RU2784486C1 - Bispecific protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to recombinant production of monoclonal antibodies to a glucagon receptor (hereinafter – GCGR); it can be used in medicine for reduction in glucose concentration in blood. Monoclonal antibodies to GCGR are proposed, containing certain combinations of a heavy chain variable region and a light chain variable region.

EFFECT: invention provides obtainment of antibodies specifically binding to GCGR and bispecific proteins based on them, also including GLP-1 peptide, effective in the treatment of diabetes.

24 cl, 5 dwg, 22 tbl, 9 ex

Description

The present application claims priority on the basis of Patent Application No. 201811573634.0, filed December 21, 2018, and Patent Application No. 201811606887.3, filed December 27, 2018; both are incorporated herein by reference.

FIELD OF TECHNOLOGY

The present invention relates to antibodies to human GCGR (glucagon receptor), GLP-1 (glucagon-like peptide-1) peptides and their mutants, as well as to bispecific proteins formed by an anti-GCGR antibody fused to a GLP-1 peptide, and methods for their preparation. and their applications.

BACKGROUND OF THE INVENTION

The descriptions in this document provide only basic information about the present invention and do not necessarily constitute state of the art.

Diabetes (diabetes mellitus, DM) is a metabolic disease characterized by high blood glucose levels due to impaired insulin secretion and/or impaired insulin function. The occurrence of the disease is mainly caused by the combined action of insulin and glucagon.

GLP-1 is one of the most important hormones affecting insulin secretion, and both GLP-1 and glucagon are synthesized from preproinsulin. Preproinsulin is composed of about 158 amino acids and can be cleaved into different peptide chains at different positions. Biologically active GLP-1 in the human body mainly includes two forms: GLP-1 (7-36) amide and GLP-1 (7-37). GLP-1 is secreted by L cells in the small intestine and lowers blood glucose levels in the body by promoting insulin secretion in a primarily glucose concentration dependent manner, protecting pancreatic islet β cells and inhibiting glucagon secretion. At the same time, GLP-1 also has an effect on suppressing gastric emptying and reducing appetite. It finds clinical use in the treatment of type II diabetes and obesity. Naturally active GLP-1 is readily cleaved by the DPPIV enzyme in the body due to its very short half-life (less than 2 minutes) and has no clinical use.

The main focus of research and development of GLP-1 drugs has always been to increase the half-life. Many GLP-1 agonists are currently available on the market, such as dulaglutide and semaglutide. Although the effectiveness of GLP-1 is fully confirmed, it has many side effects, mainly manifested in the form of gastrointestinal symptoms, hypoglycemia, pancreatitis and kidney damage.

The action of glucagon is opposite to that of insulin and mainly plays a role in raising blood glucose levels in the body. Glucagon is a 29 amino acid peptide secreted by the α-cells of the pancreatic islets. Glucagon mainly accelerates glycogenolysis, lipolysis, and gluconeogenesis by activating the downstream cAMP/PKA (Cyclic Adenosine Monophosphate/Protein Kinase A) pathway and thereby increasing blood glucose levels after binding to the GCGR receptor on the liver cell membrane.

Studies have shown that GCGR knockout mice exhibited a range of phenotypes such as increased GLP-1 levels, decreased glycogen production, increased lipid metabolism, and reduced appetite. GCGR is one of the most popular targets for the treatment of diabetes, but progress in the development of anti-GCGR antagonist drugs has slowed down. REMD-477, available from REMD Biotherapeutics, is the most advanced anti-GCGR monoclonal antibody currently in Phase II clinical trial.

Antibodies to GCGR have been disclosed in prior art sources, e.g. However, there remains a need to develop new and highly effective anti-GCGR antibodies and treatments for diabetes.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a monoclonal antibody to GCGR or its antigennegative fragment. The antibody, or antigen-binding fragment thereof, has the ability to bind to human GCGR (or the antigenic epitope contained therein).

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

a) a heavy chain variable region comprising the HCDR1 (heavy chain complementarity determining region 1), HCDR2 and HCDR3 regions shown in SEQ ID NOs: 48, 49 and 50, respectively, and a light chain variable region comprising the LCDR1 (complementarity determining region 1 light chain), LCDR2 and LCDR3 shown in SEQ ID NOs: 51, 52 and 53, respectively; or

b) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NO: 38, 39 and 54, respectively, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions set forth in SEQ ID NO: 55 , 56 and 57, respectively.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a combination of a heavy chain variable region and a light chain variable region selected from any of the following i)-vi):

i) a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 14, 15, and 16, respectively, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 17 , 18 and 19, respectively;

ii) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NOs: 20, 21 and 22, respectively, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions set forth in SEQ ID NO: 23 , 24 and 25, respectively;

iii) a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 26, 27, and 28, respectively, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 29 , 30 and 31, respectively;

iv) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NOs: 32, 33 and 34, respectively, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions set forth in SEQ ID NO: 35 , 36 and 37, respectively;

v) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NO: 38, 39 and 40, respectively, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions set forth in SEQ ID NO: 41 , 42 and 43, respectively; or

vi) a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 38, 39, and 44, respectively, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 45 , 46 and 47, respectively.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, is a mouse antibody, chimeric antibody, or humanized antibody, or antigen-binding fragment thereof.

In some embodiments of an anti-GCGR monoclonal antibody or antigen-binding fragment thereof, the humanized antibody comprises a framework region(s) derived from a human antibody, or a variant of that framework region, and the framework region variant has no more than 10 backmutations relative to the human antibody light chain framework region, and /or a framework region variant has no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 backmutation relative to the framework region heavy chain of a human antibody.

In some embodiments, a variant of the wireframe comprises:

aa) one or more amino acid back mutations from 42G, 44V, 71Y and 87F contained in the light chain variable region and/or one or more amino acid back mutations from 38K, 481, 67A, 69F, 71A, 73P, 78A and 93, contained in the variable region of the heavy chain; or

ab) one or more amino acid reverse mutations from 38L, 44V, 59S, 70E and 71Y contained in the light chain variable region and/or one or more amino acid reverse mutations from 38K, 481, 66K, 67A, 69L, 73R, 78M, and 94S contained in the variable region of the heavy chain. Next, the position of the reverse mutation site is determined according to the Kabat numbering criteria.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region present in any of SEQ ID NOs: 2, 61, 62, 63, and 64, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of SEQ ID NOs: 2, 61, 62, 63 and 64; and/or

a light chain variable region present in any of SEQ ID NOs: 3, 58, 59 and 60, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 3, 58, 59 and 60.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises the heavy chain variable region set forth in SEQ ID NO: 63 and the light chain variable region set forth in SEQ ID NO: 58.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region as set forth 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 as set forth 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.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region as set forth 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 as set forth 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.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region present in any of SEQ ID NOs: 8, 68, 69, 70, and 71, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of SEQ ID NOs: 8, 68, 69, 70 and 71; and/or

a light chain variable region present in any of SEQ ID NOs: 9, 65, 66 and 67, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 9, 65, 66 and 67.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises the heavy chain variable region set forth in SEQ ID NO: 71 and the light chain variable region set forth in SEQ ID NO: 67.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region as set forth 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 as set forth 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.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region as set forth 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 as set forth 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.

In some embodiments of the anti-GCGR monoclonal antibody or antigen-binding fragment thereof, the antibody is a full-length antibody further comprising antibody constant region(s), specifically, the heavy chain constant region of the antibody constant regions is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions, and their conventional variants, and the light chain constant region of the antibody constant regions is selected from human antibody κ- and λ-chain constant regions and conventional variants thereof, and more preferably comprising the human antibody heavy chain constant region shown in SEQ ID NO: 72 and human the light chain constant region shown in SEQ ID NO: 73.

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

the heavy chain is as 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 as 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 her.

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

the heavy chain is as 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 as 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 her.

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

the heavy chain is as 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 as 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 her.

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

the heavy chain is as 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 as 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.

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

the heavy chain is as 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 as 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.

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

the heavy chain is as 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 as 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.

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

the heavy chain is as 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 as 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.

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

the heavy chain is as 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 as 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.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain as set forth in SEQ ID NO: 78 and a light chain as set forth in SEQ ID NO: 79.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain as set forth in SEQ ID NO: 84 and a light chain as set forth in SEQ ID NO: 85.

