Bispecific proteins

Abstract

The present disclosure provides bispecific proteins. Specifically, the present disclosure provides a human GCGR antibody, GLP-1 peptide and mutants thereof, and a bispecific protein formed by fusing the GCGR antibody and the GLP-1 peptide, and a preparation method thereof, which can be used for weight loss and treatment of diabetes.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980071999.X, filed on December 20, 2019, entitled "Bispecific Protein".

[0002] This application claims priority to patent application 201811573634.0, filed on December 21, 2018, and patent application 201811606887.3, filed on December 27, 2018, both of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to human GCGR antibody, GLP-1 peptide and its mutants, as well as bispecific proteins formed by the fusion of GCGR antibody and GLP-1 peptide, their preparation methods and applications. Background Technology

[0004] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0005] Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia, which is characterized by insulin secretion defects and / or insulin action disorders. Its pathogenesis is mainly the result of the combined action of insulin and glucagon.

[0006] GLP-1 is one of the most important hormones affecting insulin secretion, and along with glucagon, it originates from proinsulin. Proinsulin consists of approximately 158 amino acids, cleaved into different peptide chains at different sites. The biologically active GLP-1 in the human body mainly exists in two forms: GLP-1(7-36)amide and GLP-1(7-37). GLP-1 is secreted by L cells in the small intestine, primarily promoting insulin secretion in a glucose concentration-dependent manner, protecting pancreatic β cells, and inhibiting glucagon secretion to lower blood glucose levels. GLP-1 also inhibits gastric emptying and reduces appetite. Clinically, it can be used to treat type II diabetes and obesity. The naturally occurring active GLP-1 in the body has a very short half-life (less than 2 minutes) and is easily degraded by DPPIV enzymes, thus lacking clinical value.

[0007] Extending the half-life has always been a major focus in the development of GLP-1 drugs, and several GLP-1 agonists are currently on the market, such as dulaglutide and semaglutide. Although the efficacy of GLP-1 has been well-established, it also has many side effects, mainly manifested as gastrointestinal symptoms, hypoglycemia, pancreatitis, and kidney damage.

[0008] Glucagon acts in the opposite way to insulin, primarily raising blood glucose levels. Glucagon is a 29-amino acid peptide secreted by pancreatic α-cells. After binding to its receptor GCGR on the hepatocyte membrane, it mainly increases blood glucose by activating the downstream cAMP / PKA pathway, accelerating glycogenolysis, lipolysis, and gluconeogenesis.

[0009] Studies have found that GCGR gene knockout mice exhibit a series of phenotypes, including elevated GLP-1 levels, decreased hepatic glucose output, increased lipid metabolism, and decreased appetite. GCGR is one of the most promising targets for diabetes treatment, but the development of GCGR antagonist drugs is currently progressing slowly. REMD Biotherapeutics' REMD-477 is currently a cutting-edge GCGR monoclonal antibody drug, in Phase II clinical trials.

[0010] Existing technologies have disclosed GCGR antibodies in patents such as CN101589062A, CN101983208A, CN102482350A, CN103314011A, CN105189560A, CN107614695A, US20180273629A1, and WO2013059531A1, but there is still a need to provide new and highly effective GCGR antibodies and treatment methods for diabetes. Summary of the Invention

[0011] This disclosure provides a monoclonal antibody against GCGR or an antigen-binding fragment thereof. The antibody or its antigen-binding fragment has the ability to bind to human GCGR (or its contained antigenic epitopes).

[0012] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a combination of a heavy chain variable region and a light chain variable region selected from a) or b):

[0013] a) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 regions as shown in SEQ ID NO: 48, 49, and 50, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 regions as shown in SEQ ID NO: 51, 52, and 53, respectively; or

[0014] b) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 38, 39 and 54 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 55, 56 and 57 respectively.

[0015] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises any combination of heavy chain variable regions and light chain variable regions selected from i) to vi):

[0016] i) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 14, 15 and 16 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 17, 18 and 19 respectively.

[0017] ii) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 20, 21 and 22 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 23, 24 and 25 respectively;

[0018] iii) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 26, 27 and 28 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 29, 30 and 31 respectively.

[0019] iv) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 32, 33 and 34 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 35, 36 and 37 respectively.

[0020] v) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 regions as shown in SEQ ID NO: 38, 39, and 40, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 regions as shown in SEQ ID NO: 41, 42, and 43, respectively; or

[0021] vi) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 38, 39 and 44 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 45, 46 and 47 respectively.

[0022] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody or its antigen-binding fragment.

[0023] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment, the humanized antibody comprising a frame region derived from a human antibody or a frame region variant thereof, the frame region variant having up to 10 reversion mutations in the light chain frame region of the human antibody, and / or, the frame region variant having up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid reversion mutations in the heavy chain frame region of the human antibody.

[0024] In some implementations, the frame region variant includes a subset selected from:

[0025] aa) The light chain variable region contains one or more amino acid reversion mutations from 42G, 44V, 71Y, and 87F, and / or the heavy chain variable region contains one or more amino acid reversion mutations from 38K, 48I, 67A, 69F, 71A, 73P, 78A, and 93S; or

[0026] ab) The light chain variable region contains one or more amino acid reversion mutations from 38L, 44V, 59S, 70E, and 71Y, and / or the heavy chain variable region contains one or more amino acid reversion mutations from 38K, 48I, 66K, 67A, 69L, 73R, 78M, and 94S. Further, the location of the reversion mutation site is determined according to the Kabat numbering rules.

[0027] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 2, 61, 62, 63, and 64; and / or

[0028] Light chain variable regions with sequences as shown in any of SEQ ID NO: 3, 58, 59 and 60 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 3, 58, 59 and 60.

[0029] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO: 63 and a light chain variable region as shown in SEQ ID NO: 58.

[0030] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the sequence shown in SEQ ID NO:4 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:4 and / or a light chain variable region having the sequence shown in SEQ ID NO:5 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:5.

[0031] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the sequence shown in SEQ ID NO:6 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:6 and / or a light chain variable region having the sequence shown in SEQ ID NO:7 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:7.

[0032] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity as shown in any of SEQ ID NO: 8, 68, 69, 70, and 71, or any of SEQ ID NO: 8, 68, 69, 70, and 71, and / or a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity as shown in any of SEQ ID NO: 9, 65, 66, and 67, or ...

[0033] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO: 71 and a light chain variable region as shown in SEQ ID NO: 67.

[0034] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the sequence shown in SEQ ID NO:10 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:10 and / or a light chain variable region having the sequence shown in SEQ ID NO:11 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:11.

[0035] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the sequence shown in SEQ ID NO:12 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:12 and / or a light chain variable region having the sequence shown in SEQ ID NO:13 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:13.

[0036] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment, wherein the antibody is a full-length antibody, further includes an antibody constant region. Specifically, the heavy chain constant region of the antibody constant region is selected from the human IgG1, IgG2, IgG3 and IgG4 constant regions and their conventional variants, and the light chain constant region of the antibody constant region is selected from the human antibody κ and λ chain constant regions and their conventional variants, more preferably including the human antibody heavy chain constant region shown in SEQ ID NO:72 and the human light chain constant region shown in SEQ ID NO:73.

[0037] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0038] The heavy chain is shown in SEQ ID NO:74, 76 or 78 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is shown in SEQ ID NO:75 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0039] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0040] The heavy chain is shown in SEQ ID NO:74, 76 or 78 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is shown in SEQ ID NO:77 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0041] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0042] The heavy chain is shown in SEQ ID NO:74, 76 or 78 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is shown in SEQ ID NO:79 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0043] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0044] The heavy chain is the one shown in SEQ ID NO:80 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is the one shown in SEQ ID NO:81 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0045] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0046] The heavy chain is the one shown in SEQ ID NO:82 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is the one shown in SEQ ID NO:83 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0047] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0048] The heavy chain is the one shown in SEQ ID NO:84 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is the one shown in SEQ ID NO:85 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0049] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0050] The heavy chain is the one shown in SEQ ID NO:86 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is the one shown in SEQ ID NO:87 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0051] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0052] The heavy chain is the one shown in SEQ ID NO:88 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, and the light chain is the one shown in SEQ ID NO:89 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0053] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO: 78 and a light chain as shown in SEQ ID NO: 79.

[0054] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO: 84 and a light chain as shown in SEQ ID NO: 85.

[0055] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment comprises any combination of heavy chain variable regions and light chain variable regions selected from ac) to ah) as follows:

[0056] ac) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 2, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions having the same sequence as the light chain variable region shown in SEQ ID NO: 3.

[0057] ad) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 4, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions having the same sequence as the light chain variable region shown in SEQ ID NO: 5.

[0058] ae) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions with the same sequence as the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions with the same sequence as the light chain variable region shown in SEQ ID NO: 7.

[0059] af) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions having the same sequence as the light chain variable region shown in SEQ ID NO: 9.

[0060] ag) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 10, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions having the same sequence as the light chain variable region shown in SEQ ID NO: 11; or

[0061] The heavy chain variable region (ah) includes HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 12, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions having the same sequence as the light chain variable region shown in SEQ ID NO: 13.

[0062] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody, dimerized V region (biantibody), and disulfide bond-stabilized V region (dsFv).

[0063] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment is characterized in that it competes with the monoclonal antibody or its antigen-binding fragment as described above for binding to human GCGR (or its epitope).

[0064] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment has at least one of the following characteristics:

[0065] i. Block the antagonistic activity of human GCGR binding to human glucagon at IC50 levels of less than 500 nM, less than 450 nM, less than 400 nM, less than 350 nM or less than 300 nM (preferably less than 300 nM);

[0066] ii. Block the antagonistic activity of GCGR binding between cynomolgus monkeys or rhesus monkeys and glucagon in cynomolgus monkeys or rhesus monkeys;

[0067] iii. Inhibits the rise in human blood glucose concentration;

[0068] iv. Block the antagonistic activity of mouse GCGR binding to mouse glucagon.

[0069] In some embodiments, the anti-GCGR monoclonal antibody or its antigen-binding fragment binds to the same antigenic epitope as described above.

[0070] On the other hand, this disclosure provides a bispecific protein. In some embodiments, a bispecific protein is provided comprising a GLP-1 peptide and a GCGR antibody, wherein the GLP-1 peptide and the polypeptide chain of the GCGR antibody are covalently linked by peptide bonds or linkers.

[0071] In some embodiments, the bispecific protein wherein the carboxyl terminus of the GLP-1 peptide is connected to the amino terminus of the heavy chain variable region of the GCGR antibody by a peptide bond or a linker, or the carboxyl terminus of the GLP-1 peptide is connected to the amino terminus of the light chain variable region of the GCGR antibody by a peptide bond or a linker.

