

[0333] In some embodiments, the dimer is a homodimer, wherein the amino acid sequences of the two fusion proteins that form the dimer are the same.

[0334] In some embodiments, the dimer is a heterodimer, wherein the amino acid sequences of the two fusion proteins that make up the dimer are different.

[0335] In some embodiments, the insulin receptor agonist is a multimer.

[0336] In some embodiments, the multimer is a homomultimer, wherein the amino acids of the multiple fusion proteins that form the multimer are the same.

[0337] In some embodiments, the multimer is a heteromultimer, wherein the amino acid sequences of the multiple fusion proteins that make up the multimer are different.

[0338] In one embodiment, insulin receptor agonists can be in the form of compounds substantially free of zinc, or in the form of zinc complexes. When the zinc complexes of the invention are provided, two Zn2+ions, three Zn2+ions, or four Zn2+ions may be linked to each hexamer.

[0339] In one aspect, disclosed herein is a nucleic acid (polynucleotide) encoding the insulin receptor agonist disclosed herein.

[0340] In some embodiments, the isolated nucleic acid (polynucleotide) encodes the complete insulin receptor agonist of the invention as disclosed herein. In other embodiments, the isolated nucleic acid (polynucleotide) encodes a polypeptide contained in the insulin receptor agonist of the invention as disclosed herein.

[0341] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide provided herein is RNA,

[0342]

[0343] in the form of messenger RNA (mRNA). The RNA provided herein may be a single- stranded or double- stranded RNA. In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 84.

[0344] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 85.

[0345] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 86.

[0346] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 87.

[0347] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 88.

[0348] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 89.

[0349] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:90.

[0350] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:91.

[0351] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:92.

[0352] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:93.

[0353] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:94.

[0354] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:95.

[0355] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:96.

[0356] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:97.

[0357] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:98.

[0358] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO:99.

[0359] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 100. In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 101.

[0360] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 102.

[0361] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 103.

[0362] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 104.

[0363] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 105.

[0364] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 106.

[0365] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 107.

[0366] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 108.

[0367] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 109.

[0368] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 110.

[0369] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 111.

[0370] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 112.

[0371] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 113.

[0372] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 114.

[0373] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 115.

[0374] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 116.

[0375] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 117. In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 118.

[0376] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 119.

[0377] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 120.

[0378] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 121.

[0379] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 122.

[0380] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 123.

[0381] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 124.

[0382] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 125.

[0383] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 126.

[0384] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 127.

[0385] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 128.

[0386] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 129.

[0387] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 130.

[0388] In one embodiment, the isolated nucleic acid encoding the insulin receptor agonist comprises the nucleotide sequence of SEQ ID NO: 131.

[0389] The insulin receptor agonists of the invention can be produced by solid-phase peptide synthesis (e.g., Merrifield solid phase synthesis), or by recombination method. For recombinant production, one or more polynucleotides encoding an insulin receptor agonist, for example, as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Said polynucleotide can be readily isolated and sequenced using conventional procedures. One object of the invention is a vector, preferably an expression vector, comprising one or more polynucleotides of the invention. Techniques well known to those of skill in the art can be used to construct expression vectors comprising the insulin receptor agonist coding sequence along with acceptable transcription / translation control signals. These methods include recombinant DNA technologies in vitro, synthetic and genetic recombination methods in vivo (see, e.g., THOMASON L. C. et al., Recombineering: genetic engineering in bacteria using homologous recombination, Current protocols in molecular biology, 2014 Apr 14: 106: 1.16). The expression vector may be a part of a plasmid or virus, or may be a nucleic acid fragment.

[0390] The expression vector includes an expression cassette in which the polynucleotide encoding the insulin receptor agonist (i.e., a coding region) is cloned to provide a functional linkage to a promoter and / or other transcription or translation control elements.

[0391] As used herein, a "coding region" is a portion of nucleic acid, which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into amino acid, it, where present, can be seen as a coding region part, however any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, etc., are not part of a coding region. Two or more coding regions can be present in an individual polynucleotide construct, e.g., an individual vector, or in separate polynucleotide constructs, e.g., separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g., the vector provided herein may encode one or more polypeptides that are post- or cotranslationally disparted into final proteins by proteolytic cleavage.

[0392] In one embodiment, a vector, polynucleotide, or nucleic acid provided in the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide that encodes the insulin receptor agonist of the invention. Heterologous coding regions include (but are not limited to) specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable linkage occurs when a coding region of a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way that the expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA fragments (such as the coding region of a polypeptide and its associated promoter) are "operably linked" if the induction of the promoter function results in transcription of the mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, or does not interfere with the ability of the DNA template to be transcribed. Thus, a promoter region must be operably linked with a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that allows for significant transcription of the DNA only in pre-selected cells. Other transcription control elements, besides promoters, e.g. enhancers, operators, repressors, and transcription termination signals, can be operably linked with the polynucleotide to provide cell- specific transcription. Acceptable promoters and other transcription control regions are provided herein. A wide variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions functioning in vertebrate cells, such as, but not limited to promoter and enhancer segments from cytomegaloviruses e.g., the immediate early promoter combined with intron A), from simian virus 40 (e.g., the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate animal genes such as the actin gene, heat shock protein, bovine growth hormone and rabbit B-globin genes, as well as other sequences capable to control gene expression in eukaryotic cells. Additional acceptable transcription control regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., promoters induced by tetracyclines). Similarly, a wide variety of translation control elements are known to those of ordinary skill in the art. These include (but are not limited to) ribosome binding sites, translation initiation codons, and termination codons, and elements derived from viral systems (in particular, the internal ribosome entry site or IRES, which is also referred to as CITE sequence).

[0393] The expression cassette may also include other characteristic structures, such as a replication initiation site and / or chromosomally integrated elements, such as retrovirus long terminal repeats (LTRs) or inverted terminal repeats (ITRs) of adeno-associated virus (AAV).

[0394] In one embodiment, the expression cassette may also include DNA or RNA encoding a short protein sequence that may be used to facilitate further purification (e.g., a histidine tag), or for labeling a fusion protein, either inside or at the ends of a polynucleotide encoding the insulin receptor agonist.

[0395] In one embodiment, the vector may further independently comprise such structural elements as a bacterial tetracycline resistance gene; a replication initiation site (origin / ori); a ROP gene which regulates a plasmid copy number; a transcription promoter (e.g., Tac-T7); a transcription terminator (e.g., rrnBl B2 T1 txn); and a LacI gene encoding a Tac promoter repressor.

[0396] Another object of the invention is a host cell comprising one or more polynucleotides of the invention. Some objects of the invention are represented by a host cell comprising one or more vectors of the invention. The polynucleotides and vectors may have any of the features recited in the present disclosure, individually or in combination. In one embodiment, the host cell comprises a vector (e.g., is transformed or transfected with a vector) comprising a polynucleotide that encodes the insulin receptor agonist polypeptide of the invention. As used herein, the term “host cell” refers to any type of cellular system that can be engineered to produce the insulin receptor agonists of the invention, or fragments thereof.

[0397] Host cells suitable for replication and for maintaining expression of insulin receptor agonists are well known in the art. Such cells can be transfected or transduced appropriately with a specific expression vector, and more vector-containing cells can be grown to be introduced into fermenters for large-scale production processes of the insulin receptor agonist in amounts sufficient for clinical applications. Suitable host cells are prokaryotic microorganisms such as E. coli, or various eukaryotic cells such as Chinese Hamster Ovary (CHO) cells, insect cells or the like. For example, polypeptides can be produced in bacteria, in particular, when there is no need for glycosylation. After expression, the polypeptide can be isolated from the bacterial cell paste into a soluble fraction and can be further purified.

[0398] In one embodiment, eukaryotic organisms such as filamentous fungi or yeast, including fungal and yeast strains with “humanized” glycosylation pathways, can be used as hosts for cloning or expressing vectors that encode a fusion polypeptide, allowing to obtain a polypeptide with a partially or fully human glycosylation scheme (see GERNGROSS T. U., Advances in the production of human therapeutic proteins in yeasts and filamentous fungi, Nature biotechnology, 2004 Nov;22(ll): 1409-14; and LI H. et al., Optimization of humanized IgGs in glycoengineered Pichia pastoris, Nature biotechnology, 2006 Feb;24(2):210-5). Host cells that can be used to express (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates).

[0399] Exemplary invertebrate cells are insect cells; plant cells may also be used. Numerous baculovirus strains and matching insect cells suitable as hosts have been identified, in particular, for transfecting Spodoptera frugiperda cells. Plant cell cultures may also be used as hosts.