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, comprises a combination of a heavy chain variable region and a light chain variable region selected from any of the following ac)-ah):

ac) a heavy chain variable region containing the same HCDR1, HCDR2 and LCDR3 regions as the heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region containing the same LCDR1, LCDR2 and LCDR3 regions as the light chain variable region shown in SEQ ID NO: 3;

ad) a heavy chain variable region containing the same HCDR1, HCDR2 and LCDR3 regions as the heavy chain variable region shown in SEQ ID NO: 4 and a light chain variable region containing the same LCDR1, LCDR2 and LCDR3 regions as the light chain variable region shown in SEQ ID NO: 5;

ae) a heavy chain variable region containing the same HCDR1, HCDR2 and LCDR3 regions as the heavy chain variable region shown in SEQ ID NO: 6 and a light chain variable region containing the same LCDR1, LCDR2 and LCDR3 regions as the light chain variable region shown in SEQ ID NO: 7;

af) a heavy chain variable region containing the same HCDR1, HCDR2 and LCDR3 regions as the heavy chain variable region shown in SEQ ID NO: 8 and a light chain variable region containing the same LCDR1, LCDR2 and LCDR3 regions as the light chain variable region shown in SEQ ID NO: 9;

ag) a heavy chain variable region containing the same HCDR1, HCDR2 and HCDR3 regions as the heavy chain variable region shown in SEQ ID NO: 10 and a light chain variable region containing the same LCDR1, LCDR2 and LCDR3 regions as the light chain variable region shown in SEQ ID NO: 11; or

ah) a heavy chain variable region containing the same HCDR1, HCDR2 and HCDR3 regions as the heavy chain variable region shown in SEQ ID NO: 12 and a light chain variable region containing the same LCDR1, LCDR2 and LCDR3 regions as light chain variable region shown in SEQ ID NO: 13.

In some embodiments of the anti-GCGR monoclonal antibody or antigen-binding fragment thereof, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain antibody, dimerized V-region (diabody), and disulfide-stabilized V-region ( dsFv).

In some embodiments, the anti-GCGR monoclonal antibody or antigen-binding fragment thereof is characterized in that it competes with the monoclonal antibody or antigen-binding fragment thereof mentioned above for binding to human GCGR (or an epitope thereof).

In some embodiments, the anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, has at least one of the following:

i. Antagonistic activity of blocking the binding of human GCGR to human glucagon with an IC50 value (concentration required to inhibit 50% of maximum activity) 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);

ii. Antagonistic activity of blocking the binding of cynomolgus or rhesus monkey GCGR to cynomolgus or rhesus monkey glucagon;

iii. Inhibition of the increase in the concentration of glucose in human blood; and

iv. Antagonistic activity of blocking the binding of mouse GCGR to mouse glucagon.

In some embodiments of the anti-GCGR monoclonal antibody or antigen-binding fragment thereof, the monoclonal antibody or antigen-binding fragment thereof binds to the same antigenic epitope as the monoclonal antibody or antigen-binding fragment thereof mentioned above.

In yet another aspect, the present invention relates to a bispecific protein. In some embodiments, the bispecific protein provided herein comprises a GLP-1 peptide and an anti-GCGR antibody, and the GLP-1 peptide is covalently linked to the anti-GCGR antibody polypeptide chain via a peptide bond or linker.

In some embodiments of the bispecific protein, the carboxyl terminus of the GLP-1 peptide is linked to the amino terminus of the heavy chain variable region of the anti-GCGR antibody via a peptide bond or linker, or the carboxyl terminus of the GLP-1 peptide is linked to the amino terminus of the light chain variable region of the anti-GCGR antibody via a peptide bond or linker. .

In some embodiments of the bispecific protein, the carboxyl terminus of the GLP-1 peptide is linked to the amino terminus of the heavy chain variable region of an anti-GCGR antibody via a peptide bond or a linker.

In some embodiments of the bispecific protein, the carboxyl terminus of the GLP-1 peptide is linked to the amino terminus of the light chain variable region of an anti-GCGR antibody via a peptide bond or a linker.

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

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

In some embodiments of the bispecific protein, the anti-GCGR antibody is selected from the anti-GCGR monoclonal antibodies or antigen-binding fragments thereof mentioned above.

In some embodiments of the bispecific protein, the GLP-1 peptide is GLP-1A as set forth in SEQ ID NO: 91, or the GLP-1 peptide is a GLP-1A peptide variant having one or more of the amino acid substitutions of Q17E, I23V, K28R, and G30R vs. GLP-1A.

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

In some embodiments of the bispecific protein, the GLP-1 peptide is GLP-1A as set forth in SEQ ID NO: 91, or the GLP-1 peptide is a GLP-1A peptide variant having Q17E and one or more amino acid substitutions from 123V, K28R, and G30R vs. GLP-1A.

In some embodiments of 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 both of Q17E and I23V relative to GLP-1A.

In some embodiments of the bispecific protein, the GLP-1A peptide variant comprises or consists of the sequence shown in SEQ ID NOs: 92, 93, 94, 95, 96, 97, 98, or 99.

In some embodiments of the bispecific protein, the anti-GCGR antibody is selected from any of the anti-GCGR monoclonal antibodies or antigen-binding fragments thereof mentioned above, and the GLP-1A peptide or GLP-1A peptide variant is any of those described above.

In some embodiments, the bispecific protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain is a polypeptide selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, and 108 , and the second polypeptide chain is the polypeptide shown in SEQ ID NO: 79.

In some embodiments, the bispecific protein comprises a first polypeptide chain comprising an anti-GCGR antibody heavy chain and a second polypeptide chain comprising an anti-GCGR antibody light chain, wherein: the first polypeptide chain is the polypeptide shown in SEQ ID NO: 109 and the second polypeptide chain is the chain is a polypeptide shown in SEQ ID NO: 81;

In some embodiments, the bispecific protein comprises a first polypeptide chain comprising an anti-GCGR antibody heavy chain and a second polypeptide chain comprising an anti-GCGR antibody light chain, wherein: the first polypeptide chain is the polypeptide shown in SEQ ID NO: 110 and the second polypeptide chain is the chain is a polypeptide shown in SEQ ID NO: 83;

In some embodiments, the bispecific protein comprises a first polypeptide chain comprising an anti-GCGR antibody heavy chain and a second polypeptide chain comprising an anti-GCGR antibody light chain, wherein: the first polypeptide chain is the polypeptide shown in SEQ ID NO: 111 and the second polypeptide chain is the chain is a polypeptide shown in SEQ ID NO: 85;

In some embodiments, the bispecific protein comprises a first polypeptide chain comprising an anti-GCGR antibody heavy chain and a second polypeptide chain comprising an anti-GCGR antibody light chain, wherein: the first polypeptide chain is the polypeptide shown in SEQ ID NO: 112 and the second polypeptide chain is the chain is a polypeptide shown in SEQ ID NO: 87; or

In some embodiments, the bispecific protein comprises a first polypeptide chain comprising an anti-GCGR antibody heavy chain and a second polypeptide chain comprising an anti-GCGR antibody light chain, wherein: the first polypeptide chain is the polypeptide shown in SEQ ID NO: 113 and the second polypeptide chain is the chain is the polypeptide shown in SEQ ID NO: 89.

In another aspect, the present invention also provides a variant GLP-1 peptide. In some embodiments, the GLP-1 peptide variant is a mutant having one or more amino acid mutations from Q17E, 123V, K28R, and G30R relative to GLP-1A as set forth in SEQ ID NO: 91.

In some embodiments, the GLP-1 peptide is GLP-1A as set forth in SEQ ID NO: 91, or the GLP-1 peptide is a GLP-1A peptide variant having Q17E or both of Q17E and I23V relative to GLP-1A.

In some embodiments, the GLP-1 peptide variant has the sequence shown in SEQ ID NOS: 92, 93, 94, 95, 96, 97, 98, or 99.

The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of an anti-GCGR monoclonal antibody, or an antigen-binding fragment thereof, as described above, or a bispecific protein, as described above, or a GLP-1 peptide variant, as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

The present invention also provides an isolated nucleic acid molecule encoding an anti-GCGR monoclonal antibody or an antigen-binding fragment thereof as described above, or a bispecific protein as described above, or a GLP-1 peptide variant as described above.

The present invention relates to a recombinant vector containing an isolated nucleic acid molecule as described above.

The present invention relates to a host cell transformed with a recombinant vector as described above, wherein said host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian or insect cells.

The present invention relates to a method for producing an anti-GCGR monoclonal antibody, or an antigen-binding fragment thereof, as described above, or a bispecific protein, as described above, or a GLP-1 peptide variant, as described above, comprising culturing a host cell, as described above, in a culture medium. a medium for obtaining and accumulating a monoclonal antibody to GCGR or its antigen-binding fragment, as described above, or a bispecific protein, as described above, or a GLP-1 peptide variant, as described above, and isolating the monoclonal antibody or its antigen-binding fragment, or the bispecific protein, or a GLP-1 peptide variant from culture.

The present invention relates to a method for detecting or measuring human GCGR in vitro, comprising the use of a monoclonal antibody or antigen-binding fragment as described above.

The human GCGR detection kit contains a monoclonal antibody, or antigen-binding fragment thereof, as described above.

In some embodiments, the invention also provides the use of an anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, as described above, for the manufacture of medical devices (such as kits, chips, test strips, multiwell plates, magnetic beads, coated particles), where the medical device contains a monoclonal antibody or antigennegative fragment, as described above. By way of example, the kit includes a multiwell plate coated with an anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, as described above.

The present invention relates to the use of a monoclonal antibody, or antigen-binding fragment thereof, as described above, for the preparation of a reagent for detecting or measuring human GCGR.

The present invention relates to a method for lowering a blood glucose concentration in a subject, comprising administering to said subject a therapeutically effective amount of an anti-GCGR monoclonal antibody, or an antigen-binding fragment thereof, as described above, 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 therapeutically effective amount is from 0.1 to 3000 mg of an anti-GCGR monoclonal antibody or an antigen-binding fragment thereof, as described above, or a bispecific protein, as described above, or a GLP-1 peptide variant, as described above, in a single dose of the composition.