[0072] In some embodiments, the bispecific protein wherein the carboxyl terminus of the GLP-1 peptide is linked to the amino terminus of the heavy chain variable region of the GCGR antibody by a peptide bond or a linker.

[0073] In some embodiments, the bispecific protein wherein the carboxyl terminus of the GLP-1 peptide is linked to the amino terminus of the light chain variable region of the GCGR antibody by a peptide bond or a linker.

[0074] In some embodiments, the bispecific protein wherein the carboxyl terminus of the GLP-1 peptide is linked to the heavy chain amino terminus of the full-length GCGR antibody by a peptide bond or a linker.

[0075] In some embodiments, the bispecific protein wherein the carboxyl terminus of the GLP-1 peptide is linked to the amino terminus of the light chain of the full-length GCGR antibody by a peptide bond or a linker.

[0076] In some embodiments, the bispecific protein, the GCGR antibody, is selected from the anti-GCGR monoclonal antibody as described above or its antigen-binding fragment.

[0077] In some embodiments, the bispecific protein, the GLP-1 peptide, is GLP-1A as shown in SEQ ID NO: 91, or the GLP-1 peptide is a GLP-1A peptide variant based on GLP-1A with one or more amino acid substitutions from Q17E, I23V, K28R, and G30R.

[0078] In some embodiments, the bispecific protein, the GLP-1 peptide, is GLP-1A as shown in SEQ ID NO: 91, or a GLP-1A peptide variant having Q17E on the basis of GLP-1A.

[0079] In some embodiments, the bispecific protein, the GLP-1 peptide is GLP-1A as shown in SEQ ID NO: 91, or the GLP-1 peptide is a GLP-1A peptide variant with a Q17E substitution and also has one or more amino acid substitutions from I23V, K28R and G30R.

[0080] In some embodiments, the bispecific protein, the GLP-1 peptide, is GLP-1A as shown in SEQ ID NO: 91, or the GLP-1 peptide is a GLP-1A peptide variant having Q17E or having Q17E and I23V.

[0081] In some embodiments, the bispecific protein, the GLP-1A peptide variant, comprises or is composed of sequences as shown in SEQ ID NO: 92, 93, 94, 95, 96, 97, 98 or 99.

[0082] In some embodiments, the bispecific protein, the GCGR antibody, is selected from monoclonal antibodies against GCGR as described in any of the preceding claims or antigen-binding fragments thereof, and GLP-1A peptides or GLP-1A peptide variants as described in any of the preceding claims.

[0083] In some embodiments, the bispecific protein has a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain is selected from any of the polypeptides shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107 and 108, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 79.

[0084] In some embodiments, the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 109, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 81;

[0085] In some embodiments, the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 110, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 83;

[0086] In some embodiments, the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 111, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 85;

[0087] In some embodiments, the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 112, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 87; or

[0088] In some embodiments, the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein the first polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 113, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 89.

[0089] On the other hand, this disclosure also provides a GLP-1 peptide variant. In some embodiments, the GLP-1 peptide variant is a mutant based on GLP-1A shown in SEQ ID NO: 91, having one or more amino acid mutations in Q17E, I23V, K28R, and G30R.

[0090] In some embodiments, the GLP-1 peptide is GLP-1A as shown in SEQ ID NO: 91, or the GLP-1 peptide is a GLP-1A peptide variant having Q17E or having Q17E and I23V.

[0091] In some embodiments, the GLP-1 peptide variants have sequences as shown in SEQ ID NO: 92, 93, 94, 95, 96, 97, 98 or 99.

[0092] This disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of the anti-GCGR monoclonal antibody or its antigen-binding fragment as described above, or the bispecific protein as described above, or the GLP-1 peptide variant as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0093] This disclosure also provides an isolated nucleic acid molecule encoding a monoclonal antibody against GCGR as described above, or an antigen-binding fragment thereof, or a bispecific protein as described above, or a GLP-1 peptide variant as described above.

[0094] This disclosure provides a recombinant vector comprising the isolated nucleic acid molecules as described above.

[0095] This disclosure provides a host cell transformed with the recombinant vector as described above, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells or insect cells.

[0096] This disclosure provides a method for producing a monoclonal antibody against GCGR as described above, or an antigen-binding fragment thereof, or a bispecific protein as described above, or a GLP-1 peptide variant as described above, the method comprising culturing host cells as described above in a culture medium to form and accumulate a monoclonal antibody against GCGR as described above, or an antigen-binding fragment thereof, or a bispecific protein as described above, or a GLP-1 peptide variant as described above, and recovering the monoclonal antibody or its antigen-binding fragment or bispecific protein or GLP-1 peptide variant from the culture.

[0097] This disclosure provides a method for detecting or determining human GCGR in vitro, the method comprising using a monoclonal antibody or an antigen-binding fragment thereof as described above.

[0098] A kit for detecting human GCGR, comprising a monoclonal antibody or its antigen-binding fragment as described above.

[0099] According to some embodiments, the use of the aforementioned anti-GCGR monoclonal antibody or its antigen-binding fragment thereof in the preparation of medical devices (e.g., kits, arrays, test strips, multi-well plates, magnetic beads, coated microparticles) is also provided, said medical devices comprising the aforementioned monoclonal antibody or its antigen-binding fragment. As an example, a kit comprises a multi-well plate coated with the aforementioned anti-GCGR monoclonal antibody or its antigen-binding fragment.

[0100] This disclosure provides the use of the monoclonal antibody or antigen-binding fragment thereof as described above in the preparation of reagents for the detection or determination of human GCGR.

[0101] This disclosure provides a method for reducing blood glucose concentration in a subject, the method comprising administering to the subject a therapeutically effective amount of a monoclonal antibody against GCGR as described above or an antigen-binding fragment thereof, or a bispecific protein as described above, or a GLP-1 peptide variant as described above, or a pharmaceutical composition as described above.

[0102] Preferably, the therapeutically effective amount is 0.1-3000 mg of the anti-GCGR monoclonal antibody or its antigen-binding fragment as described above, or the bispecific protein as described above, or according to the GLP-1 peptide variant as described above, per unit dose of the composition.

[0103] This disclosure provides a method for treating metabolic disorders, the method comprising administering to a subject a monoclonal antibody against GCGR as described above or an antigen-binding fragment thereof, or a bispecific protein as described above, or a GLP-1 peptide variant as described above, or a pharmaceutical composition as described above; preferably, the metabolic disorder is metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting glucose, dyslipidemia, atherosclerosis, or prediabetes.

[0104] This disclosure also provides the use of a monoclonal antibody against GCGR as described above, or an antigen-binding fragment thereof, or a bispecific protein as described above, or a GLP-1 peptide variant as described above, or a pharmaceutical composition as described above, in the preparation of a medicament for treating metabolic disorders or reducing blood glucose concentration in a subject.

[0105] Preferably, the metabolic disorder is metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting glucose, dyslipidemia, atherosclerosis, or prediabetes.

[0106] This disclosure also provides the use of the anti-GCGR monoclonal antibody or its antigen-binding fragment as described above, or the bispecific protein as described above, or the GLP-1 peptide variant as described above, or the pharmaceutical composition as described above, as a medicament, preferably as a medicament for treating metabolic disorders or reducing blood glucose concentration in a subject.

[0107] More preferably, the metabolic disorder is metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting glucose, dyslipidemia, atherosclerosis, or prediabetes. Attached Figure Description

[0108] Figure 1 : A schematic diagram of the structure of the bispecific protein (GLP-1 / GCGR antibody) disclosed herein.

[0109] Figure 2 : The antagonistic activity of bispecific proteins and GCGR antibodies against GCGR.

[0110] Figure 3 The activation activity of bispecific proteins and dulaglutide on GLP-1R.

[0111] Figure 4The effect of long-term drug use on random blood glucose in ob / ob mice. Vehicle (empty vector) was used as the model control group injected with phosphate-buffered saline (PBS). hu1803-9D-3mpk, hu1803-9-2.84mpk, and dulaglutide-1.16mpk were the same molar concentrations.

[0112] Figure 5 Effects of long-term drug administration on fasting blood glucose in ob / ob mice. All experimental groups significantly reduced fasting blood glucose concentration in mice, with hu1803-9D-3mpk and hu1803-9-2.84mpk showing the strongest hypoglycemic effects, and hu1803-9D-3mpk exhibiting superior hypoglycemic activity compared to hu1803-9-2.84mpk. Detailed Implementation

[0113] the term

[0114] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0115] The term "bispecific protein" refers to a protein molecule capable of binding to two target proteins or target antigens. In this disclosure, bispecific proteins specifically refer to proteins capable of binding to GCGR and GLP-1R (GLP-1 receptor), formed by the fusion of a polypeptide chain of a GLP-1 peptide and a GCGR antibody (or its antigen-binding fragment).

[0116] "GLP-1 peptide" refers to a peptide that can bind to and activate the GLP-1 receptor. The peptides described in the prior art are found in patent applications WO2008 / 071972, WO2008 / 101017, WO2009 / 155258, WO2010 / 096052, WO2010 / 096142, WO2011 / 075393, WO2008 / 152403, WO2010 / 070251, WO2010 / 070252, WO2010 / 070253, WO2010 / 070255, WO2011 / 160630, WO2011 / 006497, WO2011 / 117415, WO2011 / 117416, WO2006 / 134340, WO1997046584, and WO200712. The contents of 4461, WO2017100107, WO2007039140, CN1935261A, CN1935846A, WO2006036834, WO2005058958, WO2002046227, WO1999043705, WO1999043708, WO1999043341, CN102949730, CN104293834, CN104327187, WO2015067716, WO2015049651, WO2014096145, WO2014096148, WO2014096150, and WO2014096149 are incorporated herein by reference.This includes GLP-1, GLP-1 analogs, and GLP-1 receptor peptide agonists. Some specific GLP-1 peptides include: Lixisenatide / AVE0010 / ZP10 / Lyxumia, Exenatide / Byetta / Bydureon / ITCA 650 / AC-2993, Liraglutide / Victoza, Semaglutide, Taspoglutide, Syncria / Albiglutide, Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langennatide / HM-11260C, CM-3, GLP-1Eligen, ORMD-0901, NN-9924, NN-9 In addition to the aforementioned GLP-1 peptides, the present disclosure also includes GLP-1A and its mutants (as shown in SEQ ID NO: 92-999) based on SEQ ID NO: 91.

[0117] GPCRs (G Protein-Coupled Receptors) are a class of transmembrane proteins expressed on the cell membrane. Composed of over 800 members, GPCRs constitute the largest known family of membrane proteins in the mammalian genome. In the human body, GPCR proteins are widely distributed in organs and tissues such as the central nervous system, immune system, cardiovascular system, and retina, participating in the body's development and normal function.

