Glucagon-like peptide 1 receptor agonists and their uses

Abstract

To provide modified glucagon-like peptide 1 (GLP1) polypeptides, fusion proteins comprising modified GLP1 polypeptides, and methods of use thereof.SOLUTION: The invention provides: an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a fusion protein consisting of a specific amino acid sequence; a vector comprising the polynucleotide molecule; a cell expressing the fusion protein encoded by the polynucleotide molecule; and a pharmaceutical composition that comprises the fusion protein consisting of the specific amino acid sequence and a pharmaceutically acceptable carrier or diluent, and is for use in reducing the blood sugar level in a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to human glucagon-like peptide 1 receptor agonists and methods of treatment using said agonists. [Background technology]

[0002] Obesity is a major health problem in the United States, with two in three Americans considered overweight or obese. Obesity is a significant underlying risk factor for developing other diseases, such as heart disease, stroke, and diabetes. Even a small weight loss (5-10% of initial weight) reduces the risk of developing obesity-related diseases, such as heart disease and diabetes.

[0003] Diabetes is a chronic disease characterized by high blood sugar levels and insulin resistance. If left untreated, high blood sugar levels can lead to long-term complications, including heart disease, stroke, diabetic retinopathy, and lower limb amputation. Treatment of diabetes involves controlling and reducing blood sugar levels and includes exercise and dietary modifications along with medications such as insulin and metformin.

[0004] One approach used for obesity treatment and blood glucose control involves glucagon-like peptide (GLP)-1 receptor agonists, which target the incretin pathway. Glucagon-like peptide (GLP)-1 is a peptide hormone secreted by enteroendocrine cells in the intestinal tract. Upon oral glucose administration, GLP1 binds to its receptor, resulting in insulin secretion and a reduction in blood glucose levels (incretin effect). However, GLP1 is rapidly inactivated and degraded by the enzyme dipeptidyl peptidase 4 (DPP4) and has a very short half-life of 1.5 minutes. Therefore, long-acting derivatives of GLP1 and GLP1 receptor agonists, including fusion proteins containing GLP1, are being tested for diabetes control. GLP1 analogs, fusion proteins and GLP1 receptor agonists are disclosed, for example, in U.S. Patent No. 6,213,629, ...

[0005] However, there is a need for novel GLP1 peptide variants and GLP1 receptor agonists that are resistant to degradation by DPP4, have improved pharmacokinetics, and have high efficacy and sustained in vivo activity in glycemic control. Such GLP1 variants and GLP1 receptor agonists can be used to treat obesity and diabetes. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US7452966 [Patent Document 2] US8389689 [Patent Document 3] US8497240 [Patent Document 4] US8557769 [Patent Document 5] US8883447 [Patent Document 6] US8895694 [Patent Document 7] US9409966 [Patent Document 8] US20160194371 [Patent Document 9] US20140024586 [Patent Document 10] US20140073563 [Patent Document 11] US20120148586 [Patent Document 12] US20170114115 [Patent Document 13] US20170112904 [Patent Document 14] US20160361390 [Patent Document 15] US20150313908 [Patent Document 16] US20150259416 [Patent Document 17] WO2017074715 [Patent Document 18] , WO2016127887 [Patent Document 19] WO2015021871 [Patent Document 20] WO2014113357 [Patent Document 21] EP3034514 [Patent Document 22] EP2470198 [Patent Document 23] EP2373681 Summary of the Invention [Means for solving the problem]

[0007] According to one aspect, the present invention provides glucagon-like peptide 1 (GLP1) variants comprising at least one amino acid modification from mature GLP1(7-37) (SEQ ID NO: 4) selected from the group consisting of: (i) an amino acid addition to the N-terminus, and (ii) an amino acid deletion from the peptide sequence. In particular embodiments, the modification comprises the addition of an alanine or glutamine to the N-terminus.

[0008] According to one aspect, the present invention provides GLP1 receptor agonists, which comprise fusion proteins comprising GLP1 or variants thereof. In a particular embodiment, the GLP1 receptor agonist comprises a GLP1 peptide or a GLP1 peptide variant fused to a stabilization domain. In one embodiment, the stabilization domain is an antibody or antigen-binding fragment thereof that binds to the GLP1 receptor.

[0009] The GLP1 receptor agonists of the present invention are particularly useful for increasing the binding and / or activity of GLP1. In some embodiments, the GLP1 receptor agonists of the present invention function by activating GLP1 and reducing blood glucose levels. In some embodiments, the GLP1 peptide variants and / or GLP1 receptor agonists of the present invention are more resistant to inactivation by dipeptidyl peptidase 4 (DPP4) and exhibit improved in vivo half-life. The improved GLP1 agonists of the present invention provide significant reductions in blood glucose levels that last for more than 10 days, even with a single dose. In some embodiments, the GLP1 receptor agonists function by enhancing glucose-stimulated insulin secretion from pancreatic β cells, increasing insulin expression, inhibiting β cell apoptosis, promoting β cell neogenesis, reducing glucagon secretion, delaying gastric digestion time, promoting satiety, and increasing peripheral glucose disposal capacity. In certain embodiments, the GLP1 receptor agonists are useful for preventing, treating, or alleviating at least one symptom of a hyperglycemia-related disease or disorder (e.g., diabetes) in a subject. In certain embodiments, GLP1 receptor agonist can be administered to a subject with diabetes or a subject at risk of diabetes for prophylactic or therapeutic purposes.In certain embodiments, GLP1 receptor agonist is useful for preventing, treating or alleviating at least one symptom or sign of obesity, such as weight loss in a subject.

[0010] In a particular embodiment, the GLP1 receptor agonist is a fusion protein comprising a GLP1 variant and a stabilization domain, wherein the stabilization domain comprises an immunoglobulin or a fragment thereof. In a specific embodiment, the immunoglobulin comprises a heavy chain variable region and a light chain variable region, and the GLP1 receptor agonist is a fusion protein comprising a GLP1 variant and a stabilization domain, wherein the stabilization domain comprises an immunoglobulin or a fragment thereof. In certain embodiments, the GLP1 receptor agonist specifically binds to the GLP1 receptor, resulting in activation of the GLP1 receptor. In certain embodiments, the GLP1 receptor agonist functions by activating the GLP1 receptor, resulting in glycemic control, i.e., reduced blood glucose levels.

[0011] In one embodiment, the present invention provides a fusion protein having one or more of the following characteristics: (a) comprising a GLP1 variant domain and a stabilizing domain; (b) being a GLP1 receptor agonist; (c) the GLP1 variant domain comprising the amino acid sequence of SEQ ID NO: 5, 6, 7, or 8; (d) binding to the GLP1 receptor; (e) the stabilizing domain comprising an immunoglobulin or a fragment thereof; (f) the stabilizing domain comprising an immunoglobulin Fc fragment; (g) the stabilizing domain comprising an anti-GLP1 receptor antibody or an antigen-binding fragment thereof; (h) being resistant to degradation by blood proteases for at least 72 hours; and (i) resulting in a significant reduction in blood glucose levels sustained for more than 10 days following administration of a single dose.

[0012] In one aspect, the present invention provides nucleic acid molecules encoding GLP1 variants or portions thereof, for example, nucleic acid molecules encoding any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12, and 13, or substantially similar sequences thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] The present invention also provides nucleic acid molecules encoding any fusion protein comprising a GLP1 variant.

[0014] In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising a GLP1 variant or a fusion protein comprising a GLP1 variant described herein. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., a nucleic acid molecule encoding any GLP1 variant or a fusion protein comprising a GLP1 variant. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods for producing the protein or fragments thereof by culturing the host cells under conditions allowing for the production of the protein or fragments thereof, and recovering the proteins and fragments produced therein.

[0015] In one aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant protein or fragment thereof that specifically binds to the GLP1 receptor and a pharmaceutically acceptable carrier. In a related aspect, the present invention features a composition that is a combination of a GLP1 receptor agonist protein and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with a GLP1 receptor agonist. Exemplary agents that can be advantageously combined with a GLP1 receptor agonist include, but are not limited to, other agents that activate GLP1 receptor activity (including other proteins or metabolites, etc.) and / or agents that do not directly bind to the GLP1 receptor but that alleviate, reduce, or treat a GLP1 receptor-related disease or disorder (e.g., diabetes). Other combination therapies and co-formulations involving the GLP1 receptor agonist proteins of the present invention are disclosed elsewhere herein.

