NZ716357B2

NZ716357B2 - Antibody specifically binding to glp-1r and fusion protein thereof with glp-1

Info

Publication number: NZ716357B2
Application number: NZ716357A
Authority: NZ
Inventors: Shuqian Jing; Xiaofeng Wang; Chenjiang Yao; Cheng Zhang; Hua Zhang
Original assignee: Gmax Biopharm Llc
Priority date: 2013-08-13
Filing date: 2014-08-01
Publication date: 2022-06-28

Abstract

Disclosed in the present invention is an antibody specifically binding to GLP-1R and a fusion protein thereof with GLP-1. The fusion proteins can effectively bind to a human GLP-1R receptor and activate a receptor signalling channel, thus are useful for treating diabetes, excessive weight, obesity and related disorders thereof. nd related disorders thereof.

Description

ANTIBODY SPECIFICALLY G TO GLP-1R AND FUSION PROTEIN THEREOF WITH GLP-1 DESCRIPTION FIELD The present invention relates to the cal field of antibodies, especially relating to an dy specifically binding to GLP-1R and fusion proteins thereof with GLP-1.

BACKGROUND Typical symptoms of type II diabetes include the following three aspects: 1) the peripheral insulin ance, mainly the responsiveness of bone and muscle to insulin is reduced, leading to ed glucose output of these tissues (Kahn and Goldfine, J Diabetes Complication (1993) 7:92-105; Weyer et al., J Clin Invest. (1999) 104:787-794); 2) excessive hepatic e production, the regulation of liver cells to the responsiveness of insulin is reduced (Kahn and Goldfine, J Diabetes Complication (1993) 05; Lam et al., Am J Physiol Endocrinol Metab. (2009) -378) and the excessive secretion of glucagon (Unger and Orci, Arch Intern Med. (1977) 137:482-491); and 3) disorders of pancreatic islet beta cells, at an earlier stage of a disease, an increase in beta cell proliferation and insulin secretion compensates the impact of insulin resistance on blood sugar (Bonner- Weir, Trends Endocrinol Metab. (2000) 11:375-378), but with the se of time and the degree of insulin resistance, depletion of beta cells occurs, ed by decreased insulin ion, thus leading to type II diabetes (DeFronzo, Diabetes. (1988) 37:667-687; Kahn et al., J Nutr. (2001) 131:354S-360S).

Glucagon like peptide-1 (GLP-1) is a peptide containing 30 amino acids. It is secreted from L intestinal cells in se to the intake of glucose (Orskov et al., Diabetes (1994) 43:535-539; Drucker et al., Proc. Natl. Acad. Sci. USA (1987) 84:3431-3438). After the secretion upon stimulation, GLP-1 binds to pancreatic GLP-1R (glucagon like peptide-1 receptor) to activate the downstream adenylate cyclase signaling pathway to promote the synthesis and secretion of insulin. GLP-1 secretion also reduces gastric emptying, thereby ng the amount of glucose into the circulatory system after food digestion (Wettergren et al., Dig. Dis. Sci. (1993) 38:665-673). In mice and in patients with type I and type II diabetes, GLP-1 increases n ion and reduces blood sugar concentration (Naucket al., Diabetes. (1997) 105:187-195; Todd et al., Eur J Clin Invest. (1997) -536). Studies have shown that GLP-1 can also t apoptosis of pancreatic beta cells and promote their proliferation (Perfetti et al., Endocrinology (2000) 141:4600-4605; Hui et al., Endocrinology (2003) 144:1444-1455). The feasibility and efficacy of GLP-1 for the treatment of diabetes patients have been proved clinically (Samson and Garber, Curr Opin Endocrinol Diabetes Obes. (2013) 20:87-97). There are also patents (U.S. Pat. No 883 and U.S. Pat. No. 6,989,148) disclosing methods for the treatment of diabetes by using GLP-1 and its derivatives. r, GLP-1 has a short half-life in vivo and does not have good therapeutic effects.

SUMMARY The present invention is provides an antibody specifically binding to GLP-1R.

The present invention also provides a fusion protein of an dy specifically binding to GLP-1R with GLP-1 and uses thereof.