[0400] In one embodiment, vertebrate animal cells may also be used as hosts. For example, mammalian cell lines adapted to suspension growth may be used. Other acceptable exemplary mammalian host cell lines are SV40-transformed monkey kidney cell line CV1 (COS-7); human embryonic kidney cell line, e.g., HEK293 (GRAHAM F. L. et al., Characteristics of a human cell line transformed by DNA from human adenovirus type 5, Journal of general virology, 1977 Jul;36(l):59-72); baby hamster kidney (BHK) cells; murine Sertoli cells; monkey kidney cells (CV1); African green monkey kidney cells (VERO-76,); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); Buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); murine mammary tumor cells (MMT 060562); TRI cells (see, e.g., MATHER J. P. et al., Culture of testicular cells in hormone- supplemented serum-free medium, Annals of the New York Academy of Sciences, 1982:383:44-68); MRC 5 cells, and FS4 cells. In one embodiment, mammalian host cell lines may be represented by Chinese hamster ovary (CHO) cells, including dhfr'-CHO cells (URLAUB G. et al., Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity, Proceedings of the National Academy of Sciences, 1980 Jul;77(7):4216-20); and myeloma cell lines such as Sp2 / 0, NSO, P3X63, Y0 (see, e.g., STEINITZ M. et al. (ed.)., Human monoclonal antibodies: Methods and Protocols, New York: Humana Press, 2014).

[0401] One object of the invention is a method of preparing the insulin receptor agonist of the invention, wherein the method comprises culturing a host cell comprising one or more polynucleotides encoding the insulin receptor agonist of the invention, and optionally isolating the insulin receptor agonist from the host cell (or the host cell culture medium).

[0402] In one embodiment, the present invention also provides a method for preparing the insulin receptor agonist of the present invention using a recombinant DNA technology, said method comprising the steps of:

[0403] 1) providing a nucleotide sequence encoding the insulin receptor agonist;

[0404] 2) inserting the nucleotide sequence (1) into a suitable expression vector to produce a recombinant expression vector;

[0405] 3) introducing the recombinant expression vector (2) into a suitable host cell;

[0406] 4) culturing the transfected host cell under conditions suitable for expression;

[0407] 5) collecting and purifying the expressed product.

[0408] The coding sequence can be introduced into the host cell by a variety of methods known in the art, including, but not limited to calcium phosphate precipitation, lipofection, electroporation, microinjection, viral infection, and the alkali metal ion method.

[0409] Cultivation and expression in host cells are well-known in the prior art (see, e.g., GRAHAM, 1977; LI, 2006). Cells and debris may be removed from suspension by centrifugation, and the supernatant is collected.

[0410] In one embodiment, the product may be expressed by the cells as inclusion bodies, and then may be subject to concentration, for example by tangential flow filtration or diafiltration, and may be further purified by chromatography.

[0411] Methods for purifying recombinant proteins, including those comprising antibody Fc fragments, are known to a skilled artisan (see, e.g., SHI Y. et al., Advantages of CE-SDS over SDS-PAGE in mAb purity analysis. Anal. Methods, 2012,4, 1637-1642), and can be used to purify the insulin receptor agonists of the present disclosure. The insulin receptor agonist produced as described above can be purified substantially to homogeneity, e.g., can be represented by a single band or specific bands in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). First, the supernatant is concentrated. The concentrated supernatant may be further purified by gel chromatography or ion exchange chromatography, such as anion exchange chromatography or cation exchange chromatography. The gel matrix may be a matrix commonly used to purify proteins, such as agarose, dextran, polyamide, and the like.

[0412] Finally, the purified product may be further polished by such methods as hydroxyapatite adsorption chromatography, metal-chelate chromatography, hydrophobic interaction chromatography, and high-performance reversed phase liquid chromatography, etc. All of the aforementioned purification steps can be used in various combinations, resulting in proteins of substantially uniform purity.

[0413] The expressed insulin receptor agonist may be purified using an affinity chromatographic column comprising an antibody, receptor, or ligand specific for the Fc fragment polypeptide and / or insulin. Depending on the nature of the affinity column used, the insulin receptor agonist associated with the affinity column can be eluted using conventional methods such as high-salt buffer, pH variation, and the like.

[0414] In one embodiment, chromatographic purification of the insulin receptor agonist may be optimized by using an auxiliary domain, e.g., His-tag, fused to the N- or C-terminus of the polypeptide (KOSOBOKOVA E. N. et al., Vspomogatel'nye domeny v rekombinantnykh belkakh (obzor) (Auxiliary Domains in Recombinant Proteins (Review)), Biokhimiya (Biochemistry), 2016, V. 81(3):299-314).

[0415] In one embodiment, the auxiliary domain may be a SUMO polypeptide (SEQ ID NO:7) (see, e.g., BUTT T. R. et al., SUMO fusion technology for difficult-to-express proteins, Protein expression and purification, 2005 Sep;43(l):l-9).

[0416] The present disclosure also relates to a method for producing the insulin receptor agonist described herein, said method comprising culturing the host cell described herein in order to obtain the fusion construct based on the insulin variant fused to the physiologically active polypeptide as described above.

[0417] The present disclosure further relates to the use of the insulin receptor agonist of the present disclosure for producing a medicament.

[0418] The present disclosure further relates to the use of the insulin receptor agonist described herein for manufacturing a medicament comprising said construct as an active principle. The present disclosure further relates to a pharmaceutical composition for treating insulin- sensitive diseases or conditions, the composition comprising an effective amount of the insulin variant of the present disclosure and a pharmaceutically acceptable carrier.

[0419] Insulin-sensitive diseases or conditions include, but are not limited to, diseases or conditions associated with absolute or relative insulin deficiency, including those for which insulin replacement therapy is effective, for example, type I diabetes mellitus, type II diabetes mellitus, hyperglycemia, etc.

[0420] The present disclosure also relates to a method for preparing a pharmaceutical composition comprising an effective amount of the insulin receptor agonist of the present disclosure, and a pharmaceutically acceptable carrier, where said method comprises preparing the insulin receptor agonist as described above.

[0421] The techniques and means useful for preparing pharmaceutical compositions are known to those skilled in the art (see, e.g., Adejare A. (ed.). Remington: the science and practice of pharmacy. Academic Press, 2020).

[0422] The present invention further relates to a pharmaceutical composition comprising the insulin receptor agonist of the invention, which is soluble at physiological pH values.

[0423] In another embodiment, the present invention is related to a pharmaceutical composition comprising the insulin receptor agonist according to the invention, which agonistis soluble at pH values in the range of about 6.5 to about 8.5.

[0424] The pharmaceutical compositions comprising the insulin receptor agonist according to the invention can be administered parenterally to patients in need of such treatment. Parenteral administration may be performed by subcutaneous, intramuscular, or intravenous injection by means of a syringe, optionally a pen- like syringe. Alternatively, parenteral administration may be performed using an infusion pump. Administering the insulin receptor agonist intranasally or intrapulmonary, preferably in compositions, powders, or liquids specifically designed for this purpose, is also possible.

[0425] Injectable insulin receptor agonist compositions of the invention can be prepared using conventional pharmaceutical industry strategies that involve dissolving and mixing the ingredients to produce the desired final product.

[0426] In particular, according to one technique, the insulin receptor agonist of the present disclosure is dissolved in an amount of water which is slightly less than the final volume of the composition to be prepared. An isotonic agent, a preservative, and a buffer are added as appropriate, and pH of the solution is -adjusted, if necessary, with an acid, e.g., hydrochloric acid, or with a base, e.g., aqueous sodium hydroxide, as appropriate. Finally, the solution volume is adjusted with water to a desired concentration of the ingredients. In a further embodiment, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate and tris(hydroxymethyl) -amino methane, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.

[0427] In a further embodiment, the formulation further comprises a pharmaceutically acceptable preservative which may be selected from the group consisting of phenol, ortho-cresol, metacresol, para-cresol, methyl para-hydroxybenzoate, propyl para-hydroxybenzoate, 2-phenoxyethanol, butyl para-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol and thio mero sal, bro nopol, benzoic acid, imidourea, chlorhexidine, sodium dehydro acetate, chlorocresol, ethyl para-hydroxybenzoate, benzethonium chloride, chlorophenesine (3-para-chlorophenoxypropane-l,2-diol) or mixtures thereof. In a further embodiment, the preservative is present at a concentration of between 0.1 mg / ml to 20 mg / ml. In a further embodiment, the preservative is present at a concentration of between from 0.1 mg / ml to 5 mg / ml. In a further embodiment, the preservative is present at a concentration of between 5 mg / ml to 10 mg / ml. In a further embodiment, the preservative is present at a concentration of between 10 mg / ml to 20 mg / ml. Each of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well known to those skilled in the art (see, e.g., Adejare A. (ed.). Remington: the science and practice of pharmacy. Academic Press, 2020).

[0428] In a further embodiment, the formulation further comprises an isotonic agent which may be selected from the group consisting of a salt (e.g., sodium chloride), a sugar or sugar alcohol, an amino acid (e.g., L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g., glycerol (glycerin)), a 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol), a polyethylene glycol (e.g., PEG400), or mixtures thereof. Any sugar, such as mono-, di-, or polysaccharides, or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose, can be used. In an embodiment, the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group, and it includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment, the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, because a sugar or sugar alcohol is soluble in a liquid formulation and does not adversely affect stabilizing effects achieved through the use of the methods according to the invention. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg / mL and about 150 mg / mL. In a further embodiment, the isotonic agent is present at a concentration of between 1 mg / ml to 50 mg / mL In a further embodiment, the isotonic agent is present at a concentration of between 1 mg / ml to 7 mg / ml. In a further embodiment, the isotonic agent is present at a concentration of between 8 mg / ml to 24 mg / ml. In a further embodiment, the isotonic agent is present at a concentration of between 25 mg / ml to 50 mg / ml. Each of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well known to those skilled in the art.