[0118] The GPCR protein consists of seven transmembrane α-helical segments. The N-terminus and three loops are located extracellularly and participate in the interaction between the protein and its receptor; the C-terminus and three loops are located intracellularly, with the C-terminus and the third loop playing an important role in the interaction between the GPCR protein and downstream G proteins, thereby mediating intracellular signal transduction.

[0119] "GCGR" is the glucagon receptor, a member of the GPCR family. After glucagon binds to GCGR, it mainly accelerates glycogenolysis, lipolysis and / or gluconeogenesis by activating downstream pathways, thereby raising blood glucose levels.

[0120] The term "antibody (Ab)" refers to an antigen-binding molecule (or molecular complex) that contains at least one complementary-determining region that specifically binds to or interacts with (e.g., recognizes and / or binds to) a specific antigen (or its epitope) (e.g., GCGR).

[0121] The term "antibody" encompasses an immunoglobulin molecule consisting of four polypeptide chains linked by disulfide bonds, two heavy (H) chains, and two light (L) chains, as well as its polymers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region (CH). This heavy chain constant region contains three regions (domains): CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0122] In various embodiments of this disclosure, the FR of the anti-GCGR antibody (or its antigen-binding fragment) may be identical to the human germline sequence or may be naturally or artificially modified. The antibody may be a different subclass of antibody, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subclasses), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0123] The term "antibody" also includes the antigen-binding fragment of a complete antibody molecule.

[0124] The terms "antigen-binding moiety," "antigen-binding domain," and "antigen-binding fragment," as used herein, encompass any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody may be derived from, for example, a whole antibody molecule using any suitable standard technique, such as protein hydrolysis or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (if desired) constant regions of the antibody. This DNA is known and / or readily available from, for example, commercially available sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthesized. This DNA may be chemically or through the use of molecular biotechnologies to sequence and manipulate, for example, by arranging one or more variable and / or constant regions into a suitable configuration, or by introducing codons, generating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0125] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; and (vi) dAb fragments. Other engineered molecules, such as region-specific antibodies, single-domain antibodies, region-deleted antibodies, chimeric antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and squalane variable IgNAR regions, are also included in the term "antigen-binding fragment" as used herein.

[0126] Antigen-binding fragments typically contain at least one variable region. The variable region can be of any size or amino acid composition and generally contains a CDR adjacent to or within one or more frame sequences.

[0127] In some embodiments, the antigen-binding fragment of the antibody is configured with either variable or constant regions that can be directly linked to each other or linked by complete or partial hinge or linker regions. The hinge region may consist of at least two amino acids (e.g., 5, 10, 15, 20, 40, 60, or more), creating flexible and semi-flexible links between adjacent variable and / or constant regions within a single polypeptide molecule.

[0128] In this disclosure, "mouse-derived antibody" refers to a monoclonal antibody derived from a mouse or rat and prepared in accordance with the knowledge and skills in the art. Preparation involves injecting an antigen into the test subject, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. When the injected test subject is a mouse, the resulting antibody is a mouse-derived antibody; when the injected test subject is a rat, the resulting antibody is a rat-derived antibody.

[0129] A chimeric antibody is an antibody formed by fusing the variable region of a first-species (e.g., mouse) antibody with the constant region of a second-species (e.g., human) antibody. To create a chimeric antibody, a hybridoma that secretes the first-species (e.g., mouse) monoclonal antibody is first established. Then, the variable region gene is cloned from the hybridoma cells. Next, the constant region gene of the second-species (e.g., human) antibody is cloned as needed. The variable region gene of the first species and the constant region gene of the second species are then linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0130] In a preferred embodiment of this disclosure, the light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The heavy chain of the chimeric antibody further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprising a human IgG1, IgG2, or IgG4 heavy chain constant region, or using an IgG1, IgG2, or IgG4 heavy chain constant region variant with an amino acid mutation (such as a YTE mutation or reversion mutation, L234A and / or L235A mutation, or S228P mutation).

[0131] The term "humanized antibody," including CDR-grafted antibodies, refers to antibodies created by grafting the CDR sequence of an animal-derived antibody, such as a murine antibody, into the variable region (or framework region) of a human antibody. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies due to the presence of numerous heterologous protein components. Sequences of such framework regions can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available at http: / / www.vbase2.org / ) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition.

[0132] To avoid a decrease in activity due to reduced immunogenicity, minimal reverse or reversion mutations can be performed on the variable region framework sequence of the human antibody to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display for affinity maturation of the CDR.

[0133] Due to contact residues with the antigen, CDR transplantation can lead to a decrease in the affinity of the resulting antibody or its antigen-binding fragment for the antigen due to the framework residues in contact with the antigen. Such interactions may be a result of high somatic mutations. Therefore, it may still be necessary to transplant such donor framework amino acids into the framework of humanized antibodies. The amino acid residues involved in antigen binding from non-human antibodies or their antigen-binding fragments can be identified by examining the variable region sequence and structure of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the species can be considered relevant. If the closest species cannot be determined, the sequence can be compared with a subclass common sequence or a common sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues are thought to be a result of high somatic mutations, thus playing an important role in binding.

[0134] In one embodiment of this disclosure, the antibody or its antigen-binding fragment may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0135] "Conventional variants" of the human antibody heavy chain constant region and human antibody light chain constant region refer to variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region, as disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region. Specific substitutions include YTE mutations, L234A and / or L235A mutations, or S228P mutations known in the prior art, or mutations that obtain a knock-in-hole structure (resulting in the antibody heavy chain having a knock-Fc and hole-Fc combination). These mutations have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0136] "Human antibody" and "human-derived antibody" are used interchangeably. A human antibody can be derived from a human or from a transgenic organism "modified" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, elements of human heavy and light chain loci are introduced into cell lines derived from embryonic stem cell lines, where endogenous heavy and light chain loci are targeted and disrupted. The transgenic organism can synthesize human antibodies specific to human antigens, and can be used to produce human antibody-secreting hybridomas. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0137] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or mutations generated during the manufacture of a monoclonal antibody preparation, which are typically present in small amounts), the individual antibodies constituting said population recognize and / or bind to the same epitopes. Unlike polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody preparation (formulation) targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the manufacture of the antibody by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, as well as other exemplary methods for preparing monoclonal antibodies described herein. In the term "monoclonal antibody or antigen-binding fragment thereof," monoclonal antibody refers to a full-length antibody.

[0138] The terms “full-length antibody,” “intact antibody,” “complete antibody,” and “all antibody” are used interchangeably in this document to refer to an antibody in essentially its complete form, as defined below as an antigen-binding fragment. Specifically, this term refers to antibodies in which the heavy chain, from the amino terminus to the carboxyl terminus, contains the VH, CH1, hinge, and Fc regions, respectively, and the light chain, from the amino terminus to the carboxyl terminus, contains the VL and CL regions, respectively.

[0139] Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. The antigen-binding moiety can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0140] The antigen-binding fragment can also be incorporated into a single-chain molecule containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10): 1057-1062; and US Patent US5641870).

[0141] Fab is an antibody fragment with a molecular weight of approximately 50,000 Da obtained by treating IgG antibody molecules with papain (which cleaves the amino acid residue at position 224 of the H chain). It has antigen-binding activity, in which approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0142] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000 Da obtained by digesting the portion below two disulfide bonds in the hinge region of IgG with pepsin. It has antigen-binding activity and is contained in two Fab regions connected at the hinge position.

[0143] Fab' is an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds to antigens with a reducing agent such as dithiothreitol.

[0144] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0145] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (including 1, 2, 3, or 4) (Holliger et al. (1993), Proc Natl Acad Sci USA. 90:6444-6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur J Immuno. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J Mol Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol Immunother. 50:51-59.

[0146] "Linker" or "connector" refers to a linker polypeptide sequence used to connect protein domains. It usually has a certain degree of flexibility, and the use of the linker will not cause the original function of the protein domain to be lost.

[0147] A diabody is an antibody fragment in which scFv is dimerized; it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0148] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (Protein Engineering. 7:697 (1994)).

[0149] In some embodiments of this disclosure, the antigen-binding fragment can be produced by the following steps: obtaining cDNA encoding the VH and / or VL of the monoclonal antibody that specifically recognizes and binds to the antigen, as well as other desired domains; constructing DNA encoding the antigen-binding fragment; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into a prokaryote or eukaryote to express the antigen-binding fragment.

[0150] The "Fc region" can be a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region in the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Residues in the Fc region are numbered using the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD., 1991. The Fc region of immunoglobulins typically has two constant region domains, CH2 and CH3.

[0151] "Knob-Fc" refers to the inclusion of a T366W point mutation in the Fc region of an antibody to form a knot-like spatial structure. Correspondingly, "hole-Fc" refers to the inclusion of T366S, L368A, and Y407V point mutations in the Fc region of an antibody to form a hole-like spatial structure. Due to steric hindrance, Knob-Fc and hole-Fc are more prone to heterodimer formation. To further promote heterodimer formation, S354C and Y349C point mutations can be introduced into Knob-Fc and hole-Fc, respectively, to further promote heterodimer formation through disulfide bonds. Simultaneously, to eliminate or weaken the ADCC effect induced by antibody Fc, substitution mutations of 234A and 235A can be introduced into the Fc. In bispecific antibodies, knock-Fc or hole-Fc can be used as the Fc region of either the first polypeptide chain or the second polypeptide chain. In the same bispecific antibody, the Fc regions of the first and second polypeptide chains are not simultaneously knock-Fc or hole-Fc.

[0152] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0153] The “variable region” of an antibody refers to the variable region (VL) of the antibody light chain alone or in combination, or the variable region (VH) of the antibody heavy chain. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held together tightly by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, a CDR may refer to a CDR determined by either method or a combination of both methods.

[0154] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0155] The terms "complementarity-determining region" and "CDR" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGapAlign procedure.

[0156] The phrase “any CDR variant” in “HCDR1, HCDR2 and HCDR3 regions or any CDR variant thereof” refers to a variant obtained by mutating any one, two or three HCDRs in the HCDR1, HCDR2 and HCDR3 regions using amino acid mutations.

[0157] "Antibody constant region domains" refer to the domains derived from the constant regions of the light and heavy chains of antibodies, including CL and CH1, CH2, CH3, and CH4 domains derived from different classes of antibodies.

[0158] An epitope, or antigenic determinant, is a site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0159] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -8 M, for example, approximately less than 10 -9 M, 10 - 10 M, 10 -11 M or lower affinity (KD) binding.

