EA045412B1 - GLUCAGON-LIKE PEPTIDE 1 RECEPTOR AGONISTS AND THEIR APPLICATIONS - Google Patents

Description

Field of technology to which the present invention relates

The present invention relates to human glucagon-like peptide 1 receptor agonists and therapeutic methods for using said agonists.

BACKGROUND OF THE INVENTION

Obesity has become a major health problem in the United States, with two out of three Americans estimated to be overweight or obese. Obesity is an important risk factor for the development of other diseases such as cardiovascular disease, stroke and diabetes. Even a small decrease in body weight (5-10% of initial body weight) reduces the risk of developing diseases associated with obesity, such as cardiovascular disease and diabetes.

Diabetes mellitus is a chronic condition characterized by high blood sugar and insulin resistance. Left untreated, high blood sugar can lead to long-term complications, including heart disease, stroke, diabetic retinopathy, and lower limb amputation. Treatment of diabetes involves controlling and lowering blood sugar and includes exercise and diet modification, as well as medications such as insulin and metformin.

One approach used to treat obesity and control blood glucose 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 of the intestine. When glucose is administered orally, GLP1 binds to its receptor, which leads to insulin secretion and a decrease in blood sugar (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 min. Consequently, long-acting GLP1 derivatives as well as GLP1 receptor agonists, including GLP1-containing fusion proteins, have been studied for the control of diabetes mellitus. GLP1 analogues, fusion proteins and GLP1 receptor agonists are disclosed, for example, in US 7452966, US 8389689, US 8497240, US 8557769, US 8883447, US 8895694, US 9409966, US 2016/0194371, US 2014/002 4586, US 2014/0073563, US 2012/0148586, US 2017/0114115, US 2017/0112904, US 2016/0361390, US 2015/0313908, US 2015/0259416, WO 2017/074715, WO 2016/127887, WO 2015/021871, WO 2014/113357, EP 3034514, EP 2470198 and EP 2373681.

However, there is a need for new GLP1 peptide variants and GLP1 receptor agonists that are resistant to degradation by DPP4, have improved pharmacokinetic properties, and exhibit increased potency and sustained in vivo activity in glycemic control. Such GLP1 variants and GLP1 receptor agonists can be used to treat obesity and diabetes.

Brief Disclosure of the Present Invention

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

In one aspect, the present invention provides GLP1 receptor agonists, wherein the GLP1 receptor agonists comprise fusion proteins comprising GLP1 or a variant thereof. In certain embodiments, GLP1 receptor agonists comprise a GLP1 peptide or a variant of a GLP1 peptide fused to a stabilizing domain. In one embodiment, the stabilizing domain is an antibody or antigen-binding fragment thereof that binds to the GLP1 receptor.

The GLP1 receptor agonists of the present invention are useful, among other things, 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 lowering blood sugar.

In some embodiments, the GLP1 peptide variants and/or GLP1 receptor agonists of the present invention are more resistant to dipeptidyl peptidase 4 (DPP4) inactivation and exhibit improved in vivo half-life. The improved GLP1 agonists of the present invention result in significant reductions in blood sugar that are maintained for more than 10 days even when administered as a single dose. In some embodiments, GLP1 receptor agonists function by enhancing glucose-induced insulin secretion from pancreatic beta cells, increasing insulin expression, inhibiting beta cell apoptosis, promoting beta cell regeneration, reducing glucagon secretion, delaying gastric emptying, increasing satiety, and increasing peripheral glucose utilization. In certain embodiments, GLP1 receptor agonists are useful for preventing, treating, or reducing the intensity of at least one

- 1 045412 symptom of a hyperglycemia-related disease or disorder (eg, diabetes mellitus) in a subject. In certain embodiments, GLP1 receptor agonists can be administered prophylactically or therapeutically to a subject who has or is at risk for diabetes mellitus. In certain embodiments, GLP1 receptor agonists are useful for preventing, treating, or reducing the intensity of at least one symptom or sign of obesity, such as weight loss, in a subject.

In certain embodiments, GLP1 receptor agonists are fusion proteins comprising a GLP1 variant and a stabilizing domain, wherein the stabilizing domain comprises an immunoglobulin or fragment thereof. In a specific embodiment, the immunoglobulin contains a heavy chain variable region and a light chain variable region and specifically binds to the GLP1 receptor. In certain embodiments, GLP1 receptor agonists bind to the GLP1 receptor, resulting in activation of the GLP1 receptor. In certain embodiments, GLP1 receptor agonists function by activating the GLP1 receptor, resulting in glycemic control, i.e. reducing blood glucose levels.

In one embodiment, the present invention provides a fusion protein that has one or more of the following characteristics: (a) contains a GLP1 variant domain and a stabilization domain; (b) is a GLP1 receptor agonist; (c) the GLP1 variant domain contains the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8; (d) binds to the GLP1 receptor; (f) the stabilizing domain contains an immunoglobulin or a fragment thereof; (f) the stabilizing domain contains an immunoglobulin Fc fragment; (g) the stabilizing domain comprises an anti-GLP1 receptor antibody or an antigen-binding fragment thereof; (h) is resistant to degradation by serum proteases for at least 72 hours and (i) results in a significant reduction in serum glucose that is sustained for more than 10 days with a single dose.

In one aspect, the present invention relates to nucleic acid molecules encoding GLP1 variants or portions thereof. For example, the present invention relates to nucleic acid molecules encoding any of the amino acid sequences selected from the group consisting of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12 and 13, or substantially similar thereto a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

The present invention also relates to nucleic acid molecules encoding any of the fusion proteins containing GLP1 variants.

In a related aspect, the present invention provides recombinant expression vectors capable of expressing a GLP1 variant-containing polypeptide or a GLP1 variant-containing fusion protein as described herein. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, i.e. nucleic acid molecules encoding any of the GLP1 variants or fusion proteins containing GLP1 variants. 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 proteins or fragments thereof by culturing the host cells under conditions allowing the production of proteins or fragments thereof and recovery of the proteins and fragments so produced.

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 provides 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 may 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 with the GLP1 receptor, but nevertheless alleviate or reduce the intensity of or treat a GLP1 receptor-related disease or disorder (eg, diabetes mellitus). Additional combination therapies and co-formulations comprising the GLP1 receptor agonist proteins of the present invention are disclosed elsewhere herein.

In another aspect, the present invention provides therapeutic methods for treating a GLP1-associated disease or disorder, such as diabetes mellitus, in a subject using a GLP1 receptor agonist according to the present invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a GLP1 receptor agonist according to the present invention. invention to a subject in need. In certain embodiments, the GLP1 receptor agonist comprises a GLP1 variant or a fusion protein comprising a GLP1 variant. The disorder being treated is any disease

- 2 045412 condition or condition that is improved, reduced in intensity, inhibited or prevented by activation of GLP1 receptor activity. In certain embodiments, the present invention provides methods for preventing, treating, or ameliorating at least one symptom of a GLP1 receptor-related disease or disorder, the method 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 methods for reducing the intensity or 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 according to the present invention, wherein at least one symptom or symptom selected from the group consisting of high blood sugar, polydipsia, frequent urination, ketones in urine, fatigue, weight fluctuations, blurred vision, slow-healing ulcers, frequent infections, swollen or painful gums, obesity, heart disease -vascular system, stroke, kidney disease, eye disease, nerve damage and high blood pressure. In certain embodiments, the present invention provides methods for reducing body weight in a subject who is overweight or obese, the methods comprising administering to the subject a therapeutically effective amount of a GLP1 receptor agonist of the present invention that binds the GLP1 receptor and activates the activity of the GLP1 receptor. In certain embodiments, the present invention provides methods for lowering blood sugar in a subject, the methods comprising administering to the subject a therapeutically effective amount of a GLP1 receptor agonist of the present invention that binds the GLP1 receptor and activates the activity of the GLP1 receptor. In some embodiments, a GLP1 receptor agonist can be administered prophylactically or therapeutically to a subject having or at risk of 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 lack of exercise. exercises. In certain embodiments, the present invention provides methods for treating type 2 diabetes that is not controlled by treatment with insulin and/or metformin, the methods 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 in combination with a second therapeutic agent to a subject in need thereof. The second therapeutic agent may be selected from the group consisting of insulin or insulin analog, biguanide (eg, metformin), thiazolidinedione, sulfonylurea (eg, chlorpropamide), glinide (eg, nateglinide), alpha-glucosidase inhibitor, DPP4 inhibitor (eg, sitagliptin) , pramlintide, bromocriptine, SGLT2 inhibitor (eg, canagliflozin), antihypertensive drug, statin, aspirin, diet modification, exercise, and dietary supplement. Additional 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 may be an agent that helps neutralize or reduce any potential side effect(s) associated with the GLP1 receptor agonist of the present invention, if such(es) ) side effect(s) must occur. The GLP1 receptor agonist can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. The GLP1 receptor agonist can be administered at a dose of about 0.1 to about 100 mg/kg of the subject's body weight. In certain embodiments, the GLP1 receptor agonist of the present invention can be administered in one or more doses containing from 0.1 to 600 mg.