The present invention relates to a method for treating a metabolic disorder comprising administering to a subject an anti-GCGR monoclonal antibody or an antigen-binding fragment thereof as described above, 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 glycemia, dyslipidemia, atherosclerosis, or pre-diabetic state.

The present invention also provides the use of an anti-GCGR monoclonal antibody, or an antigen-binding fragment thereof, as described above, or a bispecific protein, as described above, or a GLP-1 peptide variant, as described above, or a pharmaceutical composition, as described above, for the preparation of a medicament for treatment of a metabolic disorder; or to lower the blood glucose concentration in a subject.

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 glycemia, dyslipidemia, atherosclerosis, or pre-diabetic state.

The present invention also provides an anti-GCGR monoclonal antibody, or an antigen-binding fragment thereof, as described above, or a bispecific protein, as described above, or a GLP-1 peptide variant, as described above, or a pharmaceutical composition, as described above, for use as a drug. , preferably for use as a drug for the treatment of a metabolic disorder or for lowering the blood glucose concentration in a subject.

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 glycemia, dyslipidemia, atherosclerosis, or pre-diabetic state.

DESCRIPTION OF GRAPHICS

Fig. 1: Schematic diagram of the structure of a bispecific protein (GLP-1/anti-GCGR antibody) according to the present invention.

Fig. 2: Antagonistic activity of a bispecific protein and an anti-GCGR anti-GCGR antibody.

Fig. 3: Activating activity of the bispecific protein and dulaglutide against GLP-1R.

Fig. 4: Effect of long-term administration on random blood glucose concentrations in ob/ob mice. Vehicle (empty vector) is a control model that was injected with sodium phosphate buffer (PBS), hu1803-9D-3 mg/kg, hu1803-9 -2.84 mg/kg and dulaglutide-1.16 mg/kg refer to the same molarity of the substance.

Fig. 5: Effect of long-term administration on fasting blood glucose concentrations in ob/ob mice. In all experimental groups, a significant decrease in fasting blood glucose concentration in mice could be achieved, among groups hu1803-9D-3mg/kg and hu1803-9-2.84mg/kg have the strongest ability to reduce blood glucose concentration, and hu1803 -9D-3mg/kg shows better ability to lower blood glucose than hul 803-9-2.84mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

Terminology

Three-letter codes and one-letter codes for amino acids used in the present description correspond to those described in J. biol. chem, 243, p3558 (1968).

The term "bispecific protein" refers to a protein molecule capable of binding to two target proteins or target antigens. The bispecific protein used in the present invention specifically refers to a protein capable of binding to GCGR and GLP-1R (GLP-1 receptor) and formed by a GLP-1 peptide fused to the polypeptide chain of an anti-GCGR antibody (or antigen-binding fragment thereof).

"GLP-1 peptide" refers to a peptide capable of binding to and activating the GLP-1 receptor. Peptides known from the prior art are described in patent applications WO 2008/071972, WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO 2011/075393, WO 2008/152403/07WO 2010 2010/070252, Wo 2010/070253, Wo 2010/070255, Wo 2011/160630, Wo 2011/006497, Wo 2011/117415, Wo 2011/117416, Wo 2006/134340, Wo 1997046584, Wo 2007124461, Wo 2017100107, Wo 2007, Wo 2001, Wo 200107, Wo 2001, Wo 200107, Wo , CN 1935261A, CN 1935846A, WO 2006036834, WO 2005058958, WO 2002046227, WO 1999043705, WO 1999043708, WO 1999043341, CN 102949730, CN 104293834, CN 104327187, WO 2015067716, WO 2015049651, WO 2014096145, WO 2014096148, WO 2014096150, WO 2014096149, the contents of which are all incorporated herein by reference. Some individual GLP-1 peptides, including GLP-1, GLP-1 analogs, and GLP-1 receptor peptide agonists, are, for example, lixisenatide/AVE0010/ZP10/Lyxumia, exenatide/exendin-4/Byetta/Bydureon/ITCA 650/ AC-2993, Liraglutide/Victoza, Semaglutide, Taspoglutide), SyncriaAlbiglutide, Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langlenatide/HM-11260C, CM-3, GLP-1 Eligen, ORMD-0901, NN-9924, NN-9926, NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, TT-401, BHM-034, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, exenatide-XTEN and glucagon-Xten. In addition to the above GLP-1 peptides, GLP-1A as shown in SEQ ID NO: 91 and its mutants (shown in SEQ ID NO: 92-99) according to the present invention are also included.

GPCR, i.e. G-protein coupled receptor is a type of transmembrane protein expressed on the cytoplasmic membrane. The GPCR has over 800 members and is the largest family of membrane proteins in the mammalian genome currently known. In the human body, GPCR proteins are widely distributed in organs and tissues such as the central nervous system, immune system, heart, blood vessels, and retina, and are involved in the development and normal functioning of the body.

The main body of the GPCR protein consists of seven segments of the transcytoplasmic alpha-helix membrane structure. The N-terminus and three loops are located outside the cell and are involved in the interaction between the protein and its receptor; The C-terminus and three loops are located within the cell, of which the C-terminus and the third loop play an important role in the intracellular signaling process mediated by the interaction between the GPCR protein and the downstream G protein.

"GCGR" is a glucagon receptor and a member of the GPCR family. Glucagon mainly accelerates glycogenolysis, lipolysis, and/or gluconeogenesis by activating the downstream pathway when bound to GCGR and thereby increasing blood glucose levels.

The term "antibody (Ab)" includes an antigen-binding molecule (or complex of molecules) containing at least one complementary region-determining antigen-binding molecule (or complex of molecules) that specifically binds to or interacts with (e.g., recognizes and/or binds to) a particular antigen (or epitope) (e.g., GCGR ).

The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bond(s), and their multimers (eg, IgM). Each heavy chain includes a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region (CH). This heavy chain constant region includes three regions (domains): CHI, CH2 and CH3. Each light chain includes a light chain variable region (hereinafter 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, also called frameworks). Each VH and VL is composed of three CDRs and four FRs, arranged from amino to carboxyl in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

In various embodiments of the present invention, the FR of an anti-GCGR antibody (or antigen-binding fragment thereof) may be the same as the FR of a human germline sequence, or may be naturally or artificially modified. Antibodies can be various subclasses of antibodies, for example, an IgG antibody (eg, a subclass of IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

The term "antibody" also encompasses antigen-binding fragments of a full-length antibody molecule.

The terms "antigen-binding portion", "antigen-binding domain", "antigen-binding fragment" of an antibody, and the like. as used herein, any naturally occurring, enzymatically, synthetically, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex is included. Antigen-binding fragments of an antibody can be obtained, for example, from a complete antibody molecule using any suitable standard techniques, such as proteolytic cleavage or recombinant genetic engineering, including the modification and expression of DNA encoding variable regions and (optionally) constant regions of the antibody. DNA is known and/or can be easily obtained, for example, from commercially available sources, a DNA database (including, for example, a database of phage antibodies), or can be synthesized. DNA can be sequenced and modified chemically or by molecular biotechnology, for example by arranging one or more variable and/or constant regions in a suitable configuration, introducing codons, generating cysteine residues, modifying, adding or deleting amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) a Fab fragment; (ii) F(ab') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); and (vi) dAb fragments. Other genetically modified molecules, such as antibodies with a specific region, single domain antibodies, antibodies with a deleted region, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (for example, monovalent nanobodies, divalent nanobodies, etc.), an immunopharmaceutical agent on small modular protein (SMIP)-based and shark IgNAR variable regions are also included in the term "antigen-binding fragment" in the context of this document.

An antigen binding fragment typically contains at least one variable region. The variable region can be a region of any size or amino acid composition, and typically contains one or more CDRs adjacent to or within framework sequences.

In some embodiments, the antigen-binding fragment of an antibody may have any variable region and constant region configuration, the variable region and constant region may be linked to each other directly or through a full or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which allows flexible and semi-flexible bonding between adjacent variable and/or constant regions within a single polypeptide molecule.

"Mouse antibody" in the context of this document refers to monoclonal antibodies of mouse or rat origin, obtained according to the knowledge and skills in this field of technology. During preparation, subjects are injected with an antigen and then a hybridoma is isolated expressing an antibody that has the desired sequence or functional characteristics. When the injected subjects are mice, the resulting antibody is a mouse-derived antibody, and when the injected subjects are rats, the resulting antibody is a rat-derived antibody.

A "chimeric antibody" is an antibody formed by fusing the variable region(s) of a first species (eg, mouse) antibody with the constant region(s) of a second species (eg, human) antibody. To create a chimeric antibody, it is necessary to create a hybridoma secreting a monoclonal antibody of the first species (for example, mice), clone the gene for the variable region (regions) from the hybridoma cell, and then clone the gene for the constant region (regions) of the second type of antibody (for example, human), as need. The first species variable region gene(s) is linked to the second species constant region gene(s) to form a chimeric gene, which is then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

In a preferred embodiment of the present invention, the antibody light chain of the chimeric antibody further comprises a human kappa, lambda light chain constant region, 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 contains a heavy chain constant region of a human IgG1, IgG2, or IgG4, or contains a heavy chain constant region variant of a human IgG1, IgG2, or IgG4 region with amino acid mutation(s) (such as YTE mutation or back mutation, L234A and/or L235A mutation or S228P mutation).