[0160] When the term "competition" is used in cases where antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) compete for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or its antigen-binding fragment) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen. Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor). Press); solid-phase direct labeling of RIA with I-125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves using a purified antigen (on a solid surface or cell surface) capable of binding to both an unlabeled detection antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the target antigen-binding protein. Typically, the target antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, wherein the two epitopes spatially interfere with each other's binding. Further details regarding methods for determining competitive binding are provided in embodiments of this disclosure. Typically, when an excess of a competing antigen-binding protein is present, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen.In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0161] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single epitope. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple amino acid sites via weak non-covalent forces; the greater the interaction, the stronger the affinity. As used herein, the term "high affinity" for an antibody or its antigen-binding fragment (e.g., the Fab fragment) generally refers to an antibody with a 1E affinity. -9 M or smaller K D (e.g., 1E) -10 M or smaller K D 1E -11 M or smaller K D 1E -12 M or smaller K D 1E -13 M or smaller K D 1E -14 M or smaller K D Antibody or antigen-binding fragments (etc.).

[0162] The term "KD" or "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, antibodies dissociate at a rate less than approximately 1E. -8 M, for example, less than approximately 1E -9 M, 1E -10 M or 1E -11 An antigen is bound by a dissociation equilibrium constant (KD) of M or smaller, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR) technology. The smaller the KD value, the greater the affinity.

[0163] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0164] The term "vector" refers to a construct capable of delivering one or more target genes or sequences and preferably expressing them in host cells. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0165] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, mice can be immunized with antigens or fragments thereof, and the resulting antibodies can be renatured, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this disclosure are prepared by adding one or more human FR regions to non-human CDR regions using genetic engineering methods. Human FR germline sequences can be obtained from the website http: / / www.imgt.org / by comparing with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0166] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), HEK293 cells (non-restrictive implementations such as HEK293E cells), and NSO cells.

[0167] Engineered antibodies or antigen-binding fragments can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As an alternative existing technology, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to the antigen. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified using conventional techniques, such as using a Sepharose FF column with adjusted buffer for protein A or protein G. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0168] "Administration," "dosage," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the provision of an exogenous drug, therapeutic agent, diagnostic agent, composition, or artificial operation (such as "euthanasia" in the examples) to an animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, and contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Giving" and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo treatment, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0169] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds described in this disclosure, to a subject who has (or is suspected of having, or is susceptible to) symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated subject or population in an amount that effectively relieves one or more disease symptoms to induce regression of such symptoms or inhibit the progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical testing method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may not be effective in alleviating symptoms of every target disease, they should reduce symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0170] "Conservative amino acid modification" or "conservative amino acid substitution" refers to the substitution of an amino acid in a protein or polypeptide with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity), thereby allowing for frequent alterations without changing the protein's or polypeptide's biological activity or other desired properties (e.g., antigen affinity and / or specificity). Those skilled in the art will recognize that, generally, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conserved amino acid substitutions are as follows:

[0171]

[0172]

[0173] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this disclosure or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of this disclosure in a subject.

[0174] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the circumstances.

[0175] "Endogenous" refers to substances that are produced in cells, organisms, or the human body, depending on the circumstances.

[0176] "Homology" and "identity" are used interchangeably in this document and refer to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of homology. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence.

[0177] The following references relate to the BLAST algorithm frequently used in sequence analysis: BLAST (BLASTALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0178] "Separated" should be changed to "detached from its original environment of existence," and in this case, it means that the specified molecule is substantially free of other non-target biomolecules. Generally, the term "separated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0179] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0180] "Pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0181] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0182] Specific examples of the term "metabolic disorder" include metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting glucose, dyslipidemia, atherosclerosis, or prediabetes.

[0183] Furthermore, this disclosure also relates to methods for immunodetection or determination of target antigens, reagents for immunodetection or determination of target antigens, methods for immunodetection or determination of cells expressing target antigens, and diagnostic agents for diagnosing diseases associated with target antigen-positive cells, comprising the monoclonal antibody or antibody fragment of this disclosure that specifically recognizes and binds to the target antigen as an active ingredient.

[0184] In this disclosure, the method for detecting or determining the amount of the target antigen can be any known method. For example, it includes immunoassay or assay methods.

[0185] Immunological detection or assay methods are methods that use labeled antigens or antibodies to detect or measure the amount of antibody or antigen. Examples of immunological detection or assay methods include radiolabeled antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0186] The aforementioned diseases associated with cells positive for the target antigen can be diagnosed by detecting or measuring cells expressing the target antigen using the monoclonal antibody or antibody fragment disclosed herein.

[0187] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0188] In this disclosure, there are no particular limitations on the live sample used for detecting or measuring the target antigen, as long as it has the potential to contain cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0189] Depending on the required diagnostic method, diagnostic reagents containing the monoclonal antibody or antibody fragment thereof of this disclosure may also contain reagents for performing an antigen-antibody reaction or for detecting the reaction. Reagents for performing the antigen-antibody reaction include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragment, or conjugates, and substrates corresponding to the labeled antibodies.

[0190] Details of one or more embodiments of the invention are set forth in the foregoing description. While the invention may be practiced or tested using any methods and materials similar to or the same as those described herein, preferred methods and materials are described below. Other features, objects, and advantages of the invention will become apparent from the description and claims. In the description and claims, the singular form includes plural references unless clearly indicated otherwise in the context. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this invention pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of the invention more fully. These embodiments should not be construed in any way as limiting the scope of the invention, which is defined by the claims.

[0191] Example

[0192] Example 1: Preparation of GCGR antigen and antibody

[0193] 1.1 Antigen Construction and Screening

[0194] Human GCGR cDNA encoding the full-length 477-amino acid glucagon receptor was subcloned into an expression vector (e.g., pcDNA3.1) and transfected into CHO-K1 cells. After screening and single-cell cloning, single clones were selected for antigen expression and characterization studies based on receptor-based cell surface expression. Unless otherwise specified, all GCGR antigens below refer to human GCGR.

[0195] Full-length GCGR: Used to construct GCGR-overexpressing cell lines, or for use as an immunoassay antigen and subsequent assays.

[0196] MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQPWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPF (SEQ ID NO: 1)

[0197] 1.2 Purification of 2GCGR Hybridoma and Recombinant Antibody

[0198] (1) Separation and Purification of Hybridoma Supernatant / Protein G Affinity Chromatography

[0199] For the purification of mouse hybridoma supernatant, Protein G affinity chromatography is the preferred method. After centrifuging the cultured hybridomas, collect the supernatant and adjust its pH by adding 10-15% of 1M Tris-HCl (pH 8.0-8.5). Wash the Protein G column with 6M guanidine hydrochloride for 3-5 column volumes, followed by 3-5 column volumes of pure water. Equilibrate the column with 1×PBS (pH 7.4) buffer for 3-5 column volumes. Load the cell supernatant at a low flow rate, controlling the flow rate to maintain a retention time of approximately 1 min or longer. Wash the column with 1×PBS (pH 7.4) for 3-5 column volumes until the UV absorption returns to baseline. Elute the sample with 0.1M acetate / sodium acetate (pH 3.0) buffer, collecting the elution peak based on UV detection. Quickly adjust the pH of the eluted product to 5-6 with 1M Tris-HCl (pH 8.0) for temporary storage. The elution products can be replaced using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and replacement of the solution with the desired buffer system, or desalting using size exclusion columns such as G-25 to replace the desired buffer system, or removing aggregate components from the elution products using high-resolution size exclusion columns such as Superdex 200 to improve sample purity.

[0200] (2) Protein A affinity chromatography is used to extract Fc-tagged bispecific proteins or antibodies:

[0201] First, the cell culture supernatant expressing Fc bispecific protein or antibody is collected by high-speed centrifugation. The Protein A affinity column is washed with 3-5 column volumes of 6M guanidine hydrochloride, followed by 3-5 column volumes of pure water. The column is then equilibrated with 3-5 column volumes of 1×PBS (pH 7.4) buffer. Cell supernatant is loaded for binding at a low flow rate, controlling the flow rate to maintain a retention time of approximately 1 minute or longer. After binding, the column is washed with 3-5 column volumes of 1×PBS (pH 7.4) until the UV absorbance returns to baseline. Sample elution is performed using 0.1M acetate / sodium acetate (pH 3.0-3.5) buffer. The elution peak is collected based on UV detection. The elution product is then rapidly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0) for temporary storage. The elution products can be replaced using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and replacement of the solution with the desired buffer system, or desalting using size exclusion columns such as G-25 to replace the desired buffer system, or removing aggregate components from the elution products using high-resolution size exclusion columns such as Superdex 200 to improve sample purity.

[0202] Example 2: Preparation of anti-human GCGR monoclonal antibody

[0203] 2.1 Immunity

[0204] (1) Mouse immunization: Anti-human GCGR monoclonal antibody is produced by immunizing mice. SJL white mice, female, 6 - 8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., Animal Production License No.: SCXK(Beijing)2012 - 0001) are used in the experiment. Feeding environment: SPF level. After the mice are purchased, they are raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle adjustment, temperature 20 - 25 °C; humidity 40 - 60%. The mice that have adapted to the environment are immunized according to the following protocol. The immunization antigen is GCGR CHO-K1 stable transfected cells.

[0205] Immunization protocol: Use adjuvant Gold Adjuvant (Sigma Cat No.T2684) to pre-immunize mice, intraperitoneal (IP) injection of 0.1 ml / mouse (primary immunization). 15 minutes later, intraperitoneal (IP) injection of GCGR CHO-K1 stable transfected cells, 1E7 cells / mouse. The inoculation times are days 0, 14, 28, 42, 56, 70, 84, 98, 112. Blood is taken on days 35, 63, 91, and the antibody titer in the mouse serum is determined by ELISA method. After the 6th - 9th immunization, mice with high antibody titer in the serum and a titer tending to plateau are selected for splenocyte fusion. 3 days before splenocyte fusion, booster immunization is carried out by intraperitoneal (IP) injection of 1E7 cells / mouse of GCGR CHO-K1 stable transfected cells.

[0206] (2) Rat immunization: Use DNA (encoding full-length hGCGR, the coding sequence is shown in Genbank accession number: NM_000160) to immunize 6 - 8 week-old SD rats, and the antibody titer in the rat serum is determined by FACS method. After the 3rd - 4th immunization, rats with high antibody titer in the serum and a titer tending to plateau are selected for splenocyte fusion. 3 days before splenocyte fusion, booster immunization is carried out.

[0207] 2.2 Splenocyte fusion

[0208] Adopt an optimized PEG-mediated fusion procedure to fuse splenic lymphocytes with myeloma cells Sp2 / 0 cells ( CRL-8287 TMHybridoma cells were obtained by fusion. The fused hybridoma cells were resuspended at a density of 0.5-1E6 / ml in complete medium (IMEM medium containing 20% ​​FBS, 1×HAT, and 1×OPI), and seeded at 100 μl / well in 96-well plates. After incubation at 37°C and 5% CO2 for 3-4 days, 100 μl / well of HAT complete medium was added, and the plates were cultured for another 3-4 days until clones formed. The supernatant was removed, and 200 μl / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. The plates were then cultured at 37°C and 5% CO2 for 3 days before ELISA detection.