The present invention also provides for the use of a GLP1 receptor agonist of the present invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from stimulation of GLP1 receptor binding and/or activity (eg, diabetes mellitus, including type 2 diabetes mellitus).

Other embodiments will become apparent from a review of the following detailed disclosure.

Detailed Disclosure of the Present Invention

Before the present methods are described, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

- 3 045412

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

Definitions

The term GLP1, also called glucagon-like peptide 1, refers to a 31-amino acid peptide hormone released from intestinal L cells after nutrient consumption. GLP1 binds to the GLP1 receptor and enhances glucose-induced insulin secretion from pancreatic beta cells, increases insulin expression, inhibits beta cell apoptosis, promotes beta cell regeneration, reduces glucagon secretion, delays gastric emptying, promotes satiety, and increases peripheral glucose utilization. In certain embodiments, the term GLP1 refers to a mature 31 amino acid peptide hormone (SEQ ID NO: 4) comprising amino acids 7 to 37 of the full length GLP1 peptide (SEQ ID NO: 3). The term also includes GLP1 variants, where the variants contain 1, 2, 3, 4, 5 or 6 amino acid substitutions, additions or deletions. For example, the term includes variants that contain the amino acid sequences of SEQ ID NO: 5, 6, 7 or 8.

As used herein, the term stabilizing domain is any macromolecule that, when fused to a peptide, increases the activity and/or stability of the peptide in vivo. For example, the stabilizing domain may be a polypeptide containing an immunoglobulin CH3 domain. In certain embodiments, the stabilizing domain increases the serum half-life of the peptide. In certain embodiments, the stabilizing peptide increases the activity of the peptide in vivo. A non-limiting example of a stabilizing domain is the Fc portion of an immunoglobulin, for example the IgG Fc domain selected from the IgG1, IgG2, IgG3 and IgG4 isotypes, as well as any allotype within each isotype group. In certain embodiments, the stabilizing domain is an Fc fragment or amino acid sequence ranging in length from 1 to about 200 amino acids containing at least one cysteine residue. As another example, the stabilizing domain may be an immunoglobulin or an antigen-binding fragment thereof. In certain embodiments, the stabilizing domain is an immunoglobulin comprising a heavy chain variable region and a light chain variable region, wherein the immunoglobulin binds a specific antigen. In certain embodiments, the stabilizing domain comprises an antigen binding domain and an Fc domain (e.g., IgG1 or IgG4 antibodies) or may contain only an antigen binding portion (e.g., Fab, F(ab')2, or scFv) and may be modified to affect functionality . In a specific embodiment, the stabilizing domain is an immunoglobulin comprising a heavy chain variable region and a light chain variable region, wherein the immunoglobulin binds to the GLP1 receptor. In other embodiments, the stabilizing domain is a cysteine residue or a short cysteine-containing peptide. Other stabilizing domains include peptides or polypeptides containing or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

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 containing GLP1 or a GLP1 variant fused to a stabilization domain. In certain embodiments, the term includes fusion proteins comprising a GLP1 variant fused to an immunoglobulin or fragment thereof. In specific embodiments, the term includes fusion proteins comprising GLP1 or a GLP1 variant fused to the N-terminus of an immunoglobulin light chain variable region (VL). In one specific embodiment, the term includes GLP1 or a GLP1 variant fused to the N-terminus of a VL antibody or antigen binding fragment thereof that binds to the GLP1 receptor.

As used herein, the term antibody is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as multimers thereof (e.g., IgM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain constant region (consisting of the CH1, CH2 and CH3 domains) of an Ig variable region, which may be a heavy chain variable region (HCVR or _VH ) or a light chain variable region (LCVR or VL). Each light chain consists of a light chain variable region (LCVR or VL) and a light chain constant region ( _CL ). The VH and _VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved called frameworks.

- 4 045412 mi regions (FR). Each VH and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FR antibody (or antigen binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on parallel comparative analysis of two or more CDRs. As used herein, the term antigen binding protein also includes antibodies.

In addition, it is possible to replace one or more CDR residues or omit one or more CDRs. The scientific literature describes antibodies that can dispense with one or two CDRs to bind. Padlan et al. (1995, FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only approximately one-fifth to one-third of the CDR residues actually contact the antigen. Padlan has also found many antibodies in which one or two CDRs are not characterized by the presence of amino acids in contact with the antigen (see also Vajdos et al., 2002, J. Mol. Biol. 320:415-428).

Methods and techniques for identifying CDRs within VR amino acid sequences are well known in the art and can be used to identify CDRs within given VR amino acid sequences disclosed herein. Illustrative conventions that may be used to identify the boundaries of the CDR include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, 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); Al-Lazikam et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA, 56:9268-9272 (1989). Public databases are also available to identify CDR sequences within the antigen binding domain of an antigen binding protein or antibody.

Non-antigen contact CDR residues can be identified based on previous studies (eg, residues H60-H65 in CDRH2 are often not required) from Kabat CDR regions outside the Chothia CDR, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is typically replaced by an amino acid occupying the corresponding position in another human antibody sequence or consensus of such sequences. The positions to be replaced within the CDR and the amino acid to be replaced can also be selected empirically. Empirical substitutions may be conservative or non-conservative substitutions.

As used herein, the terms antigen binding portion of an antigen binding protein, antigen binding fragment of an antigen binding protein, and the like. include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms antigen binding protein fragment or antigen binding protein fragment refer to one or more antigen binding protein fragments that retain the ability to specifically bind to the GLP1 receptor. An antigen binding protein fragment may include a Fab fragment, an F(ab') ₂ fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term antigen binding fragment refers to a polypeptide or a fragment thereof of a multispecific antigen binding molecule. Antigen-binding fragments of an antigen-binding protein or antibody can be prepared, for example, from complete protein molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving DNA manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antigen-binding protein . 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. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example to arrange one or more variable and/or constant domains into a suitable configuration or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. .

Non-limiting examples of antigen binding fragments include the following: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as the CDR3 peptide) or a conformationally restricted peptide FR3-CDR3-FR4 . Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, mini-bodies, nanoantibodies (e.g. monovalent nanoantibodies, bivalent nanoantibodies, etc. .d.), small modular protein (SMIP) immunopharmaceuticals, and shark IgNAR variable domains are also referred to as antigen binding fragment as used herein.

An antigen binding fragment of an antigen binding protein or antibody according to the present invention will generally contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent or in the same reading frame as one or more framework sequences. In antigen binding fragments containing a _VH domain associated with a _VL domain, the VH and _VL domains may be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and may contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen binding fragment of the antigen binding protein may comprise a monomeric VH or VL domain.