More particularly, the t invention provides the following: An antibody specifically binding to GLP-1R comprises an amino acid sequence selected from: (a) a light chain CDR3 sequence selected from: light chain CDR3 sequences differing by no more than three amino acid additions, substitutions and/or deletions in total from one of L1-L13 light chain CDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53; preferably, light chain CDR3 sequences differing by no more than two amino acid additions, substitutions and/or deletions in total from one of L1-L13 light chain CDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53; and more preferably, light chain CDR3 sequences differing by one amino acid addition, tution and/or deletion from one of L1-L13 light chain CDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53; (b) a heavy chain CDR3 sequence selected from: heavy chain CDR3 sequences differing by no more than four amino acid additions, substitutions and/or deletions in total from one of H1-H13 heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27; preferably, heavy chain CDR3 sequences differing by no more than three amino acid additions, substitutions and/or deletions in total from one of H1-H13 heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27; more preferably, heavy chain CDR3 sequences differing by no more than two amino acid ons, substitutions and/or deletions in total from one of H1-H13 heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27; and further ably, heavy chain CDR3 sequences differing by one amino acid addition, substitution and/or deletion in total from one of H1-H13 heavy chain CDR3 sequences SEQ ID NO: 20 to SEQ ID NO: 27; and (c) a light chain CDR3 sequence from (a) and a heavy chain CDR3 sequence from Preferably, the antibody further comprises one or more amino acid sequences selected from: (a) a light chain CDR1 sequence selected from: light chain CDR1 sequences differing by no more than three amino acid additions, substitutions and/or deletions from one of L1-L13 light chain CDR1 sequences: SEQ ID NO: 28 to SEQ ID NO: 37; preferably, light chain CDR1 sequences ing by no more than two amino acid additions, tutions and/or deletions in total from one of L1-L13 light chain CDR1 sequences: SEQ ID NO: 28 to SEQ ID NO: 37; and more ably, light chain CDR1 sequences differing by one amino acid addition, substitution and/or deletion from one of L1- L13 light chain CDR1 ces: SEQ ID NO: 28 to SEQ ID NO: 37; (b) a light chain CDR2 sequence selected from: light chain CDR2 sequences differing by no more than two amino acid ons, substitutions and/or deletions from one of L1-L13 light chain CDR2 sequences: SEQ ID NO: 38 to SEQ ID NO: 45; and preferably, light chain CDR2 sequences ing by one amino acid addition, substitution and/or deletion from one of L1-L13 light chain CDR2 sequences SEQ ID NO: 38 to SEQ ID NO: 45; (c) a heavy chain CDR1 sequence selected from: heavy chain CDR1 ces differing by no more than two amino acid additions, substitutions and/or deletions from one of H1-H13 heavy chain CDR1 sequences: SEQ ID NO: 6 to SEQ ID NO: 12; and preferably, heavy chain CDR1 sequences differing by one amino acid addition, substitution and/or deletion from one of H1-H13 heavy chain CDR1 sequences: SEQ ID NO: 6 to SEQ ID NO: 12; and (d) a heavy chain CDR2 selected from: heavy chain CDR2 sequences differing by no more than three amino acid additions, substitutions and/or deletions from one of H1-H13 heavy chain sequences: SEQ ID NO: 13 to SEQ ID NO: 19; preferably, heavy chain CDR2 sequences differing by no more than two amino acid additions, substitutions and/or deletions in total from one of H1-H13 heavy chain CDR2 sequences: SEQ ID NO: 13 to SEQ ID NO: 19; and more preferably, heavy chain CDR2 sequences differing by one amino acid addition, tution and/or deletion from one of H1- H13 heavy chain CDR2 sequences: SEQ ID NO: 13 to SEQ ID NO: 19.

An antibody specifically binding to GLP-1R comprises an amino acid ce selected from: (a) one or more light chain variable regions selected from: i. light chain CDR1 sequences: SEQ ID NO: 28 to SEQ ID NO: 37; ii. light chain CDR2 sequences: SEQ ID NO: 38 to SEQ ID NO: 45; and iii. light chain CDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53; (b) one or more heavy chain variable regions selected from: i. heavy chain CDR1 sequences: SEQ ID NO: 6 to SEQ ID NO: 12; ii. heavy chain CDR2 ces: SEQ ID NO: 13 to SEQ ID NO: 19; and iii. heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27; and (c) a light chain variable domain sequence from (a) and a heavy chain variable domain sequence from (b).

An antibody specifically binding to GLP-1R comprises an amino acid sequence selected from: (a) a light chain variable region selected from: i. amino acid sequences that are at least 80% cal to any of L1-L13 light chain variable region sequences: SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105; and ii. amino acid ces encoded by polynucleotide sequences that are at least 80% identical to any of the polynucleotide sequences encoding for L1-L13 light chain le region sequences: SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104; (b) a heavy chain variable domain sequence selected from: i. amino acid sequences that are at least 80% identical to any of H1-H13 heavy chain variable region sequences: SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79; and ii. an amino acid sequences encoded by polynucleotide sequences that are at least 80% identical to any of the polynucleotide sequences encoding for H1-H13 heavy chain variable region sequences: SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78; and (c) a light chain variable region sequence from (a) and a heavy chain le region sequence from (b). ably, the antibody further comprises an amino acid sequence selected from: (a) L1-L13 light chain variable region ces: SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: (b) H1-H13 heavy chain variable region sequences: SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79; and (c) a light chain le region sequence from (a) and a heavy chain le region sequence from (b).

Preferably, the combination (c) of a light chain variable region sequence (a) and a heavy chain variable region sequence (b) is selected from L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13.

In particular aspect the present invention provides an antibody specifically binding to glucagon-like peptide-1 receptor ("GLP-1R"), comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

Preferably the antibody of this aspect comprises: (a) a light chain variable domain ce comprising: i. an amino acid sequence at least 80% identical to SEQ ID NO: 105; or ii. an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 104; and (b) a heavy chain variable domain sequence comprising: i. an amino acid sequence at least 80% identical to SEQ ID NO: 79; or ii. an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 78.

Still more preferably, the antibody of this aspect comprises a light chain le region sequence SEQ ID NO: 105 and a heavy chain variable domain sequence SEQ ID NO: 79.