[0209] 2.3 Hybridoma cell screening

[0210] Based on the hybridoma cell growth density, the hybridoma culture supernatant was analyzed using a cell-binding ELISA method. Positive cells were promptly expanded, cryopreserved, and subjected to two to three subcloning processes until single-cell clones were obtained.

[0211] Each subcloned cell was also subjected to GCGR cell binding ELISA assay. Hybridoma clones were obtained through experimental screening, and antibodies were further prepared using serum-free cell culture. The antibodies were purified according to the purification examples for use in the test cases.

[0212] 2.4 Sequence determination of hybridoma-positive clones

[0213] The procedure for cloning sequences from positive hybridomas is as follows: Log-growing hybridoma cells were collected, and RNA was extracted using Trizol (Invitrogen, Cat No. 15596-018) according to the kit instructions. RNA was then cloned using PrimeScript. TM Reverse transcription was performed using a Reverse Transcriptase kit (Takara, Cat No. 2680A). The cDNA obtained from reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced.

[0214] Positive clones were obtained from the obtained DNA sequences, and antibodies were prepared using serum-free cell culture. The antibodies were purified according to the purification examples, and multiple rounds of screening were performed using cell-based GCGR binding blocking experiments in the test cases to obtain hybridoma clones 1803, 1805, 1808 and 1810 from mice, and hybridoma clones 1817 and 1822 from rats.

[0215] Table 1. Sequences of the light chain and heavy chain variable regions of GCGR antibody

[0216]

[0217]

[0218] Table 2. CDR sequence of Kabat numbering

[0219]

[0220]

[0221] Sequence alignment and computer simulation revealed high homology between the light and heavy chain CDR sequences of the mouse-derived antibodies m1803, m1805, m1808, and m1810, and high homology between the light and heavy chain CDR sequences of the rat-derived antibodies rat1817 and rat1822. Their shared sequences are shown in the table below.

[0222] Table 3. Common sequences of heavy chain and light chain CDR regions

[0223]

[0224]

[0225] Example 3: Humanization of mouse-derived anti-human GCGR antibody

[0226] Using MOE software, germline genes of the heavy and light chain variable regions of human antibodies were selected from the IMGT human antibody heavy and light chain variable regions, respectively, based on high homology with the murine antibody. The CDRs of the murine antibody were then transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, some amino acids in the backbone sequence were reverted to the amino acids corresponding to those in the murine antibody, thus obtaining the humanized anti-GCGR monoclonal antibody. The amino acid residues in the CDR region were determined and annotated using the Kabat numbering system.

[0227] The chimeric antibody is formed by linking the variable regions of the light and heavy chains of the mouse antibody with the constant regions of the light and heavy chains of the human antibody. The chimeric antibody corresponding to clone 1803 is named ch1803, and the same applies to other antibodies.

[0228] 3.1 Humanization of Hybridoma Clone 1803

[0229] (1) Selection of humanized framework for hybridoma clone 1803:

[0230] The humanized light chain templates for the murine antibody m1803 are IGKV1-39*01 and hjk4.1, and the humanized heavy chain templates are IGHV1-3*01 and hjh6.1. The humanized variable region sequence is as follows:

[0231] hu1803VH-CDR grafting: (SEQ ID NO: 61)

[0232]

[0233] hu1803VL-CDR grafting: (SEQ ID NO: 58)

[0234]

[0235] Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italicized parts in the sequence are FR sequences, and the underlined parts are CDR sequences.

[0236] (2) The selection of the humanized template and the design of the reverse mutation for hybridoma clone 1803 are shown in the table below:

[0237] Table 4. Template selection and reversal mutation

[0238]

[0239]

[0240] Note: P44V indicates that, according to the Kabat numbering system, the 44th P position of the Kabat number has been mutated back to V. Grafting represents the direct insertion of mouse antibody CDR into the human FR region sequence.

[0241] (3) The specific humanized sequence of hybridoma clone 1803 is as follows:

[0242] >hu1803_VL.1 (grafted from Hu1803VL-CDR): (SEQ ID NO: 58)

[0243]

[0244] >hu1803_VL.1A: (SEQ ID NO: 59)

[0245]

[0246] >hu1803_VL.1B: (SEQ ID NO: 60)

[0247]

[0248] >hu1803_VH.1 (grafted from Hu1803VH-CDR): (SEQ ID NO: 61)

[0249]

[0250] >hu1803_VH.1A: (SEQ ID NO: 62)

[0251]

[0252] >hu1803_VH.1B: (SEQ ID NO: 63)

[0253]

[0254] >hu1803_VH.1C: (SEQ ID NO: 64)

[0255]

[0256] (4) The humanized antibody light chain variable region and heavy chain variable region sequence combination of the antibody derived from hybridoma clone 1803 is as follows:

[0257] Table 5. Combinations of light and heavy chain variable regions of different humanized antibodies

[0258] hu1803_VL.1 hu1803_VL.1A hu1803_VL.1B hu1803_VH.1 hu1803-1 hu1803-5 hu1803-9 hu1803_VH.1A hu1803-2 hu1803-6 hu1803-10 hu1803_VH.1B hu1803-3 hu1803-7 hu1803-11 hu1803_VH.1C hu1803-4 hu1803-8 hu1803-12

[0259] The antibody light and heavy chain variable regions referred to by the antibody names in the table above can be linked to the antibody light and heavy chain constant regions respectively to form full-length antibodies. Unless otherwise specified in this disclosure, when forming full-length antibodies, the light chain variable region is linked to the Kappa chain constant region shown in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to the IgG4-AA shown in SEQ ID NO: 72 to form the antibody heavy chain.

[0260] 3.2 Humanization of Hybridoma Clone 1810

[0261] (1) Selection of humanized framework for hybridoma clone 1810:

[0262] The humanized light chain templates for the murine antibody m1810 are IGKV1-39*01 and hJK4.1, and the humanized heavy chain templates are IGHV1-69*02 and hJH4.1. The humanized variable region sequence is as follows:

[0263] Hu1810VH-CDR grafting: (SEQ ID NO: 68)

[0264]

[0265] Hu1810VL-CDR grafting: (SEQ ID NO: 65)

[0266]

[0267] Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italicized parts in the sequence are FR sequences, and the underlined parts are CDR sequences.

[0268] (2) The selection of the humanized template and the design of the reverse mutation for hybridoma clone 1810 are shown in the table below:

[0269] Table 6. Template selection and reversal mutation

[0270]

[0271] Note: P44V indicates that, according to the Kabat numbering system, the 44th P position of the Kabat number has been mutated back to V. Grafting represents the direct insertion of mouse antibody CDR into the human FR region sequence.

[0272] (3) The specific humanized sequence of hybridoma clone 1810 is as follows:

[0273] >hu1810_VL.1 (grafted from Hu1810VL-CDR): (SEQ ID NO: 65)

[0274]

[0275] >hu1810_VL.1A: (SEQ ID NO: 66)

[0276]

[0277] >hu1810_VL.1B: (SEQ ID NO: 67)

[0278]

[0279] >hu1810_VH.1 (grafted from Hu1810VH-CDR): (SEQ ID NO: 68)

[0280]

[0281] >hu1810_VH.1A: (SEQ ID NO: 69)

[0282]

[0283] >hu1810_VH.1B: (SEQ ID NO: 70)

[0284]

[0285] >hu1810_VH.1C: (SEQ ID NO: 71)

[0286]

[0287] (4) The humanized antibody light and heavy chain variable region sequence combination of hybridoma clone 1810 is as follows:

[0288] Table 7. Combinations of light and heavy chain variable regions of different humanized antibodies

[0289] hu1810_VL.1 hu1810_VL.1A hu1810_VL.1B hu1810_VH.1 hu1810-1 hu1810-5 hu1810-9 hu1810_VH.1A hu1810-2 hu1810-6 hu1810-10 hu1810_VH.1B hu1810-3 hu1810-7 hu1810-11 hu1810_VH.1C hu1810-4 hu1810-8 hu1810-12

[0290] The antibody light and heavy chain variable regions referred to by the antibody names in the table above can be linked to the antibody light and heavy chain constant regions to form full-length antibodies. Unless otherwise specified in this disclosure, when forming full-length antibodies, the light chain variable region is linked to the Kappa chain constant region shown in SEQ ID NO: 73 to form the antibody light chain, and the heavy chain variable region is linked to the IgG4-AA shown in SEQ ID NO: 72 to form the antibody heavy chain.

[0291] Example 4: Construction and expression of IgG4-AA form of GCGR chimeric / humanized antibody

[0292] Primers were designed, and PCR was used to construct the VH / VK gene fragments of each chimeric / humanized antibody. These fragments were then homologously recombinated with the expression vector pHr (containing a signal peptide and constant region gene (CH1-FC / CL) fragment) to construct the full-length antibody expression vectors VH-CH1-FC-pHr / VK-CL-pHr. The IgG4-AA antibody form can be obtained through simple point mutations in the IgG4 antibody form. IgG4-AA represents mutations in F234A, L235A, and S228P. Mutations in F234A and L235A reduce the binding affinity of IgG4-Fc to FcγR, further reducing ADCC / CDC. S228P represents a mutation from S to P at amino acid position 228 of the wild-type IgG4 hinge region; this mutation avoids mismatches caused by Fab-exchange in vivo with natural IgG4 antibodies.

[0293] ch1803, ch1805, ch1808, ch1810, ch1817 and ch1822 are chimeric antibodies formed by linking the variable regions of the light and heavy chains of the animals shown in Table 1 to the kappa chain of human antibody and the constant region of the heavy chain of human antibody IgG4-AA, respectively.