The term "humanized antibody", including an antibody with grafted CDRs, refers to an antibody obtained by grafting the CDR sequences of an animal-derived antibody, such as a mouse antibody, onto human antibody variable region framework regions (i.e., framework regions). Humanized antibodies can avoid the heterologous responses induced by chimeric antibodies that carry a large number of heterologous protein components. Such framework sequences may be obtained from a public DNA database covering germline antibody gene sequences or from published sources. For example, the germline DNA sequences of the 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 also in Kabat, E.A. ., et al. 1991 Sequences of Proteins of Immunological Interest, 5th Ed.

To avoid a decrease in activity caused by reduced immunogenicity, framework sequences in the variable region of a human antibody can be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibodies of the present invention also include humanized antibodies in which CDR affinity maturation is performed by phage display.

Due to the fact that the residues are in contact with the antigen, the grafting of the CDR can lead to a decrease in the affinity of the antibody or its antigen-binding fragment for this antigen due to the fact that the residues of the frame are in contact with the antigen. Such interactions may be the result of highly somatic mutations. Therefore, it may still be necessary to graft the donor framework amino acids onto the humanized antibody framework regions. Antigen-binding amino acid residues derived from a non-human antibody or antigen-binding fragment thereof can be identified by examining the sequence and structure of the variable region of an animal monoclonal antibody. Amino acid residues of the CDR donor backbone that differ from the germline can be considered as cognate. If it is not possible to determine the most closely related germline, the sequence can be compared to a sequence common to subtypes or an antibody sequence from an animal with a high percentage of similarity. Rare framework residues are believed to be the result of extensive mutation in somatic cells and play an important role in binding.

In one embodiment, the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a human or mouse k-, X-chain, or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4, or her version.

"Conventional variants" of a human antibody heavy chain constant region and a human antibody light chain constant region refer to variants of the human heavy or light chain constant region disclosed in the art that do not alter the structure and function of the antibody variable regions. Examples of variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants generated by site-directed modification and amino acid substitutions in the heavy chain constant region. Specific substitutions are, for example, a YTE mutation, L234A and/or L235A mutations, an S228P mutation, or mutations resulting in a ridge-in-gap structure (causing the heavy chain of an antibody to form a combination of ridge-Fc and trough-Fc), etc. .d. These mutations have been shown to confer novel properties on the antibody without altering the function of the antibody's variable region.

"Human antibody" and "human-derived antibody" may be used interchangeably and may refer to antibodies of human origin or antibodies derived from a transgenic organism that has been "genetically modified" and obtained by any method known in the art to produce certain human antibodies, in response to antigen stimulation. In some technologies, elements of the human heavy and light chain loci are introduced into body cell strains derived from embryonic stem cell lines in which endogenous heavy and light chain loci are targeted for destruction. Transgenic organisms can synthesize human antibodies specific for human antigens, and these organisms can be used to produce hybridomas that secrete human antibodies. A human antibody can also be one 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 by gene or chromosome transfection techniques and phage display technology, or constructed from in vitro activated B cells, all of which are known in the art.

"Monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. the individual antibodies that make up the population are identical and/or bind to the same epitope, with the exception of possible variant antibodies (e.g., variants having naturally occurring mutations or mutations that occur during the manufacture of monoclonal antibody preparations, and these mutations are usually present in minimal amounts). Unlike preparations of polyclonal antibodies, which usually contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in the preparation (composition) of monoclonal antibodies is directed against one determinant on the antigen. Therefore, the term "monoclonal" refers to the characteristics of an antibody derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring any specific method for producing the antibody. For example, monoclonal antibodies used in accordance with the present invention can be obtained by various methods, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals having all or part of the human immunoglobulin loci. Such methods and other exemplary methods for producing monoclonal antibodies are described herein. Monoclonal antibodies included in the term "monoclonal antibody or antigen-binding fragment" refer to full-length antibodies.

The terms "full antibody", "complete antibody", "whole antibody" and "whole antibody" are used interchangeably herein and refer to an antibody in substantially whole form, as opposed to antigen-binding fragments defined below. The term specifically refers to an antibody in which the heavy chain includes the VH region, the CH1 region, the hinge region and the Fc region in amino-terminal to carboxyl-terminal order, and the light chain includes the VL region and the CL region in amino-terminal to carboxyl-terminal order.

In addition, the VL domain and VH domain of the Fv fragment are encoded by two separate genes, however, they can be linked with a synthetic linker using recombinant techniques to create a single protein chain, where a monovalent molecule is formed by pairing the VL and VH domain (referred to as single-stranded Fv ( scFv); see, for example, Bird et al. (1988): 423-426; Science 242 and Huston et al (1988) Proc. Natl. Acad. Sci USA85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibodies are prepared using conventional techniques known in the art and are screened for functional fragments using the same method as for an intact antibody. Antigen-binding portions can be obtained using recombinant DNA technology or enzymatic or chemical destruction of intact immunoglobulin.

Antigen binding fragments can also be incorporated into a single chain molecule containing a pair of tandem Fv fragments (VH-CH1-VH-CH1) and the pair of tandem Fv fragments form a pair of antigen binding regions together with complementary light chain polypeptides (Zapata et al., 1995 Protein Eng 8(10): 1057-1062 and U.S. Patent No. 5,641,870).

Fab is an antibody fragment obtained by treating an IgG antibody molecule with papain (which cleaves the amino acid residue at position 224 of the H chain) and this antibody fragment has a molecular weight of approximately 50,000 and has antigen binding activity, with about half of the N-terminal side of the H chain and the entire L chain is linked to each other by disulfide bond(s).

F(ab')2 is an antibody fragment having a molecular weight of approximately 100,000 Da and having antigen-binding activity, and containing two Fab regions that are linked at the hinge position, it can be obtained by hydrolysis with pepsin of the part located below the two disulfide bonds in the hinge IgG regions.

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

In addition, a Fab' can be obtained by inserting the DNA encoding the Fab' of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryote or eukaryote to express the Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" refers to a molecule containing the variable domain (or region) of the heavy chain of the antibody (VH), connected to the variable domain (or region) of the light chain of the antibody (VL) using a linker. Such scFv molecules have the general structure NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable linkers in the art consist of the repeat amino acid sequence GGGGS or a variant thereof, such as a variant with 1-4 (including 1, 2, 3 or 4) repeats (Holliger et al. (1993), Proc Natl Acad Sci USA. 90: 6444 -6448). Other linkers that can be used in the present invention 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.

"Linker" refers to a connecting peptide sequence used to connect protein domains, usually with some degree of flexibility, and the use of linkers does not cause the protein domain to lose its original functions.

A diabody is an antibody fragment in which scFv is dimerized and is an antibody fragment having bivalent antigen-binding activity. With bivalent antigen-binding activity, the two antigens may be the same or different.

dsFv is produced by replacing one amino acid residue in each of VH and VL with a cysteine residue, and then linking the substituted polypeptides via a disulfide bond between the two cysteine residues. Amino acid residues to be replaced by a cysteine residue can be selected based on the prediction of the three-dimensional structure of an antibody according to known methods (Protein Engineering, 7, 697 (1994)).

In some embodiments of the present invention, an antigen-binding fragment can be obtained using the following steps: obtaining cDNA (complementary DNA) encoding the VH and/or VL of a monoclonal antibody of the present invention that specifically recognizes and binds to an antigen, and cDNA encoding other desired domains; constructing a DNA encoding an antigen-binding fragment; inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the antigen-binding fragment.

An "Fc region" may be a naturally occurring sequence or variant of an Fc region. The boundaries of the immunoglobulin heavy chain Fc region can vary, however, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus. The numbering of the residues in the Fc region corresponds to the numbering of the EU index according to Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically has two constant domains, CH2 and CH3.

"Knub-Fc" refers to a three-dimensional knuckle-like structure formed by the incorporation of the T366W point mutation into the Fc region of an antibody. Accordingly, "trough-Fc" refers to a trough-like spatial structure formed by the incorporation of T366S, L368A, and Y407V point mutations into the Fc region of an antibody. The ridge-Fc and trough-Fc are more likely to form heterodimers due to steric hindrance. To further promote the formation of heterodimers, point mutations S354C and Y349C, respectively, can be introduced into the ridge-Fc and trough-Fc, respectively, to further promote the formation of heterodimers via disulfide bonds. Meanwhile, in order to eliminate or reduce the effect of ADCC (Antibody Dependent Cellular Cytotoxicity) caused by Fc antibody, replacement mutations 234A and 235A can also be introduced into Fc. In a bispecific antibody, the Fc ridge or Fc trough can be used either as the Fc region of the first polypeptide chain or as the Fc region of the second polypeptide chain. For a single bispecific antibody, the Fc regions of the first and second polypeptide chain cannot be both Fc ridge and Fc trough.

The term "amino acid difference" or "amino acid mutation" refers to amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including one or more amino acid insertions, deletions or substitutions relative to the original protein or polypeptide.