In certain embodiments, the antigen binding fragment may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen binding fragment of an antibody of the present invention include the following: (i) V _H - C _h 1; (ii) VH - _CH 2; (iii) VH - _CH 3; (iv) Vh - Ch1 - Ch2; (v) Vh - Ch1 - Ch2 - Ch3; (vi) Vh - Ch2 - Ch3; (vii) Vh - Cl; (viii) Vl - Ch1; (ix) Vl - Ch2; (x) Vl - Ch3; (xi) Vl - Ch1 - Ch2; (xii) Vl - Ch1 - Ch2 - Ch3; (xiii) Vl - Ch2 - Ch3 and (xiv) Vl - Cl. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly linked to each other or can be linked through a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, the antigen binding fragment of the antigen binding protein of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric VH or _VL domains (eg via disulfide bond(s)).

As with complete protein molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen binding fragment of an antigen binding protein will typically contain at least two different variable domains, each variable domain being capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any format of multispecific antigen binding proteins, including the exemplary bispecific antigen binding protein formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antigen binding protein according to the present invention using standard techniques available in the art.

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 containing variable and constant regions derived from human germline immunoglobulin sequences. The human antigen-binding proteins of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the CDR and, in particular , CDR3. However, as used herein, the term human antigen binding protein is not intended to include antigen binding proteins in which CDR sequences derived from the germ line of another mammalian species (eg, mouse) have been grafted onto human FR sequences. The term includes antigen-binding proteins or antibodies produced recombinantly in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antigen binding proteins or antibodies isolated or generated in a human subject.

As used herein, the term recombinant refers to the fusion proteins or fragments thereof of the present invention generated, expressed, isolated or produced by technologies or methods known in the art as recombinant DNA technology, which includes, for example, DNA splicing and transgene expression. The term refers to fusion proteins expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice) or in a cellular (e.g., CHO cells) expression system or isolated from

- 6 045412 combinatorial library of recombinant human antibodies.

The term specifically binds or specifically binds to or the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may have an equilibrium dissociation constant of at least about 1x10 ^-8 M or less (eg, a lower K _D value indicates tighter 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.

As used herein, the terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like. include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. As used herein, the terms antigen binding fragment of an antigen binding protein or antibody or antibody fragment refer to one or more immunoglobulin protein fragments that retain the ability to bind to the GLP1 receptor.

The term K _D as used herein is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

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

As applied to polypeptides, the term substantially similar or substantially similar means that the two peptide sequences, when optimally aligned, for example using the GAP or BESTFIT programs using default gap penalties, have at least 90% sequence identity, even more preferably at least 95, 98 or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to adjust for the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994), Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include the following: 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) main side chains: lysine, arginine and histidine; 6) acid side chains: aspartate and glutamate and 7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992), Science, 256:1443 45, incorporated herein by reference. A moderately conservative substitution is any change characterized by a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity scores assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology

- 7 045412 similarity or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query sequence and the search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the present invention to a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410 and (1997), Nucleic Acids Res. 25:33893402, each of which is incorporated herein by reference.

By the phrase a therapeutically effective amount is meant an amount that provides the desired effect for which it is administered. The exact amount will depend on the purpose of treatment and will be determined by one skilled in the art using known techniques (see, for example, Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term subject refers to an animal, preferably a mammal, more preferably a human, in need of mitigation, prevention and/or treatment of a disease or disorder associated with GLP1. The term includes human subjects who have or are at risk of a GLP1-related disease or disorder. For example, the term includes subjects who have or are at risk of developing diabetes mellitus (eg, type 2 diabetes mellitus). In certain embodiments, the term includes subjects who have or are at risk of developing obesity, stroke, or myocardial infarction. The term also includes subjects who have high blood sugar and/or elevated levels of one or more diabetes biomarkers, such as HbA1c. The term also includes subjects with diabetes mellitus for whom standard treatment (eg, metformin) is contraindicated or intolerable, or whose disease is uncontrolled despite treatment (eg, metformin).

As used herein, the terms treat, administer a treatment, or treat refer to reducing or ameliorating the severity of at least one symptom or sign of a GLP1-related disease or disorder due to the administration of a therapeutic agent, such as a GLP1 receptor agonist of the present invention, to a subject in need thereof. The terms include inhibition of disease progression or worsening of symptoms. The terms also include a positive prognosis of the disease, i.e. the subject may be characterized by the absence of symptoms or signs, or may be characterized by a reduced intensity of symptoms or signs upon administration of a therapeutic agent, such as the GLP1 receptor agonist protein of the present invention. For example, a subject with diabetes mellitus may experience a decrease in blood glucose upon administration of a GLP1 receptor agonist according to the present invention. The therapeutic agent can be administered to a subject at a therapeutic dose.

The terms prevent, prophylaxis or prevention refer to inhibition of the occurrence of any symptoms or signs of a hyperglycemia-related disease or disorder upon administration of a GLP1 receptor agonist according to the present invention. The term includes inhibiting the expression of a symptom or sign of a GLP1 receptor-related disease or disorder in a subject at risk of developing such disease or disorder.

GLP1(7-37) (SEQ ID NO: 4) has a very short half-life in the bloodstream (1-2 min) due to its rapid inactivation by the enzyme dipeptidyl peptidase 4 (DPP4). Previous work has shown that various amino acid substitutions at position 8 of GLP1 (7-37) make it more resistant to DPP4, thereby providing a longer half-life. However, there is a residual sensitivity of these molecules to DPP4 cleavage (Deacon et al., 1998, Diabetologia, 41:271-278). Thus, there is a need to develop new molecules that have increased resistance to degradation by DPP4.

The present inventors hypothesized that to confer better resistance to DPP4, the first step was to introduce mutations that lengthen or shorten the amino terminus of GLP1, either by adding an amino acid (i.e., Ala, Gln) to the N terminus, or by deleting His or Ala in the peptide sequences to provide better resistance to DPP4 cleavage. The present inventors have shown herein that these new GLP1 variants are indeed very resistant to degradation by DPP4. The second step was to compensate for any weakened or reduced GLP1 activity by fusing GLP1 with an anti-GLP1 receptor antibody, which binds the weakened GLP1 to the GLP1 receptor and thereby increases its activity. In addition, the present inventors found that these fusion proteins, likely because they contain an Fc domain, had an increased serum half-life and resulted in

- 8 045412 increased decrease in blood sugar, which was maintained for more than 10 days. The novel GLP1 variants and fusion proteins disclosed herein exhibit significantly improved resistance to degradation by DPP4 in vitro and in vivo and demonstrate significantly improved efficacy in glycemic control as demonstrated herein. The terms significantly improved or enhanced or increased as used herein in the context of resistance to degradation by DPP4 refer to increased resistance to degradation when incubated with DPP4 for more than 4 hours, more than 8 hours, more than 16 hours, more 24 hours, more than 36 hours, or more than 70 hours, as measured herein. The terms significantly improved or enhanced or increased as used herein in the context of blood sugar reduction refer to a sustained reduction in blood sugar 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 in a subject when administered a GLP1 receptor agonist according to the present invention.

The GLP1 receptor agonists of the present invention bind to the GLP1 receptor with high affinity and result in activation of the GLP1 receptor. In some embodiments, the proteins are useful for treating a subject suffering from diabetes mellitus. The proteins, when administered to a subject in need thereof, may lower the subject's blood sugar. They can be used alone or as adjunctive therapy with other therapeutic moieties or methods known in the art for the treatment of hyperglycemia.

Certain GLP1 receptor agonist proteins of the present invention are capable of binding to and stimulating the activity of the GLP1 receptor, as determined by in vitro or in vivo assays. The ability of the proteins of the present invention to bind and enhance the activity of the GLP1 receptor can be measured using any standard method known to those skilled in the art, including binding assays or activity assays as described herein.

Antigen binding proteins specific for the GLP1 receptor may not contain any additional tags or fragments, or they may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the tag or moiety is biotin. In a binding assay, the location of the tag (if present) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if the surface is coated with avidin, the peptide containing N-terminal biotin will be oriented such that the C-terminal region of the peptide is distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or an MRI detectable label. In certain embodiments, such labeled antigen binding proteins can be used in diagnostic assays, including imaging assays.