[0016] In another aspect, the present invention provides a method of treatment for a disease or disorder associated with GLP1, such as diabetes, in a subject using a GLP1 receptor agonist of the present invention, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a GLP1 receptor agonist of the present invention. In a particular embodiment, the GLP1 receptor agonist comprises a GLP1 variant or a fusion protein comprising a GLP1 variant. The disorder to be treated is any disease that is ameliorated, alleviated, inhibited, or prevented by activation of GLP1 receptor activity. or condition. In certain embodiments, the present invention provides methods for preventing, treating, or alleviating at least one symptom of a GLP1 receptor-related disease or disorder, comprising administering a therapeutically effective amount of a GLP1 receptor agonist of the present invention to a subject in need thereof. In some embodiments, the present invention provides a method for alleviating or reducing the severity of at least one symptom or sign of a GLP1 receptor-related disease or disorder in a subject by administering a therapeutically effective amount of a GLP1 receptor agonist protein of the present invention, wherein the at least one symptom or sign is selected from the group consisting of high blood sugar levels, excessive thirst, frequent urination, the presence of ketone bodies in the urine, fatigue, weight fluctuations, blurred vision, slow-healing pain, frequent infections, swollen or tender gums, obesity, heart disease, stroke, kidney disease, eye disease, nerve damage, and high blood pressure. In certain embodiments, the present invention provides methods for reducing weight in an overweight or obese subject, comprising administering to the subject a therapeutically effective amount of a GLP1 receptor agonist of the present invention that binds to the GLP1 receptor and activates GLP1 receptor activity. In certain embodiments, the present invention provides methods for reducing blood glucose levels in a subject, comprising administering to the subject a therapeutically effective amount of a GLP1 receptor agonist of the present invention that binds to the GLP1 receptor and activates GLP1 receptor activity. In some embodiments, the GLP1 receptor agonist can be administered prophylactically or therapeutically to subjects with hyperglycemia or at risk of having hyperglycemia. Subjects at risk include, but are not limited to, elderly subjects, pregnant women, subjects with high HbA1c levels, and subjects with one or more risk factors, including obesity, high blood cholesterol, smoking, excessive alcohol consumption, and / or physical inactivity. In certain embodiments, the present invention provides methods for treating type 2 diabetes uncontrolled by treatment with insulin and / or metformin, comprising administering a therapeutically effective amount of a GLP1 receptor agonist of the present invention to a subject in need thereof. In certain embodiments, the GLP1 receptor agonist of the present invention is administered to a subject in need thereof in combination with a second therapeutic agent.The second therapeutic agent can be selected from the group consisting of insulin or insulin analogs, biguanides (e.g., metformin), thiazolidinediones, sulfonylureas (e.g., clopropamide), glinides (e.g., nateglinide), α-glucosidase inhibitors, DPP4 inhibitors (e.g., sitagliptin), pramlintide, bromocriptine, SGLT2 inhibitors (e.g., canagliflozin), antihypertensive drugs, statins, aspirin, dietary modification, exercise, and nutritional supplements. Other therapeutic agents that can be used in combination with the GLP1 receptor agonist fusion proteins of the present invention are described elsewhere herein. In certain embodiments, the second therapeutic agent can be an agent that helps prevent or reduce any possible side effect(s) associated with the GLP1 receptor agonists of the present invention, if such side effect(s) may occur. The GLP1 receptor agonist can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. The GLP1 receptor agonist may be administered in a dose of about 0.1 mg / kg of the subject's body weight to about 100 mg / kg of the subject's body weight. In certain embodiments, the GLP1 receptor agonist of the present invention may be administered in one or more doses including 0.1 mg to 600 mg.

[0017] The present invention also includes the use of a GLP1 receptor agonist of the present invention in the manufacture of a medicament for treating a disease or disorder that may be caused by stimulation of GLP1 receptor binding and / or activity (e.g., diabetes, including type 2 diabetes).

[0018] Other embodiments will become apparent from a summary of the detailed description that follows. DETAILED DESCRIPTION OF THE INVENTION

[0019] Before describing the methods of the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should also be understood that it is not intended that the scope of the present invention be limited solely by the appended claims.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described here. All publications mentioned herein are incorporated by reference in their entirety.

[0021] definition The term "GLP1," also known as "glucagon-like peptide 1," refers to a 31-amino acid peptide hormone released from intestinal L-cells after nutrient ingestion. GLP1 binds to the GLP1 receptor and enhances glucose-stimulated insulin secretion from pancreatic β-cells, increases insulin expression, inhibits β-cell apoptosis, promotes β-cell neogenesis, reduces glucagon secretion, delays gastric digestion, promotes satiety, and increases peripheral glucose disposal. In certain embodiments, the term "GLP1" refers to the mature 31-amino acid peptide hormone (SEQ ID NO: 4), which contains amino acids 7-37 of the full-length GLP1 peptide (SEQ ID NO: 3). The term also includes variants of GLP1, which contain one, two, three, four, five, or six amino acid substitutions, additions, or deletions. For example, the term includes variants containing the amino acid sequence of SEQ ID NO: 5, 6, 7, or 8.

[0022] As used herein, a "stabilization domain" refers to any macromolecule that, when fused to a peptide, increases the in vivo activity and / or stability of the peptide. For example, a stabilization domain can be used to enhance the in vivo activity and / or stability of an immunoglobulin C HThe stabilized peptide may be a polypeptide comprising three domains. In certain embodiments, the stabilized domain increases the serum half-life of the peptide. In certain embodiments, the stabilized peptide increases the in vivo efficacy of the peptide. A non-limiting example of a stabilized domain is the Fc portion of an immunoglobulin, e.g., the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group. In certain embodiments, the stabilized domain is an Fc fragment or amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. As another example, the stabilized domain may be an immunoglobulin or an antigen-binding fragment thereof. In certain embodiments, the stabilized domain is an immunoglobulin comprising a heavy chain variable region and a light chain variable region, and the immunoglobulin binds to a specific antigen. In certain embodiments, the stabilized domain may comprise an antigen-binding domain and an Fc domain (e.g., of an IgG1 or IgG4 antibody), or may comprise only the antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), which can be modified to affect function. In a specific embodiment, the stability domain is an immunoglobulin comprising a heavy chain variable region and a light chain variable region, and the immunoglobulin binds to the GLP1 receptor. In another embodiment, the stability domain is a cysteine ​​residue or a short cysteine-containing peptide. Other stability domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0023] As used herein, the term "GLP1 receptor agonist" refers to a protein that binds to the GLP1 receptor. In the context of the present invention, this term refers to a fusion protein comprising GLP1 or a GLP1 variant fused to a stabilizing domain. In a particular embodiment, this term includes a fusion protein comprising a GLP1 variant fused to an immunoglobulin or a fragment thereof. In a specific embodiment, this term includes a fusion protein comprising GLP1 or a GLP1 variant fused to the N-terminus of the light chain variable region (VL) of an immunoglobulin. In a specific embodiment, this term includes a GLP1 or a GLP1 variant fused to the N-terminus of the VL of an antibody or antigen-binding fragment thereof that binds to the GLP1 receptor.

[0024] As used herein, the term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) and two light (L) chains inter-connected by disulfide bonds, and multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain comprises a domain (C H 1. C H 2 and C H 3), and the heavy chain variable region ("HCVR" or "V H ") or light chain variable region ("LCVR" or "V L Each light chain is composed of an Ig variable region, which may be a light chain variable region ("LCVR" or "V"). L ") and the light chain constant region (C L ) V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of an antibody (or antigen-binding fragment thereof) can be identical to human germline sequences, or can be naturally occurring, or can be artificially modified. A consensus sequence of amino acids can be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antigen-binding protein" also includes antibodies.

[0025] Substitution of one or more CDR residues or deletion of one or more CDR residues is also contemplated. Antibodies that can omit one or two CDRs for binding have been described in the scientific literature. Padlan et al. (1995 FASEBJ. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al., 2002, J Mol Biol 320:415-428).

[0026] Methods and techniques for identifying CDRs within a VR amino acid sequence are well known in the art and can be used to identify CDRs within the designated VR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence diversity, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); AI-Lazikani et al., J. Mol. Biol. 273:927-948 (1997), and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available to identify CDR sequences within the antigen-binding domain of antigen-binding proteins or antibodies.