[0429] Typical isotonic agents are sodium chloride, mannitol, dimethylsulfone, and glycerol, and typical preservatives are phenol, meta-cresol, methyl para-hydroxybenzoate, and benzyl alcohol.

[0430] Examples of suitable buffers are sodium acetate, glycylglycine, HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), and sodium phosphate.

[0431] In a preferred embodiment, the invention provides:

[0432] 1. An insulin receptor agonist, wherein the insulin receptor agonist is a fusion protein having the B-L1-A-L2-F structure, wherein

[0433] B is the insulin B chain or an insulin analog B chain comprising the amino acid sequence XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 set forth in SEQ ID NO:24, wherein

[0434] Xi is F or H,

[0435] Xi6 is H, Y, E, or D,

[0436] X24 is H or F,

[0437] X25 is F or H,

[0438] X26is Y or H,

[0439] X27 is T or absent,

[0440] X28is P or absent,

[0441] X29 is K or absent;

[0442] A is the insulin or insulin analog A chain comprising the amino acid sequence GIVEQCCX8SICSLXi4QLENYCX2i set forth in SEQ ID NO: 15, wherein

[0443] X8is T or H, X14 is D, E, or Y, and

[0444] X2iis G or N;

[0445] F is a human IgG Fc fragment polypeptide comprising the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX208HEALHX214HYTQKSLSLSX225G as set forth in SEQ ID NO: 34, wherein

[0446] Xi is E or D,

[0447] X2 is K or absent,

[0448] X3is T or absent,

[0449] X4is H or absent,

[0450] X5is W or absent,

[0451] X13 is P or E,

[0452] X14 is F or absent,

[0453] X15 is V or L,

[0454] Xi6 is A or G,

[0455] X17 is G or M,

[0456] X19 is S or E,

[0457] X30is T or Q,

[0458] X32is M, Y, or W,

[0459] X34 is S or T,

[0460] X36 is T or E,

[0461] X44 is V, E, or D,

[0462] X48is H or Q,

[0463] Xes is H or E,

[0464] X66 is N or E, X77 is N or D,

[0465] X80is F or Y,

[0466] X88is V or F,

[0467] X89is V or L,

[0468] X110is A, F, or S,

[0469] X111is P or S,

[0470] X119 is T or A,

[0471] X135 is R or Q,

[0472] X177 is M or V,

[0473] X189is K or R,

[0474] X199 is Q or E,

[0475] X208is M or L,

[0476] X214 is N, Y, or W, and

[0477] X225 is P or L; and

[0478] LI and L2 are peptide linkers.

[0479] 2. The insulin receptor agonist of item 1, wherein the LI linker is selected from the peptides of SEQ ID NOs:4-6.

[0480] 3. The insulin receptor agonist of item 1, wherein the L2 linker is a peptide of SEQ ID NOs:8-9.

[0481] 4. The insulin receptor agonist of item 1, wherein B is selected from the peptides of SEQ ID NOs: 16-23.

[0482] 5. The insulin receptor agonist of item 1, wherein A is selected from the peptides of SEQ ID NOs: 10-14.

[0483] 6. The insulin receptor agonist of item 1, wherein F is selected from the peptides of SEQ ID NOs:25-33.

[0484] 7. An insulin receptor agonist comprising the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO 83, wherein:

[0485] Xi is F or H,

[0486] Xi6 is Y, H, D, or E,

[0487] X24 is H or F,

[0488] X25 is F or H,

[0489] X26is H or Y,

[0490] X27 is T or absent,

[0491] X28is P or absent,

[0492] X29 is K or absent,

[0493] X35is S or Q,

[0494] X40 is G or absent,

[0495] X48is T or H,

[0496] X54 is Y, E, or D,

[0497] Xei is N or G,

[0498] X82 is D or E,

[0499] X83is K or absent,

[0500] X84is T or absent,

[0501] X85is H or absent,

[0502] X86 is W or absent,

[0503] X97 is M or G,

[0504] X99 is S or E,

[0505] X110is Q or T, X112is Y, M, or W,

[0506] X114is S or T,

[0507] X116is T or E,

[0508] X124 is D, E, or V,

[0509] X190 is A or F,

[0510] X288is M or L,

[0511] X294 is N or Y.

[0512] 8. An insulin receptor agonist comprising an amino acid sequence selected from SEQ ID NOs:35-82.

[0513] 9. A nucleic acid encoding the insulin receptor agonist according to any one of items 1 to 8.

[0514] 10. The nucleic acid of item 9, comprising a nucleotide sequence selected from SEQ ID NOs:84-131.

[0515] 11. An expression cassette comprising the nucleic acid of item 9.

[0516] 12. A vector comprising the nucleic acid of item 9 or the expression cassette of item 11.

[0517] 13. A host cell for expression of the insulin receptor agonist according to any one of items 1 to 8 comprising the nucleic acid according to item 9, the expression cassette according to item 11, or the vector according to item 12.

[0518] 14. The host cell of item 13, wherein the host cell is an E. coli cell.

[0519] 15. A method for preparing the insulin receptor agonist according to any one of items 1-8, wherein the method comprises culturing the cell according to item 13 under conditions allowing the expression of the insulin receptor agonist according to any one of items 1-8.

[0520] 16. A pharmaceutical composition for the treatment of an insulin-sensitive disease or condition, comprising an effective amount of the insulin receptor agonist of any of items 1 to 8 and a pharmaceutically acceptable carrier or diluent.

[0521] 17. A method of treating an insulin-sensitive disease or condition in a subject in need thereof, wherein the method comprises administering to the subject the insulin receptor agonist of any one of items 1-8 or the pharmaceutical composition of item 16. 18. Use of the insulin receptor agonist of any one of items 1-8 for the manufacture of a medicament for treating an insulin- sensitive disease or condition.

[0522] BRIEF DESCRIPTION OF THE DRAWINGS

[0523] Figure 1. General structure of a plasmid for expressing insulin analogs fused with the Fc fragment polypeptide.

[0524] Figure 2. Comparison of the pH dependence of the interaction of insulin receptor agonists with IR-A vs human insulin.

[0525] Figure 3. Comparison of the pH dependence of the interaction of insulin receptor agonists with IR-B vs human insulin.

[0526] Figure 4. 10-day glycemia profiles in animals treated with the L3-01 molecule at the doses of 100 nM / kg, 250 nM / kg, and 400 nM / kg compared to placebo. The graph displays mean, standard error of the mean, and individual values.

[0527] Figure 5. 10-day glycemia profiles in animals treated with L3-11 molecules at the doses of 100 nM / kg, 250 nM / kg, and 400 nM / kg compared to placebo. The graph displays mean, standard error of the mean, and individual values.

[0528] Figure 6. 10-day glycemia profiles in animals treated with L3-12 molecules at the doses of 100 nM / kg, 250 nM / kg, and 400 nM / kg compared to placebo. The graph displays mean, standard error of the mean, and individual values.

[0529] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0530] Example 1. Producing genetic constructs of single-chain insulins conjugated to Fc fragments: novel insulin receptor agonists.

[0531] Single chain insulin structures comprising A and B insulin chains joined by a 10 amino acid flexible linker (SEQ ID NOs:4-5) were prepared in order to produce novel insulin receptor agonists. The amino acid structures of the A chains are set forth in SEQ ID NOs:10-14. The amino acid structures of the B chains are set forth in SEQ ID NOs: 16-23.

[0532] The resulting single-chain agonists are conjugated to modified Fc fragments which have increased affinity for FcRn at pH 6.0. The following combinations of mutations in the Fc fragment according to the EU classification were used to create Fc-insulins: M252Y, S254T, T256E (YTE); M252W, T256E, M428L, N434Y (WELY), S239E, T256E, V264E, A33OF, N434Y (EEEFY); ins221D, V222K, ins223T, E224H, ins225W, G237M, T250Q, M252Y, V264D (DKTHWMQYD).

[0533] The nucleotide sequences encoding the insulin receptor agonists are shown in Table 1 and SEQ ID NOs:84-131. Table 1. Nucleotide sequences of the single- stranded human insulin analogs, the sequences conjugated to Fc fragments.