Bioequivalents.

The GLP1 receptor agonists of the present invention encompass proteins with amino acid sequences that differ from the amino acid sequences of the described GLP1 receptor agonists, but which retain the ability to bind to the GLP1 receptor. Such variant GLP1 receptor agonists contain one or more insertions, deletions or substitutions of amino acids from the parent sequence, but exhibit biological activity that is substantially equivalent to that of the described GLP1 receptor agonists. Likewise, GLP1 receptor agonist-encoding DNA sequences of the present invention include sequences that contain one or more insertions, deletions, or nucleotide substitutions relative to the disclosed sequence, but that encode a GLP1 receptor agonist that is substantially bioequivalent to the GLP1 receptor agonist of the present invention.

Two proteins are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show significant differences when administered at the same molar dose under similar experimental conditions, either as a single dose or as several doses. Some proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and may still be considered bioequivalent because such differences in rate of absorption are intentional and reflected on the label are not significant for achieving effective concentrations of the drug in the body, for example, with chronic use, and are considered medically insignificant for the particular drug being studied.

In one embodiment, two GLP1 receptor agonist proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

In one embodiment, two GLP1 receptor agonist proteins are bioequivalent if the patient can switch one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including

- 9 045412 clinically significant change in immunogenicity or reduced effectiveness compared to continuation of therapy without such a switch.

In one embodiment, two GLP1 receptor agonist proteins are bioequivalent if they both act through a common mechanism or mechanisms of action for the condition or conditions of use, to the extent such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo assay in humans or other mammals, in which the concentration of a protein or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro assay that has been correlated with and is reasonably predictive of in vivo bioavailability data in humans; (c) an in vivo assay in humans or other mammals, in which the relevant acute pharmacological effect of the protein (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding protein.

Bioequivalent variants of the GLP1 receptor agonist proteins of the present invention can be constructed, for example, by making various residue or sequence substitutions or removing terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or irregular intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent proteins may include variants containing amino acid substitutions that modify the characteristics of the proteins, such as mutations that eliminate or remove glycosylation.

Biological characteristics of GLP1 receptor agonists.

In general, the GLP1 receptor agonists of the present invention function by binding to the GLP1 receptor and promote activation of the GLP1 receptor upon binding. In certain embodiments, the proteins of the present invention bind with high affinity to the GLP1 receptor. For example, the present invention includes GLP1 receptor agonists that result in activation of the GLP1 receptor (eg, at 25 or 37°C), as measured by a luciferase reporter gene assay, for example, using the assay format as defined in Example 2 in this document. In certain embodiments, GLP1 receptor agonists activate the GLP1 receptor with an EC ₅₀ of less than 10 nM, less than 500 pM, or less than 250 pM, as measured by a luciferase reporter gene assay, for example, using the assay format as defined in Example 2 in herein, or by substantially similar analysis.

The present invention also includes GLP1 receptor agonists that reduce blood sugar levels in vivo after administration to a subject in need thereof, for example, as shown in Example 3, or using a substantially similar assay. GLP1 receptor agonists act to enhance glycemic control when administered, resulting in a decrease in blood glucose. In certain embodiments, even a single therapeutically effective dose of the GLP1 receptor agonists of the present invention results in a significant reduction in blood sugar that is maintained for more than 10 days.

The present invention also includes GLP1 receptor agonists that exhibit increased resistance to degradation by serum proteases/peptidases, as measured by mass spectroscopy, for example, as shown in Example 4 herein, or in a substantially similar manner. In certain embodiments, GLP1 receptor agonists are 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 in this document.

The GLP1 receptor agonists of the present invention may have one or more of the above biological characteristics or any combinations thereof. Other biological characteristics of the proteins of the present invention will be apparent to one skilled in the art from a review of the present disclosure, including the worked examples provided herein.

Therapeutic administration and formulations.

The present invention relates to therapeutic compositions containing GLP1 receptor agonists according to the present invention. The therapeutic compositions of the present invention are formulated with suitable carriers, excipients and other agents which are formulated to provide improved transport, delivery, tolerability and the like. Many acceptable formulations can be found in the formulary 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, lipid containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates,

- 10 045412 anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. See also Powell et al., Compendium of excipients for parenteral formulations PDA (1998),

J. Pharm. Sci. Technol. 52:238-311.

The dose of GLP1 receptor agonist administered to a patient may vary depending on the age and size of the subject being administered, the target disease, conditions, route of administration, and the like. When the antigen binding protein of the present invention is used to treat a disease or disorder in an adult patient or for the prevention of such a disease, it is advantageous to administer the antigen binding protein of the present invention, typically in a single dose of about 0.001 to about 100 mg/kg body weight, more preferably about 0.001 to about 60, about 0.01 to about 10, or about 0.01 to about 1 mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, the antigen binding protein or antigen binding fragment thereof of the present invention may be administered at an initial dose of at least about 0.001 mg to about 100 mg, about 0.001 to about 50 mg, about 0.005 to about 50 mg, about 0.01 - approximately 40 mg, up to approximately 30 mg or up to approximately 10 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the GLP1 receptor agonist in an amount that is approximately the same as or less than the amount of the initial dose, with the interval between subsequent doses being at least 1-3 days; at least 1 week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 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.

Various delivery systems are known that can be used to administer the pharmaceutical composition of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) , J Biol Chem 262:4429–4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucous membranes (for example, through the oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biological active means. Administration may be systemic or local. The pharmaceutical composition can also be delivered in a vesicle, in particular a liposome (see, for example, Langer (1990), Science, 249:1527-1533).

The use of nanoparticles to deliver GLP1 receptor agonists according to the present invention is also provided herein. Protein-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Nanoparticles can be formulated and conjugated to antigen-binding proteins contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery are also described, for example, in US Pat. No. 8,257,740 or 8,246,995, each of which is incorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. According to one embodiment, a pump may be used. According to another embodiment, polymeric materials can be used. In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the above-described antigen binding protein or a salt thereof in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, physiological saline solution, isotonic solution containing glucose and other auxiliaries, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (eg ethanol), polyhydric alcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant [eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl

- 11 045412 alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, with respect to subcutaneous delivery, the pen delivery device easily finds use in delivering the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of 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 that contains the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen delivery device does not have a replaceable cartridge. In contrast, a disposable pen delivery device comes pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and auto-injector delivery devices are used in the subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, without limitation, the following: AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, pen HUMALOG™, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Inn.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark ), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices useful in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the following: SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly ), SURECLICK™ auto-injector (Amgen, Thousand Oaks, Calif), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMTRA™ Pen (Abbott Labs, Abbott Park, IL), to name a few of them.

The pharmaceutical compositions for oral or parenteral use described above are advantageously prepared in unit dose dosage forms that correspond to the dose of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of GLP1 receptor agonist contained is typically from about 0.001 to about 100 mg per unit dose dosage form; especially in injection form, it is preferred that the GLP1 receptor agonist is contained in an amount of from about 0.001 to about 100 mg and from about 0.01 to about 100 mg for other dosage forms.

Therapeutic applications of GLP1 receptor agonists.

The GLP1 receptor agonists of the present invention are useful for treating and/or preventing a disease or disorder or condition associated with hyperglycemia, such as diabetes mellitus, and/or for reducing the intensity of at least one symptom associated with such a disease, disorder or condition. condition. In one embodiment, the GLP1 receptor agonist of the present invention can be administered at a therapeutic dose to a patient with diabetes mellitus (eg, type 2 diabetes mellitus).

In certain embodiments, the GLP1 receptor agonists of the present invention are useful for treating subjects suffering from a disease or disorder selected from the group consisting of diabetes mellitus, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes mellitus, type 1 diabetes mellitus, prediabetes , cardiovascular disease, atherosclerosis, congestive heart failure, coronary artery disease, arteriosclerosis, peripheral artery disease, stroke, respiratory dysfunction, kidney disease, fatty liver disease, non-alcoholic steatohepatitis (NASH) and metabolic syndrome.