[0294] The heavy chain constant region sequence of IgG4-AA is as follows: (SEQ ID NO: 72)

[0295] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0296] The constant region sequence of the antibody's light chain (Kappa chain) is as follows: (SEQ ID NO: 73)

[0297] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0298] Examples of constructed antibody sequences are listed below:

[0299] ch1803: Antibody form IgG4AA

[0300] ch1803 heavy chain sequence: (SEQ ID NO: 74)

[0301] QFQLHQSGAELVKPGASVKLSCKATGYTFTDYWIEWVKQRPGHGLEWIGEILPGSTYTNYNEKFKGRATFTAEPSSSSAYMQLSGLTTEDSAIYYCSRGLSTLMAVDYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0302] ch1803 light chain sequence: (SEQ ID NO: 75)

[0303] DIQMTQTTSSLSASLGDRVTINCRASQDISNYLNWYQQKPDGTVKLLIYYSSTLHSGVPSRFSGSGSGTDYSLTISHLEQEDIATYFCQQTNIFPWTFGGGTKLEIRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0304] hu1803-1: Antibody form IgG4AA

[0305] hu1803-1 heavy chain sequence:: (SEQ ID NO: 76)

[0306] EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWIEWVRQAPGQGLEWMGEILPGSTYTNYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLSTLMAVDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0307] The light chain sequence of hu1803-1: (SEQ ID NO: 77)

[0308] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYSSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNIFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0309] The heavy chain sequence of hu1803-9: (SEQ ID NO: 78)

[0310] EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWIEWVRQAPGQGLEWMGEILPGSTYTNYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLSTLMAVDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0311] hu1803-9 light chain sequence: (SEQ ID NO: 79)

[0312] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYSSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQTNIFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0313] ch1805 heavy chain sequence: (SEQ ID NO: 80)

[0314] EVQLQQSGPELVKPGASVKIPCKTSGYTFT DYNMD WVKQSHGRSLEWIG SIDPDNGGTIYNQKFKG KATLTVDKSSSTAYMELRSLTSEDTAVYYCTR DYYGSSSWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0315] Light chain sequence of ch1805: (SEQ ID NO: 81)

[0316] DVVMTQSPATLSVTPGDRVSLSC RASQSISDYLH WYQQKSHESPRLLIK YASQSIS GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC QNGHSFPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; Heavy chain sequence of ch1808: (SEQ ID NO: 82)

[0317] QVQLQQSGAELARPGASVKLSCKASGDTFT TNGIS WVKQRIGQGLEWIG EIYPRSGNTYYNENFKG KATLTADKSSTTAYMELRRLTSEDSAVYFCAR SITSVIGADYFDY WGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0318] ch1808 light chain sequence: (SEQ ID NO: 83)

[0319] DIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQKKPDGTVKLLIY YSSTLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTFPWT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0320] hu1810-12 heavy chain sequence: (SEQ ID NO: 84)

[0321]

[0322] hu1810-12 light chain sequence: (SEQ ID NO: 85)

[0323]

[0324] ch1817 heavy chain sequence: (SEQ ID NO: 86)

[0325] EVQLVESGGDLVQPGRSMKLSCAASGFTFS NYYMA WVRQAPTKGLEWVA SISTGGVNTYYRDSVKG RFTISRDNAKNNLYLQMDSLRSEETATYYCAR HTTADYFYGIYFALDAWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0326] Light chain sequence of ch1817: (SEQ ID NO: 87)

[0327] QFTLTQPKSVSGSLRSTITIPC ERSSGDIGDSYVN WYQQHLGRPPLNVIY ADVQRPS EVSDRFSGSIDSSSNSASLTITNLQMDDEADYFC QSYDTNIDII FGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0328] Heavy chain sequence of ch1822: (SEQ ID NO: 88)

[0329] EVRLVESGGDFVQPGRSVKLSCAASGFTFS NYYMA WVRQAPTKGLEWVG SISTGGVNTYYRDSVKG RFTISRDNAESTLYLQMDSLRSEETATYYCAR HTTPDYHYGIYFAMDA WGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0330] ch1822 light chain sequence: (SEQ ID NO: 89)

[0331] QVTLTQPKSVSGSLRSTITIPC ERSSGDIGESYVN WYQQHLGRPPINVIY ADDQRPS EVSDRFSGSIDSSSNSASLTITNLQVDDEADYFC QSYDSSIDIF FGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0332] Positive control: Dulaglutide is in double-chain form, with the single chain being GLP-1 / hIgG4 Fc (SEQ ID NO: 90).

[0333] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0334] Example 5: Cloning and expression of antibody bispecific protein

[0335] Human GLP-1 peptide was used as the GLP-1 receptor agonist part of the bispecific protein, and GCGR antibody was used as the GCGR antagonist part of the bispecific protein to form a GLP-1 / GCGR antibody bispecific protein.

[0336] Studies have found that specific amino acid mutations at certain sites in GLP-1 (such as Q17E, I23V, K28R, or G30R) result in novel GLP-1 / GCGR antibody bispecific proteins with higher in vitro stability. The highest stability was observed when GLP-1A was mutated from Q at position 17 to E and from I at position 23 to V (GLP-1C). The non-limiting sequences of GLP-1 and its mutant forms disclosed in this invention are as follows:

[0337] Table 8. Sequences of GLP-1A peptides and their variants

[0338]

[0339] Using homologous recombination technology, the C-terminal amino acid of the disclosed GLP-1 peptide was linked to the N-terminal amino acid of the GCGR antibody heavy chain via a peptide bond or linker. Conventional expression using the 293 expression system yielded the bispecific protein pattern structure shown in Table 9.

[0340] Table 9. Structural patterns of GLP-1 / GCGR dual-specific proteins

[0341]

[0342]

[0343] *Note: Ab refers to the GCGR antibody described in this disclosure. The GLP-1 peptide can be linked to the N-terminus of the heavy chain variable region or the N-terminus of the light chain variable region of the GCGR antibody. Experimental verification has shown that the bispecific protein with the GLP-1 peptide linked to the N-terminus of the heavy chain variable region of the GCGR antibody exhibits better stability than that linked to the N-terminus of the light chain variable region of the GCGR antibody. A schematic diagram of the bispecific protein structure in some embodiments of this disclosure, in which the GLP-1 peptide is linked to the heavy chain variable region of the full-length GCGR antibody, is shown below. Figure 1 As shown.

[0344] Different GLP-1 peptides are linked to the heavy chain amino acids of different antibodies using linkers (e.g., (GGGGS)3) to form the following proteins:

[0345] Table 10. Bispecific protein sequences

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352] The adapter in this bispecific protein can be (GGGGS)3, but in other embodiments it can also be a peptide bond or other adapters commonly used for peptide linking. The use of (GGGGS)3 is not a limitation on the bispecific protein adapter of this disclosure. The nucleotide sequences encoding GLP-1, the nucleotide sequences encoding the GCGR antibody, and the nucleotide sequences of the adapter protein fragment ((GGGGS)3) were obtained using conventional techniques in the art. The C-terminal nucleotide of GLP-1 was linked to the N-terminal nucleotide of the GCGR antibody via the adapter protein using homologous recombination technology and cloned into the Phr-BsmbI vector. The recombinant GLP-1 / GCGR antibody bispecific protein was expressed in 293 cells and purified using the method of Example 6. The purified protein can be used in the following examples.

[0353] Example 6: Purification of Bispecific Antibody Protein

[0354] Cell culture medium was centrifuged at high speed, and the supernatant was collected for the first step of purification using affinity chromatography. The chromatography medium was Protein A or a derivative of Fc, such as GE's Mabselect. The equilibration buffer was 1×PBS (137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na₂HPO₄, 2 mmol / L KH₂PO₄, pH 7.4). After equilibration to 5 column volumes, the cell supernatant was loaded, with the flow rate controlled so that the sample retention time on the column was ≥1 min. After loading, the column was washed with 1×PBS (pH 7.4) until the A280 UV absorbance dropped to baseline. The column was then washed with 0.1 M glycine (pH 3.0) elution buffer, and the elution peak was collected based on the A280 UV absorption peak. The collected eluted sample was neutralized with 1 M Tris (pH 8.5).

[0355] The neutralized eluted sample was concentrated by ultrafiltration and then subjected to size exclusion chromatography. The buffer was 1×PBS, the chromatography column was an XK26 / 60 Superdex 200 (GE), the flow rate was controlled at 4 ml / min, and the sample loading volume was less than 5 ml. The target protein peak was combined based on A280 UV absorption. The collected protein was identified as having a purity greater than 95% by SEC-HPLC and confirmed to be correct by LC-MS before being aliquoted and used. The GLP-1 / GCGR antibody bispecific protein was obtained.

[0356] Test case

[0357] Test Example 1: ELISA experiment of GCGR chimeric antibody binding to human, mouse and cynomolgus monkey GCGR

[0358] The binding affinity of anti-GCGR antibodies was assessed using an antibody-CHO cell overexpressing GCGR. Human, mouse, and cynomolgus monkey full-length GCGR plasmids were transfected into CHO cells, and after two weeks of pressure selection, GCGR expression levels were measured. Overexpressing cells were fixed at the bottom of 96-well plates, and the intensity of the signal after antibody addition was used to determine the binding activity between the antibody and GCGR-overexpressing CHO cells. The GCGR antibody binding assay was performed using the same method for all three genera. Taking the assay for GCGR antibody binding to human GCGR as an example, the specific experimental method is as follows:

[0359] Cells were sputtered at a concentration of 0.9–1.0 × 10⁻⁶. 6 At a density of 1 / ml, 100 μl / well was seeded into 96-well plates and cultured overnight. The supernatant was discarded, and the plates were washed three times with PBS. Then, 100 μl / well of cell immunofixation solution (Beyotime, Cat Wo. P0098) was added and the plates were fixed at room temperature for 1 hour, followed by four washes with PBS. After discarding the liquid, 200 μl / well of blocking buffer (5% skim milk diluted with PBS, Cat Wo. 232100) was added and the plates were incubated at 37°C for 3 hours for blocking. After blocking, the blocking buffer was discarded, and the plates were washed three times with PBST buffer (pH 7.4, PBS containing 0.05% TweeeW-20). Then, 50 μl / well of different concentrations of the test antibody (hybridoma purified antibody, chimeric antibody, or humanized antibody) diluted with sample diluent was added and the plates were incubated at 37°C for 2 hours. After incubation, wash the plate three times with PBST, add 50 μl / well of HRP-labeled goat anti-mouse secondary antibody (JacksoWImmuWo Research, CatWo.115-035-003) or goat anti-human secondary antibody (JacksoWImmuWo Research, CatWo.109-035-003) diluted with sample dilution buffer, and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 50 μl / well of TMB chromogenic substrate (KPL, CatWo.52-00-03), incubate at room temperature for 10 min, and stop the reaction by adding 50 μl / well of 1M H2SO4. Read the absorbance at 450 nm using a Thermoscientific Multiskan MK3 microplate reader, analyze the data using GraphPad Prism 5, and calculate the EC50 value of GCGR chimeric antibody binding to GCGR-overexpressing CHO cells. The results are shown in the table below.

[0360] Table 11. Chimeric antibody binding activity

[0361]

[0362]

[0363] The results showed that all chimeric antibodies ch1803, ch1805, ch1808, ch1810, ch1817 and ch1822 had good cell surface binding activity with human GCGR, and also had good cross-affinity activity with mouse GCGR and cynomolgus monkey GCGR.

[0364] Test Example 2: Assay for blocking GCGR ligand binding to GCGR with GCGR chimeric antibody

[0365] 1. Purpose of the test:

[0366] The antagonistic activity of GCGR chimeric antibodies was evaluated by blocking the binding of GCGR ligand glucagon to GCGR.