[0027] Molecular modeling and / or experiments can identify CDR residues that are not in contact with the antigen based on previous testing from regions of the Kabat CDRs located outside the Chothia CDRs (e.g., residues H60-H65 in CDRH2 are often not required). If a CDR or its residue(s) is missing, it is typically replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus sequence of such sequences. The substitution positions within the CDR and amino acids for substitution can also be selected experimentally. Experimental substitutions can be conservative or non-conservative.

[0028] As used herein, the term "antigen-binding portion" of an antigen-binding protein refers to an antigen-binding An "antigen-binding fragment" of a protein, etc., includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antigen-binding protein, or "antigen-binding protein fragment," refers to one or more fragments of an antigen-binding protein that retain the ability to specifically bind to the GLP1 receptor. Antigen-binding protein fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDRs, or fragments containing isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. An antigen-binding fragment of an antigen-binding protein or antibody can be derived, for example, from the complete protein molecule, using any appropriate standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antigen-binding protein variable domains and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques to place one or more variable and / or constant domains into the appropriate conformation, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0029] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the minimal recognition unit consisting of amino acid residues similar to a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0030] Antigen-binding proteins or antibody antigen-binding fragments of the invention typically comprise at least one immunoglobulin (Ig) variable domain. Variable domains may be of any size or amino acid composition and generally comprise at least one CDR adjacent to or in frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer, V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antigen-binding protein may contain a dimer of monomer V. H or V L May contain domains.

[0031] In certain embodiments, the antigen-binding fragment may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary conformations of variable and constant domains that may be found in antigen-binding fragments of antibodies of the invention include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C L In any conformation of the variable and constant domains, including any of the exemplary conformations described above, the variable and constant domains may be either directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which may form the link between adjacent variable domains in a polypeptide molecule. and / or constant domains. Furthermore, antigen-binding fragments of the antigen-binding proteins of the present invention may be linked (e.g., by disulfide bond(s)) to each other and / or to one or more monomeric V H or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the aforementioned variable and constant domain configurations in non-covalent association.

[0032] Like intact protein molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antigen-binding proteins typically comprise at least two different variable domains, each capable of specifically binding to a different antigen or a different epitope on the same antigen. Any multispecific antigen-binding protein format, including the exemplary bispecific antigen-binding protein formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antigen-binding proteins of the present invention using routine techniques available in the art.

[0033] As used herein, the terms "fully human antibody," "human antibody," "fully human antigen-binding protein," or "human antigen-binding protein" are intended to include antigen-binding proteins having variable and constant regions derived from human germline immunoglobulin sequences. Human antigen-binding proteins of the invention may include amino acid residues (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation), e.g., in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antigen-binding protein," as used herein, is not intended to include antigen-binding proteins in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences. This term includes antigen-binding proteins or antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. This term is not intended to include antigen-binding proteins or antibodies isolated from or produced in a human subject.

[0034] As used herein, the term "recombinant" refers to a fusion protein of the invention or a fragment thereof that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term also refers to fusion proteins expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse), or cellular (e.g., CHO cell) expression system, or isolated from a recombinant combinatorial human antibody library.

[0035] The terms "specifically bind," or "specifically bind to," and the like, mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is defined as binding to an antigen with a binding affinity of at least about 1 x 10 -8 can be characterized by an equilibrium dissociation constant less than or equal to M (e.g., a low K Dindicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, isothermal titration calorimetry, and the like.

[0036] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antigen-binding protein or antibody, or "antibody fragment" refers to one or more fragments of an immunoglobulin protein that retain the ability to bind to the GLP1 receptor.

[0037] As used herein, the term "K D " refers to the equilibrium dissociation constant of a particular protein-antigen interaction.

[0038] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that, with appropriate nucleotide insertions or deletions, when optimally aligned with another nucleic acid (or its complementary strand), there is at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0039] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using default gap weights, such as with the programs GAP or BESTFIT, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Non-identical residue positions preferably differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and aspartic acid-glutamic acid. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0040] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of closest overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is An example algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0041] By the phrase "therapeutically effective amount" is meant the amount that produces the desired effect for which it is administered. The precise amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using well-known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0042] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention, and / or treatment of a disease or disorder associated with GLP1. This term includes human subjects having or at risk of having a disease or disorder associated with GLP1. For example, this term includes subjects with diabetes (e.g., type 2 diabetes) or subjects at risk of developing diabetes (e.g., type 2 diabetes). In certain embodiments, this term includes subjects with obesity, stroke, or myocardial infarction or subjects at risk of developing these. This term also includes subjects with high blood glucose levels and / or high levels of one or more biomarkers for diabetes, such as HbA1c. This term also includes subjects with diabetes for whom standard of care therapy (e.g., metformin) is contraindicated or intolerant, or whose disease is uncontrolled by treatment (e.g., with metformin).

[0043] As used herein, the terms "treat," "treating," or "treatment" refer to the reduction or alleviation of the severity of at least one symptom or sign of a GLP1-related disease or disorder by administering a therapeutic agent, such as a GLP1 receptor agonist of the present invention, to a subject in need thereof. These terms include the inhibition of disease progression or worsening of symptoms. These terms also include a favorable prognosis of the disease. That is, upon administration of a therapeutic agent, such as a GLP1 receptor agonist of the present invention, the subject may have no symptoms or signs, or may have low-intensity symptoms or signs. For example, a subject with diabetes may experience a reduction in blood glucose levels by administration of a GLP1 receptor agonist of the present invention. The therapeutic agent can be administered to the subject at a therapeutic dose.

[0044] The terms "prevent", "preventing" or "prevention" refer to the inhibition of the onset of any symptom or sign of a disease or disorder associated with hyperglycemia by administering a GLP1 receptor agonist of the present invention. This term includes the inhibition of the onset of a symptom or sign of a GLP1 receptor-related disease or disorder in a subject at risk of developing such a disease or disorder.

[0045] Due to its rapid inactivation by the enzyme dipeptidyl peptidase 4 (DPP4), GLP1(7-37) (SEQ ID NO: 4) has a very short circulating half-life (1-2 minutes). Previous studies have shown that various amino acid substitutions at position 8 of GLP1(7-37) confer increased resistance to DPP4, thereby resulting in a longer half-life. However, these molecules remain susceptible to DPP4 cleavage (Deacon et al., 1998, Diabetologia 41:271-278). Therefore, there is a need to develop new molecules that are highly resistant to degradation by DPP4.

[0046] The inventors have found that the first step is the addition of an amino acid (i.e., Ala, Gln) to the N-terminus, or His or Gln within the peptide sequence, to provide higher resistance to DPP4. We hypothesized that the first step would be to introduce mutations that extend or shorten the amino terminus of GLP1, either by deletion of an Ala, thereby conferring greater resistance to DPP4 cleavage. We demonstrate herein that these novel GLP1 variants are indeed highly resistant to degradation by DPP4. The second step was to compensate for any weakened or reduced GLP1 activity and thereby increase its potency by fusing GLP1 to an anti-GLP1 receptor antibody that binds the weakened GLP1 to the GLP1 receptor. Furthermore, we discovered that these fusion proteins had long serum half-lives, likely due to the inclusion of an Fc domain, and resulted in further reductions in blood glucose levels that were sustained for more than 10 days. As shown herein, the novel GLP1 variants and fusion proteins disclosed herein have significantly improved resistance to degradation by DPP4 in vitro and in vivo, demonstrating greatly improved efficacy in glycemic control. As used herein, the term "significantly improved" or "enhanced" or "increased," in the context of resistance to degradation by DPP4, refers to increased resistance to degradation upon incubation with DPP4 for more than 4 hours, more than 8 hours, more than 16 hours, more than 24 hours, more than 36 hours, or more than 70 hours, as measured by the assays described herein. As used herein, the term "significantly improved" or "enhanced" or "increased," in the context of reducing blood glucose levels, refers to a sustained reduction in blood glucose levels in a subject for more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, or more than 10 days upon administration of a GLP1 receptor agonist of the present invention.

[0047] The GLP1 receptor agonists of the present invention bind to GLP1 receptor with high affinity, resulting in GLP1 receptor activation. In some embodiments, these proteins are useful for treating subjects suffering from diabetes. When administered to a subject in need thereof, these proteins can reduce the blood glucose level in the subject. They can be used alone or as adjunctive therapy with other therapeutic components or modalities known in the field of hyperglycemia treatment.