In certain embodiments, the GLP1 receptor agonists of the present invention are useful for treating overweight or obese subjects and/or preventing or treating one or more obesity-related disorders, such as cardiovascular disease, stroke, and diabetes mellitus.

In certain embodiments, the GLP1 receptor agonists of the present invention are useful for treating subjects suffering from diabetes mellitus and/or preventing one or more complications of diabetes mellitus, such as cardiovascular disease, stroke, kidney disease, retinopathy, blindness, and nerve damage.

- 12 045412

Also provided herein is the use of one or more GLP1 receptor agonist proteins of the present invention prophylactically for subjects at risk of developing diabetes mellitus (eg, type 2 diabetes mellitus). 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 lack of exercise.

In a further embodiment, the proteins of the present invention are used to prepare a pharmaceutical composition or medicament for treating patients suffering from a disease or disorder such as diabetes mellitus and obesity. In another embodiment of the present invention, the GLP1 receptor agonists of the present invention are used as adjunctive therapy to any other agent or therapy known to those skilled in the art useful for treating or ameliorating a disease or disorder associated with hyperglycemia, such as diabetes mellitus (for example, type 2 diabetes mellitus).

Types of combination therapy.

Combination therapies may include a GLP1 receptor agonist of the present invention and any additional therapeutic agent that can be effectively combined with a GLP1 receptor agonist of the present invention or a biologically active fragment thereof of the present invention. The GLP1 receptor agonists of the present invention can be synergistically combined with one or more drugs or therapies used to treat any disease or disorder associated with hyperglycemia (eg, diabetes mellitus). In some embodiments, the GLP1 receptor agonists of the present invention can be combined with a second therapeutic agent to lower blood sugar in a subject or to reduce the intensity of one or more symptoms of diabetes mellitus.

The GLP1 receptor agonists of the present invention can be used in combination with the following: insulin (insulin or insulin analog), insulin sensitizers such as biguanides (eg, metformin) and thiazolidinediones (eg, rosiglitazone), insulin secretagogues such as sulfonylureas (eg, chlorpropamide) and glinides (eg, nateglinide), alpha-glucosidase inhibitors (eg, acarbose), dipeptidyl peptidase 4 (DPP4) inhibitors (eg, sitagliptin), pramlinitide, bromocriptine, sodium-glucose cotransporter 2 (SGL) inhibitors (eg, canagliflozin) , antihypertensive drug (eg, angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, diuretic, calcium channel blocker, alpha-adrenergic blocker, endothelin-1 receptor blocker, organic nitrate, and protein kinase C inhibitor), statin, aspirin, other GLP1 receptor agonist , dietary supplement, or any other therapy (such as exercise) for the treatment or management of diabetes mellitus. In certain embodiments, the GLP1 receptor agonists of the present invention can be administered in combination with a second therapeutic agent or therapy selected from the group consisting of the following: insulin, insulin analog, metformin, rosiglitazone, pioglitazone, chlorpropamide, glibenclamide, glimepiride, glipizide, tolazamide, tolbutamide, nateglinide, repaglinide, acarbose, miglitol, exenatide, liraglutide, albiglutide, dulaglutide, sitagliptin, saxagliptin, linagliptin, alogliptin, pramlinitide, immediate-release bromocriptine, canagliflozin, dapagliflozin, empagliflozin, diet modifications and exercise.

As used herein, in combination with, means that additional therapeutically active component(s) may be administered before, simultaneously with, or after administration of the GLP1 receptor agonist of the present invention. The term in combination with also includes sequential or simultaneous administration of a GLP1 receptor agonist and a second therapeutic agent.

Additional therapeutically active component(s) may be administered to the subject prior to administration of the GLP1 receptor agonist of the present invention. For example, the first component may be considered administered before the second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before or less than 1 minute before the introduction of the second component. In other embodiments, additional therapeutically active component(s) may be administered to a subject following administration of a GLP1 receptor agonist of the present invention. For example, the first component may be considered administered after the second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after the introduction of the second component. In other embodiments, additional therapeutically active component(s) may be administered to a subject concurrently with administration of a GLP1 receptor agonist of the present invention. Simultaneous administration for the purposes of this

- 13045412 of the invention includes, for example, administering a GLP1 receptor agonist and an additional therapeutically active component to a subject in a single dosage form or in separate dosage forms administered to the subject at intervals of about 30 minutes or less. When administered in separate dosage forms, each dosage form may be administered by the same route (eg, both the GLP1 receptor agonist and the additional therapeutically active component may be administered intravenously, etc.); alternatively, each dosage form may be administered by a different route (eg, the GLP1 receptor agonist may be administered intravenously and the additional therapeutically active component may be administered orally). In any case, all of the following: administration of components in the same dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes, is considered simultaneous administration for purposes of this disclosure. For purposes of this disclosure, administration of a GLP1 receptor agonist before, simultaneously with, or after (as those terms are defined hereinbefore) administration of an additional therapeutically active component is considered to be administration of a GLP1 receptor agonist in combination with an additional therapeutically active component.

The present invention includes pharmaceutical compositions in which the GLP1 receptor agonist of the present invention is formulated in conjunction with one or more additional therapeutically active components as described elsewhere herein.

Administration schemes.

In 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 of the additional therapeutically active agents mentioned herein) can be administered to a subject in need thereof. In certain embodiments of the present invention, multiple doses of a GLP1 receptor agonist (or a pharmaceutical composition comprising a combination of a GLP1 receptor agonist and any of the additional therapeutically active agents mentioned herein) can be administered to a subject over a period of time. The methods of this aspect of the present invention involve sequentially administering multiple doses of a GLP1 receptor agonist of the present invention to a subject.

As used herein, the term sequential administration means that each dose of the GLP1 receptor agonist is administered to the patient at different points in time, for example on different days separated by a predetermined interval (eg, hours, days, weeks or months). The present invention provides methods that include sequentially administering to a patient one initial dose of a GLP1 receptor agonist, followed by one or more secondary doses of a GLP1 receptor agonist, and optionally followed by one or more tertiary doses of a GLP1 receptor agonist.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of the GLP1 receptor agonist according to the present invention. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the initial dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary and tertiary doses may contain the same amount of GLP1 receptor agonist, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of GLP1 receptor agonist contained in the initial, secondary, and/or tertiary doses varies relative to each other (eg, it is adjusted higher or lower depending on the situation) during the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered less frequently (eg, maintenance doses).

According to one illustrative embodiment of the present invention, each secondary and/or tertiary dose is administered after 1-48 hours (for example, after 1.11/ ₂ , 2, 21/2, 3, 31/2 _, 4, 41/2, 5, 51/2, 6, 61/ ₂ , 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/ 2, 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22, 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2 or more) after the immediately preceding dose. As used herein, the phrase immediately preceding dose in a sequence of administrations means the dose of a GLP1 receptor agonist that is administered to a patient before the administration of the immediately next dose in a sequence without any intervening doses. In certain embodiments, each secondary and/or tertiary dose is administered every other day, every 2, 3, 4, 5, 6, or 7 days after the immediately preceding dose. In certain embodiments, each secondary and/or tertiary dose is administered every 0.5, 1, 2, 3, or 4 weeks after the immediately preceding dose.

Methods according to this aspect of the present invention may involve administering to the patient any number of secondary and/or tertiary doses of a GLP1 receptor agonist. For example, in certain embodiments, only one secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

In certain embodiments of the present invention, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of treatment. The frequency of administration may also be adjusted during the course of treatment by the physician depending on the needs of the individual patient after clinical examination.

Dosage.

The amount of GLP1 receptor agonist administered to a subject in accordance with the methods of the present invention will generally be a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means an amount of a GLP1 receptor agonist that results in one or more of the following: (a) reducing high blood sugar to normal levels (eg, preprandial blood glucose of 80-130 mg/day). d) and/or (b) a detectable improvement in one or more symptoms or signs of diabetes mellitus.