Preferably, the antibody of the invention also comprises an amino acid sequence selected from: (a) light chain nt region amino acid sequence: SEQ ID NO 106; (b) light chain constant region amino acid sequence: SEQ ID NO 107; (c) heavy chain constant region amino acid sequence: SEQ ID NO 108; (d) heavy chain constant region amino acid sequence: SEQ ID NO 109; (e) light chain constant region amino acid sequence: SEQ ID NO 106 and heavy chain constant region amino acid sequence: SEQ ID NO 108; (f) light chain constant region amino acid sequence: SEQ ID NO 107 and heavy chain nt region amino acid sequence of SEQ ID NO 108; (g) light chain nt region amino acid sequence: SEQ ID NO 106 and heavy chain constant region amino acid sequence: SEQ ID NO 109; and (h) light chain constant region amino acid sequence: SEQ ID NO 107 and heavy chain constant region amino acid sequence: SEQ ID NO 109.

Preferably, the antibody is selected from murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, doublechain dies, triple-chain antibodies, tetra-chain dies, Fab fragments, x fragments, domain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 dies, IgG3 antibodies, and IgG4 antibodies.

A GLP-1 fusion protein comprising GLP-1 and an antibody of the present invention, wherein GLP-1 comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:127.

Preferably, GLP-1 is fused with the light chain and/or heavy chain of the antibody of the present invention in the form N’-R1-L-R2-C’, N’-R2-L-R1-C’ or N’-R2-L-R1r-C’; wherein L is a peptide linker sequence, comprising a full-length, partial or repeated amino acid sequence ed from LK1-LK3 amino acid sequences: SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112; R1 is an amino acid ce of GLP-1; R1r is a reverse amino acid sequence of GLP-1; R2 is an amino acid ce of the light chain or heavy chain of the antibody of the present ion; C’ represents the yl al of the GLP-1 fusion protein polypeptide chain; N’ represents the amino terminal of the GLP-1 fusion protein ptide chain.

A polynucleotide or combination of polynucleotides encoding a GLP-1 fusion protein of the present invention.

A vector comprising a polynucleotide or combination of cleotides of the present invention.

A host cell comprising a vector of the present invention. In certain embodiments the host cell is not within a human.

A pharmaceutical ition comprising a GLP-1 fusion protein of the present invention and a pharmaceutically acceptable carrier.

Use of a GLP-1 fusion protein of the present invention, or a pharmaceutical composition comprising or based on an antibody or GLP-1 fusion protein of the present invention in the preparation of a medicament for preventing or treating non-insulin-dependent diabetes.

Use of a GLP-1 fusion protein of the present ion, or a pharmaceutical composition comprising or based on an antibody or GLP-1 fusion protein of the present invention in the preparation of a medicament for treating obesity or subjects whose body weight is about 25% above normal body weight for the subject’s height and body fluid. (followed by 7A) Given that the key role of GLP-1R plays in the use of glucagon like peptide-1 for the tion and control of blood glucose levels in type II diabetes patients, and that its significant therapeutic characteristic is the ability of stimulating insulin secretion without the associated risk of ycaemia. GLP-1 is fused with an antibody specifically binding to GLP-1R in the present invention, thereby prolonging the half-life of GLP-1in vivo to retain the biological activity of GLP-1. At the same time, the fusion protein formed by GLP-1 and the antibody ically binding to GLP-1R has the molecular targeting properties provided by the antibody.

Furthermore, the genicity of the antibody is also lower than that of other fusion partners.

DETAILED DESCRIPTION The present ion is directed to the disadvantage that GLP-1 is quickly removed by dipeptidyl peptidase (DPP-IV) in vivo and has insufficient efficacy, and s antibodies of GLP-1R to fuse with GLP-1 so as to enhance the half-life and biological activity of GLP-1.

Antibodies used for the fusion do not hinder the binding of GLP-1 with receptors, can ically facilitate the biological activity of GLP-1R, and due to their high affinity to receptors and stability, are capable of enhancing the long lasting local concentrations of GLP- 1 around the receptors and thereby significantly increasing its effective time and potency for binding to the receptor. At the same time, the fusion with the antibody increases the steric hindrance for DPP-IV to ize or capture GLP-1, thus reducing the elimination rate of GLP-1 in vivo and increasing the effective time of GLP-1. According to literature reports (Lin and Wang, J of Molecular Modeling (2009) 15:53-65), the release of the articulation state n the N-terminal ellular region of GLP-1R and the (followed by 8) transmembrane region thereof is an essential step for GLP-1 to enter the binding site to GLP- 1R and become biologically active. As described in the present invention, the binding of the antibody to GLP-1R is largely involved in the N-terminal extracellular region of the receptor, and the binding thereof to the receptor helps the release of said articulation state and can tate the access of GLP-1. Accordingly, the fusion of GLP-1 with the antibody targeting GLP-1R increases the half-life and affinity potency of GLP-1 to result a stronger biological activity, and thus is an important innovation superior to the GLP-1 y. More importantly, some of the antibodies themselves used in the present invention have the biological characteristics of enhancing GLP-1 activation of GLP-1R in the presence of GLP- 1. Because of some of the s above, the GLP-1 fusion protein in the present invention may be a more effective activator of GLP-1R than GLP-1.