[0048] The specific GLP1 receptor agonist proteins of the present invention can be used in vitro or in It can bind to the GLP1 receptor and stimulate its activity, as measured by an in vivo assay. The ability of the proteins of the present invention to bind to the GLP1 receptor and stimulate its activity can be measured using any standard method known to those skilled in the art, including the binding assays or activity assays described herein.

[0049] Antigen-binding proteins specific to the GLP1 receptor may contain no other labels or moieties, or they may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it binds. For example, when the surface is coated with avidin, a peptide containing N-terminal biotin is adapted so that the C-terminal portion of the peptide is away from the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or a label detectable by MRI. In certain embodiments, such labeled antigen-binding proteins can be used in diagnostic assays, including imaging assays.

[0050] bioequivalence The GLP1 receptor agonists of the present invention include proteins that have an amino acid sequence that differs from that of the disclosed GLP1 receptor agonists, but retain the ability to bind to the GLP1 receptor. Such variant GLP1 receptor agonists contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity approximately equal to that of the disclosed GLP1 receptor agonists. Similarly, the GLP1 receptor agonist-encoding DNA sequences of the present invention may contain one or more nucleotide changes when compared to the disclosed sequences. The present invention also encompasses sequences that encode GLP1 receptor agonists that contain the above additions, deletions, or substitutions but are substantially biologically equivalent to the GLP1 receptor agonists of the present invention.

[0051] Two proteins are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes whose rate and extent of absorption do not differ significantly when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some proteins can be considered pharmaceutical equivalents or pharmaceutical substitutes, and even bioequivalent, if they are comparable in their extent of absorption but not their rate of absorption. Such differences in absorption rate are intentional, reflected in the label, are not necessary to achieve effective body drug concentrations, for example, during chronic use, and are not considered medically significant for the particular drug being tested.

[0052] In one embodiment, two GLP1 receptor agonist proteins are bioequivalent if they have no clinically meaningful differences in their safety, purity, or potency.

[0053] In one embodiment, two GLP1 receptor agonist proteins are bioequivalent if a patient can be switched between the reference formulation and the biologic one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continued therapy without such switching.

[0054] In one embodiment, two GLP1 receptor agonist proteins are bioequivalent if they both act by one or more mechanisms of action common to one or more conditions of use, to the extent such mechanisms are known.

[0055] Bioequivalence can be demonstrated by in vitro and / or in vivo methods. Studies to measure bioequivalence include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of the protein or its metabolites in blood, plasma, serum, or other body fluids as a function of time; (b) in vitro studies that correlate with and reasonably predict in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals that measure the relevant acute pharmacological effect of the protein (or its target) as a function of time; and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0056] Biologically equivalent variants of the GLP1 receptor agonist proteins of the present invention can be constructed, for example, by various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences not required for biological activity. For example, cysteine ​​residues not required for biological activity can be deleted or replaced with other amino acids to prevent unnecessary or inappropriate intramolecular disulfide bond formation during renaturation. In other situations, biologically equivalent proteins may include variants containing amino acid changes that modify the glycosylation characteristics of the protein, for example, mutations that eliminate or remove glycosylation.

[0057] Biological characteristics of GLP1 receptor agonists Generally, GLP1 receptor agonists of the present invention function by binding to the GLP1 receptor and, upon binding, promote activation of the GLP1 receptor. In particular embodiments, proteins of the present invention bind to the GLP1 receptor with high affinity. For example, the present invention includes GLP1 receptor agonists that result in activation of the GLP1 receptor as measured by a luciferase assay (e.g., at 25°C or 37°C), e.g., using the assay format defined in Example 2 herein. In particular embodiments, GLP1 receptor agonists are measured using the assay format defined in Example 2 herein, or a substantially similar assay, e.g., Activates the GLP1 receptor with an EC50 of less than 10 nM, less than 500 pM, or less than 250 pM as measured by luciferase assay.

[0058] The present invention also includes GLP1 receptor agonists that, upon administration to a subject in need thereof, reduce blood glucose levels in vivo, e.g., as shown in Example 3 or in a substantially similar assay. Upon administration, GLP1 receptor agonists affect improved glycemic control, resulting in reduced blood glucose levels. In certain embodiments, even a single therapeutically effective dose of the GLP1 receptor agonists of the present invention results in significant blood glucose reductions that last for more than 10 days.

[0059] The present invention also includes GLP1 receptor agonists that exhibit improved resistance to degradation by blood proteases / peptidases, as measured by mass spectrometry, or a substantially similar method, for example, as shown in Example 4 herein. In particular embodiments, the GLP1 receptor agonist is resistant to degradation by dipeptidyl peptidase 4 (DPP4) for more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 70 hours, as measured by the assay described in Example 4 herein.

[0060] The GLP1 receptor agonists of the present invention may have one or more of the aforementioned biological characteristics, or any combination thereof. Other biological characteristics of the proteins of the present invention will be apparent to those skilled in the art from a review of this disclosure, including the working examples herein.

[0061] Therapeutic Administration and Formulation The present invention provides therapeutic compositions comprising the GLP1 receptor agonists of the present invention. The therapeutic compositions of the present invention are administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved mobility, delivery, tolerance, etc. Many suitable formulations can be found in formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, (cationic or anionic) lipid-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbable pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci See also Technol 52:238-311.

[0062] The dose of a GLP1 receptor agonist may vary depending on the age and size of the subject, the target disease, condition, route of administration, etc. When using antigen-binding proteins of the present invention to treat or prevent a disease or disorder in an adult patient, it is generally advantageous to administer the antigen-binding proteins of the present invention at a single dose of about 0.001 mg to about 100 mg per kg of body weight, more preferably about 0.001 mg to about 60 mg, about 0.01 mg to about 10 mg, or about 0.01 mg to about 1 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In specific embodiments, the antigen-binding proteins of the present invention or antigen-binding fragments thereof can be administered as an initial dose of at least about 0.001 mg to about 100 mg, about 0.001 mg to about 50 mg, about 0.005 mg to about 50 mg, about 0.01 mg to about 40 mg, about 30 mg, or about 10 mg. In certain embodiments, the initial dose can be followed by administration of a second or more subsequent doses of the GLP1 receptor agonist in an amount that can be about the same as or less than that of the initial dose, and the subsequent doses can be administered for at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or at least The intervals may be 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0063] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis are possible (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249:1527-1533).

[0064] The use of nanoparticles for delivering the GLP1 receptor agonist of the present invention is also contemplated herein.Protein-conjugated nanoparticles can be used for both therapeutic and diagnostic applications.Several nanoparticles may be developed, and can be conjugated to antigen-binding proteins contained in pharmaceutical compositions to target cells.Nanoparticles for drug delivery are also described in, for example, US8257740 or US8246995, each of which is incorporated herein by reference in its entirety.

[0065] In certain situations, the pharmaceutical composition can be delivered in a sustained release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the sustained release system can be placed near the target of the composition, thereby requiring a very small systemic dose.

[0066] Injectable preparations include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injections, infusions, and the like. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared by, for example, dissolving, suspending, or emulsifying the antigen-binding protein or its salt described above in a sterile aqueous or oily medium conventionally used for injections. Aqueous injectable media include, for example, saline, an isotonic solution containing glucose, and other adjuvants that can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) addition product of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable solutions prepared in this manner are preferably filled into appropriate ampoules.

[0067] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen-type delivery device is easily utilized to deliver the pharmaceutical composition of the present invention. Such a pen-type delivery device may be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. After all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Thus, the pen-type delivery device can be reused. In a disposable pen-type delivery device, there is no replaceable cartridge. Instead, a disposable pen-type delivery device The delivery device is pre-filled with the pharmaceutical composition and held in a reservoir within the device, after which the reservoir is emptied of pharmaceutical composition and the entire device is discarded.

[0068] A number of reusable pen and autoinjector delivery devices find some use in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOGMIX75 / 25™ pen, the HUMALOG™ pen, the HUMALIN70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin), to name just a few. Certain examples of disposable pen delivery devices that have some utility in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ Pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0069] The above-described pharmaceutical compositions for oral or parenteral use are advantageously prepared in unit dosage forms suitable for containing a fixed dose of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injection solutions (ampoules), suppositories, etc. The amount of GLP1 receptor agonist contained is generally about 0.001 to about 100 mg per unit dosage form, particularly in the form of injection solutions. For other dosage forms, the amount of GLP1 receptor agonist contained is preferably about 0.001 to about 100 mg, or about 0.01 to about 100 mg.