For a GLP1 receptor agonist, a therapeutically effective amount may be from about 0.001 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, approximately 0.009 mg, approximately approximately approximately approximately approximately approximately approximately approximately approximately approximately approximately

0.01 0.05 0.09 0.4 0.8 3 7 15 35 55 75 mg, mg, mg, mg, mg, mg, mg, mg, mg, mg, mg, approximately 0.02 approximately 0, 06 approximately approximately approximately approximately approximately approximately approximately approximately approximately

0.1

0.5 mg, mg, mg, mg, approximately approximately approximately approximately

0.03 0.07 0.2 0.6 mg, approximately mg, approximately

0.9 mg, approximately 1 mg, mg, mg, mg, mg, mg, approximately approximately approximately approximately approximately approximately

9 25 45 65 85 mg, mg, mg, mg, mg, mg, mg, mg, mg, approximately 95 mg or approximately approximately approximately approximately approximately approximately approximately approximately approximately approximately

0.04

0.08

0.3 0.7 2 6 mg, mg, mg, mg, mg, mg, mg, mg, mg, mg, mg,

100 mg GLP1 receptor agonist. In certain embodiments, 0.005-50 mg, 0.005-30 mg, 0.005-10 mg, 0.1-10 mg, or 0.1-5 mg of a GLP1 receptor agonist is administered to a subject in need thereof.

The amount of GLP1 receptor agonist contained in individual doses can be expressed as mg of antibody per kg of subject body weight (ie, mg/kg). For example, a GLP1 receptor agonist can be administered to a subject at a dose of from about 0.0001 to about 100 mg/kg of the subject's body weight.

Selected embodiments.

According to 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 the following: (i) adding an amino acid to the N-terminus and (ii) deleting the amino acid from the peptide sequence; wherein the GLP1 variant is characterized by enhanced resistance to proteolytic degradation and/or enhanced ability to lower blood glucose.

According to Embodiment 2, the present invention relates to the GLP1 variant of Embodiment 1, wherein the amino acid modification comprises adding an amino acid selected from the group consisting of alanine (Ala) and glutamine (Gln) to the N-terminus.

According to 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.

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

According to Embodiment 5, the present invention provides a GLP1 variant according to any of Embodiments 1-4 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8.

According to Embodiment 6, the present invention provides a GLP1 variant according to Embodiment 5 comprising the amino acid sequence of SEQ ID NO: 6.

In embodiment 7, the present invention provides a fusion protein comprising a GLP1 variant according to any one of embodiments 1-6 fused to a stabilizing

- 15 045412 domain, wherein the stabilizing domain is an antigen binding protein or an antigen binding fragment thereof that specifically binds to the GLP1 receptor and which contains a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

According to 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 an HCVR antigen binding protein or an antigen binding fragment thereof.

According to 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 an LCVR antigen binding protein or an antigen binding fragment thereof.

According to embodiment 10, the present invention provides a fusion protein comprising a GLP1 variant according to any of embodiments 1-6 fused to a stabilizing domain, wherein the stabilizing domain is an immunoglobulin (Ig) or a fragment thereof.

According to 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.

According to Embodiment 12, the present invention provides a fusion protein comprising a GLP1 variant fused to a stabilizing domain, wherein the stabilizing domain is an antigen binding protein or an antigen binding fragment thereof, wherein the antigen binding protein or fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region circuits (LCVR).

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 an HCVR antigen binding protein or fragment thereof.

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 an LCVR antigen binding protein or fragment thereof.

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

In embodiment 16, the present invention provides a fusion protein according to any of embodiments 12-15 comprising a GLP1 variant according to any of the above embodiments.

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

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

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

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 an HCVR antigen binding protein or fragment thereof.

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 an LCVR antigen binding protein or fragment thereof.

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

According to Embodiment 23, the present invention provides a GLP1 receptor agonist according to any of Embodiments 19-22 comprising the GLP1 variant according to Embodiment 1.

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

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.

According to Embodiment 26, the present invention relates to a receptor agonist

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GLP1 containing a GLP1 variant, wherein the GLP1 variant is fused to a stabilizing domain, wherein the stabilizing domain is an immunoglobulin (Ig) or a fragment thereof.

According to 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.

According to embodiment 28, the present invention provides a pharmaceutical composition comprising a protein according to any of embodiments 1-27 and a pharmaceutically acceptable carrier or diluent.

According to embodiment 29, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a GLP1 variant as presented in any of embodiments 1-6.

In embodiment 30, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a fusion protein as provided in any of embodiments 10, 11.

In embodiment 31, the present invention provides an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a GLP1 receptor agonist as provided in any of embodiments 26, 27.

According to embodiment 32, the present invention provides a vector containing a polynucleotide sequence according to any of embodiments 29-31.

According to Embodiment 33, the present invention provides a cell expressing the vector according to Embodiment 32.

In embodiment 34, the present invention provides a method for lowering blood sugar comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a protein according to any of embodiments 1-27 to a subject in need thereof.

According to embodiment 35, the present invention relates to the method of embodiment 34, wherein the subject is characterized by having a disease or disorder selected from the group consisting of the following: diabetes mellitus, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes mellitus, type 1 diabetes mellitus type, prediabetes, cardiovascular disease, atherosclerosis, congestive heart failure, coronary artery disease, arteriosclerosis, peripheral arterial disease, stroke, respiratory dysfunction, kidney disease, fatty liver disease, non-alcoholic steatohepatitis (NASH) and metabolic syndrome.

In embodiment 36, the present invention provides a method for preventing, treating, or reducing the intensity of at least one symptom, sign, or complication of type 2 diabetes mellitus, the method comprising administering a pharmaceutical composition containing a therapeutically effective amount of a protein according to any of embodiments 1-27. to the subject in need.

In embodiment 37, the present invention relates to the method of embodiment 36, wherein at least one symptom, sign, or complication is selected from the group consisting of high blood sugar, polydipsia, urinary frequency, urinary ketones, fatigue, weight fluctuations, blurred vision, slow-healing ulcers, frequent infections, swollen or painful gums, obesity, cardiovascular disease, stroke, kidney disease, eye disease, nerve damage and high blood pressure.

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

In embodiment 39, the present invention relates to the method of embodiment 38, wherein the second therapeutic agent or therapy is selected from the group consisting of the following: insulin or insulin analog, biguanide (eg, metformin), thiazolidinedione, sulfonylurea (eg, chlorpropamide), glinide (eg, nateglinide), alpha-glucosidase inhibitor, DPP4 inhibitor (eg, sitagliptin), pramlintide, bromocriptine, SGLT2 inhibitor (eg, canagliflozin), antihypertensive drug, statin, aspirin, diet modification, exercise, and dietary supplement.

According to embodiment 40, the present invention relates to a method according to any of embodiments 34-39, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally or intramuscularly.

Examples

The following examples are presented so that those skilled in the art will have full disclosure and description of how to make and apply the methods and compositions of the present invention, and are not intended to limit the scope of what the present inventors believe to be their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is approximately 25° C., and pressure is at or near atmospheric pressure.

Example 1 Exemplary GLP1-containing fusion proteins.

GLP1(7-37) has a very short half-life in the circulation (1-2 min) due to its rapid inactivation by the enzyme dipeptidyl peptidase 4 (DPP4). Previous work has shown that various amino acid substitutions at position 8 of GLP1 (7-37) make it more resistant to DPP4, thereby providing a longer half-life (Deacon et al., 1998, Diabetologia, 41:271-278). However, there is residual sensitivity of these molecules to DPP4 cleavage. Thus, there is a need to develop new molecules that are even more resistant to DPP4.

To confer better resistance to DPP4, the first part of the technology is to introduce mutations that lengthen or shorten the amino terminus of GLP1 either by adding an amino acid (i.e. Ala, Gln) to the N-terminus or by deleting His7 or Ala8 in the peptide sequence to provide better resistance to cleavage by DPP4. These modifications also attenuate the activity of GLP1, and the second part of the technology is to compensate for the reduced activity by attaching a weakened agonist to the receptor using an anti-GLP1R antibody by fusing the peptide to the N-terminus of the light chain sequence of the anti-GLP1R antibody. As a proof of concept, modified GLP1 ligand sequences (7-37) were fused to the N-terminus of the GLP1R antibody light chain as described below.