[0534] B-chain Linker A-chain Fc fragment SEQ ID mutations mutations mutations NO:

[0535] GP20091-L3-01 F25H, Y26H, GGGGGS - M252Y, S254T, 84 T30del GGGG T256E

[0536] GP20091-L3-02 F24H, Y26H, GGGGGS M252Y, S254T, 85 T27del, GGGG T256E

[0537] P28del,

[0538] K29del,

[0539] T30del

[0540] GP20091-L3-03 Y16H, F25H, GGGGGS - M252Y, S254T, 86 T30del GGGG T256E

[0541] GP20091-L3-04 F1H, F25H, GGGGGQ - M252Y, S254T, 87 T30del GGGG T256E

[0542] GP20091-L3-05 F25H, Y26H, GGGGGQ T8H M252Y, S254T, 88 T30del GGGG T256E

[0543] GP20091-L3-06 Y16E, F25H, GGGGGS T8H M252Y, S254T, 89 T30del GGGG T256E

[0544] GP20091-L3-07 Y16H, F25H, GGGGGS YUE M252Y, S254T, 90 T30del GGGG T256E

[0545] GP20091-L3-08 Y16H, F25H, GGGGGS T8H, YUE M252Y, S254T, 91 T30del GGGG T256E

[0546] GP20091-L3-09 Y16H, F24H, GGGGGS YUE M252Y, S254T, 92 T27del, GGGG T256E

[0547] P28del,

[0548] K29del,

[0549] T30del

[0550] GP20091-L3-10 Y16E, F25H, GGGGGS T8H, YUE M252Y, S254T, 93 T30del GGGG T256E

[0551] GP20091-L3-11 Y16D, F25H, GGGGGS Y14D, M252Y, S254T, 94 T30del GGGG N21G T256E

[0552] GP20091-L3-12 F25H, GGGGGS T8H, YUE M252Y, S254T, 95 T27del, GGGG T256E

[0553] P28del,

[0554] K29del,

[0555] T30del

[0556] GP20091-L3-15 F25H, Y26H, GGGGGS - M252W, T256E, 96 T30del GGGG M428L, N434Y GP20091-L3-16 F25H, Y26H, GGGGGS - S239E, T256E, 97 T30del GGGG V264E, A33OF,

[0557] N434Y (EEEFY) GP20091-L3-17 F25H, Y26H, GGGGGS ins221D, V222K, 98 T30del GGGG ins223T, E224H,

[0558] ins225W, G237M, T250Q, M252Y, V264D

[0559] (DKTHWMQYD)

[0560]

[0561] GP20091-L3-18 F24H, Y26H, GGGGGS - M252W, T256E, 99 T27del, GGGG M428L, N434Y P28del, (WELY)

[0562] K29del,

[0563] T30del

[0564] GP20091-L3-19 F24H, Y26H, GGGGGS S239E, T256E, 100

[0565] T27del, GGGG V264E, A33OF, P28del, N434Y (EEEFY) K29del,

[0566] T30del

[0567] GP20091-L3-20 F24H, Y26H, GGGGGS ins221D, V222K, 101

[0568] T27del, GGGG ins223T, E224H, P28del, ins225W, G237M, K29del, T250Q, M252Y, T30del V264D (DKTHWMQYD) GP20091-L3-21 Y16H, F25H, GGGGGS - M252W, T256E, 102

[0569] T30del GGGG M428L, N434Y (WELY)

[0570] GP20091-L3-22 Y16H, F25H, GGGGGS - S239E, T256E, 103

[0571] T30del GGGG V264E, A33OF,

[0572] N434Y (EEEFY) GP20091-L3-23 Y16H, F25H, GGGGGS ins221D, V222K, 104

[0573] T30del GGGG ins223T, E224H,

[0574] ins225W, G237M, T250Q, M252Y, V264D (DKTHWMQYD) GP20091-L3-24 F1H, F25H, GGGGGQ - M252W, T256E, 105

[0575] T30del GGGG M428L, N434Y (WELY)

[0576] GP20091-L3-25 F1H, F25H, GGGGGQ - S239E, T256E, 106

[0577] T30del GGGG V264E, A33OF,

[0578] N434Y (EEEFY) GP20091-L3-26 F1H, F25H, GGGGGQ ins221D, V222K, 107

[0579] T30del GGGG ins223T, E224H,

[0580] ins225W, G237M, T250Q, M252Y, V264D (DKTHWMQYD) GP20091-L3-27 F25H, Y26H, GGGGGQ T8H M252W, T256E, 108

[0581] T30del GGGG M428L, N434Y (WELY)

[0582] GP20091-L3-28 F25H, Y26H, GGGGGQ T8H S239E, T256E, 109

[0583] T30del GGGG V264E, A33OF,

[0584] N434Y (EEEFY) GP20091-L3-29 F25H, Y26H, GGGGGQ T8H ins221D, V222K, 110

[0585] T30del GGGG ins223T, E224H,

[0586] ins225W, G237M, T250Q, M252Y,

[0587] V264D

[0588]

[0589] (DKTHWMQYD) GP20091-L3-30 Y16E, F25H, GGGGGS T8H M252W, T256E, 111 T30del GGGG M428L, N434Y (WELY)

[0590] GP20091-L3-31 Y16E, F25H, GGGGGS T8H S239E, T256E, 112

[0591] T30del GGGG V264E, A33OF,

[0592] N434Y (EEEFY) GP20091-L3-32 Y16E, F25H, GGGGGS T8H ins221D, V222K, 113

[0593] T30del GGGG ins223T, E224H,

[0594] ins225W, G237M, T250Q, M252Y, V264D (DKTHWMQYD) GP20091-L3-33 Y16H, F25H, GGGGGS Y14E M252W, T256E, 114

[0595] T30del GGGG M428L, N434Y (WELY)

[0596] GP20091-L3-34 Y16H, F25H, GGGGGS Y14E S239E, T256E, 115

[0597] T30del GGGG V264E, A33OF,

[0598] N434Y (EEEFY) GP20091-L3-35 Y16H, F25H, GGGGGS Y14E ins221D, V222K, 116

[0599] T30del GGGG ins223T, E224H,

[0600] ins225W, G237M, T250Q, M252Y, V264D (DKTHWMQYD) GP20091-L3-36 Y16H, F25H, GGGGGS T8H, Y14E M252W, T256E, 117

[0601] T30del GGGG M428L, N434Y (WELY)

[0602] GP20091-L3-37 Y16H, F25H, GGGGGS T8H, Y14E S239E, T256E, 118

[0603] T30del GGGG V264E, A33OF,

[0604] N434Y (EEEFY) GP20091-L3-38 Y16H, F25H, GGGGGS T8H, Y14E ins221D, V222K, 119

[0605] T30del GGGG ins223T, E224H,

[0606] ins225W, G237M, T250Q, M252Y, V264D (DKTHWMQYD) GP20091-L3-39 Y16H, F24H, GGGGGS Y14E M252W, T256E, 120

[0607] T27del, GGGG M428L, N434Y P28del, (WELY)

[0608] K29del,

[0609] T30del

[0610] GP20091-L3-40 Y16H, F24H, GGGGGS Y14E S239E, T256E, 121

[0611] T27del, GGGG V264E, A33OF, P28del, N434Y (EEEFY) K29del,

[0612] T30del

[0613] GP20091-L3-41 Y16H, F24H, GGGGGS Y14E ins221D, V222K, 122

[0614] T27del, GGGG ins223T, E224H, P28del, ins225W, G237M, K29del, T250Q, M252Y,

[0615]

[0616] T30del V264D (DKTHWMQYD) GP20091-L3-42 Y16E, F25H, GGGGGS T8H, YUE M252W, T256E, 123

[0617] T30del GGGG M428L, N434Y

[0618] (WELY)

[0619] GP20091-L3-43 Y16E, F25H, GGGGGS T8H, YUE S239E, T256E, 124

[0620] T30del GGGG V264E, A33OF,

[0621] N434Y (EEEFY) GP20091-L3-44 Y16E, F25H, GGGGGS T8H, YUE ins221D, V222K, 125

[0622] T30del GGGG ins223T, E224H,

[0623] ins225W, G237M, T250Q, M252Y,

[0624] V264D

[0625] (DKTHWMQYD) GP20091-L3-45 Y16D, F25H, GGGGGS Y14D, M252W, T256E, 126

[0626] T30del GGGG N21G M428L, N434Y

[0627] (WELY)

[0628] GP20091-L3-46 Y16D, F25H, GGGGGS Y14D, S239E, T256E, 127

[0629] T30del GGGG N21G V264E, A33OF,

[0630] N434Y (EEEFY) GP20091-L3-47 Y16D, F25H, GGGGGS Y14D, ins221D, V222K, 128 T30del GGGG N21G ins223T, E224H,

[0631] ins225W, G237M, T250Q, M252Y,

[0632] V264D

[0633] (DKTHWMQYD) GP20091-L3-48 F25H, GGGGGS T8H, YUE M252W, T256E, 129

[0634] T27del, GGGG M428L, N434Y

[0635] P28del, (WELY)

[0636] K29del,

[0637] T30del

[0638] GP20091-L3-49 F25H, GGGGGS T8H, YUE S239E, T256E, 130

[0639] T27del, GGGG V264E, A33OF,

[0640] P28del, N434Y (EEEFY) K29del,

[0641] T30del

[0642] GP20091-L3-50 F25H, GGGGGS T8H, YUE ins221D, V222K, 131 T27del, GGGG ins223T, E224H,

[0643] P28del, ins225W, G237M, K29del, T250Q, M252Y,

[0644] T30del V264D

[0645]

[0646] (DKTHWMQYD)

[0647] Example 2. Production of the insulin receptor agonists.