It is a common concern that the repetitive administration of a fusion protein for a long time may elicit nicity. This is especially a concern in the case of GLP-1 fusion protein therapy, because once a patient is diagnosed with diabetes, the patient is to e a life- long ent for the disease. In addition, if the Fc parts of the immune globulin retain undesirable effector function, the Fc fusion protein therapy can be a concern. Via computerassisted 3D structure prediction of an immune globulin, and antibody sequence optimization and humanization, the identified specific GLP-1 fusion proteins no longer have or on and thus have reduced risk of inducing immune response after repeated and long- term administration. As discussed in the t ion, the amino acid of GLP-1 moiety is preferably fused with light and heavy chains of the antibodies through glycine and serine rich peptide linker. e of having smaller side chains, glycine and serine enable the peptide linker sequence considerably flexible, reducing the rigidity between GLP-1 and the corresponding positions of the antibody, thus GLP-1 can interact with GLP-1R freely. At the same time, the presence of the peptide linker tes GLP-1 from the antibody, thus ng the interaction of the two domains. The glycine and serine appear alternately to avoid excessive repetition, in order not to uce undesirable immunogenicity to the fusion protein, r, the peptide linker inevitably increases the immunogenicity of the fusion protein in vivo, and it is of great importance to select the length of the peptide linker so as to balance structure flexibility and immunogenicity. Accordingly, the present invention provides three ent lengths of peptide linkers for fusion. At the same time, the present invention provides different ways of linking GLP-1 with the antibody by using peptide linkers for fusion, and the patterns of the formed GLP-1 fusion proteins would include: 1) a fusion protein with GLP-1 and a light chain linked in the form of N’-R1-LR2-C 2) a fusion protein with GLP-1 and a light chain linked in the form of N’-R2-LR1-C 3) a fusion protein with GLP-1 and a light chain linked in the form of N’-R2-LR1r-C 4) a fusion protein with GLP-1 and a heavy chain linked in the form of N’-R1-LR2-C ) a fusion protein with 1) and 4) at the same time; 6) a fusion protein with 2) and 4) at the same time; 7) a fusion protein with 3) and 4) at the same time.

Within the scope of the present invention, the DNA ng the GLP-1 is linked to full length/variable region/fragment light chain or full length/variable region/full length heavy chain DNA of said antibody, via the DNA encoding the e linker sequence, forming a fused light chain or fused heavy chain DNA, furthermore, at the 5' end of the light chain DNA, the DNA encoding the signal peptide is also introduced to form a gene based on which the mutant/wild type GLP-1 can be linked to antibody sequences. In the present invention, the GLP-1 ces obtained by the method of gene synthesis are linked to the peptide linker as well as antibody light or heavy chain DNA through the method of PCR.

The light or heavy chain variable region sequences of the antibodies to GLP-1R are obtained from specific hybridoma cells h the method of PCR followed by being linked to the constant region DNA of specific antibody subtype. The constant region DNA of the wild type antibody subtype can be obtained from a specific clone library and used as the basis of sequence optimization. After cloned into an expression vector, genes used for expressing the fusion n described herein are used for producing and expressing the fusion ns.

After the light chain and heavy chain expression vectors are paired during expression, the DNA carrying the genes are co-transfected or transformed into a host cell. The promoter is induced by optimal adaption. The transformants or genes for amplifying desired sequences are cultured in a proper medium at an appropriate pH and temperature. DNA is usually introduced by commonly used methods, such as CaPO4, electroporation, and PEI etc.

The suitable host cells, suitable for the expression of the nucleic acid within the vector described herein, include higher eukaryotic cells, and the examples of ian host cell line for expression include e hamster ovary cell line (CHO) and human embryonic kidney cell (HEK293, or HEK293 cell line cultured in a suspension), and the signal peptide at the N-terminal of the light chain guides the secretion of recombinant fusion ns from the mammalian host cell line. The vector for expression or cloning carries the selection marker that enables its continuously replication in host cells and is used to screen cells capable of integrating the fusion protein encoding c acid, and promoter that effectively links with the fusion protein encoding ce and guides mRNA synthesis. One e is to use a vector carrying otics ance and Hepatitis B virus and simian virus promoter (SV40) to select CHO host cells stably sing the fusion proteins.

After host cell lines have expressed fusion proteins, the present ion adopts an affinity chromatographic method to purify the part secreted thereby in the cell e atant. In an example of the invention, the fusion protein fused with full-length antibody is captured by a protein G affinity chromatography column and then eluted from the chromatography column by low pH followed by collection. Mild elution conditions help to t denaturing of the protein.

The fusion protein of the present invention can be formulated with one or more excipients. The fusion protein of the present invention can be combined with a pharmaceutically acceptable buffer having adjusted pH that provides acceptable stability and is suitable for administration (such as parenteral administration). Optionally, one or more pharmaceutically acceptable antimicrobial reagents may be added. Preferred pharmaceutically acceptable antimicrobial reagents are m-cresol and phenol. One or more pharmaceutically acceptable salt solution can be added to adjust the ionic strength or tension.

One or more excipients may be added to further adjust the isotonicity of the formulation. in is an example of excipients for ing the isotonicity. "Pharmaceutically acceptable" means being suitable for administration to human or other animals, and therefore free of toxic ingredients or undesirable pollutants and not interfere with the activity of the active compounds therein.