[0367] 2. Test principle:

[0368] cAMP binding to CRE initiates the expression of the downstream luciferase gene. Luciferase emits fluorescence upon binding to its substrate, and changes in fluorescence signal reflect the inhibition efficiency. CRE was cloned upstream of the luciferase gene, and CHO-K1 cells were co-transfected with a plasmid containing the GCGR gene to select monoclonal cells that simultaneously highly expressed both CRE and GCGR. The GLP-1 / GCGR antibody bispecific protein and glucagon competitively bind to GCGR, blocking downstream signal transduction and affecting downstream cAMP expression. Changes in fluorescence signal can be used to assess the antagonistic activity of the GLP-1 / GCGR antibody bispecific protein against GCGR.

[0369] 3. Test Samples:

[0370] Chimeric antibodies ch1803, ch1805, ch1808, ch1810, ch1817, ch1822.

[0371] 4. Experimental steps:

[0372] a. Prepare a cell suspension using fresh cell culture medium, add 20,000 cells / well to a 96-well cell culture plate with 80 μl of culture system, and incubate at 37°C for 16 hours with 5% carbon dioxide.

[0373] b. Add 10 μl of the prepared test protein to each well, then add 10 μl of the prepared glucagon, 5% carbon dioxide, and incubate at 37°C for 5 hours.

[0374] c. Add 100 μl of ONE Glo (Promega) detection solution to each well and incubate at room temperature in the dark for 7 minutes.

[0375] d. Fluorescence was detected on a Victor3 microplate reader, and the IC50 value and blocking efficiency (Imax) of the GCGR chimeric antibody fusion against the binding of human, mouse, and cynomolgus monkey GCGR ligand glucagon were calculated.

[0376] Table 12. Antagonistic Activity of Chimeric Antibodies

[0377]

[0378]

[0379] Test Example 3: Blocking Assay of GCGR Humanized Antibody on the Binding of GCGR Ligand to GCGR

[0380] The antagonistic activity of GCGR antibodies was evaluated by blocking the binding of GCGR ligand glucagon to GCGR using anti-GCGR antibodies. The experimental principle and procedure were the same as in Test Example 2, and the results are shown in the table below:

[0381] Table 13. Anti-antagonistic activity of humanized antibodies

[0382]

[0383] In vitro bioactivity evaluation

[0384] Test Example 4: Stability of GLP-1 / GCGR antibody bispecific protein in PBS

[0385] 200 μg of the bispecific protein of the test antibody was dissolved in 1 ml of 1×PBS (pH 7.4) and stored at 37°C. Samples were taken at 0 and 14 days, and the retention of the intact heavy chain was detected by LC-MS using an Agilent 6530Q-TOF analyzer. The results are shown in Table 14 below. The bispecific proteins containing each mutant GLP-1 peptide showed significantly improved stability compared to the bispecific protein containing GLP-1A (SEQ ID NO: 91), and hu1803-9B, hu1803-9D, and hu1803-9G showed even better stability.

[0386] Table 14. Stability detection of GLP-1 / GCGR antibody bispecific protein

[0387]

[0388]

[0389] Test Example 5: Cell-based GCGR binding blocking experiment

[0390] The experimental principle and procedure are the same as in Test Example 2.

[0391] Test sample:

[0392] ①GCGR antibodies (hu1803-9, hu1810-12)

[0393] ② Bispecific proteins hu1803-9B and hu1803-9D.

[0394] Table 15. Antagonistic activity of bispecific proteins against GCGR

[0395]

[0396] Table 16. Antagonistic activity of humanized antibodies against GCGR in mice and cynomolgus monkeys

[0397]

[0398] Figure 2 Table 15 shows that both hu1803-9B and hu1803-9D completely inhibited the antagonistic activity of GCGR, and had comparable efficacy and IC50 (the concentration required to inhibit 50% of the maximum activity) to GCGR monoclonal antibodies, indicating that hu1803-9B and hu1803-9D retained partially complete biological activity of the GCGR antibody. In addition to verifying that hu1803-9 and bispecific proteins containing hu1803-9 have antagonistic activity against human GCGR, further evidence (see Table 16) demonstrates that hu1803-9 and hu1810-12 have antagonistic activity against mouse GCGR and cynomolgus monkey GCGR.

[0399] Test Example 6: Cell-based GLP-1R binding activation experiment

[0400] 1. Purpose of the test:

[0401] To evaluate the activation activity of the GLP-1 moiety of the GLP-1 antibody bispecific protein on GLP-1R.

[0402] 2. Test principle:

[0403] cAMP binding to CRE initiates the expression of the downstream luciferase gene. Luciferase fluoresces upon binding to its substrate, and changes in fluorescence signal reflect the inhibition efficiency. CRE was cloned upstream of the luciferase gene, and CHO-K1 cells were co-transfected with a plasmid containing the GLP-1R gene to select monoclonal cells that simultaneously highly expressed both CRE and GLP-1R. The GLP-1 / GCGR antibody bispecific protein and the positive control dulaglutide can bind to GLP-1R, activating downstream GLP-1R signal transduction and stimulating downstream cAMP expression. Changes in fluorescence signal can be used to assess the activation activity of the GLP-1 / GCGR antibody bispecific protein on GLP-1R.

[0404] 3. Test Samples:

[0405] ① Positive control: dulaglutide

[0406] ②hu1803-9B, hu1803-9D.

[0407] 4. Experimental steps:

[0408] a. Prepare a cell suspension using fresh cell culture medium, add 25,000 cells / well to a 96-well cell culture plate containing 90 μl of culture system, and incubate at 37°C for 16 hours with 5% carbon dioxide.

[0409] b. Add 10 μl of the prepared test protein to each well, incubate at 37°C for 5 hours with 5% carbon dioxide.

[0410] c. Add 100 μl of ONE Glo (Promega) detection solution to each well and incubate at room temperature in the dark for 7 minutes.

[0411] d. Detect fluorescence on a Victor3 microplate reader and calculate the EC50 value of the activation of GLP-1R by the binding of the GLP-1 / GCGR antibody bispecific protein.

[0412] Table 17. Activation activity of bispecific proteins on GLP-1R

[0413] protein EC50(nM) Emax (%) Dulaglutide 0.21 100 hu1803-9B 0.22 100 hu1803-9D 0.32 100

[0414] Figure 3 Table 17 shows that both hu1803-9B and hu1803-9D can fully activate GLP-1R and have comparable efficacy and EC50 (the concentration required to activate 50% of the maximum activity) to the positive control dulaglutide, indicating that both hu1803-9B and hu1803-9D retain some of the complete biological activity of GLP-1.

[0415] Table 18. Activation activity of bispecific proteins on human GLP-1R

[0416] sample EC50(nM) Emax% ch1805-D 0.11 108 ch1808-D 0.14 107 ch1817-D 0.30 106

[0417] Table 18 shows that the chimeric antibodies ch1805-D, ch1808-D, and ch1817-D, when conjugated with the GLP1 peptide, can completely activate GLP-1R and have comparable efficacy and EC50 (the concentration required to activate 50% of the maximum activity) to the positive control dulaglutide. This indicates that the bispecific proteins formed by conjugating different GCGR antibodies with the GLP1 peptide in the manner disclosed herein do not affect the biological activity of the GLP1 peptide.

[0418] Pharmacokinetic evaluation

[0419] Test Example 7: Pharmacokinetic Detection in C57 Mice

[0420] Four female C57 mice were used in the experiment. They were kept under 12 / 12-hour light / dark cycles, maintained at a constant temperature of 24±3℃ and humidity of 50-60%, and had free access to food and water. They were purchased from JessJet Laboratory Animal Co., Ltd. On the day of the experiment, the C57 mice were injected intravenously with an equal molar dose of hu1803-9D and the positive control drug dulaglutide, respectively, at doses of 6 mg / kg and 2.35 mg / kg, with an injection volume of 10 ml / kg.

[0421] Because the GLP-1 / GCGR antibody bispecific protein and the positive control dulaglutide disclosed herein both exhibit cross-activity with mice, their metabolism time in mice is relatively short. The selected blood collection time points were: 0h, 1h, and 24h after administration on day 1 (day 2), and on day 3. Blood was collected from the fundus vein of mice, 150μl each time (1.5μl of DPP-4 inhibitor was added to the blood collection tube before collection). The collected blood samples were incubated at 4°C for half an hour until agglutination, and then centrifuged at 14000×g for 5 minutes at 4°C. The supernatant (approximately 80μl) was collected and immediately stored at -80°C.

[0422] The testing process is described as follows:

[0423] a. Plate 1 μg / mL anti-GLP1 (Novus, NBP1-05180) antibody and incubate overnight at 4°C.

[0424] b. Wash 250 μl of 1×PBST 4 times, add 200 μl of PBS containing 5% skim milk, and block at 37°C for 3 hours.

[0425] c. Wash 250 μl of 1×PBST 4 times, add 100 μl of serially diluted test drug in mouse blank serum, and incubate at 37°C for 2 hours.

[0426] Wash 3 times with 250 μl of 1×PBST.

[0427] e. Add 100 μl of horseradish peroxidase-labeled secondary antibody anti-human IgG Fc to each well and incubate at 37°C for 1 hour.

[0428] f. Wash 250 μl of 1×PBST three times.

[0429] g. Add 100 μl TMB to each well, incubate at room temperature for 10 minutes, and then add 100 μl 1M H2SO4 to terminate the reaction.

[0430] h. The absorbance at 450 nm was measured using an ELISA reader, and the data was analyzed using Graphpad Prism5.

[0431] Table 19. T1 / 2 of bispecific proteins in mice

[0432] test drug Administration method T1 / 2(h) hu1803-9D IV (6 mg / kg) 23.4 Dulaglutide IV (2.35 mg / kg) 10

[0433] PK analysis results showed that the half-life of the bispecific protein molecule hu1803-9D disclosed herein in mice was approximately 23.4 h, which is twice the half-life of the positive control drug.

[0434] In vivo bioactivity evaluation

[0435] Test Example 8: In vivo drug efficacy experiment in ob / ob mice

[0436] 1. The mouse strains used in this experiment were diabetic ob / ob mice and age-matched wild-type mice (Nanjing Institute of Model Animals, Nanjing University). The purpose was to observe the therapeutic effect of repeated administration of GLP-1 / GCGR antibody bispecific protein on diabetes-related indicators such as blood glucose, glycated hemoglobin, body weight, and food intake in ob / ob mice.

[0437] Before the experiment, the model group animals were divided into 6 groups according to their random and fasting body weight and random and fasting blood glucose on the day of the experiment, as follows:

[0438] The model control group, the GCGR monoclonal antibody group (hu1803-9) with a dose of 2.84 mg / kg and 1.42 mg / kg, the positive control group (dulaglutide) with a dose of 1.16 mg / kg, and the hu1803-9D group with a dose of 3 mg / kg and 1.5 mg / kg were administered phosphate-buffered saline (SC) subcutaneously. All groups of mice were administered SC once a week (9:00 AM) for a total of 4 weeks (Table 20).