[0648] The insulin receptor agonists were produced by heterologous expression of plasmids in Escherichia coli cells. In addition to the target protein nucleotide sequence, a SUMO peptide was added to the genetic construct as a leader sequence, and a ULP protease was added to remove SUMO during fermentation. Each plasmid consists of the following elements:

[0649] •A bacterial tetracycline resistance gene, a replication initiation site (origin / ori), and the ROP gene which regulates a plasmid copy number;

[0650] •The Tac-T7 transcription promoter initiating the recombinant protein expression;

[0651] • Sequences of the genes encoding the SUMO-GP20091.01-L3-XX molecule (Table 1) and the Ulp protease, which are under the Tac-T7 promoter control and representing an operon;

[0652] •Transcription terminator rrnB 1 B2 T1 txn;

[0653] •LacI gene encoding a Tac promoter repressor;

[0654] •SgrAI, EcoRI, and Spel are restriction sites where the gene is inserted;

[0655] •Pr 1082 and Prl66 are sequencing primers.

[0656] General plasmid structure is shown in Figure 1.

[0657] Bacterial cells of Escherichia coli strain BL21 (NEB, C2530H) were used as producer cells for insulin receptor agonists. The strain was transformed with the plasmids using the Eporator electroporator (Eppendorf).

[0658] Tetracycline 2 pg / ml (Sigma, Cat. No. T7660-5G) was used as a selective marker. Fermentation was carried out in a Biostat B bioreactor (Sartorius), induction was started at OD60024±4, with using 0.05 mM IPTG as an inducer (Panreac, lot 65010055). After induction, the fermentation continued for 6 hours. Insulin receptor agonists were produced as inclusion bodies.

[0659] Cell biomass was isolated by centrifugation method using the Avanti J-HC floor- standing centrifuge (Beckman Coulter) and JLA-8.1000 rotor (Beckman Coulter) as per standard protocol (20 min, 8000 rpm). Cell biomass was disintegrated using the PandaPLUS 1000 high pressure homogenizer (GEA), the inclusion bodies were subsequently precipitated by centrifugation according to the same protocol as for the isolation of cell biomass. The inclusion bodies were dissolved in the buffer 7 M GuHCl, 100 mM Tris, 15 mM DTT, pH 9.5 for 30 min. Refolding and formation of the Fc-insulin dimers was performed in the renaturation buffer 1.5 M Urea, 300 mM Arg, 20 mM Tris, 5 mM EDTA pH 8.25, using L-cysteine reducing agent (1.5 mM) and L-cystamine oxidizing agent (1.5 mM).

[0660] Purification of the target proteins was performed using two-step chromatography in the AKTA pure and AKTA avant chromatography systems (GE Healthcare). On the first step, affinity chromatography with Absolute® High Cap sorbent (Novasep, Cat. No. 124O1C803-002) was used. As a second purification step, hydrophobic interaction chromatography was performed with using Butyl Sepharose™ 4 Fast Flow (GE Healthcare, Cat. No. 17-0980-01) as a sorbent.

[0661] The identity of the resulting proteins was confirmed by comparing the theoretical and experimental molecular weights determined using the Acquity UPLC Waters (MS) liquid chromatography system. The purity of the Fc-insulins was assessed by size exclusion high performance chromatography (SE-HPLC) using the Prominence-i 2030C liquid chromatography system (Shimadzu) and TSKgel G3000SW, 300x7.5 mm, 5 um chromatography column (Tosoh Bio science).

[0662] In this way, the insulin receptor agonists with the amino acid sequences set forth in SEQ ID NOs: 35-82 were obtained.

[0663] Example 3. Binding to the type A insulin receptor.

[0664] The insulin receptor type A (IR-A) binding kinetics was assessed by the label-free biolayer interferometry using the Octet Red96 96-well plate interferometer (Pall ForteBio). Streptavidin sensors (Sartorius, Cat. No. 18-5019) hydrated for 10 min in 0.05% PBST solution and biotinylated IR-A receptors, 25 pg / ml (Sino Biological, Cat. No. 11086-H08H) were used for the analysis. The IR-A binding kinetics was assessed in neutral (pH 7.4) and acidic (pH 6.0) conditions. All tests are performed in comparison with human insulin (Rinsulin R, GEROPHARM).

[0665] All insulin receptor agonist molecules have reduced affinity for IR-A under physiological conditions when compared to human insulin (Table 2).

[0666] Table 2. Ligand-receptor interaction of the test molecules with IR-A at pH 7.4 and pH 6.0 compared to human insulin. KD: dissociation constant, Mean KD: mean KD for independent experiments, n: No. of independent experiments, SEM: standard error of the mean for independent experiments.

[0667] Mean KD IR-A n, KD IR- SEM, KD IR-A SEQ ID NO: 7.4, nM A 7.4 7.4, nM

[0668] Human Insulin 412 11 124

[0669] GP20091-L3-01 21,685 4 10,843 35 GP20091-L3-03 6,432 2 4,548 37 GP20091-L3-04 8,473 3 4,892 38 GP20091-L3-05 1,430 3 826 39 GP20091-L3-06 2,533 1 40 GP20091-L3-07 18,240 2 12,898 41 GP20091-L3-08 6,161 2 4,356 42

[0670]

[0671] GP20091-L3-09 >100,000 3 >100,000 43 GP20091-L3-10 5,847 3 3,376 44 GP20091-L3-11 56,750 1 45 GP20091-L3-12 9,898 3 5,715 46 GP20091-L3-17 3,036 1 49 GP20091-L3-47 8,840 1 79

[0672]

[0673] GP20091-L3-50 5,617 1 82

[0674] When the medium is acidified, the binding of human insulin to IR-A decreases 6.4-fold. The affinity of the insulin receptor agonists for IR-A is also reduced in acidic environment, with the exception of the GP20091-L3-04 and GP20091-L3-06 molecules, which show 2.9-fold and 47-fold increase in affinity, respectively (Table 3).

[0675] Table 3. Ligand-receptor interaction of the test molecules with IR-A at pH 7.4 and pH 6.0 compared to human insulin. KD: dissociation constant, Mean KD: mean KD for independent experiments, n: No. of independent experiments, SEM: standard error of the mean for independent experiments.

[0676] Mean KD IR-A n, KD IR-A SEM, KD IR- SEQ ID 6.0 6.0 A 6.0

[0677] Human Insulin 2,654 9 885

[0678] GP20091-L3-01 >1,000,000 3 >1,000,000 35 GP20091-L3-03 35,205 2 24,894 37 GP20091-L3-04 2,937 2 2,077 38 GP20091-L3-06 54 1 40 GP20091-L3-08 89,550 2 63,321 42 GP20091-L3-09 No binding 3 43 GP20091-L3-11 No binding 3 45 GP20091-L3-12 No binding 2 46 GP20091-L3-17 No binding 1 49 GP20091-L3-47 No binding 1 79

[0679]

[0680] GP20091-L3-50 No binding 1 82

[0681] A decrease in binding due to acidic conditions, which is typical of early endosomes, contributes to the insulin-insulin receptor complex dissociation after its internalization and a longer-lasting effect of the drug. The higher the difference between the molecule binding at neutral and acidic pH, the more pronounced is the potential of the molecule for recycling. For the insulin receptor agonists GP20091-L3-09, GP20091-L3-50, GP20091-L3-17, and GP20091-L3- 01, the pH-dependence of the interaction with IR-A is more distinct compared to human insulin (Figure 2).

[0682] Example 4. Binding to the type B insulin receptor. The insulin receptor type B (IR-B) binding kinetics was accessed by the label-free biolayer interferometry method using the Octet Red96 96-well plate interferometer (Pall ForteBio). Streptavidin sensors (Sartorius, Cat. No. 18-5019) hydrated for 10 min in 0.05% PBST solution and biotinylated IR-B, 2.5 µg / mL (Sino Biological, Cat. No. 11081-H08H-B) were used in the assay. For the analysis of pH-dependent interactions, affinity tests were performed both under physiological conditions (pH 7.4) and in acidic environment (pH 6.0). All tests are performed in comparison with human insulin (Rinsulin R, GEROPHARM).

[0683] Under physiological conditions, all of the insulin receptor agonist molecules have lower affinity for IR-B (KD: 1,930 nM - >1,000,000 nM) compared to human insulin (KD=287 nM), as shown in Table 4.

[0684] Table 4. Ligand-receptor interaction of the test molecules with IR-B at pH 7.4 compared to human insulin. KD: dissociation constant, Mean KD: mean KD for independent experiments, n: No. of independent experiments, SEM: standard error of the mean for independent experiments.

[0685] Mean KD IR-B n, KD SEM, KD IR-B SEQ ID 7.4 IR-B 7.4 7.4 NO:

[0686] Human Insulin 287 3 166

[0687] GP20091-L3-01 22,787 3 13,156 35 GP20091-L3-03 6,936 4 3,468 37 GP20091-L3-04 9,304 3 5,372 38 GP20091-L3-05 1,930 3 1,114 39 GP20091-L3-06 3,306 1 40 GP20091-L3-07 10,610 2 7,502 41 GP20091-L3-08 5,167 2 3,654 42 GP20091-L3-09 >1,000,000 5 >1,000,000 43 GP20091-L3-10 3,522 3 2,033 44 GP20091-L3-11 54,338 5 24,301 45 GP20091-L3-12 7,593 3 4,384 46 GP20091-L3-17 4,954 1 49 GP20091-L3-47 10,820 1 79

[0688]

[0689] GP20091-L3-50 6,624 1 82

[0690] A decrease in the medium pH leads to a decrease in the affinity of human insulin to IR-B by 4.6 times. Most insulin receptor agonists also show an affinity decrease in acidic environment (Table 5).