The "variable region" of an antibody refers to the variable region of the light chain (VL) of an antibody or the variable region of the heavy chain (VH) of an antibody, alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of three complementarity determining regions (CDRs) (also referred to as hypervariable regions) associated with four framework regions (FRs). The CDRs in each strand are held tightly together by the FR and contribute to the formation of an antigen-binding site along with CDR regions from the other strand. There are at least two methods for determining CDRs: (1) a method based on interspecies sequence variation (eg, Kabat et al. Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda MD)); and (2) a method based on crystallographic examination of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). In the context of this document, CDRs may refer to CDRs determined by either or a combination of these two methods.

The term "antibody scaffold" or "FR region" refers to the part of the variable domain, either VL or VH, that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. Essentially, it is a variable domain without a CDR.

The terms "complementarity determining region" and "CDR" refer to one of six hypervariable regions present in the variable domain of an antibody that primarily contribute to antigen binding. There are typically three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. CDR amino acid sequence boundaries can be defined using any of a variety of well known schemes, including the "Kabat" numbering criteria (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering criteria (Al-Lazikani et al., (1997) JMB 273:927-948) and ImmunoGenTics (IMGT) numbering criteria (Lefranc MP, Immunologist, 7, 132-136 (1999) Lefranc, MP, etc., Dev. Comp. Immunol., 27,55-77 (2003), and the like. chains (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3) According to the Chothia criteria, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). When Kabat and Chothia are combined to determine CDRs, CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3) in human VL. According to the IMGT criteria, CDR amino acid residues in VH are roughly numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and CDR amino acid residues in VL are roughly numbered 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). According to IMGT criteria, antibody CDR regions can be determined using the IMGT/DomainGap Align program.

"Any CDR variant thereof" in the "HCDR1, HCDR2 and HCDR3 regions or any variant thereof" refers to a variant obtained by subjecting one, two or three regions of HCDR1, HCDR2 and HCDR3 to amino acid mutations.

An "antibody constant region domain" refers to domains derived from antibody light and heavy chain constant regions, comprising CL and CH1, CH2, CH3, and CH4 domains derived from various types of antibodies.

An "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

The term "specifically bind to", "selectively bind to", "selectively binds to" or "specifically binds to" refers to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, or 10 ^-11 M, or even less.

When the term "competition" is used in the context of antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) that compete for the same epitope, this means that there is competition between antigen-binding proteins, which is determined using assays where the subject to be tested an antigen-binding protein (eg, an antibody or antigen-binding fragment thereof) prevents or inhibits (eg, reduces) specific binding of a reference antigen-binding protein (eg, ligand or reference antibody) to a common antigen. Numerous types of competitive binding assays are available to determine if a given antigen binding protein competes with another. These assays are, for example, solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), competitive sandwich 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), direct labeled solid phase, direct labeled sandwich solid phase (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct-labeled solid phase RIA with 1-125 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 direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of a purified antigen capable of binding to both the unlabeled antigen-binding protein being tested and the labeled reference antigen-binding protein (the antigen is located on a solid or cell surface). Competitive inhibition is determined by measuring the amount of label bound to a solid surface or cell surface in the presence of the antigen binding protein being tested. Typically, the antigen binding protein being tested is present in excess. Antigen-binding proteins identified by competitive analysis (competing with an antigen-binding protein) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an epitope sufficiently close to the epitope to which the reference antigen-binding protein binds, where the two epitopes spatially interfere with each other to prevent binding. Additional details regarding methods for determining competitive binding are provided in the examples provided herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (eg, reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70% , 70-75% or 75% or even greater percentage of specific binding of the reference antigen-binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or 97%, or even more.

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 antibody "arm" variable region interacts with the antigen at multiple amino acid sites through weak non-covalent forces; the greater the interaction, the stronger the affinity. As used herein, the term "high affinity" of an antibody or antigen-binding fragment (e.g., Fab fragment) thereof generally refers to an antibody or antigen-binding fragment with a K _D 1E ^-9 M or less (e.g., K _D 1E ^-10 M or less, K _D 1E ^-11 M or less, K _D 1E ^-12 M or less, K _D 1E ^-13 M or less, K _D IE ^-14 M or less, etc.).

The term "KD" or "K _D " refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. Typically, an antibody will bind to an antigen with an equilibrium dissociation constant (KD) of less than about IE ^-8 M, such as less than about 1E ^-9 M, 1E ^-10 M, or 1E ^-11 M, or even less, such as as determined by surface sensing technology. plasma resonance (SPR) on the Biacore instrument. The smaller the KD value, the greater the affinity.

The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single or double stranded, but is preferably double stranded DNA. A nucleic acid is "operably linked" when it is operably linked to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.

The term "vector" means a construct capable of delivering one or more target genes or sequences and preferably expressing them in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors coupled to cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and some eukaryotic cells such as producer cells.

Methods for preparing and purifying antibodies and antigen-binding fragments are well known in the art, see, for example, A Laboratory Manual for Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15. For example, mice can be immunized with an antigen or fragment thereof, and then the resulting antibodies can be renatured, purified and sequenced for amino acid sequences using conventional methods. Antigen binding fragments can also be obtained by conventional methods. Antibodies or antigen-binding fragments of the present invention are designed to include one or more human framework regions in CDR regions derived from a non-human antibody. Human FR germline sequences can be obtained from the website http://imgt.cines.fr or from The Immunoglobulin Facts Book, 2001, ISBN 012441351, by alignment with the IMGT human antibody germline variable gene database and software MY.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria suitable for transformation include representatives of enterobacteria such as strains of Escherichia coli or Salmonella; Bacillaceae 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-limiting examples such as HEK293E cells), and NS0 cells.

Genetically modified antibodies or antigen-binding fragments can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As an alternative to the prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminal sites of the Fc region. Stable clones were obtained by expression of an antibody that specifically binds to an antigen. Positive clones can be expanded in serum-free culture medium in bioreactors to produce antibodies. The culture medium into which the antibody has been secreted can be purified by conventional methods. For example, purification can be carried out on a Protein A FF or Protein G Sepharose column containing a modified buffer. Non-specific binding components are washed out. The bound antibody is eluted with a pH gradient and the antibody fragments are detected by SDS-PAGE and then pooled. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and aggregates can be effectively removed by conventional methods such as gel filtration or ion exchange. The resulting product must be immediately frozen, for example, at minus 70°C, or lyophilized.

"Administration", "administration", "dosing", and "treatment", as applied to animals, humans, research subjects, cells, tissues, organs, or biological fluids, refers to the administration of exogenous pharmaceutical agents, therapeutic agents, diagnostic agents, compositions, or the implementation of artificial manipulation (eg, "euthanasia" as used in the examples) of animals, humans, objects, cells, tissues, organs, or body fluids. "Administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes bringing the reagent into contact with the cell, as well as bringing the reagent into contact with a liquid, where the liquid is in contact with the cell. "Administration" or "treatment" also means in vitro or ex vivo treatment of, for example, a cell with a reagent, diagnostic, binding compound, or other cell. "Treatment", as applied to a human, subject of veterinary treatment or research object, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treat" means administering a therapeutic agent, such as a composition containing any of the compounds of the present invention, orally or topically to a subject who has (or is suspected of having, or is susceptible to developing) one or more of the symptoms of a disease for which the agent has a known therapeutic benefit. activity. Typically, the agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population being treated by inducing regression or inhibiting the progression of such symptom(s) to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary depending on various factors such as the pathological condition, age and body weight of the subject, and the ability of the drug to elicit the desired response. at the subject. Whether a symptom of a disease has been alleviated can be assessed using any clinical parameter commonly used by physicians or other qualified healthcare professionals to assess the severity or progression status of that symptom. Although an embodiment of the present invention (e.g., a method of treatment or article of manufacture) is not effective in alleviating the target symptom(s) of the disease in each subject, it should alleviate the target symptom(s) of the disease in a statistically significant number of subjects, as determined by any statistical criterion, known in the art, such as Student's t-test, chi-square test, Mann-Whitney U-test, Kruskal-Wallis (H-test), Jonkheer-Terpstra test, and Wilcoxon test.

"Conservative amino acid modification" or "conservative amino acid substitution" means that amino acids in a protein or polypeptide are replaced by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) so that changes can often be made without changing the biological activity or other desired characteristics (such as affinity and/or antigen specificity) of the protein or polypeptide. It will be recognized by those skilled in the art that, in general, single amino acid substitution in non-essential regions of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., p.224 (4th Ed.)). In addition, substitutions with structurally or functionally similar amino acids are less likely to impair biological activity. Illustrative conservative amino acid substitutions are as follows:

An "effective amount" or "effective dose" refers to the amount of drug, compound, or pharmaceutical composition required to produce any one or more beneficial or desired results. For prophylactic purposes, beneficial or desirable outcomes include the elimination or reduction of risk, reduction in severity, or delay in onset of disease, including the biochemical, histological, and behavioral manifestations of the condition, its complications, and intermediate pathological phenotypes during the development of the condition. For therapeutic purposes, beneficial or desirable results include clinical results such as a reduction in the incidence of various conditions associated with the target antigen of the present invention, or an improvement in one or more of the symptoms of the condition, a reduction in the dosage of other agents needed to treat the condition, an increase in the efficacy of another agent, and /or delaying the progression of a condition associated with a target antigen of the present invention in subjects.