[0070] Therapeutic uses of GLP1 receptor agonists The GLP1 receptor agonists of the present invention are useful for treating and / or preventing diseases, disorders, or conditions associated with hyperglycemia, such as diabetes, and / or alleviating at least one symptom associated with such diseases, disorders, or conditions. In one embodiment, the GLP1 receptor agonists of the present invention can be administered in therapeutic doses to patients with diabetes (e.g., type 2 diabetes).

[0071] In certain embodiments, the GLP1 receptor agonists of the present invention are useful for treating a subject suffering from a disease or disorder selected from the group consisting of diabetes, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes, type 1 diabetes, prediabetes, cardiovascular disease, atherosclerosis, congestive heart failure, coronary heart disease, arteriosclerosis, peripheral arterial disease, stroke, respiratory dysfunction, renal disease, fatty liver disease, nonalcoholic steatohepatitis (NASH), and metabolic syndrome.

[0072] In certain embodiments, the GLP1 receptor agonists of the present invention are useful for treating a subject who is overweight or obese, and / or for preventing or treating one or more obesity-related disorders, such as heart disease, stroke, and diabetes.

[0073] In certain embodiments, the GLP1 receptor agonists of the present invention are useful for treating patients with diabetes and / or preventing one or more complications, such as heart disease, stroke, kidney disease, retinopathy, blindness, and nerve damage.

[0074] It is also contemplated herein that one or more of the GLP1 receptor agonist proteins of the present invention can be used prophylactically in subjects at risk of developing diabetes (e.g., type 2 diabetes). Subjects at risk include, but are not limited to, elderly subjects, pregnant women, and subjects with one or more risk factors, including a family history of obesity, high blood cholesterol, smoking, excessive alcohol consumption, and / or physical inactivity.

[0075] In a further embodiment, the proteins of the present invention are useful for preparing pharmaceutical compositions or medicaments for treating patients suffering from diseases or disorders such as diabetes and obesity. In another embodiment of the present invention, the GLP1 receptor agonists of the present invention are used as adjunctive therapeutic agents together with any other agent or any other therapeutic agent known to those skilled in the art to treat or alleviate diseases or disorders associated with hyperglycemia, such as diabetes (e.g., type 2 diabetes).

[0076] Combination therapy The combination therapy can include the GLP1 receptor agonist of the present invention and any other therapeutic agent that can be advantageously combined with the GLP1 receptor agonist of the present invention, or its bioactive fragment of the present invention. The GLP1 receptor agonist of the present invention can be synergistically combined with one or more drugs or therapeutic agents used to treat any disease or disorder associated with hyperglycemia (e.g., diabetes). In some embodiments, the GLP1 receptor agonist of the present invention can be combined with a second therapeutic agent to reduce blood glucose levels in a subject or alleviate one or more symptoms of diabetes.

[0077] The GLP1 receptor agonists of the present invention include insulin (insulin or insulin analogues), insulin sensitizers such as biguanides (e.g. metformin), and insulin secretion stimulants such as thiazolidinediones (e.g. rosiglitazone), sulfonylureas (e.g. clopropamide), and glinides (e.g. nateglinide), alpha-glucosidase inhibitors (e.g. acarbose), dipeptidyl peptidase 4 (DPP4) inhibitors (e.g. sitagliptin), pramlintide, bromocriptine, sodium It can be used in combination with glucose cotransporter type 2 (SGLT-2) inhibitors (e.g., canagliflozin), antihypertensive drugs (e.g., angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, diuretics, calcium channel blockers, alpha-adrenergic receptor antagonists, endothelin-1 receptor antagonists, organic nitrates, and protein kinase C inhibitors), statins, aspirin, different GLP1 receptor agonists, nutritional supplements, or any other therapy (e.g., exercise) to treat or manage diabetes. In certain embodiments, the GLP1 receptor agonists of the present invention can be administered in combination with a second therapeutic agent or treatment selected from the group consisting of insulin, insulin analogs, metformin, rosiglitazone, pioglitazone, clopamide, glibenclamide, glimepiride, glipizide, tolazamide, tolbutamide, nateglinide, repaglinide, acarbose, miglitol, exenatide, liraglutide, albiglutide, dulaglutide, sitagliptin, saxagliptin, linagliptin, alogliptin, pramlinitide, immediate-release bromocriptine, canagliflozin, dapagliflozin, empagliflozin, dietary modification, and exercise.

[0078] As used herein, the term "in combination with" means that the other therapeutically active ingredient(s) can be administered before, simultaneously with, or after the administration of the GLP1 receptor agonist of the present invention. The term "in combination with" does not include the sequential administration of a GLP1 receptor agonist and a second therapeutic agent. This also includes administration or simultaneous administration.

[0079] Other therapeutic active ingredient(s) can be administered to the subject before administering the GLP1 receptor agonist of the present invention.For example, if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or more than 1 minute before the administration of the second component, the first component can be considered to be administered "before" the second component.In other embodiments, other therapeutic active ingredient(s) can be administered to the subject after administering the GLP1 receptor agonist of the present invention. For example, a first component can be considered to be administered "after" a second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after the administration of the second component. In yet other embodiments, the other therapeutically active component(s) can be administered to a subject simultaneously with the administration of a GLP1 receptor agonist of the present invention. "Concurrent administration," for purposes of the present invention, includes, for example, the administration of a GLP1 receptor agonist and another therapeutically active component to a subject in a unit dosage form or separate dosage forms administered to the subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form can be administered via the same route (e.g., both the GLP1 receptor agonist and the other therapeutically active ingredient can be administered intravenously), or each dosage form can be administered via a different route (e.g., the GLP1 receptor agonist can be administered intravenously and the other therapeutically active ingredient can be administered orally). In any case, administration of the ingredients in a unit dosage form or separate dosage forms via the same route, or in separate dosage forms via different routes, is considered "co-administration" for the purposes of this disclosure. For the purposes of this disclosure, administration of a GLP1 receptor agonist "before," "concurrently with," or "after" the administration of the other therapeutically active ingredient (as these terms are defined hereinabove) is considered administration of a GLP1 receptor agonist "in combination with" the other therapeutically active ingredient.

[0080] The present invention includes pharmaceutical compositions in which the GLP1 receptor agonists of the present invention are co-formulated with one or more other therapeutically active ingredient(s) as described elsewhere herein.

[0081] Dosing regimen According to certain embodiments, a single dose of a GLP1 receptor agonist of the present invention (or a pharmaceutical composition comprising a combination of a GLP1 receptor agonist and any other therapeutically active ingredient mentioned herein) can be administered to a subject in need thereof. According to certain embodiments of the present invention, multiple doses of a GLP1 receptor agonist of the present invention (or a pharmaceutical composition comprising a combination of a GLP1 receptor agonist and any other therapeutically active ingredient mentioned herein) can be administered to a subject over a defined time course. The method according to this aspect of the present invention comprises sequentially administering multiple doses of a GLP1 receptor agonist of the present invention to a subject. As used herein, "sequentially administering" means administering each dose of a GLP1 receptor agonist to a subject at different time points, for example, on different days separated by a predetermined interval (e.g., several hours, days, weeks, or months). The present invention includes methods comprising sequentially administering to a patient one initial dose of a GLP1 receptor agonist, then one or more secondary doses of a GLP1 receptor agonist, and optionally then one or more tertiary doses of a GLP1 receptor agonist.

[0082] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the chronological administration of the GLP1 receptor agonist of the present invention. Thus, the "initial dose" is the dose administered at the beginning of a treatment regimen (also called the "baseline dose"), the "secondary dose" is the dose administered after the initial dose, and the "tertiary dose" is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of GLP1 receptor agonist. Although there are some, generally, they may be different from each other in terms of frequency of administration.However, in certain embodiments, the amount of GLP1 receptor agonist contained in the first, second and / or third dose is different from each other during the course of treatment (for example, be adjusted upward or downward appropriately).In certain embodiments, one or more (for example, 2, 3, 4 or 5) doses are administered as "loading dose" at the beginning of treatment regimen, and then subsequent doses (for example, "maintenance dose") are administered less frequently.