[0691] Table 5. Ligand-receptor interaction of the test molecules with IR-B at pH 7.4 and pH 6.0 compared to human insulin. KD: dissociation constant, Mean KD: mean KD for independent experiments, n: No. of independent experiments, SEM: standard error of the mean for independent experiments.

[0692] Mean KD IR-B n, KD IR-B 6.0 SEM, KD IR-B SEQ ID 6.0 6.0 NO:

[0693] Human Insulin 1,331 11 401.2

[0694] GP20091-L3-01 119,967 3 69,262.8 35 GP20091-L3-03 19,790 4 9,895.0 37 GP20091-L3-04 4,618 3 2,666.4 38 GP20091-L3-05 17,510 3 10,109.2 39 GP20091-L3-06 5,231 1 40 GP20091-L3-07 34,225 2 24,200.7 41 GP20091-L3-08 27,325 2 19,321.7 42 GP20091-L3-09 >1,000,000 3 >1,000,000 43 GP20091-L3-10 15,308 3 8,838.3 44 GP20091-L3-11 47,698 4 23,848.8 45 GP20091-L3-12 553,350 2 391,277.5 46 GP20091-L3-17 116,100 1 49 GP20091-L3-47 17,210 1 79

[0695]

[0696] GP20091-L3-50 177,300 1 82

[0697] The results of the tests comparing the dependence of the interactions between insulin molecules and IR-B on environmental conditions showed that the insulin receptor agonists GP20091-L3-12, GP20091-L3-50, GP20091-L3-17, GP20091-L3-05, GP20091-L3-08, and GP20091-L3-01 are more pH-dependent compared to human insulin (Figure 3).

[0698] Example 5. Binding of the insulin receptor agonists to FcRn.

[0699] The biotinylated Fc neonatal receptor (FcRn) binding kinetics was assessed by the label- free biolayer interferometry using the OCTET RED96 96-well plate interferometer (Pall ForteBio). Rituximab solution (Rituxara, Nanolek, Russia) was used as a positive control.

[0700] Streptavidin sensors (Sartorius, Cat. No. 18-5019) hydrated for 10 min in 0.05% PBST solution, pH 7.4, were used in the analysis. Test samples were diluted in 0.05% PBST solution, pH 6.0. The biotinylated FcRn receptor was used at the concentration of 2.5 µg / mL. The following cascade dilutions of rituximab were used in the experiment: 1.75 nmol / ml, 0.44 nmol / ml, 0.11 nmol / ml, 0.03 nmol / ml, and 0.007 nmol / ml. The following concentrations were used for the test samples: 0.625 nmol / mL, 0.25 nmol / mL, 0.1 nmol / mL, 0.04 nmol / mL, and 0.016 nmol / mL. A 1:1 interaction model was used for data processing. The results of the experiments are shown in Table 6. Table 6. Ligand-receptor interaction of the test molecules with FcRn at pH 6.0 as compared to rituximab.

[0701] Mean KD FcRn pH SEQ ID NO:

[0702] 6.0, nM n SEM

[0703] Rituximab 16.47 17 6.76

[0704] GP20091-L3-01 0.78 3 0.34 35

[0705] GP20091-L3-02 1.33 1 36

[0706] GP20091-L3-03 0.80 1 37

[0707] GP20091-L3-04 0.84 2 0.06 38

[0708] GP20091-L3-05 0.38 3 0.06 39

[0709] GP20091-L3-06 1.06 1 40

[0710] GP20091-L3-07 0.75 2 0.26 41

[0711] GP20091-L3-08 0.87 3 0.25 42

[0712] GP20091-L3-09 0.68 3 0.03 43

[0713] GP20091-L3-10 0.99 3 0.04 44

[0714] GP20091-L3-11 1.31 3 0.25 45

[0715] GP20091-L3-12 1.58 2 0.25 46

[0716] GP20091-L3-17 0.63 1 49

[0717] GP20091-L3-47 1.16 1 79

[0718]

[0719] GP20091-L3-50 0.75 1 82

[0720] Based on the obtained data (Table 6), it can be concluded that the produced insulin receptor agonists have a high affinity for FcRn.

[0721] Example 6. Glucose uptake induced by insulin receptor agonists.

[0722] To assess the biological activity of the tested insulin receptor agonists, an insulin-induced glucose uptake study was performed on the rat myoblast cell line L6J1 (INC RAS). The cells were dispersed into 96-well plates at 8,000 cells / well in the medium DMEM + 2% horse serum (HS). Cells dispersed in the medium DMEM + 10% FBS were used as a control. After 72 hours, the medium was exchanged to DMEM + 2% HS in the experimental wells, and DMEM + 10% in the control wells. On day 7 of the experiment, cells were starved for 24 hours by adding DMEM glu 4.5 g / 1, w / o phenol red, s / f to all wells.

[0723] The test sample was added to the wells at concentrations of 2 nM, 20 nM, 100 nM, 200 nM, 500 nM, 800 nM, 1600 nM, 3200 nM, and 5000 nM. As a control, human insulin (Rinsulin R, GEROPHARM, Russia) was added at concentrations of 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 400 nM, 800 nM, 1600 nM, and 2500 nM. For all experiments, each test sample concentration was tested in four replicate wells to evaluate the activity. Cells were incubated with the test and control samples for 72 hours. Glucose concentration was measured by enzymatic colorimetric method using the Glucose GOD-PAP kit by adding culture medium to the Glucose GOD-PAP working solution (ABRIS+, Cat. No 304.3.250). Detection was performed using the CLARIOstar multimodal microplate reader (BMG LABTECH). The activity of the test molecules was evaluated by the average residual glucose content in the medium (Table 7). The produced insulin receptor agonists demonstrate high biological activity.

[0724] Table 7. Results of the insulin- induced glucose uptake by L6J1 cells. Emaxis the lowest residual glucose concentration, EC50 is half-maximal concentration of the test sample. Mean is the mean for independent experiments, n is the number of independent experiments, SEM is the standard error of the mean for independent experiments.

[0725] Emax EC50

[0726] Mean, n SEM, Mean, nM n SEM, mmol / L mmol / L nM Human Insulin 11.10 11 0.86 163.72 11 17.21 GP20091-L3-01 14.67 4 1.98 394.23 4 145.02 GP20091-L3-03 15.69 2 0.15 1,194.50 2 71.50 GP20091-L3-04 14.61 1 921.70 1

[0727] GP20091-L3-05 10.49 3 2.89 885.49 3 174.42 GP20091-L3-06 8.88 1 1,783

[0728] GP20091-L3-07 12.13 2 0.10 198.48 2 140.35 GP20091-L3-09 16.97 5 0.87 821.66 5 147.79 GP20091-L3-10 11.70 3 0.87 363.97 3 16.86 GP20091-L3-11 13.93 1 608.30 1

[0729] GP20091-L3-12 10.62 2 3.32 993.65 2 385.35 GP20091-L3-17 10.48 1 954.4 1

[0730] GP20091-L3-47 9.70 1 1,189

[0731]

[0732] GP20091-L3-50 11.66 1 753.5 1

[0733] Example 7. Hypoglycemic activity of the GP20091.01-L3-01, GP20091.01-L3-11, GP20091.01-L3-12 in the therapy of STZ-induced DM in rats.

[0734] The study was conducted on male rats of Wistar line (‘Stolbovaya’ Branch of the Federal State Budgetary Institution of Science “Scientific Center for Biomedical Technologies”, Russia) weighing 300-400 g. All animals were quarantined for 14 days prior to study entry. Intact animals were allocated to the pathology control group immediately after the quarantine period (group #1, n=5). Diabetes mellitus was induced in the remaining animals by a single intraperitoneal injection of streptozotocin (STZ) (Sisco Research Laboratories (Sri), India) at the dose of 65 mg / kg after 48 hours of food deprivation. Animals with the pathology were allocated for the treatment step with test drugs if they had a glycemia level of 20-33 mmol / 1 and were in a satisfactory clinical condition.

[0735] Rats with the pathology were randomly assigned to the treatment groups based on their glycemia level. Animals in the placebo group were administered vehicle (group #2, n=5), animals in the active control group were dosed 10 IU / kg insulin degludec (Tresiba®, Novo Nordisk A / S, Denmark) (group #3, n=5), animals in the test drug groups were dosed 100 nM / kg, 250 nM / kg, and 400 nM / kg Fc-insulin molecules (groups # 3-14, n=10). Animals from the pathology control group were administered the same vehicle as the placebo group (group #1, n=5). The design of the experiment is shown in Table 8. All test drugs and placebo were administered to the animals as a single intraperitoneal subcutaneous injection.