"Exogenous" refers to substances produced outside organisms, cells, or humans, as the case may be.

"Endogenous" refers to substances produced in cells, organisms, or human bodies, as the case may be.

"Homology" and "identity" are used interchangeably herein and refer to sequence similarity between two polynucleotide sequences or between two polypeptide sequences. When a position in both of the two sequences to be compared is occupied by the same base or subunit of an amino acid monomer, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent 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 to be compared, and then multiplied by 100. For example, when two sequences are optimally aligned, if 6 out of 10 positions in the two sequences are the same or homologous , then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences are the same or homologous, then the two sequences are 95% homologous. Typically, when two sequences are aligned, a comparison is made to obtain the maximum percentage of homology. For example, the comparison can be performed by the BLAST algorithm, in which the algorithm parameters are chosen to provide the maximum match between each sequence over the entire length of each reference sequence.

The following references refer to the BLAST algorithm often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T.L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms, such as those available from NCBI BLAST, are also well known to those skilled in the art.

"Isolated" refers to the state of "separated from its original environment", and in this case means that the specified molecule essentially does not contain other non-target biomolecules. In general, the term "isolated" is not intended to mean the complete absence of these substances, or the absence of water, buffers, or salts, unless they are present in an amount that substantially interferes with the experimental or therapeutic use of the compound as described herein.

"Optional" or "optional" means that the subsequent event or circumstance may, but need not, occur, and the description includes instances in which the event or circumstance occurs or does not occur.

"Pharmaceutical composition" refers to a mixture containing one or more compounds of the present invention, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components such as physiologically/pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to speed up the introduction into the body, facilitate the absorption of the active ingredient and thus provide a biological effect.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a composition for the delivery of antibodies or antigen-binding fragments. The carrier may be an anti-adhesive agent, adhesive agent, coating agent, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyhydric alcohol (for example, glycerol, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oil (for example, olive oil), saline, buffer, buffered saline, and isotonic agent, for example, sugars, polyhydric alcohol, sorbitol, and sodium chloride.

Specific examples of the term "metabolic disorder" are metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting glycemia, dyslipidemia, atherosclerosis, and pre-diabetic state.

In addition, another aspect of the present invention relates to methods for immunodetection or detection of target antigens, reagents for immunodetection or detection of target antigens, methods for immunodetection or detection of cells expressing target antigens, and diagnostic agents for diagnosing diseases associated with cells positive for target antigens. containing monoclonal antibodies or antibody fragments according to the present invention specifically recognizing and binding to the target antigen as active ingredients.

In the present invention, the method for detecting or measuring the amount of the target antigen may be any known method. For example, it includes an immunoassay or immunodetection method.

An immunoassay or immunodetection method is a method for detecting or measuring the amount of an antibody or antigen using a labeled antigen or antibody. Examples of immunoassay or immunodetection methods include radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), fluorescent immunoassay, Western blotting, physicochemical method, and the like.

The aforementioned diseases associated with antigen-positive target cells can be diagnosed by detecting or measuring cells expressing the target antigen using the antibodies or antibody fragments of the present invention.

Cells expressing the polypeptide can be detected by known immunodetection methods, preferably by immunoprecipitation, cell fluorescence staining, tissue immunostaining, and the like. In addition, a method such as the fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present invention, live samples to be detected or measured for the target antigen are not particularly limited, as long as they may contain cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces , tissue fluid or culture medium.

Depending on the required diagnostic method, a diagnostic agent containing a monoclonal antibody or antibody fragment according to the present invention may also contain reagents for carrying out an antigen-antibody reaction or reagents for detecting such a reaction. Reagents for carrying out an antigen-antibody reaction include buffers, salts, and the like. Detection reagents include agents commonly used in immunoassay or immunodetection methods, such as a labeled secondary antibody that recognizes a monoclonal antibody, antibody fragment or conjugate, and a substrate matching the label.

The details of one or more embodiments of the present invention are set forth in the description above. Preferred methods and materials are described below, although any method and material similar or identical to those described herein may be used in the practice or testing of the present invention. Other features, objects and advantages of the present invention will become apparent from the description and claims. In the specification and claims, the singular forms include aspects of the plural, unless the context clearly dictates otherwise. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to further illustrate preferred embodiments of the present invention. These examples should not be construed as limiting the scope of the present invention in any way, and the scope of the present invention is defined by the claims.

EXAMPLES

Example 1 Preparation of GCGR Antigen and Antibody Thereto

1.1 Construction and screening of antigens

The human GCGR cDNA encoding the full length 477 amino acid glucagon receptor was subcloned into an expression vector (such as pcDNA3.1) and transfected into CHO-K1 cells. After screening and cloning of individual cell progeny, individual clones were selected to study antigen and receptor-based expression characteristics on the cell surface. The following GCGR antigens refer to human GCGR unless otherwise noted.

Full-length GCGR: used to construct cell lines overexpressing GCGR or used as antigens for immunization and subsequent detection:

1.2 Purification of hybridomas and recombinant anti-GCGR antibodies

(1) Isolation and purification of hybridoma supernatant/Protein G affinity chromatography:

Protein G affinity chromatography is preferred for purification of mouse hybridoma supernatant. The harvested hybridoma culture was centrifuged, the supernatant was collected and, based on the volume of the supernatant, 10-15% by volume of 1 M Tris-HCl (pH 8.0-8.5) was added to adjust supernatant pH. The Protein G column was washed with 3-5 CVs of 6M guanidine hydrochloride and then washed with 3-5 CVs of pure water; the column was equilibrated with 3-5 times the column volume of an equilibrating buffer such as 1×PBS buffer system (pH 7.4); the cell supernatant was loaded at a low flow rate for binding, and the flow rate was controlled so that the retention time was about 1 minute or more; the column was washed with 3-5 times the column volume of 1×PBS (pH 7.4) until the UV absorbance dropped to the original level; samples were eluted with 0.1 M acetic acid/sodium acetate buffer (pH 3.0), elution peaks were pooled according to UV detection. The eluted product was temporarily preserved after adjusting the pH to 5-6 with 1 M Tris-HCl (pH 8.0). The eluted product may be subjected to a solution exchange according to methods well known to those skilled in the art, for example by replacing the solution with the desired buffer system by ultrafiltration-concentration with an ultrafiltration column or by replacing the solution with the desired buffer system by using molecular exclusion such as desalting G-25, or removing aggregation components from the eluted product to improve sample purity using a high resolution molecular exclusion column such as Superdex 200.

(2) Extraction of bispecific proteins or Fc-labeled antibodies by protein A affinity chromatography:

First, the cell culture supernatant containing the expressed Fc bispecific protein or antibody was centrifuged at high speed to collect the supernatant. The protein A affinity column was washed with 3-5 x column volume of 6M guanidine hydrochloride and then washed with 3-5 x column volume of pure water. The column was equilibrated with 3-5 column volumes of a column of 1xPBS buffer system (pH 7.4) as equilibration buffer. The cell supernatant was loaded at a low flow rate for binding, and the flow rate was controlled so that the retention time was about 1 min or more; after binding was completed, the column was washed with 3-5 times the column volume of 1×PBS (pH 7.4) until the UV absorbance dropped to the original level; samples were eluted with 0.1 M acetic acid/sodium acetate buffer (pH 3.0-3.5), the elution peak was pooled according to UV detection. The eluted product was temporarily preserved after adjusting the pH to 5-6 with 1 M Tris-HCl (pH 8.0). The eluted product may be subjected to a solution exchange according to methods well known to those skilled in the art, for example, by replacing the solution with the desired buffer system by ultrafiltration-concentration with an ultrafiltration column, or by replacing the solution with the desired buffer system by using molecular exclusion such as desalting G-25, or removing aggregation components from the eluted product to improve sample purity using a high resolution molecular exclusion column such as Superdex 200.

Example 2: Obtaining a monoclonal antibody to human GCGR

2.1 Immunization

(1) Immunization of mice: Monoclonal antibodies to human GCGR were obtained by immunization of mice. White mice SJL, female, aged 6-8 weeks (Beijing Charles River Experimental Animal Technology Co., Ltd., animal husbandry license number: SCXK (Jing) 2012-0001) were used for the experiment. Feeding environment: SPF level (free from pathogenic microflora). After purchase, mice were adapted to laboratory conditions for 1 week, with a light/dark cycle of 12/12 hours at a temperature of 20-25°C; humidity 40-60%. Then mice adapted to the environment, were immunized according to the following schemes. The antigen for immunization was CHO-K1 cells stably transfected with GCGR.

Immunization schedule: Mice were pre-immunized with the adjuvant, TiterMax® Gold Adjuvant (Sigma, cat. no. T2684), by intraperitoneal injection (ip), 0.1 ml/mouse (first immunization). Fifteen minutes later, mice were intraperitoneally injected (ip) with CHO-K1 cells stably transfected with GCGR, 1E7 cells per mouse. Vaccination was performed at 0, 14, 28, 42, 56, 70, 84, 98 and 112 days. Blood samples were collected on days 35, 63, and 91, and antibody titers in mouse serum were determined by ELISA. After 6-9 immunizations, mice with high serum antibody titers tending to plateau were selected for splenocyte fusion. Three days prior to splenocyte fusion, GCGR-stably transfected CHO-K1 cells were injected intraperitoneally (IP) at 1E7 cells per mouse for booster immunization.