The fusion protein of the invention can be prepared in a solution preparation or in a lyophilized powder that can be reconstituted with appropriate diluent. Lyophilized dosage form is one of the formulation types in which the fusion protein is stable, with or without the buffering capacity to maintain the pH over its intended in-use shelf-life of the tituted product. The solution comprising fusion proteins discussed herein is preferably isotonic before lyophilization to enable the formation of an isotonic solution after reconstitution.

A pharmaceutically acceptable salt form of the fusion proteins of the present invention is within the scope of the present invention. Commonly employed acids to form acid addition salts are inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and oric acid; and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, ophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Preferable acid addition salts are those formed with inorganic acids, such as hydrochloric acid and hydrobromic acid.

Base addition salts include those derived from inorganic bases, such as ammonium, base or alkali earth metal hydroxide, carbonate, and onate. Such bases useful in preparing the salt solution of the present invention thus e sodium hydroxide, potassium ide, ammonium hydroxide, potassium carbonate, and the like.

The fusion proteins of the present invention have biological activity. Biological activity refers to the ability of the fusion proteins to bind and activate the GLP-1R in vivo and ate stress response. Responses include but not limited to, increased secretion of insulin, suppression of secretion of on, inhibition of appetite, weight loss, induction of satiety, inhibition of apoptosis, and induction of pancreatic beta cell proliferation and pancreatic beta cell differentiation. A number of representative GLP-1 fusion proteins are tested for in vitro and in vivo activities. First, step 4 (Figure 1) provides data on a fluorescence detection assay of the fusion protein to interact with the GLP-1R. Then, step 12 es in vitro activity test of the fusion protein interacting with and activating human . In this set of experiments, CHO cells over-expressing human GLP-1R were used.

Activation of the GLP-1R in these cells causes adenylyl cyclase tion which in turn induces expression of a reporter gene driven by a cAMP response element (CRE). Step 12 (Figure 2) provides the data where the reporter gene is luciferase. In vitro experimental data indicate that the fusion proteins are capable of binding and ting GLP-1R and appear to be more effective than the native GLP-1 in vitro. Step 13 (Figure 3) provides the data of blood glucose concentration change of the mice 16 hours (hr) after being intraperitoneally administrated with one of the fusion proteins of the present invention. The in vivo data generated on mice of step 13 demonstrate the activity of the fusion protein and its longer half-life than the native GLP-1.

Administration of the fusion protein may be via any route known to be effective by the physician of ry skill. Peripheral eral administration is one of such methods.

Parenteral administration is commonly tood in medical literature as the injection of a dosage form into the body with a sterile syringe or other mechanical device such as an infusion pump. Peripheral parenteral routes include intravenous, intramuscular, subcutaneous, and eritoneal routes of administration.

The fusion proteins of the present invention can also be strated by oral, rectal, nasal, or lower respiratory routes, which are non-parenteral routes. Of these non-parenteral routes, the lower respiratory route and the oral route are preferred.

The fusion proteins of the present invention can be used to treat a wide variety of diseases and conditions. The fusion proteins of the present invention primarily exert their biological effects by acting at GLP-1R. Subjects with diseases and/or conditions that d favorably to GLP-1R stimulation or to the administration of GLP-1 nds can ore be treated with the GLP-1 fusion ns of the present ion. These subjects are referred to as subjects "in need of treatment with GLP-1 compounds" or "in need of GLP- 1R stimulation". ed are subjects with sulin dependent diabetes, insulin dependent diabetes, stroke (see WO 00/16797), myocardial infarction (see WO 98/08531), obesity (see WO 98/19698), catabolic changes after surgery (see US Patent No. 6,006,753), functional dyspepsia and irritable bowel syndrome (see WO 99/64060). Also included are subjects requiring prophylactic ent with a GLP-1 compound, e.g., subjects at risk of developing non-insulin ent diabetes (see WO 00/07617). Subjects with impaired glucose nce or impaired fasting glucose, subjects whose body weight is about 25% above normal body weight for the subject’s height and body fluid, subjects with a partial pancreatectomy, subjects having one or both s with non-insulin dependent diabetes, subjects who have had gestational diabetes and subjects who have had acute or chronic pancreatitis are at risk of developing non-insulin dependent diabetes. An effective amount of the fusion proteins described herein is the dosage which results in a desired therapeutic and/or prophylactic effect without causing unacceptable side-effects when administrated to a subject in need of GLP-1 receptor stimulation. A "desired therapeutic effect" includes one or more of the followings: an amelioration of the symptom(s) associated with the disease or condition; a delay in the onset of symptoms associated with the disease or condition; increased longevity compared with the absence of the treatment; and better quality of life ed with that in the absence of the treatment. An "effective amount" of the GLP-1 fusion proteins for the treatment of diabetes is the amount that would result in better control of blood glucose concentration compared with that in the e of the treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy or kidney diseases. An "effective amount" of the GLP-1 fusion n for the prevention of diabetes is the amount that would delay, compared with that in the absence of treatment, the onset of elevated blood e levels that requires treatment with anti-hyperglycaemic drugs such as sulfonyl urea, thiazolidinedione, insulin and/or bisguanidine. The dosage of fusion proteins effective to normalize a patient’s blood glucose will depend on a number of factors, among which are included, without limitation, the subject’s sex, weight and age, the severity of inability to regulate blood glucose, the route of administration and bioavailability, the pharmacokinetic profile of the fusion n, the potency, and the formulation. Doses may be in the range of 0.01 to 1 mg/kg body weight, preferably in the range of 0.05 to 0.5 mg/kg body weight. It is able that the fusion proteins of the present ion are administered either once a week or twice a week. Depending on the disease to be treated, it may be necessary to administrate the fusion protein more frequently such as three or more times per week.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a flow cytometry (FACS) showing the specific binding of a recombinant expressed GLP-1 fusion protein (GLP-1(A8G)-LK-L13H13) with human GLP-1R (hGLP- 1R) stably expressed in the Chinese hamster ovary cell line (solid line peak, marked with *) in comparison with the Chinese hamster ovary cell line itself (dotted line peak).