Mature GLP1 is a 31 amino acid peptide hormone containing amino acids 7-37 of full-length GLP1 (SEQ ID NO: 3), and is characterized by the amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 4).

Mature GLP1 has been modified by amino acid deletions or additions at the amino terminus to create GLP1 variants. Exemplary GLP1 variants are presented below:

Des-Ala-GLP1 containing amino acid

HEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 5);

Q-GLP1 containing amino acid

QHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO; 6);

A-GLP1 containing amino acid

AHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID NO: 7);

desH-GLP 1 containing

AEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 8).

sequence sequence sequence sequence

To compensate for the possible reduction in activity of the above GLP1 variants, they were coupled to the GLP1 receptor (GLP1R) using an anti-GLP1R antibody or an Fc portion of the antibody. Exemplary fusion proteins comprising mature GLP1 or a GLP1 variant are prepared by fusing mature GLP1 or a GLP1 variant to the N-terminus of the light chain of an anti-GLP1 receptor antibody comprising a heavy chain variable region according to SEQ ID NO: 2 and a light chain variable region (LCVR) according to SEQ ID NO: 1 (hereinafter referred to as mAb1; US Patent Application Publication No. 20060275288 [Abbott Laboratories]) and are listed below:

Des-Ala-GLP1-mAb1: Des-Ala-GLP1 (SEQ ID NO: 5) fused to the N-terminus of mAb1 light chain;

Q-GLP1-mAb1: Q-GLP1 (SEQ ID NO: 6) fused to the N-terminus of mAb1 light chain;

A-GLP1-mAb1: A-GLP1 (SEQ ID NO: 7) fused to the N-terminus of mAb1 light chain.

In addition, fusion proteins containing mature GLP1 or GLP1 variant and immunoglobulin Fc fragment have been 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).

Control construct.

Comparative product: a GLP1 analogue with the amino acid sequence characteristics of LY2189265 fused to the Fc domain of hIgG4 (dulaglutide; Eli Lilly), as disclosed in Glaesner et al., 2010 (Diabetes Metab. Res. Rev. 26:287-296), was used in as a reference product (SEQ ID NO: 14) in the following examples.

Example 2: Luciferase assay.

GLP1 fusion proteins were tested for their ability to stimulate cAMP production in the 293/FSC11/Cre-Luc reporter cell line, which stably expresses the human GLP1 receptor along with a luciferase coding sequence under the control of the Cre promoter, which

- 18 045412 responds to cAMP.

For the luciferase bioassay, stable 293/FSC11/Cre-Luc GLP1R cells were seeded into 96-well assay plates at a density of 30,000 cells/well in OPTIMEM supplemented with 0.1% FBS and then incubated at 37°C in 5% CO ₂ during the night. The next day, human GLP1 (Phoenix, cat. no. 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 compounds tested 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. Material in the culture media was quantified using ELISA. Test samples were added to cells at concentrations ranging from 0.02 pM to 100 nM.

After 5.5 hours of incubation or after incubation overnight at 37°C in 5% CO ₂ , OneGlo reagent (Promega, cat. no. E6051) was added to the samples and luciferase activity was then measured using a Victor X plate reader (Perkin Elmer ). Results were analyzed using nonlinear regression (three parameters) using Prism 6 software (GraphPad) to obtain EC50 values.

As shown in table. 1, Q- and A-modified mAb1 antibody fusions showed EC ₅₀ values of 204 and 312 pM, respectively, for GLP1R activation.

Table 1

EU ₅₀ for GLP1 fusion proteins

GLP1 fusion proteins EC50 des-Ala-GLP 1 -mAb 1 10 nM Q-GLPl-mAbl 0.204 nM A-GLPl-mAbl 0.312 nM GLPl-hFc 0.147 nM A-GLPl-hFc 120 nM Q-GLPl-hFc 135 nM Des-Ala-GLPl-hFc Not detected Des-H-GLPl-hFc 2560 nM Comparison product 0.059 nM

The EC ₅₀ value for des-Ala-GLP1-mAb1 was 10 nM. The EC ₅₀ value for Q- and A-GLP1-hFc is only 135 and 120 nM, while the EC ₅₀ value for Des A-GLP1-hFc is undetectable.

Example 3 Effect of Q-GLP1 fused to an anti-GLP1R antibody on blood glucose and glucose tolerance in GLP1R humanized mice.

The effect 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 was determined in genetically engineered mice expressing human GLP1R protein (GLP1R humanized mice). Thirty-one GLP1R humanized mice were divided into four groups of seven to eight animals. Each group received one subcutaneous injection of isotype control, Q-GLP1-hFc, mAb1, or Q-GLP1-mAb1 at a concentration of 194 nmol/kg. Mice were bled postprandially on days 0, 1, 4, 7, 11, 14, 16, 18, and 22 to measure blood glucose. The mean ± SEM of blood glucose at each time point was calculated for each group and is shown in Table. 2.

table 2

Blood glucose level

Time (days) Isotypic control Q-GLPl-hFc mAbl Q-GLPlmAbl Blood glucose level (mg/dL) 0 188±6 188 ±5 188 ±6 186 ±8 1 185 ±3 184 ±8 193 ±8 133 ±4 4 182 ±9 193 ±9 180 ±7 128 ±5 7 187±6 199 ±9 178 ±7 132 ±4 eleven 180±4 192 ±6 183 ±8 152 ±4 14 174 ±6 185 ±8 184 ±7 145 ±4 16 179 ±6 193 ±7 183 ±7 156 ±4 18 172 ±7 184 ±8 161 ±7 154 ±6 22 174 ±6 188 ±7 181±9 171±7

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Oral glucose tolerance tests (oGTT) were performed on days 3 and 9 after an overnight fast with blood glucose measurements taken at 0, 15, 30, 60 and 120 min after the glucose bolus. The mean ±SEM blood glucose at each time point and the area under the glucose curve (AUC) were calculated for each group and are shown in Table 1. 3 and 4.

Table 3

Blood glucose and glucose AUC on day 3

Time (min) Isotypic control Q-GLPl-hFc mAbl Q-GLPlmAbl Blood glucose level (mg/dL) 0 143 ±6 147 ±5 143 ±8 111 ± 5 15 242 ± 13 252 ± 19 233 ± 16 193 ± 11 thirty 233 ±9 235 ±8 217 ± 8 160 ±6 60 187 ±8 195 ±7 200±12 129 ±4 120 151 ± 7 160 ±7 159 ± 10 115 ± 4 Blood glucose AUC (mg/dL* 120 min) 22884 ± 594 23756 ±681 23229 ± 959 16556 ±386

Table 4

Blood glucose levels and glucose AUC on day 9_______

Time (min) Isotypic control Q-GLPlhFc mAbl Q-GLPl-mAbl Blood glucose level (mg/dL) 0 143 ±4 151 ±3 147 ±5 116 ± 4 15 281 ±17 269 ± 15 253 ± 15 203 ± 13 thirty 207 ±5 228 ± 15 223 ± 14 167 ± 14 60 200 ±7 187 ±5 207 ±8 139 ± 10 120 155 ±5 173 ± 11 157 ± 6 131 ±7 Blood glucose AUC (mg/dL* 120 min) 23589 ±610 23869± 795 23959 ±696 17852 ±920

A single administration of Q-GLPl-mAbl in normoglycemic mice humanized for GLP1R led to a significant decrease in glucose levels within 14 days, while Q-GLPl-hFc or shAA had no effect on blood glucose levels (Table 2). Q-GLPl-mAbl reduced fasting glucose and improved glucose tolerance on days 3 and 9 in mice, whereas Q-GLPl-hFc and mAbl did not. These data suggest that Q-GLP1 or mAbl antibody alone does not alter glycemic control, but the fusion molecule composed of them may exhibit glucose-lowering effects that last for 2 weeks after a single injection in normoglycemic animals.