(2) Immunization of rats: SD rats aged 6-8 weeks were immunized with DNA (encoding full-length hGCGR, coding sequence can be Genbank accession number: NM_000160), and rat serum antibody titers were determined by FACS. After 3-4 immunizations, rats with a high titer of serum antibodies tending to a plateau were selected for splenocyte fusion. Booster immunization was performed 3 days before splenocyte fusion.

2.2 Splenocyte fusion

Hybridoma cells were generated by fusion of spleen lymphocytes with Sp2/0 myeloma cells (ATCC® CRL-8287™) using an optimized PEG-mediated fusion procedure. Merged hybridoma cells were resuspended in complete medium (IMEM medium containing 20% FBS, 1×HAT, 1×OPI) at a density of 0.5-1E6/ml, plated in a 96-well plate at 100 μl/well, incubated at 37°C C and 5% CO ₂ for 3-4 days, with the addition of 100 μl/well of complete HAT medium, and continued to culture for 3-4 days until clones were formed. The supernatant was removed, 200 μl/well of complete HT medium (IMDM medium containing 20% FBS, 1×HT and 1×OPI) was added to each well, incubated at 37°C, 5% CO ₂ for 3 days and then subjected to determination ELISA method.

2.3 Screening for hybridoma cells

According to the growth density of the hybridoma cells, the hybridoma culture supernatant was determined by cell binding ELISA. The cells in the wells that tested positive were promptly expanded, cryopreserved and subcloned 2-3 times to obtain a single cell clone.

Cells after each subcloning were also tested using ELISA with the binding of GCGR cells. Hybridoma clones were screened by assay. Antibodies were additionally obtained by the method of serum-free cell culture. Antibodies were purified according to the purification example described in the test examples.

2.4 Sequencing of hybridoma positive clones

The procedures for cloning sequences from a positive hybridoma were as follows. Collected hybridoma cells in the logarithmic phase of growth. RNA was extracted with Trizol (Invitrogen cat. no. 15596-018) according to the kit instructions and reverse transcribed using the PrimeScript™ Reverse Transcription Kit (Takara cat. no. 2680A). cDNAs resulting from reverse transcription were amplified by PCR using a mouse Ig primer kit (Novagen, TB326 Rev. B 0503)) and sequenced after PCR amplification.

Positive clones were obtained from the obtained DNA sequences, and antibodies were obtained by a serum-free cell culture method. Antibodies were purified according to the purification example and subjected to several rounds of screening using a cellular GCGR binding blocking assay, thus mouse hybridoma clones 1803, 1805, 1808 and 1810 and rat hybridoma clones 1817 and 1822 were obtained.

By sequence alignment and computer modeling, it was found that the light and heavy chain CDR sequences of m1803, m1805, m1808 and m1810 mouse antibodies have higher homology, and the light and heavy chain CDR sequences of rat rat1817 and rat1822 antibodies have higher homology. Their consensus sequences are given in the following table:

Example 3 Humanization of a Mouse Anti-Human GCGR Antibody

Heavy and light chain variable region germline genes with high homology to mouse antibodies were selected as templates by alignment with the human IMGT heavy and light chain variable region germline gene database by analysis using MOE software. Mouse antibody CDRs were grafted onto an appropriate human template to form the variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, some amino acids in the framework sequence were subjected to reverse mutation to the amino acids corresponding to the mouse antibody, to obtain a humanized monoclinal antibody to GCGR. Amino acid residues in the CDR regions were identified and annotated using the Kabat numbering system.

The aforementioned mouse light and heavy chain variable regions were linked to human light and heavy chain constant regions, respectively, to form a chimeric antibody. The chimeric antibody corresponding to clone 1803 was named ch1803, and other antibodies were named similarly.

3.1 Humanization of hybridoma clone 1803

(1) Scaffold selection for humanization of hybridoma clone 1803:

For the m1803 mouse antibody, light chain humanization templates were IGKV1-39*01 and hjk4.1, and heavy chain humanization templates were IGHV1-3*01 and hjh6.1. The sequences of the humanized variable regions are as follows:

hu1803VH-graft CDR: (SEQ ID NO: 61)

hu1803VL-graft CDR: (SEQ ID NO: 58)

Note: Arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italicized sequences are FRs and the underlined sequences are CDRs.

(2) Humanization template selection and backmutation design for hybridoma clone 1803 are shown in the table below:

(3) The specific humanized sequences of hybridoma clone 1803 are as follows:

> hu1803_VL.1 (same as Hu1803VL-graft CDR): (SEQ ID NO: 58)

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

> hu1803_VL.1B: (SEQ Yu NO: 60)

> hu1803_VH.1 (same as Hu1803VH-graft CDR): (SEQ ID NO: 61)

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

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

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

(4) The sequence combinations of the light chain variable region of the humanized antibody and the heavy chain variable region of the antibody derived from hybridoma clone 1803 are as follows:

The antibody light and heavy chain variable regions shown in the table above can be linked to the corresponding antibody light and heavy chain constant region to form full length antibodies. Unless otherwise stated herein, for a full-length antibody, the antibody light chain is formed from the light chain variable region linked to the kappa chain constant region as shown in SEQ ID NO: 73, and the antibody heavy chain is formed from the heavy chain variable region linked to IgG4 -AA as shown in SEQ ID NO: 72.

3.2 Humanization of hybridoma clone 1810

(1) Choice of scaffolds for humanization of hybridoma clone 1810:

For the m1810 mouse antibody, the light chain humanization templates were IGKV1-39*01 and hJK.4.1, and the heavy chain humanization templates were IGHV1-69*02 and hJH4.1. The sequences of the humanized variable regions are as follows:

Hu1810VH-grafting CDR: (SEQ ID NO: 68)

Hu1810VL-Grafting CDR: (SEQ ID NO: 65)

Note: Arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italicized sequences are FRs and the underlined sequences are CDRs.

(2) Humanization template selection and backmutation design for hybridoma clone 1810 are shown in the table below:

(3) The specific humanized sequences of hybridoma clone 1810 are as follows:

> hu1810_VL.1 (same as Hu1810VL-graft CDR): (SEQ ID NO: 65)

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

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

> hu1810_VH.1 (same as Hu1810VH-graft CDR): (SEQ ID NO: 68)

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

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

> hu1810_VH.1C: (SEQID NO: 71)

(4) The combinations of the light and heavy chain variable region sequences of the humanized hybridoma clone 1810 antibody are as follows:

The antibody light and heavy chain variable regions shown in the table above can be linked to the corresponding antibody light and heavy chain constant region to form full length antibodies. Unless otherwise noted herein, for a full-length antibody, the antibody light chain is formed from the light chain variable region linked to the kappa chain constant region as shown in SEQ ID NO: 73 and the antibody heavy chain is formed from the heavy chain variable region linked to IgG4 -AA as shown in SEQ ID NO: 72.

Example 4: Construction and expression of the IgG4-AA format of a chimeric/humanized anti-GCGR antibody

Primers were designed, the VH/VK gene fragment of each chimeric/humanized antibody was amplified by PCR, and then inserted into the pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC/CL)) by homologous recombination to construct the vector expression for the full length antibody VH-CH1-FC-pHr/VK-CL-pHr. The IgG4-AA antibody format can be derived from the IgG4 antibody format by simple point mutations. IgG4-AA are F234A, L235A and S228P mutations. F234A and L235A mutations can reduce the binding ability of IgG4-Fc to FcγR and further reduce ADCC/CCC (complement dependent cytotoxicity). S228P indicates that the S amino acid at position 228 in the wild-type IgG4 hinge region is mutated to P. A mutation at this position can prevent native IgG4 antibody mismatches caused by in vivo Fab exchange.

ch1803, ch1805, ch1808, ch1810, ch1817, and ch1822 are chimeric antibodies formed by linking animal light and heavy chain variable regions to a human antibody kappa chain and a heavy chain constant region of a human IgG4-AA antibody, respectively.

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

The sequence of the light chain constant region (kappa chain) of an antibody is as follows: (SEQ ID NO: 73)

Illustrative engineered antibody sequences are as follows:

ch1803: IgG4-AA antibody format

ch1803 heavy chain sequence: (SEQ ID NO: 74)

ch1803 light chain sequence: (SEQ ID NO: 75)

hu1803-1: IgG4AA antibody format

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

hu1803-1 light chain sequence: (SEQ ID NO: 77)

hu1803-9 heavy chain sequence: (SEQ ID NO: 78)

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

ch1805 heavy chain sequence: (SEQ ID NO: 80)

ch1805 light chain sequence: (SEQ ID NO: 81)

ch1808 heavy chain sequence: (SEQ ID NO: 82)

ch1808 light chain sequence: (SEQ ID NO: 83)

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

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

ch1817 heavy chain sequence: (SEQ ID NO: 86)

ch1817 light chain sequence: (SEQ ID NO: 87)

ch1822 heavy chain sequence: (SEQ ID NO: 88)

ch1822 light chain sequence: (SEQ ID NO: 89)

Positive Control: Dulaglutide was present in the double chain form and the single chain form is GLP-1/hIgG4 Fc (SEQ ID NO: 90)

Example 5: Cloning and expression of a bispecific antibody protein

The human GLP-1 peptide was used as the GLP-1 receptor agonist bispecific protein part, and the anti-GCGR antibody was used as the GCGR antagonist part of the bispecific protein to form the GLP-1 bispecific protein/anti-GCGR antibody.