Figure 2 shows the reporter gene assay dose-responsive curves of GLP-1 wild type (circles) and GLP-1(A8G)-LK-L13H13 (triangles) activating hGLP-1R stably expressed in Chinese hamster ovary cell line.

Figure 3 is the result of a mouse (ICR) glucose nce test, showing the glucose tolerance of fasting mice 16 hr single i.p. injection of GLP-1(A8G)-LK-L13H13 at 5 micrograms per mouse (square) and 15 micrograms per mouse gles).

Figure 4 is the result of a mouse (C57BL) e tolerance test, g the glucose tolerance of fasting mice 40 hr single i.p. injection of GLP-1(A8G)-LK-L13H13 at 15 micrograms per mouse gles).

Figure 5 is the blood e concentration-time curve of the type II diabetic mice (db/db mice), reflecting the blood glucose concentration change over the experiment period of the type II diabetic mice after a single i.p. injection of GLP-1(A8G)-LK-L13H13 at a concentration of 10 nmol/kg (inverted triangles).

Figure 6 is the daily food intake-time curve of the type II diabetic mice (db/db mice), reflecting the mice daily food intake change of the type II diabetic mice after i.p. injection of A8G)-LK-L13H13 at 10 nmol/kg (inverted triangles), during the time period from 3 days before injection of the fusion protein to 5 days after injection. Figure 6 and Figure 5 are the results of two parallel experiments.

SPECIFIC EMBODIMENTS OF THE INVENTIION Through the following specific embodiments in combination with the figures, the technical solutions of the t invention are further illustrated.

In this invention, unless referred specifically, the employed raw materials, equipments and the like can all be purchased from the market or are commonly used in the art. The methods of the following embodiments, if not indicated ically, are all conventional methods in the art.

Step 1: Construction of stable antigen cell line for immunization CHO-DHFR minus cells are transferred into a 6-well plate and transfected with the pYS plasmid carrying R gene (see SEQ ID NO: 113 for the nucleotide ce, and see SEQ ID NO: 114 for the amino acid sequence) after 24 hr culture. The medium is changed before transfection, and it is carried out by following the recommended ection ions provided by the manufacturer of Lipofectamine 2000 (Invitrogen). 48 hr after transfection, the medium of the culture is replaced by the complete medium containing 10 nM MTX. The medium is changed every 3 days for about two weeks, until stable clones appear. The dispersed cell colonies are detached from the plate and collected. After cells grow to about 50% confluence, lly sing concentrations of MTX (up to a concentration of 10 μM MTX) are added for pressure selection. The constructed stable cell lines are tested by FACS analysis using antibodies (Abcam) against hGLP-1R to identify cell clones after pressure selection. There is a lot of hGLP-1R expression in the selected CHO-DHFR-hGLP-1R cell nes after MTX selection. Finally six high-expression and stable cell lines of hGLP-1R are identified through ning.

Step 2: Preparation of antibodies Freund’s adjuvant emulsified CHO-DHFR-hGLP-1R whole-cells are used at 2 x 106 cells/mouse dosage for subcutaneous injection into BALB/c mice (6-8 weeks). After 2 weeks, the immunity of the mice is boosted with incomplete Freund’s adjuvant emulsified immunogen, and then once a week. The blood samples are collected from the clipped tail end and centrifuged to collect the serum for detecting the serum titers by FACS analysis.

After the acceptable antibody titers are achieved, the mice are sacrificed and their spleen cells are harvested under aseptic condition. SP2/0 cells are collected at the logarithmic phase of growth with 3 min fugation at 2000 rpm. The precipitation is resuspended with serum-free culture medium, then fuged and resuspended for a second time, and counted. Spleen cells and SP2/0 cells are mixed at ratio of SP2/0 cells : spleen cells ≥ 1:1, followed by 3 rounds of washing-centrifugation. After the precipitation from the last centrifugation is detached, 1 ml of the 50 armed to 37°C) is added drop wise (finished in 30 s), after pipette-mixing for 1 min, 30 ml (pre-warmed to 37°C) serum-free medium (Invitrogen) is added slowly to terminate the PEG fusion. After 5 min centrifugation at 1500 rpm, the cell s are resuspended and RPMI1640 (Invitrogen) containing HAT (sarcine, pterin and thymidine; Invitrogen) and 20% FBS (Bioind) is added as the fusion culture medium. 20000 spleen cells and 5000 feeder layer cells in 100 μl volume are plated into each well of 96-well plates. Fused hybridoma cells and feeder layer cells are cocultured in 96-well plates with HAT selection to get rid of the non-fused cells. After 10 days, the supernatant of the hybridoma cells in the culture plates is collected for ELISA test.