[0736] Table 8. Design of the experiment for the GP20091-L3-01, GP20091-L3-11, and GP20091-L3-12 molecules.

[0737] Group No. Induction of No. of Animals,

[0738] Drug Dose, U / kg

[0739] pathology n

[0740] 1 - Placebo 5

[0741] 0 nmol / kg

[0742] 2 Placebo 5 3 Insulin degludec

[0743] 10 IU / kg 5 (positive control)

[0744] 7 100 nmol / kg 10 8 GP20091-L3-01 250 nmol / kg 10 9 400 nmol / kg 10

[0745] +

[0746] 10 100 nmol / kg 10 11 GP20091-L3-11 250 nmol / kg 10 12 400 nmol / kg 10 13 100 nmol / kg 10

[0747] GP20091-L3-12

[0748] 14 250 nmol / kg 10

[0749]

[0750] During the entire treatment period, animals had access to water and food ad libitum. The hypoglycemic activity of the drugs was assessed by the glycemia dynamics compared to baseline and placebo. Glucose level was measured using a StarStrip ® handheld blood glucose meter (Nova, USA) in blood samples collected from the sublingual vein. The glycemia level was assessed immediately before administration, 1 h, 4 h, 8 h, 12 h, 24 h, and then every day up to 168 h after administration (Table 9). Table 9. Glycemia dynamics in rats after a single administration of the GP20091-L3-01, GP20091-L3-11, and GP20091-L3-12 molecules, insulin degludec, and placebo.

[0751] Glycemia level, mmol / L

[0752] befo

[0753] Group,

[0754] re 120 144 168 dose Ih 4 h 8 h 12h 24 h 48 h 72 h 96 h

[0755] treat h h h ment

[0756] 5.2+ 5.6+ 5.5+ 6+0. 6.4+ 6.4+ 6.1+ 6.3+ 6.6+ 6.2+ 5.8+ 6.8+ Intact 0.2 0.2 0.1 3 0.2 0.1 0.2 0.1 0.1 0.3 0.4 0.3 0 nm / kg

[0757] 30+1 28+2 28.2 28.4 29.2 29+1 28.1 27.9 29.9 30.5 29.5 30.2 Placebo.5.3 +2.2 +1.7 +2.7 +2.3 +1.6 +1.8 +1.5 +1.5 +1.7 0 nm / kg #### #### #### #### #### #### #### #### #### #### #### ####

[0758] 29.7 23.9 16.2 21.1 27.2 29.5 28.5 29.8 29.1 30.9 26.8 29+2 Insulin +1.4 +2.8 +3.1 +3 +2.4 +1.8 +2.1 +1.9 +2.5 +2 +2.3.1 degludec ** &

[0759] 10 IU / kg &&

[0760] &

[0761] GP20091 30.5 31.4 29.1 27.8 25.2 26.4 29+1 29.9 29.3 30.5 30.4 31.5 -L3-01 +0.9 +1.4 +2.2 +2.3 +2 +2.3.8 +1.9 +1.4 +1.4 +1.5 +1.1 100

[0762] nm / kg

[0763] GP20091 30.1 30.9 23.9 22.8 25.6 24.9 26.1 29.2 28.8 30.4 30.4 31.8 -L3-01 +1.5 +1.9 +2.7 +3.5 +3.1 +2.5 +2.4 +1.6 +2 +1.5 +1.5 +0.9 250nm / k

[0764] g

[0765] GP20091 30.7 30.2 26.5 25.5 23.7 22.4 19.1 26.1 27.1 28.4 28.1 28.8 -L3-01 +1.1 +1.5 +2.6 +2.3 +2.5 +2.1 +2.1 +2.4 +1.9 +1.4 +1.6 +1.4 400nm / k

[0766] g

[0767] GP20091 30+1 30.4 28.6 28.7 28.8 27.3 26.1 26.6 26.5 27.9 28+2 28.3 -L3-11.3 +1.4 +1.9 +1.5 +1.4 +2.2 +2.5 +2.6 +2.7 +2.1.4 +1.9 100

[0768] nm / kg

[0769] GP20091 30+1 31.2 29.7 28.4 25.3 25.4 19.9 20.1 19.9 21.7 23.9 26+2 -L3-11 +1.2 +1.3 +1.2 +1.2 +2 +2.3 +3 +3.2 +3.6 +3.3 250nm / k && && * * &

[0770] g && && && &&

[0771] && &

[0772] GP20091 30.4 32+1 28.8 26.5 25.3 25.4 18.6 18.3 16.1 17.7 20+3 23.8 -L3-11 +1 +1.9 +2.2 +2 +2.2 +3.3 +2.9 +3.2 +3.1.1* +2.1 400nm / k && * *** *** && && g && && && && &&

[0773]

[0774] && && && Glycemia level, mmol / L

[0775] befo

[0776] Group,

[0777] re 120 144 168 dose Ih 4 h 8 h 12h 24 h 48 h 72 h 96 h

[0778] treat h h h ment

[0779] GP20091 30.2 29.1 28.1 24.7 24.9 25.2 18±2 18.5 16.2 21.3 26.5 30±l -L3-12 ±1.4 ±2 ±1.5 ±2.6 ±2.7 ±2.5.4* ±2.4 ±2.9 ±2.5 ±2.1.3 lOOnm / k & & && * *** *

[0780] g && && && &&

[0781] && && &&

[0782] GP20091 30.1 31.3 24.3 17±3 19.6 19.6 11.5 5.2± 5.3± 5.1± 15.3 26.2 -L3-12 ±1.4 ±1.1 ±2.4 * ±2.6 ±2.1 ±2.9 0.8 1 0.7 ±2.2 ±1.6 250nm / k & && * * **** **** **** **** ***

[0783] g && && && && && && && &&

[0784] && && && && && && && 30.2 27.2 20.2 18.9 16.9 16±2 9.4± 7.4± 5.9± 4.9± 12.9 24.4 GP20091

[0785] ±1.1 ±2.1 ±3 ±3.4 ±3.3 9** 2.7 1.9 1.7 2 ±2.9 ±1.7 -L3-12

[0786] && && ** * **** **** **** **** **** & 400nm / k

[0787] && && && && && && && && && g

[0788]

[0789] && && && && && && &&

[0790] Notes: #### denotes statistically significant differences compared to the “Intact” group at p<0.0001; Dunnett's test (one-way analysis of variance); *, **, ***, **** - statistically significant differences compared to the " Placebo" group at p<0.05, p<0.01, p<0.001, p<0.0001, respectively, Dunnett's test (one-way analysis of variance); &, &&, &&&& - statistically significant differences compared to baseline glycemia levels before treatment at p<0.05, p<0.01, p<0.0001, respectively, Dunnett's test (two-way analysis of variance with repeated measures) 10-day glycemia profiles in animals treated with GP20091-L3-01, GP20091-L3-11, and GP20091-L3-12 at the doses 100 nM / kg, 250 nM / kg, and 400 nM / kg compared to placebo are shown in Figures 4-6.

[0791] Example 8. Hypoglycemic activity of GP20091-L3-47, GP20091-L3-50 in the therapy of STZ-induced DM in rats.

[0792] The study was conducted on male rats of Wistar line (KroltInfo LLC, Russia) weighing 300-350 g. All animals were quarantined for 14 days prior to study entry. Intact animals were allocated to the pathology control group immediately after the quarantine period (group #1, n=5). Diabetes mellitus was induced in the remaining animals by a single intraperitoneal injection of streptozotocin (STZ) (Sisco Research Laboratories (Sri), India) at the dose of 65 mg / kg after 48 hours of food deprivation. Animals with the pathology were allocated for the treatment step with test drugs if they had a glycemia level of 20-33 mmol / 1 and were in a satisfactory clinical condition.

[0793] Rats with the pathology were randomly assigned to the treatment groups based on their glycemia level. Animals in the placebo group were administered vehicle (group #2, n=5), animals in the active control group were administered insulin degludec (Tresiba®, Novo Nordisk A / S, Denmark) at the dose of 10 IU / kg (group #3, n=5). The animals were administered the GP20091.01-L3-47 molecule at the doses of 100 nM / kg, 175 nM / kg, and 250 nM / kg (groups #16-21, n=10). All test drugs and placebo were administered to the animals as a single intraperitoneal subcutaneous injection. The design of the experiment is shown in Table 11.

[0794] Table 11. The design of the experiment for the GP20091.01-L3-47 and GP20091.01-L3- 50 molecules.

[0795] Group Induction of

[0796] Drug Dose No. of Animals pathology

[0797] 1 - Placebo 5

[0798] 0 nmol / kg

[0799] 2 Placebo 5 3 Insulin degludec

[0800] 10 IU / kg 5 (positive control)

[0801] 16 250 nmol / kg 10 17 GP20091.01-L3-47 400 nmol / kg 10

[0802] +

[0803] 18 550 nmol / kg 10 19 100 nmol / kg 10 20 GP20091.01-L3-50 175 nmol / kg 10 21 250 nmol / kg 10

[0804]

[0805] During the entire treatment period, the animals had access to water and food ad libitum. The hypoglycemic activity of the drugs was assessed based on glycemia level dynamics compared to the baseline and placebo. Glucose level was measured using a StarStrip ® handheld blood glucose meter (Nova, USA) in blood samples collected from the sublingual vein. The glycemia level was assessed immediately before administration, 1 h, 4 h, 8 h, 12 h, 24 h and then every day up to 168 h after administration (Table 12).