Example 4: Stability of GLP1 Variants.

The stability of various GLP1 variants and fusion proteins was tested by incubating them with serum proteases and analyzing the digested peptides using mass spectrometry.

In the first experiment, 0.5 μg of each GLP1 fusion protein was added to 50 μl of naïve mouse serum, respectively. The mixtures were then incubated at 37°C for 6 and 24 h, respectively. 1 μl of the serum mixture at 0 min, 6 and 24 h was applied to a Tris-glycine gel.

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

For each experiment, gel slices corresponding to the molecular weight of each construct were excised and subjected to in-gel trypsin digestion. Excised gel pieces were decolorized in 50:50 acetonitrile: _NH4HCO3 (50 mM), reduced with 65 mM dithiothreitol (Sigma) at 37°C for 30 _min , followed by alkylation with 135 mM iodoacetamide (Sigma) at room temperature. temperature in the dark for 30 minutes. Proteins were then digested overnight with sequence-grade modified porcine trypsin (Promega) at 37°C. Peptides were extracted twice with extraction buffer (50% ACN, 5% formic acid in _H2O ). The extracted peptides from each band were dried completely in a SpeedVac and reduced with 0.1% tetrafluoroacetic acid (TFA) before nano-LC-MS/MS analysis.

Reduced peptide mixtures were separated using online capillary reverse phase (RP) liquid chromatography (Easy-nLC1000, Thermo Fisher Scientific) and analyzed using

-20045412 electrospray tandem mass spectrometry (Orbitrap Elite, Thermo Fisher Scientific). Peptide mixtures were injected into a 75 μm i.d. PepMar RSLC column (C18, 25 cm, 100 A, 2 μm, Thermo Fisher Scientific) at a flow rate of 250 nL/min and then eluted with 2% to 35% ACN in 0. 1% formic acid in a 60-minute gradient. The mass spectrometer was operated in a data-dependent mode to automatically switch between MS and MS/MS data acquisition. Survey full-scan MS spectra (m/z 350 to 2000) were acquired in the Orbitrap at 120,000 resolution. The most intense ions (up to ten) were sequentially isolated for hybrid ion trap fragmentation using collision-induced dissociation (CID) with normalized collision energy of 35% with a target value of 5000. Target ions already selected for MS/MS were dynamically excluded for 30 s.

MS and MS/MS peak lists were extracted and checked against an in-house protein database using ProteomeDiscoverer 1.4 (Thermo Fisher Scientific). All searches assumed trypsin treatment and considered cysteine carboxymethylation as a fixed modification and methionine oxidation as a variable modification. A peptide mass tolerance of 10 ppm, an MS/MS mass tolerance of 0.8 Da, and a tolerance of up to 1 undercut were used. Ion extraction areas were calculated from the resulting ion chromatograms (XIC) using Thermo Xcaliber software (Thermo Fisher Scientific).

Results.

To characterize the susceptibility of each construct to serum enzyme cleavage, the intact peptide (N-terminal peptide), digested peptide (N-terminal peptide after cleavage), and one internal reference peptide (stable peptide not susceptible to any modification in the construct) for each construct were monitored with using nano-LC-MS/MS. The reduced ratio of intact peptide to reference peptide and the concomitant increased ratio of degraded peptide to reference peptide indicates enzyme-mediated degradation of the construct over time. The percentage of cleavage was calculated using the following formula

100 x area of cleaved peptide/(area of cleaved peptide + area of non-cleaved peptide).

GLP1-hFc and A-GLP1-hFc were completely cleaved by 6 h, while desH-GLP1-hFc showed significant cleavage (2%) by 6 h. Q-GLP1-hFc and the reference product did not show any cleavage after 24 h. hour incubation (Table 5).

Table 5

Percentage of cleavage of selected GLP1 receptor agonists

% degradation after 6 hours % degradation after 24 hours GLPl-hFc 100% 100% desH-GLPl-hFc 2% 5% Q-GLPl-hFc 0% 0% A-GLPl-hFc 100% 100% Comparison product 0% 0%

To further differentiate the stability of Q-GLP1-hFc and the reference product, two constructs were mixed with recombinant human DPP4 and the intact peptide (N-terminal peptide), cleaved peptide (N-terminal peptide after cleavage) and one internal reference peptide (stable peptide, immune to any modification in the construct) for any construct were monitored using nano-LC-MS/MS.

Table 6

Stability of selected GLP1 receptor agonists to DPP4

Splitting in % through 4 hours Splitting in % through 72 hours Q-GLPl-hFc 0% 0% Comparison product 4% 41%

The comparison product showed noticeable degradation (4%) at 4 hours and more than 40% at 72 hours (Table 6). In contrast, Q-GLP1-hFc did not show any cleavage even after 72 h incubation with DPP4 at 37°C.

The present invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art.

Claims (14)

standing art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. CLAIM 1. A fusion protein comprising a glucagon-like peptide 1 (GLP1) variant, wherein the GLP1 variant consists of SEQ ID NO: 6 and a stabilizing domain that is an antibody or an antigen-binding fragment thereof that contains a light chain variable region (LCVR) and a heavy chain variable region chain (HCVR) that specifically binds to the GLP1 receptor, wherein the fusion protein is characterized by enhanced resistance to proteolytic cleavage and/or enhanced ability to lower blood glucose. 2. The fusion protein of claim 1, wherein the GLP1 variant is fused to the N-terminus or C-terminus of an HCVR antibody or an antigen-binding fragment thereof. 3. The fusion protein of claim 1, wherein the GLP1 variant is fused to the N-terminus or C-terminus of the LCVR antibody or an antigen binding fragment thereof. 4. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or diluent. 5. An isolated polynucleotide molecule containing a polynucleotide sequence that encodes a fusion protein as presented in any of claims 1-3. 6. A vector containing a polynucleotide molecule according to claim 5. 7. A cell expressing a fusion protein encoded by the polynucleotide molecule according to claim 6. 8. A method for lowering blood sugar, comprising administering a pharmaceutical composition containing a therapeutically effective amount of the fusion protein according to any one of claims 1 to 3 to a subject in need thereof. 9. The method of claim 8, wherein the subject is characterized by having a disease or disorder selected from the group consisting of the following: diabetes mellitus, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes mellitus, type 1 diabetes mellitus, prediabetes, cardiovascular disease , atherosclerosis, congestive heart failure, coronary artery disease, arteriosclerosis, peripheral artery disease, stroke, respiratory dysfunction, kidney disease, fatty liver disease, non-alcoholic steatohepatitis (NASH) and metabolic syndrome. 10. A method of preventing, treating or reducing the intensity of at least one symptom, sign or complication of type 2 diabetes mellitus, the method comprising administering a pharmaceutical composition containing a therapeutically effective amount of the fusion protein according to any one of claims 1 to 3 to a subject in need thereof. 11. The method of claim 10, wherein at least one symptom, sign or complication is selected from the group consisting of high blood sugar, polydipsia, frequent urination, presence of ketones in urine, pathological fatigue, weight fluctuations, blurred vision , slow-healing ulcers, frequent infections, swollen or painful gums, obesity, heart disease, stroke, kidney disease, eye disease, nerve damage and high blood pressure. 12. The method according to any one of claims 8 to 11, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent or therapy. 13. The method of claim 12, wherein the second therapeutic agent or therapy is selected from the group consisting of the following: insulin or insulin analogue, metformin, thiazolidinedione, sulfonylurea, biguanide, chlorpropamide, glinide, alpha-glucosidase inhibitor, nateglinide, DPP4 inhibitor, pramlintide, sitagliptin, bromocriptine, SGLT2 inhibitor, canagliflozin, antihypertensive drug, statin, aspirin, diet modification, exercise and dietary supplement. 14. The method according to any one of claims 8 to 13, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally or intramuscularly. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2