Step 3: ELISA screening of the whole cells FR-hGLP-1R cells over-expressing hGLP-1R and CHO-DHFR minus cells not sing hGLP-1R, were separately erred into a 96-well plate, and kept growing to 90% confluent. The supernatant of the culture medium is d and attached cells are washed twice with PBS, then 100 μl 100% methanol is added to fix the cells for 10 min at 4°C. Then 100 μl freshly made 0.6% H2O2-PBS is added, and after incubation at room temperature for 20 min, the cells are washed twice with PBS. After blocking with PBS-1% BSA solution, the hybridoma supernatant is added and incubated for 90 min at 4°C. After several washes, 100 μl of the secondary antibody GxM-HRP-Fc (Sigma-Aldrich) (5000- times diluted) is added into each well and incubated at 37°C for 0.5 hr. After washing for five times, 100 μl of TMB chromogenic substrate is added into each well and incubated at 37°C for 15 min, and then 2M H2SO4 is added to terminate the reaction for reading of OD450 values. Positive control is the mouse serum after immunization; negative control is the cell culture supernatant. Hybridoma clones secreting GLP-1R antibody are screened and the stable secretory cell lines against hGLP-1R are obtained after cloning. Lastly, antibody supernatant secreted by hybridoma is verified by FACS analysis.

Step 4: Flow analysis (FACS) of the supernatant of the positive hybridoma cells PBS containing 10 mM EDTA is used to detach and collect 105 CHO DHFR-hGLP- 1R cells into a 1.5 ml EP tube. The supernatant is removed after centrifugation and the negative control sample is resuspended with a loading buffer (PBS, 2% FBS). For positive control, 200 μl antibody supernatant is added to end the cells with incubation at room temperature; the cells are then centrifuged at 1500 rpm to remove the supernatant, washed with loading buffer and centrifuged again. The cells are resuspended with addition of FITC labeled goat anti-mouse fluorescent antibody at 1:50 dilution (BD ngen, 200 μl/well) and incubated at room temperature for 30 min in the dark. Supernatant is removed after centrifugation, cells are washed with loading buffer, centrifuged again and resuspended with loading buffer for analysis. The oma supernatant and CHO-DHFR-hGLP-1R cells have specific binding: gray peak and dotted line peak are ve ls; the solid line peak (marked with *), corresponding to the hybridoma supernatant, moves to the right sly (Figure 1).

Step 5: Cloning and subcloning of dy genes Hybridoma cells secreting antibody are collected. Hybridoma mRNA is extracted according to the manufacturer protocol of QIAGEN mRNA extraction kit. Then the ted mRNA is transcribed ely into cDNA. The reverse transcription primers are specific primers for the light and heavy chain constant regions of mouse, with the heavy chain reverse transcription primer being (5’-TTTGGRGGGAAGATGAAGAC-3’) (SEQ ID NO: 115), the light chain reverse transcription primers being (5’-TTAACACTCTCCCCTGTTGAA-3’) (SEQ ID NO: 116) and (5’-TTAACACTCATTCCTGTTGAA-3’) (SEQ ID NO: 117). RTPCR reaction conditions are as following: 25°C for 5 min, 50°C for 60 min, and 70°C for 15 min. Reversely transcribed cDNA is diluted with 0.1 mM TE to 500 μl, added into the ultrafiltration centrifuge tube n Ultra-0.5) and centrifuged at 2000 g for 10 min. The filtrate is removed, 500 μl of 0.1 mM TE is added and centrifuged at 2000 g for 10 min. The filtrate is d and the preparation tube is placed in inversion to the new centrifugal tube, and centrifuged at 2000 g for 10 min to obtain the ed cDNA. 10 μl of purified cDNA serves as the template. Add 4 μl 5x tailing buffer, 4 μl dATP (1 mM) and 10 U terminal transferase (Promega), mix uniformly and incubate at 37°C for 5 min and at 65°C for 5 min.

The PolyA tail cDNA is used as templates and PCR is performed to amplify light and heavy chain variable region genes of antibodies. Upstream primers are all OligodT, with heavy chain downstream s being (5’-TGGACAGGGATCCAGAGTTCC-3’) (SEQ ID NO: 118) and (5’-TGGACAGGGCTCCATAGTTCC-3’) (SEQ ID NO: 119), and light chain downstream primer being (5’-ACTCGTCCTTGGTCAACGTG-3’) (SEQ ID NO: 120). The PCR reaction conditions are as following: 95°C for 5 min; 95°C 30s, 56°C for 30s, 72°C for 1 min, 40 cycles; and 72°C for 7 min. The PCR products are connected to the PMD 18-T vector for sequencing. The resulting sequences of the light and heavy chain variable regions of the antibody after sequencing are listed in the attached Sequence Listing.

PCR primers are designed based on the sequenced DNA ces of the dy, thus the complete light chain, heavy chain signal es and variable domains and mouse IgG1 constant region are connected with expression vector pTM5.