[0083] In certain exemplary embodiments of the invention, each secondary and / or tertiary dose is administered 1 to 48 hours (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 1 The term "immediately preceding dose" refers to a dose of a GLP1 receptor agonist administered to a patient immediately prior to administration of the next dose in a series of multiple doses, with no intervening doses. In certain embodiments, each secondary and / or tertiary dose is administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days after the immediately preceding dose. In particular embodiments, each secondary and / or tertiary dose is administered every 0.5 weeks, every week, every 2 weeks, every 3 weeks, or every 4 weeks after the immediately preceding dose.

[0084] The method according to this aspect of the invention can include administering any number of secondary and / or tertiary doses of a GLP1 receptor agonist to the patient. For example, in certain embodiments, only one secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0085] In certain embodiments of the invention, the frequency with which a patient receives secondary and / or tertiary doses may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by a physician during the course of treatment depending on the needs of an individual patient following clinical trials.

[0086] dosage The amount of GLP1 receptor agonist administered to a subject according to the methods of the present invention is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to an amount of GLP1 receptor agonist that results in one or more of: (a) a reduction in hyperglycemia to normal levels (e.g., a preprandial blood glucose level of 80 to 130 mg / dL), and / or (b) a detectable improvement in one or more symptoms or signs of diabetes.

[0087] In the case of a GLP1 receptor agonist, the therapeutically effective amount is about 0.001 mg to about 100 mg, for example, about 0.001 mg, about 0.002 mg, about 0.003 mg, about 0.004 mg, about 0.005 mg, about 0.006 mg, about 0.007 mg, about 0.008 mg, about 0.009 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg g, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about In certain embodiments, 0.005 mg to 50 mg, 0.005 mg to 30 mg, 0.005 mg to 10 mg, 0.1 mg to 10 mg, or 0.1 mg to 5 mg of the GLP1 receptor agonist is administered to a subject in need thereof.

[0088] The amount of GLP1 receptor agonist contained in each dose can be expressed in milligrams of antibody per kilogram of subject body weight (i.e., mg / kg). For example, the GLP1 receptor agonist can be administered to a subject at a dose of about 0.0001 to about 100 mg per kilogram of subject body weight.

[0089] Selected embodiments In embodiment 1, the present invention provides a glucagon-like peptide 1 (GLP1) variant comprising mature GLP1(7-37) (SEQ ID NO: 4) with at least one amino acid modification selected from the group consisting of (i) an addition of an amino acid to the N-terminus, and (ii) a deletion of an amino acid from the peptide sequence, wherein the GLP1 variant has high resistance to proteolytic cleavage and / or high blood glucose lowering ability.

[0090] In embodiment 2, the present invention provides a GLP1 variant according to embodiment 1, wherein the amino acid modification comprises the addition of an amino acid selected from the group consisting of alanine (Ala) and glutamine (Gln) to the N-terminus.

[0091] In embodiment 3, the present invention provides a GLP1 variant according to embodiment 1 or 2, wherein the amino acid modification comprises the addition of Gln to the N-terminus.

[0092] In embodiment 4, the present invention provides a GLP1 variant according to embodiment 1, wherein the amino acid modification comprises a deletion of a histidine (His1) or an alanine (Ala2) from SEQ ID NO:4.

[0093] In embodiment 5, the present invention provides a GLP1 variant according to any one of embodiments 1 to 4, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8.

[0094] In embodiment 6, the present invention provides a GLP1 variant according to embodiment 5, comprising the amino acid sequence of SEQ ID NO:6.

[0095] In embodiment 7, the present invention provides a fusion protein comprising a GLP1 variant described in any one of embodiments 1 to 6 fused to a stabilization domain, wherein the stabilization domain is an antigen-binding protein or an antigen-binding fragment thereof that specifically binds to the GLP1 receptor and comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

[0096] In embodiment 8, the present invention provides a fusion protein according to embodiment 7, wherein the GLP1 variant is fused to the N-terminus or C-terminus of the HCVR of the antigen-binding protein or antigen-binding fragment thereof.

[0097] In embodiment 9, the present invention provides a fusion protein according to embodiment 7, wherein the GLP1 variant is fused to the N-terminus or C-terminus of the LCVR of the antigen-binding protein or antigen-binding fragment thereof.

[0098] In embodiment 10, the present invention provides a fusion protein comprising a GLP1 variant according to any one of embodiments 1 to 6 fused to a stabilization domain, wherein the stabilization domain is an immunomodulatory agent. Fusion proteins that are globulins (Ig) or fragments thereof are provided.

[0099] In embodiment 11, the present invention provides a fusion protein according to embodiment 11, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12 and 13.

[0100] In embodiment 12, the present invention provides a fusion protein comprising a GLP1 variant fused to a stabilization domain, wherein the stabilization domain is an antigen-binding protein or an antigen-binding fragment thereof, and the antigen-binding protein or fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

[0101] In embodiment 13, the present invention provides a fusion protein according to embodiment 12, wherein the GLP1 variant is fused to the N-terminus or C-terminus of the HCVR of the antigen binding protein or fragment thereof.

[0102] In embodiment 14, the present invention provides a fusion protein according to embodiment 12, wherein the GLP1 variant is fused to the N-terminus or C-terminus of the LCVR of the antigen binding protein or fragment thereof.

[0103] In embodiment 15, the present invention provides a fusion protein of any one of embodiments 12 to 14, wherein the antigen-binding protein or fragment thereof specifically binds to the GLP1 receptor.

[0104] In embodiment 16, the present invention provides a fusion protein according to any one of embodiments 12 to 15, comprising a GLP1 variant according to any one of embodiments 1 to 15.

[0105] In embodiment 17, the present invention provides a fusion protein according to any one of embodiments 12 to 16, wherein the GLP1 variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8.

[0106] In embodiment 18, the present invention provides the fusion protein of embodiment 16 or 17, wherein the GLP1 variant comprises the amino acid sequence of SEQ ID NO:6.

[0107] In embodiment 19, the present invention provides a GLP1 receptor agonist comprising a GLP1 variant, wherein the GLP1 variant is fused to a stabilization domain, the stabilization domain is an antigen-binding protein or an antigen-binding fragment thereof, and the antigen-binding protein or fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

[0108] In embodiment 20, the present invention provides a GLP1 receptor agonist according to embodiment 19, wherein the GLP1 variant is fused to the N-terminus or C-terminus of the HCVR of the antigen binding protein or fragment thereof.

[0109] In embodiment 21, the present invention provides a GLP1 receptor agonist according to embodiment 19, wherein the GLP1 variant is fused to the N-terminus or C-terminus of the LCVR of the antigen binding protein or fragment thereof.

[0110] In embodiment 22, the present invention provides a GLP1 receptor agonist according to any one of embodiments 19 to 21, wherein the antigen-binding protein or fragment thereof specifically binds to the GLP1 receptor.

[0111] In embodiment 23, the present invention provides a GLP1 receptor agonist according to any one of embodiments 19 to 22, which comprises a GLP1 variant according to embodiment 1.

[0112] In embodiment 24, the present invention provides a GLP1 receptor agonist according to any one of embodiments 19 to 23, wherein the GLP1 variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8.

[0113] In embodiment 25, the present invention provides a GLP1 receptor agonist according to embodiment 23 or 24, wherein the GLP1 variant comprises the amino acid sequence of SEQ ID NO:6.

[0114] In embodiment 26, the present invention provides a GLP1 receptor agonist comprising a GLP1 variant, wherein the GLP1 variant is fused to a stabilization domain, and the stabilization domain is an immunoglobulin (Ig) or a fragment thereof.

[0115] In embodiment 27, the present invention provides a GLP1 receptor agonist according to embodiment 26, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12 and 13.

[0116] In embodiment 28, the present invention provides a pharmaceutical composition comprising the protein of any one of embodiments 1 to 27 and a pharmaceutically acceptable carrier or diluent.

[0117] In embodiment 29, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding a GLP1 variant according to any one of embodiments 1 to 6.

[0118] In embodiment 30, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the fusion protein of any one of embodiments 10-11.

[0119] In embodiment 31, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the GLP1 receptor agonist of any one of embodiments 26-27.

[0120] In embodiment 32, the present invention provides a vector comprising the polynucleotide sequence of any one of embodiments 29 to 31.