[0439] Table 20. Trial Groups and Drug Administration

[0440] Group deal with dose Dosage frequency Administration method 1 ob / ob mouse model control group PBS Once a week for 4 weeks SC 2 GCGR monoclonal antibody low-dose group 1.42mpk Once a week for 4 weeks SC 3 GCGR monoclonal antibody high-dose group 2.84mpk Once a week for 4 weeks SC 4 Positive control high-dose group 1.16mpk Once a week for 4 weeks SC 4 hu1803-9D low-dose group 1.5mpk Once a week for 4 weeks SC 5 hu1803-9D high-dose group 3mpk Once a week for 4 weeks SC

[0441] 2. Experimental steps:

[0442] a. Measure fasting and random body weight once a week, and measure food intake and water intake once a day.

[0443] b. Random blood glucose should be measured before the first dose and on days 1, 2, 3, and 7 after the first dose, and then weekly thereafter. 6-hour fasting blood glucose should be measured before the first dose and on days 3 and 7 after the first dose, and then weekly thereafter.

[0444] c. On day 26 of drug administration, after fasting for 6 hours (8:00-14:00), the animals were given a single intraperitoneal administration of 2 g / kg of glucose solution, and the time of glucose administration was recorded as 0:00. Blood glucose was measured in the animals at 0 min before glucose administration and at 15, 30, 60, 90 and 120 min after glucose administration. Based on the time, a glucose tolerance curve was plotted on the blood glucose data, and the area under the curve (AUC) was calculated.

[0445] d. After the experiment, the mice were fasted for 6 hours (8:00-14:00) and then euthanized. Blood was collected from the heart and the whole blood was divided into two parts. One part, about 30 μl, was injected into a centrifuge tube with anticoagulant and stored on wet ice for glycated hemoglobin determination. The other part was centrifuged after standing and the serum was used for the determination of TG, FFA, CHOL, HDL and LDL levels.

[0446] e. Data were analyzed using GraphPad Prism 6 software, and the Student-t test was used for statistical analysis.

[0447] 3. Experimental Results:

[0448] 1) Effects of long-term drug use on random blood glucose in ob / ob mice:

[0449] like Figure 4 As shown, throughout the experiment, the random blood glucose levels of the ob / ob mice in the model control group remained at a high level. After subcutaneous injection of different doses of each drug once a week, the random blood glucose levels of the mice in each group decreased to varying degrees, showing a good dose-response effect and significantly lower than those in the model control group. The hypoglycemic effects of 2.84 mg / kg hu1803-9 and 3 mg / kg hu1803-9D were significantly better than those of other experimental groups, and the random blood glucose effect was even better on days 3, 6, 14, and 30 after administration of 3 mg / kg hu1803-9D.

[0450] 2) Effects of long-term drug use on glycemic blood glucose in ob / ob mice: such as Figure 5As shown, throughout the experiment, the random blood glucose levels of the ob / ob mice in the model control group remained at a high level. After subcutaneous injection of different doses of each drug once a week, the blood glucose levels of the mice in each group decreased to varying degrees, showing a good dose-response effect and significantly lower than those in the model control group. Similar to the random blood glucose concentration test, the hypoglycemic effects of 2.84 mg / kg hu1803-9 and 3 mg / kg hu1803-9D were significantly better than those of other experimental groups, and the hypoglycemic effect of 3 mg / kg hu1803-9D was even better on days 3, 6, 13, and 30.

[0451] 3) Effects of long-term drug use on glycated hemoglobin (HbA1c) in ob / ob mice:

[0452] The results are shown in Table 21. After subcutaneous injection of different doses of drugs once a week for 30 days in ob / ob mice, the glycated hemoglobin levels decreased to varying degrees and were significantly lower than those in the model control group (P<0.05). The glycated hemoglobin levels in the 3 mg / kg and 1.5 mg / kg hu1803-9D groups were 5.5±0.2% and 4.7±0.1%, respectively, showing a clear dose-response relationship. Among them, the 3 mg / kg hu1803-9D group was lower than that of the equimolar 1.16 mg / kg positive control dulaglutide and 2.84 mg / kg GCGR monoclonal antibody hu1803-9 (P<0.05).

[0453] Table 21. Effects of long-term drug use on HbA1c% in ob / ob mice

[0454]

[0455] Test Example 9: Competitive ELISA assay using GCGR antibody

[0456] The binding of GCGR to CHO cells overexpressing GCGR was detected by an ELISA assay using biotin-labeled antibodies and different concentrations of standard antibodies. Epitopes of GCGR binding with the GCGR antibody were then classified. The cell plate preparation method was the same as in Test Example 1. Subsequent experimental procedures are as follows:

[0457] Antibodies were labeled according to the instructions of the biotinylated assay kit (Dojindo Molecular Technologies, Inc. LK03). Different concentrations of unlabeled antibody were diluted with sample diluent at 50 μl / well in a 96-well cell culture plate and incubated at 37°C for 2 hours. After incubation, the plate was washed three times with PBST, and 50 μl / well of biotinylated antibody diluted to 0.1 μg / ml with sample diluent was added. The plate was incubated at 37°C for 2 hours, washed three times with PBST, and HRP-labeled goat anti-human secondary antibody (JacksoW ImmuWo Research, Cat Wo.109-035-003) was added. The plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, add 50 μl / well of TMB chromogenic substrate (KPL, Cat Wo.52-00-03), incubate at room temperature for 10 min, and stop the reaction by adding 50 μl / well of 1M H2SO4. Read the absorbance at 450 nm using a Thermo Scientific Multiskan MK3 microplate reader, and analyze the data using a GraphPad Prism 5.

[0458] The lower the concentration of biotin-labeled antibody competitively binding to the cell plate, the lower the OD value, and vice versa. The IC% value is calculated using the formula: IC% = (Highest OD value of the antibody to be tested) / (Highest OD value of the antibody to be tested). 450nm -Lowest OD of the antibody to be tested 450nm ) / (Highest OD to be measured 450nm - Labeled antibody has the lowest OD 450nm The competition efficiency was calculated, and the results are shown in Table 22 below.

[0459] Table 22. Competitive binding relationships among antibodies

[0460]

[0461] The results showed that ch1817 and ch1822 had very similar epitopes, as did ch1808, hu1803-9, and hu1810-12; and the epitopes of ch1808, hu1803-9, and hu1810-12 were inferred to be within the epitope range of ch1805.

Claims

1. A monoclonal antibody against GCGR or an antigen-binding fragment thereof, comprising a combination of heavy chain variable regions and light chain variable regions selected from the following: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 regions as shown in SEQ ID NO: 20, 21, and 22, respectively. The light chain variable region includes LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 23, 24 and 25, respectively.

2. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 1, wherein it is a murine antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment.

3. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 2, wherein the humanized antibody comprises a frame region derived from a human antibody or a frame region variant thereof, wherein: The frame region variant has a reversion mutation of up to 10 amino acids in the light chain frame region and / or heavy chain frame region of the human antibody, respectively.

4. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 2, comprising a combination of light chain variable regions and heavy chain variable regions selected from the following: Heavy chain variable region, the sequence of which is as shown in SEQ ID NO:4 or has at least 90% sequence identity with the sequence shown in SEQ ID NO:4; and The light chain variable region has a sequence as shown in SEQ ID NO: 5 or has at least 90% sequence identity with the sequence shown in SEQ ID NO:

5.

5. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the antibody is a full-length antibody and further includes an antibody constant region.

6. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 5, wherein the antibody constant region comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3 and IgG4 and conventional variants thereof, and a light chain constant region selected from human antibody κ and λ chains and conventional variants thereof.

7. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 6, comprising the human antibody heavy chain constant region shown in SEQ ID NO: 72 and the human antibody light chain constant region shown in SEQ ID NO:

73.

8. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 5, comprising a combination of heavy and light chains selected from the following: The heavy chain is shown in SEQ ID NO: 80 or has at least 85% sequence identity with it, and the light chain is shown in SEQ ID NO: 81 or has at least 85% sequence identity with it.

9. The anti-GCGR monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody, biantibody, and disulfide bond-stabilized V region (dsFv).

10. A bispecific protein comprising a GLP-1 peptide and an anti-GCGR antibody or an antigen-binding fragment thereof, The carboxyl terminus of the GLP-1 peptide is connected to the amino terminus of the heavy chain variable region of the anti-GCGR antibody or its antigen-binding fragment via a peptide bond or a linker, wherein the anti-GCGR antibody or its antigen-binding fragment is the anti-GCGR monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 9.

11. The bispecific protein according to claim 10, wherein: The GLP-1 peptide is a variant of the GLP-1 peptide shown in SEQ ID NO: 91, wherein the sequence of the GLP-1 peptide variant is shown in SEQ ID NO:

94.

12. The bispecific protein according to claim 10, wherein the bispecific protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the heavy chain of the anti-GCGR monoclonal antibody according to any one of claims 1 to 9; the second polypeptide chain comprising the light chain of the anti-GCGR monoclonal antibody according to any one of claims 1 to 9; wherein: The first polypeptide chain comprises the polypeptide shown in SEQ ID NO: 109, and The second polypeptide chain contains the polypeptide shown in SEQ ID NO:

81.

13. A pharmaceutical composition comprising: A therapeutically effective amount of the anti-GCGR monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 9, or the bispecific protein according to any one of claims 10 to 12, and One or more pharmaceutically acceptable carriers.

14. An isolated nucleic acid molecule encoding a monoclonal antibody against GCGR as described in any one of claims 1 to 9 or an antigen-binding fragment thereof, or a bispecific protein as described in any one of claims 10 to 12.

15. A recombinant vector comprising the isolated nucleic acid molecule of claim 14.

16. A host cell transformed with the recombinant vector of claim 15, wherein the host cell is selected from prokaryotic cells and eukaryotic cells.

17. The host cell according to claim 16, wherein it is a eukaryotic cell.

18. The host cell according to claim 16, wherein it is a mammalian cell or an insect cell.

19. A method for preparing a monoclonal antibody against GCGR according to any one of claims 1 to 9, or an antigen-binding fragment thereof, or a bispecific protein according to any one of claims 10 to 12, the method comprising: Culture the host cells according to claims 16 to 18 to form the anti-GCGR monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 9, or the bispecific protein according to any one of claims 10 to 12, and The monoclonal antibody or its antigen-binding fragment, or bispecific protein, is recovered from the culture.

20. Use of the anti-GCGR monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 9, or the bispecific protein as described in any one of claims 10 to 12, or the pharmaceutical composition as described in claim 13, in the preparation of a medicament for treating metabolic disorders, comprising: The subject is given a therapeutically effective amount of the anti-GCGR monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 9, or the bispecific protein as described in any one of claims 10 to 12, or the pharmaceutical composition as described in claim 13; The metabolic disorders mentioned are selected from: obesity and diabetes.

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