Step 6: Transient expression of anti-GLP-1R antibodies in HEK293 suspension host cell line The suspension HEK293 or CHO expressing cell line are inoculated to a shaker flask, and after 24 hr rotation at 37°C, the cells are ready for transfection. Polyethylenimine (PEI) is used as a transfection reagent during transfection, and its mixture with DNA is added into the cell culture. The mixing optimization ratio of PEI to DNA is 1:1 to 5:1. PEI/DNA e treated cells is d for more than 96 hr at 37°C to express the antigen g protein, meanwhile 0.5% of tryptone is added into the cell culture as the source of amino acids required by expression, and finally the cell supernatant is collected for the purification and separation of the antigen binding protein.

Step 7: Antibody humanization and optimization First of all, the sequences of light and heavy chain variable regions of the screened mouse antibody are aligned with the homologous dies, using NCBI online antibody variable region sequence ent tool (Ig Blast) to search the germline gene sequences of a humanized antibody (Ig Germline Gene ce) homologous to the selected antibodies variable region sequence for humanization, and the humanized gene sequence with highest homology except CDR sequences is used as template for CDR grafting to get the humanized dy variable region sequences and to synthesize humanized antibody light and heavy chain genes. According to the sequence, PCR primers are designed and at the 5’ end and 3’ end of the synthetic sequence, appropriate restriction enzyme sites are introduced. By PCR, the humanized antibody variable s are ied and then combined with the human

Claims

WHAT WE CLAIM IS:

1. An antibody specifically binding to glucagon-like peptide-1 receptor (“GLP1R”), comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

2. The antibody of claim 1, wherein the antibody comprises: (a) a light chain variable domain sequence comprising: i. an amino acid sequence at least 80% identical to SEQ ID NO: 105; or ii. an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 104; and (b) a heavy chain variable domain sequence comprising: i. an amino acid sequence at least 80% identical to SEQ ID NO: 79; or ii. an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 78.

3. The antibody of claim 1 or 2, wherein the antibody comprises a light chain variable region sequence SEQ ID NO: 105 and a heavy chain variable region sequence SEQ ID NO: 79.

4. The antibody of any one of claims 1 to 3, wherein the antibody further comprises an amino acid sequence selected from: (a) light chain constant region amino acid sequence: SEQ ID NO 106; (b) light chain constant region amino acid sequence: SEQ ID NO 107; (c) heavy chain constant region amino acid sequence: SEQ ID NO 108; (d) heavy chain constant region amino acid sequence: SEQ ID NO 109; (e) light chain constant region amino acid sequence: SEQ ID NO 106 and heavy chain constant region amino acid sequence: SEQ ID NO 108; (f) light chain constant region amino acid sequence: SEQ ID NO 107 and heavy chain constant region amino acid sequence: SEQ ID NO 108; 23 (g) light chain constant region amino acid sequence: SEQ ID NO 106 and heavy chain constant region amino acid sequence: SEQ ID NO 109; and (h) light chain constant region amino acid sequence: SEQ ID NO 107 and heavy chain constant region amino acid sequence: SEQ ID NO 109.

5. The antibody of any one of claims 1 to 4, wherein the antibody is selected from murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, double-chain antibodies, triple-chain antibodies, tetrachain antibodies, Fab fragments, domain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies.

6. A glucagon-like peptide 1 (“GLP-1”) fusion protein comprising GLP-1 and the antibody of any one of claims 1 to 5, wherein the GLP-1 comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 127.

7. The GLP-1 fusion protein of claim 6, wherein the GLP-1 is fused with the light chain and/or heavy chain of the antibody in the form of N’-R1-L-R2-C’, N’-R2-L-R1-C’ or N’-R2-L-R1r-C’, wherein L is a peptide linker, comprising a full-length, partial, or repeated amino acid sequence selected from SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112; R1 is an amino acid sequence of GLP-1; R1r is a reverse amino acid sequence of GLP-1; R2 is an amino acid sequence of the light chain or heavy chain of the antibody; C’ represents a carboxyl terminal of the GLP-1 fusion protein polypeptide; and N’ represents an amino terminal of the GLP-1 fusion protein polypeptide.

8. A polynucleotide or combination of polynucleotides encoding the GLP-1 fusion protein of claim 6 or 7. 24

9. A vector comprising the polynucleotide or combination of polynucleotides of claim 8.

10. A host cell comprising the vector of claim 9, provided said host cell is not within a human.

11. A pharmaceutical composition comprising the GLP-1 fusion protein of claim 6 or 7 and a pharmaceutically acceptable carrier.

12. Use of the GLP-1 fusion protein according to claim 6 or 7, or the pharmaceutical composition of claim 11 in the preparation of a medicament for preventing or treating noninsulin-dependent diabetes.

13. Use of the GLP-1 fusion protein according to claims 6 or 7, or the pharmaceutical composition according to claim 11, in the preparation of a medicament for treating obesity or subjects whose body weight is about 25% above normal body weight for the subject’s height and body fluid.

14. An antibody according to claim 1 substantially as herein described or exemplified.

15. A GLP-1 fusion protein according to claim 6 substantially as herein described or exemplified.

16. A polynucleotide according to claim 8 substantially as herein described or exemplified.

17. A vector according to claim 9 substantially as herein described or exemplified.

18. A host cell according to claim 10 substantially as herein described or exemplified.

19. A pharmaceutical composition according to claim 11 substantially as herein described or exemplified.

20. A use according to claim 12, substantially as herein described or exemplified.

21. A use according to claim 13 substantially as herein described or exemplified.