[0121] In embodiment 33, the present invention provides a cell expressing the vector of embodiment 32.

[0122] In embodiment 34, the present invention provides a method for reducing blood glucose levels, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a protein described in any one of embodiments 1 to 27.

[0123] In embodiment 35, the present invention provides the method of embodiment 34, wherein the subject has a disease or disorder selected from the group consisting of diabetes, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes, type 1 diabetes, prediabetes, cardiovascular disease, atherosclerosis, congestive heart failure, coronary heart disease, arteriosclerosis, peripheral artery disease, stroke, respiratory dysfunction, kidney disease, fatty liver disease, nonalcoholic steatohepatitis (NASH), and metabolic syndrome.

[0124] In embodiment 36, the present invention provides a method for preventing, treating, or ameliorating at least one symptom, sign, or complication of type 2 diabetes, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a protein described in any one of embodiments 1 to 27.

[0125] In embodiment 37, the present invention provides the method of embodiment 36, wherein the at least one symptom, sign, or complication is selected from the group consisting of high blood sugar levels, excessive thirst, frequent urination, presence of ketones in the urine, fatigue, weight fluctuations, blurred vision, slow-healing sores, frequent infections, swollen or tender gums, obesity, heart disease, stroke, kidney disease, eye disease, nerve damage, and high blood pressure.

[0126] In embodiment 38, the present invention provides a method according to any one of embodiments 34 to 37, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent or treatment.

[0127] In embodiment 39, the present invention provides the method of embodiment 38, wherein the second therapeutic agent or treatment is selected from the group consisting of insulin or an insulin analog, a biguanide (e.g., metformin), a thiazolidinedione, a sulfonylurea (e.g., clopropamide), a glinide (e.g., nateglinide), an alpha-glucosidase inhibitor, a DPP4 inhibitor (e.g., sitagliptin), pramlintide, bromocriptine, an SGLT2 inhibitor (e.g., canagliflozin), an antihypertensive agent, a statin, aspirin, a modified diet, exercise, and a nutritional supplement.

[0128] In embodiment 40, the present invention provides a method according to any one of embodiments 34 to 39, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. [Example]

[0129] The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of this invention, but are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is atmospheric or normal pressure. [Example]

[0130] Exemplary Fusion Proteins Comprising GLP1 Due to its rapid inactivation by the enzyme dipeptidyl peptidase 4 (DPP4), GLP1(7-37) has a very short circulating half-life (1-2 minutes). Previous studies have shown that various amino acid substitutions at position 8 of GLP1(7-37) confer increased resistance to DPP4, thereby resulting in a longer half-life (Deacon et al., 1998, Diabetologia 41:271-278). However, these molecules remain susceptible to DPP4 cleavage. Therefore, there is a need to develop new molecules that are even more resistant to DPP4.

[0131] To confer higher resistance to DPP4, the first part of the technique is to introduce mutations that lengthen or shorten the amino terminus of GLP1, either by adding amino acids (i.e., Ala, Gln) to the N-terminus or deleting His7 or Ala8 within the peptide sequence, to confer higher resistance to DPP4 cleavage. These modifications also weaken GLP1 activity, and the second part of the technique is to compensate for the reduced activity by using an anti-GLP1R antibody to bind the receptor with a weakened agonist by fusing a peptide to the N-terminus of the anti-GLP1R antibody light chain sequence. As a proof of concept, as described below, , a modified GLP1(7–37) ligand sequence was fused to the N-terminus of the light chain of a GLP1R antibody.

[0132] Mature GLP1 is a peptide hormone of 31 amino acids, including amino acids 7 to 37 of full-length GLP1 (SEQ ID NO: 3), and has the amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 4).

[0133] Mature GLP1 was modified by amino acid deletion or addition at the amino terminus to generate GLP1 variants. Exemplary GLP1 variants are provided below: Des-Ala-GLP1 containing the amino acid sequence HEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 5) Q-GLP1 containing the amino acid sequence QHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 6) A-GLP1 containing the amino acid sequence AHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 7) desH-GLP1 containing the amino acid sequence AEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 8)

[0134] To compensate for the possible reduced activity of the aforementioned GLP1 variants, anti-GLP1R antibodies were used to conjugate them to the GLP1 receptor (GLP1R) or antibody Fc fragments. Exemplary fusion proteins containing mature GLP1 or GLP1 variants were prepared using an anti-GLP1 receptor antibody (hereinafter referred to as "mAb1") comprising the heavy chain variable region of SEQ ID NO:2 and the light chain variable region (LCVR) of SEQ ID NO:1, as described in U.S. Patent Application Publication No. 20060275288 [Abbott These were generated by fusion of mature GLP1 or GLP1 variants to the N-terminus of the light chain of [Gibberish Laboratories], and are listed below: Des-Ala-GLP1-mAb1 fused to the N-terminus of the light chain of mAb1: Des-Ala-GLP1 (SEQ ID NO: 5) Q-GLP1-mAb1 fused to the N-terminus of the light chain of mAb1: Q-GLP1 (SEQ ID NO: 6) A-GLP1-mAb1 fused to the N-terminus of the light chain of mAb1: A-GLP1 (SEQ ID NO: 7)

[0135] Fusion proteins comprising mature GLP1 or GLP1 variants and immunoglobulin Fc fragments were also generated and are listed below: GLP1-hFc (SEQ ID NO: 9) A-GLP1-hFc (SEQ ID NO: 10) Q-GLP1-hFc (SEQ ID NO: 11) Des-Ala-GLP1-hFc (SEQ ID NO: 12) desH-GLP1-hFc (SEQ ID NO: 13)

[0136] Control constructs Comparator: A GLP1 analog (dulaglutide; Eli Lilly) with an amino acid sequence characteristic of LY2189265 fused to a hIgG4 Fc domain, as disclosed in Glaesner et al., 2010 (Diabetes Metab. Res. Rev. 26:287-296), was used as a comparator (SEQ ID NO: 14) in the following examples. [Example]

[0137] Luciferase assay GLP1 fusion proteins were tested for their ability to stimulate cAMP production in the reporter cell line 293 / FSC11 / Cre-Luc, which stably expresses the human GLP1 receptor and luciferase coding sequences under the control of the cAMP-responsive cre promoter.

[0138] For the luciferase bioassay, 293 / FSC11 / Cre-LucGLP1R stable cells were seeded into 96-well assay plates at 30,000 cells / well in OPTIMEM supplemented with 0.1% FBS and then incubated overnight at 37°C in 5% CO2. The following day, human GLP1 (Phoenix #028-13), des-Ala-GLP1-mAb1, Q-GLP1-mAb1, or A-GLP1-mAb1 were tested in the assay to determine the dose response of the test proteins. All test compounds were purified proteins except for A-GLP1-mAb1, which was used directly from the culture medium after transient transfection of CHO cells with a vector encoding the modified antibody. Materials in the culture medium were quantified by ELISA. Test samples were added to cells at concentrations ranging from 0.02 pM to 100 nM.

[0139] After 5.5 hours or overnight incubation at 37°C in 5% CO2, OneGIo reagent (Promega, #E6051) was added to the samples, and luciferase activity was then measured using a VictorX (Perkin Elmer) plate reader. Results were analyzed using nonlinear regression (3 parameters) and Prism 6 software (GraphPad) to calculate EC 50 got the value.

[0140] As shown in Table 1, the Q- and A-modified mAb1 antibody fusions exhibited EC50 values ​​of 204 pM and 312 pM, respectively, for GLP1R activation.

[0141] [Table 1]

[0142] The EC50 for des-Ala-GLP1-mAb1 was 10 nM. The EC50 for Q- and A-GLP1-hFc were 135 nM and 120 nM, while the EC50 for DesA-GLP1-hFc was undetectable. [Example]

[0143] Effects of GLP1R antibody fusion Q-GLP1 on blood glucose and glucose tolerance in GLP1R-humanized mice The effects of Q-GLP1 fused to the N-terminus of the light chain of an anti-GLP1R antibody (Q-GLP1-mAb1) on blood glucose and glucose tolerance were investigated using human GLP1R protein. The GLP1R-expressing GLP1R-humanized mice were genetically engineered to express GLP1R ("GLP1R-humanized mice"). Thirty-one GLP1R-humanized mice were divided into four groups of 7-8 animals. Each group received a single subcutaneous injection of isotype control, Q-GLP1-hFc, mAb1, or Q-GLP1-mAb1 at 194 nmol / kg. Mice were bled in captivity on days 0, 1, 4, 7, 11, 14, 16, and 18, and blood glucose was measured on day 22. The mean ± SEM of blood glucose levels at each time point was calculated for each group and is shown in Table 2.