[0806] Table 12. Dynamics of glycemia in rats during 7 days after administering the test molecules. Glycemia level, mmol / L

[0807] befo

[0808] Group,

[0809] re 24 120 168 dose Ih 4 h 8 hr 12h 48 h 72 h 96 h 144h treat hr h h ment

[0810] Intact 5.2+ 5.6+ 5.5+ 6+0. 6.4+ 6.4+ 6.1+ 6.3+ 6.6+ 6.2+ 5.8+ 6.8+ 0 nm / kg 0.2 0.2 0.1 3 0.2 0.1 0.2 0.1 0.1 0.3 0.4 0.3

[0811] 30+1 28+2 28.2 28.4 29.2 29+1 28.1 27.9 29.9 30.5 29.5 30.2 Placebo

[0812] .5.3 +2.2 +1.7 +2.7 +2.3 +1.6 +1.8 +1.5 +1.5 +1.7 0 nm / kg

[0813] #### #### #### #### #### #### #### #### #### #### #### #### Insulin 29.7 23.9 16.2 21.1 27.2 29.5 28.5 29.8 29.1 30.9 26.8 29+2 degludec +1.4 +2.8 +3.1 +3 +2.4 +1.8 +2.1 +1.9 +2.5 +2 +2.3.1 10 IU / kg *

[0814] GP20091- 28.9 28.6 24.6 22.6 20.4 22.6 25+1 24.8 29.8 30.1 28.6 31.2 L3-47 +1 +1.7 +1.7 +2.4 +2.5 +2.4.7 +1.5 +1.6 +1.1 +1.1 +1.1 50 nm / kg

[0815] GP20091- 29+1 25.6 18.1 20.8 18.2 17.2 22.1 21.9 24.5 29+2 24.5 28.3 L3-47.4 +2.2 +3.4 +2.9 +2.7 +2.9 +2.6 +2.1 +2.5 +1.6 +1.6 00 nm / kg * * *

[0816] GP20091- 28.8 24.8 15.9 16.6 11.1 17.3 18.3 20.3 23.4 26.3 24.8 26.7 L3-47 +1.3 +2.5 +2.9 +3* +2.1 +3.3 +2.7 +2.6 +2.1 +2.1 +2.1 +1.7 550 nm / kg ** **** * *

[0817] GP20091- 28.8 23.1 18.9 18.5 18.5 20.5 24.8 22.7 28.4 26.3 26+2 28+2 L3-50 +1.2 +2.2 +2.3 +2* +2.5 +2.6 +2.1 +1.5 +1.9 +1.8.1 100 nm / kg *

[0818] GP20091- 29+1 25.1 13.6 17.3 18+3 19.1 21.4 20.9 24.1 26.6 24.2 26.9 L3-50.3 +2.8 +2.9 +3.1.2* +3.1 +2.7 +2.7 +2.3 +2.4 +2.2 +1.8 175 nm / kg ** *

[0819] GP20091- 29+1 22.4 10.3 10.7 16.8 17.9 20.5 22.8 24.8 26.4 23.8 27.7 L3-50.2 +1.6 +1.7 +2.3 +2.4 +3 +1.7 +1.6 +1.5 +1.5 +1.5 50 nm / kg **** ** ** *

[0820]

[0821] Notes: #### denotes statistically significant differences compared to the “Intact” group at p<0.0001; Dunnett's test (one-way analysis of variance); *, **, ***, **** - statistically significant differences compared to the " Placebo" group at p<0.05, p< 0.01, p< 0.001, p<0.0001, respectively, Dunnett's test (one-way analysis of variance).

Claims

CLAIMS1. An insulin receptor agonist, wherein the insulin receptor agonist is a fusion protein having the structure of B-L1-A-L2-F, whereinB is an insulin or insulin analog B chain comprising the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29 set forth in SEQ ID NO:24, whereinXi is F or H,Xi6 is H, Y, E, or D,X24 is H or F,X25 is F or H,X26is Y or H,X27 is T or absent,X28is P or absent,X29 is K or absent;A is an insulin or insulin analog A chain comprising the amino acid sequence of GIVEQCCX8SICSLXi4QLENYCX2i set forth in SEQ ID NO: 15, whereinX8is T or H,X14 is D, E, or Y, andX2iis G or N;F is a human IgG Fc fragment polypeptide comprising the amino acid sequence of X1X2X3X4X5CPPCPAPX13X14X15X16X17PX19VFLFPPKPKDX30LX32IX34RX36PEVTCVVX44D VSX48EDPEVQFNWYVDGVEVX65X66AKTKPREEQFX77STX80RVVSVLTX88X89HQDWLN GKEYKCKVSNKGLPXIi0Xi 11IEKTISKX119KGQPREPQVYTLPPSX135EEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPXi77LDSDGSFFLYSXi89LTVDKSRWQXi99GNV FSCSVX2O8HEALHX214HYTQKSLSLSX225G set forth in SEQ ID NO:34, whereinXi is E or D,X2 is K or absent,X3is T or absent,X4 is H or absent, X5is W or absent, X13 is P or E, X14 is F or absent, X15 is V or L, Xi6 is A or G, X17 is G or M, X19 is S or E, X30is T or Q, X32is M, Y, or W, X34 is S or T, X36 is T or E, X44 is V, E, or D, X48is H or Q, Xes is H or E, X66 is N or E, X77 is N or D, X80is F or Y, X88is V or F, X89is V or L, X110is A, F, or S, X111is P or S, X119 is T or A, X135 is R or Q, X177 is M or V,Xi89 is K or R,X199 is Q or E,X208is M or L,X214 is N, Y, or W, andX225 is P or L, wherein the IgG Fc fragment polypeptide is not a wild-type IgG Fc fragment polypeptide;and L1 and L2 are peptide linkers.

2. The insulin receptor agonist of claim 1, wherein the LI linker is selected from the peptides of SEQ ID NOs:4-6.

3. The insulin receptor agonist of claim 1, wherein the L2 linker is a peptide of SEQ ID NOs:8-9.

4. The insulin receptor agonist of claim 1, wherein B is selected from the peptides of SEQ ID NOs: 16-23.

5. The insulin receptor agonist of claim 1, wherein A is selected from the peptides of SEQ ID NOs: 10- 14.

6. The insulin receptor agonist of claim 1, wherein F is selected from the peptides of SEQ ID NOs:25-33.

7. An insulin receptor agonist comprising the amino acid sequence of XiVNQHLCGSHLVEALXi6LVCGERGX24X25X26X27X28X29GGGGGX35GGGGX4oGIVEQCC X48SICSLX54QLENYCX61GGGGQGGGGQGGGGQGGGGGX82X83X84X85X86CPPCPAPPVA X97PX99VFLFPPKPKDX110LX112IX114RX116PEVTCVVX124DVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPX190PIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVX288HEALHX294HYTQKSLSLSPG set forth in SEQ ID NO:83, whereinXi is F or H,Xi6 is Y, H, D, or E,X24 is H or F,X25 is F or H,X26is H or Y,X27 is T or absent,X28 is P or absent,X29 is K or absent,X35is S or Q,X40 is G or absent,X48 is T or H,X54 is Y, E, or D,Xei is N or G,Xs2 is D or E,X83 is K or absent,X84 is T or absent,X85 is H or absent,X86 is W or absent,X97 is M or G,X99 is S or E,X110is Q or T,X112 is Y, M, or W,X114 is S or T,X116is T or E,X124 is D, E, or V,X190 is A or F,X288is M or L,X294 is N or Y, wherein the IgG Fc fragment polypeptide is not a wild-type IgG Fc fragment polypeptide.

8. An insulin receptor agonist comprising an amino acid sequence selected from SEQ ID NOs:35-82.

9. A nucleic acid encoding the insulin receptor agonist according to any one of claims 1 to 8.

10. The nucleic acid of claim 9, said nucleic acid comprising a nucleotide sequence selected from SEQ ID NOs:84-131.

11. An expression cassette comprising the nucleic acid of claim 9.

12. A vector comprising the nucleic acid of claim 9 or the expression cassette of claim 11.

13. A host cell for expression of the insulin receptor agonist according to any one of claims 1 to 8, wherein the host cell comprises the nucleic acid of claim 9, the expression cassette of claim 11, or the vector of claim 12.

14. The host cell of claim 13, wherein the host cell is an E. coli cell.

15. A method for preparing the insulin receptor agonist according to any one of claims 1 to 8, wherein the method comprises culturing the cell according to claim 13 under conditions allowing for the expression of the insulin receptor agonist according to any one of claims 1 to 8.

16. Use of the insulin receptor agonist according to any one of claims 1 to 8 for manufacturing a medicament for treating an insulin- sensitive disease or condition.

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