[0144] [Table 2]

[0145] Oral glucose tolerance tests (oGTTs) were performed on days 3 and 9 after an overnight fast, with blood glucose measurements taken at 0, 15, 30, 60, and 120 minutes after bolus glucose gavage. The mean ± SEM blood glucose levels and area under the glucose curve (AUC) at each time point were calculated for each group and are shown in Tables 3 and 4.

[0146] [Table 3]

[0147] [Table 4]

[0148] A single administration of Q-GLP1-mAb1 in normoglycemic GLP1R-humanized mice resulted in significant glucose reduction for 14 days, whereas Q-GLP1-hFc or mAb1 had no effect on blood glucose levels (Table 2). Q-GLP1-mAb1 reduced fasting glucose levels and improved glucose tolerance in mice on days 3 and 9, whereas Q-GLP1-hFc and mAb1 did not. These data suggest that the Q-GLP1 or mAb1 antibodies alone do not alter glycemic control; however, the two fusion molecules may exert a glucose-lowering function lasting for 2 weeks after a single injection in normoglycemic animals. [Example]

[0149] Stability of GLP1 mutants The stability of various GLP1 variants and fusion proteins was tested by incubating them with blood proteases and analyzing the cleaved peptides by mass spectrometry.

[0150] In the first experiment, 0.5 μg of each GLP1 fusion protein was added to 50 μL of naive mouse serum. The mixture was then incubated at 37°C for 6 and 24 hours, respectively. 1 μL of the serum mixture was loaded onto a Tris-glycine gel at 0 minutes, 6 hours, and 24 hours.

[0151] In a second experiment, to further differentiate the stability, 2 μg of each GLP1 fusion protein was incubated with 500 ng of recombinant human DPP4 (R and D system) in PBS (pH 7.4) for 0 min, 1 h, 4 h, and 72 h at 37° C. One-fifth of the mixture (corresponding to 400 ng of construct) was loaded onto a Tris-glycine gel.

[0152] For each experiment, a gel slice corresponding to the molecular weight of each construct was excised and subjected to in-gel trypsin digestion. The excised gel slice was destained in 50:50 acetonitrile:NH4HCO3 (50 mM), reduced with 65 mM dithiothreitol (Sigma) for 30 min at 37°C, and then alkylated with 135 mM iodoacetamide (Sigma) for 30 min at room temperature in the dark. Proteins were then digested overnight with sequencing-grade modified porcine trypsin (Promega) at 37°C. Peptides were extracted twice with extraction buffer (50% ACN, 5% formic acid in H2O). Peptides extracted from each band were completely dried in a SpeedVac and renatured with 0.1% tetrafluoroacetic acid (TFA) before nanoLC-MS / MS analysis.

[0153] The renatured peptide mixture was analyzed by online reversed-phase (RP) nanoscale capillary liquid chromatography (Easy-nLC1000, Thermo Fisher Scientific). Peptides were separated by electrospray tandem mass spectrometry (Orbitrap Elite, Thermo Fisher Scientific) and analyzed by electrospray tandem mass spectrometry. The peptide mixture was injected onto a 75 μm i.d. "PepMap RSLC" column (C18, 25 cm, 100 Å, 2 μm, Thermo Fisher Scientific) at a flow rate of 250 nL / min and then eluted with a 60-minute gradient of 2% to 35% ACN, 0.1% formic acid. The mass spectrometer was operated in data-dependent mode to automatically switch between MS and MS / MS acquisition. Survey full-scan MS spectra (m / z 350–2000) were collected in the Orbitrap at a resolution of 120,000. The most intense ions (up to 10) were sequentially isolated for fragmentation in the hybrid ion trap using collision-induced dissociation (CID) with a target value of 5000 and 35% normal collision energy. Target ions previously selected for MS / MS were dynamically excluded for 30 s.

[0154] MS and MS / MS peak lists were extracted and searched against an internal protein database using ProteomeDiscoverer 1.4 (Thermo Fisher Scientific). Trypsin digestion was assumed by the full search, and carboxymethylation of cysteine ​​was considered as a fixed modification, and oxidation of methionine as a variable modification. A peptide mass tolerance of 10 ppm, an MS / MS mass tolerance of 0.8 Da, and a maximum truncation tolerance of 1 were used. Extracted ion areas were computed based on extracted ion chromatograms (XICs) using Thermo Xcaliber software (Thermo Fisher Scientific).

[0155] result To characterize the susceptibility of each construct to cleavage by serum enzymes, the intact peptide (N-terminal peptide), cleaved peptide (N-terminal peptide after cleavage), and one internal reference peptide (a stable peptide unaffected by any modifications in the construct) were monitored for each construct by nanoLC-MS / MS. A decreased ratio of intact peptide to reference peptide and a concomitant increase in the ratio of cleaved peptide to reference peptide suggested enzyme-mediated cleavage of the construct over time. The cleavage rate was calculated using the following formula: 100 × area of ​​cleaved peptide / (area of ​​cleaved peptide + area of ​​uncleaved peptide).

[0156] GLP1-hFc and A-GLP1-hFc were completely cleaved by 6 hours, while desH-GLP1-hFc showed significant cleavage (2%) by 6 hours. Q-GLP1-hFc and the comparator did not show any cleavage after 24 hours of incubation (Table 5).

[0157] [Table 5]

[0158] To further differentiate the stability of Q-GLP1-hFc and the comparator, the two structures were The constructs were mixed with recombinant human DPP4, and for each construct, the intact peptide (N-terminal peptide), the truncated peptide (N-terminal peptide after cleavage), and one internal reference peptide (a stable peptide unaffected by any modifications in the construct) were monitored by nanoLC-MS / MS.

[0159] [Table 6]

[0160] The comparator showed significant cleavage (4%) by 4 hours and over 40% cleavage by 72 hours (Table 6). In contrast, Q-GLP1-hFc did not show any cleavage even after 72 hours of incubation with DPP4 at 37°C.

[0161] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding a fusion protein consisting of the amino acid sequence shown in SEQ ID NO:

11.

2. A vector comprising the polynucleotide molecule of claim 1.

3. A cell that expresses the fusion protein encoded by the polynucleotide molecule in the vector of claim 2.

4. A pharmaceutical composition for use in reducing blood glucose levels in a subject, comprising a fusion protein consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a pharmaceutically acceptable carrier or diluent.

5. 5. The pharmaceutical composition of claim 4, wherein the subject has a disease or disorder selected from the group consisting of diabetes, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes, type 1 diabetes, prediabetes, cardiovascular disease, atherosclerosis, congestive heart failure, coronary heart disease, arteriosclerosis, peripheral arterial disease, stroke, respiratory dysfunction, kidney disease, fatty liver disease, nonalcoholic steatohepatitis (NASH), and metabolic syndrome.

6. A pharmaceutical composition for use in preventing, treating or alleviating at least one symptom, sign or complication of type 2 diabetes, comprising a fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 11 and a pharmaceutically acceptable carrier or diluent.

7. 7. The pharmaceutical composition of claim 6, wherein the at least one symptom, sign or complication is selected from the group consisting of high blood sugar levels, excessive thirst, frequent urination, presence of ketone bodies in the urine, fatigue, weight fluctuations, blurred vision, slow-healing sores, frequent infections, swollen or tender gums, obesity, heart disease, stroke, kidney disease, eye disease, nerve damage and high blood pressure.

8. 8. The method of claim 4, wherein the method is administered in combination with a second therapeutic agent or treatment. The pharmaceutical composition described above.

9. 9. The pharmaceutical composition of claim 8, wherein the second therapeutic agent or treatment is selected from the group consisting of insulin or insulin analogs, metformin, thiazolidinediones, sulfonylureas, biguanides, clopropamide, glinides, alpha-glucosidase inhibitors, nateglinide, DPP4 inhibitors, pramlintide, sitagliptin, bromocriptine, SGLT2 inhibitors, canagliflozin, antihypertensive drugs, statins, aspirin, dietary modification, exercise, and nutritional supplements.

10. The pharmaceutical composition according to any one of claims 4 to 9, which is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly.

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