Studies have shown that a novel GLP-1 bispecific protein/anti-GCGR antibody with amino acid mutation(s) at a specific GLP-1 position(s) (e.g., in Q17E, 123V, K28R, or G30R) has increased in vitro stability, with stability is highest when the Q at position 17 of GLP-1A is mutated to E and the I at position 23 of GLP-1A is mutated to V(GLP-1C). Non-limiting examples of the sequences of GLP-1 and its mutant forms according to the present invention are as follows:

The C-terminal amino acid of the GLP-1 peptide of the present invention was linked to the N-terminal amino acid of the heavy chain of an anti-GCGR antibody via a peptide bond or a linker using homologous recombination technology. Expression was performed using the 293 expression system and structural models of the bispecific protein were generated as shown in Table 9:

The following proteins are formed by linking various GLP-1 peptides to the heavy chain amino acids of various antibodies via linkers (e.g.,

In this case, the linker (GGGGS) ₃ can preferably be used as the linker in the bispecific protein. In other embodiments, a peptide bond or other linkers commonly used to join polypeptides can also be used. The linkers for the bispecific protein of the present invention are not limited to the use of (GGGGS) ₃ . Nucleotide sequences encoding GLP-1, anti-GCGR antibodies, and a linker protein fragment ((GGGGS)3) were obtained by conventional techniques in the art. The C-terminal nucleotide of GLP-1 was linked to the N-terminal nucleotide of an anti-GCGR antibody via a linker protein using homologous recombination technology and cloned into the Phr-BsmbI vector. Recombinant bispecific GLP-1/anti-GCGR antibodies were expressed in 293 cells and purified by the method described in Example 6. The purified protein can be used in each of the following examples.

Example 6 Purification of a Bispecific Antibody Protein

The cell culture medium was centrifuged at high speed, the supernatant was collected and subjected to affinity chromatography as the first purification step. The chromatographic medium was protein A, which interacts with Fc, or a derived excipient such as Mabselect, GE. The balancing buffer was 1×pBS (137 mmol/l NaCl, 2.7 mmol/l KCl, 10 mmol/l Na ₂ HPO ₄ , 2 mmol/nL KH ₂ PO ₄ , pH 7.4). After the column was equilibrated with 5 times the column volume, the cell culture supernatant was loaded for binding, and the flow rate was controlled so that the sample retention time on the column was 1 minute or more. After loading the sample, the column was washed with 1×PBS (pH 7.4) until the UV absorbance at 280 nm dropped to the original level. The column was then washed with 0.1 M glycine (pH 3.0) elution buffer, the elution peak was collected based on the UV absorption peak at 280 nm, and the collected eluted sample was neutralized with 1 M Tris (pH 8.5).

The neutralized eluted sample was ultrafiltered and concentrated, and then subjected to size exclusion chromatography using 1×pBS buffer, the chromatography column was XK26/60 Superdex200 (GE), the flow rate was controlled at 4 ml/min, and the loading volume was less than 5 ml. The target protein peak was pooled based on UV absorbance at 280 nm. The assembled protein was identified by SEC-HPLC and was above 95% pure. After identification by LC-MS, the desired protein was aliquoted for use. Bispecific GLP-1 proteins/anti-GCGR antibodies have been generated

Test Cases

Test Example 1: ELISA Assay (Enzymatic ImmunoAssay) Binding of Chimeric Anti-GCGR Antibody to Human, Mouse, and Cynomolgus Macaque GCGR

The binding capacity of antibodies to GCGR was tested by assaying the binding of the antibody to CHO cells overexpressing GCGR. Full-length human, mouse, and cynomolgus GCGR plasmids were transfected into CHO cells, respectively. GCGR expression levels were determined after two weeks of pressure screening. After cells overexpressing GCGR were fixed on the bottom of a 96-well plate, antibodies were added and signal strength was used to determine antibody binding activity to GCGR-overexpressing CHO cells. The binding abilities of the antibody to the three GCGR species were determined using the same binding assay. For example, binding of GCGR antibodies to human GCGR was determined using an assay detailed as follows:

Cells were seeded in a 96-well plate at a density of 0.9-1.0×10 ⁶ /ml, 100 μl/well and cultured overnight. The supernatant was discarded, the plate was washed three times with PBS, 100 μl/well of cell immune fixative solution (Beyotime, cat. no. P0098) was added, fixed for one hour at room temperature, then washed four times with PBS. The liquid was discarded, 200 μl/well of blocking solution (5% skimmed milk (BD skim milk, cat. #232100) diluted with PBS) was added and incubated in an incubator at 37° C. for 3 hours to block. After blocking was completed, the blocking solution was discarded, the plate was washed 3 times with PBST buffer (PBS containing 0.05% tweenW-20, pH 7.4), 50 µl/well of various concentrations of each of the tested antibodies (antibodies purified from hybridoma, chimeric antibodies or humanized antibodies) diluted with Sample Diluent and incubated in an incubator at 37°C for 2 hours. After completion of the incubation, the plate was washed 3 times with PBST, 50 µl/well HRP-labeled secondary goat anti-mouse antibody (Jacksow ImmuWo Research, cat. no. 115-035-003) or secondary goat anti-human antibody (Jacksow ImmuWo Research, cat. no. 109-035-003) diluted with Sample Diluent and incubated at 37°C for 1 hour. The plate was washed 3 times with PBST, 50 µl/well TMB chromogenic substrate (KPL, Cat #52-00-03) was added, incubated at room temperature for 10 minutes, and 50 µl/well 1M H ₂ SO ₄ was added to stop reactions. The absorbance value was read with a microplate reader (Thermo scientific Multiskan MK3) at a wavelength of 450 nm, and the data was analyzed using a GraphPad Prism 5. The binding of anti-GCGR chimeric antibodies to GCGR-overexpressing CHO cells was calculated as an EC50 value.

The results demonstrate that the chimeric antibodies ch1803, ch1805, ch1808, ch1810, ch1817, and ch1822 all have favorable binding activity to human GCGR on the cell surface, and also have favorable cross-affinity to mouse and cynomolgus monkey GCGR.

Test Example 2 Chimeric Anti-GCGR Antibody Blocking Assay of GCGR Ligand Binding to GCGR

1. Purpose:

The antagonistic activities of chimeric anti-GCGR antibodies were assessed by a chimeric anti-GCGR antibody blocking assay of GCGR ligand (glucagon) binding to GCGR.

2. The principle of the test:

Expression of the luciferase (luciferase) gene below CRE (cAMP response element) can be induced by cAMP binding to CRE. The change in the fluorescence signal emitted by the binding of a luciferase to its substrate may reflect the effectiveness of the inhibition. CRE was cloned upstream of the luciferase gene and co-transfected into CHO-K1 cells with a plasmid containing the GCGR gene. Monoclonal cells expressing both CRE and GCGR were selected. The bispecific GLP-1/anti-GCGR protein can compete with glucagon for binding to GCGR, block signaling downstream of GCGR, and affect downstream cAMP expression. Antagonistic activities of anti-GCGR GLP-1 bispecific proteins/anti-GCGR antibodies can be assessed by measuring changes in the fluorescence signal.

3. Samples tested:

Chimeric antibodies ch1803, ch1805, ch1808, ch1810, ch1817 and ch1822.

4. Experimental technique

a. The cell suspension was prepared with fresh cell culture medium and added to a 96-well cell culture plate with 80 μl culture system, 20,000 cells/well, and incubated at 5% CO2, 37° C. for 16 hours.

b. 10 µl of each of the prepared test proteins was added to each well, then 10 µl of prepared glucagon was added and incubated at 5% CO2, 37°C for 5 hours.

With. 100 μl of ONE Glo detection solution (Promega) was added to each well and placed in the dark at room temperature for 7 minutes.

d. Fluorescence was determined on a Victor3 microplate reader and the blocking efficiency IC50 and Imax were calculated in relation to the fusion blocking of the chimeric anti-GCGR antibody from the binding of the GCGR ligand (glucagon) to human, mouse and cynomolgus monkey GCGR.

Test Example 3 Humanized Anti-GCGR Antibody Blocking Assay of GCGR Ligand Binding to GCGR

The antagonistic activities of anti-GCGR antibodies were assessed by an anti-GCGR antibody blocking assay of GCGR ligand (glucagon) binding to GCGR. The principle and steps of the experiment are the same as in Test Case 2, and the results are presented in the following table:

Biological evaluation in vitro

Test Example 4: Stability of GLP-1 Bispecific Protein/Anti-GCGR Antibody in PBS

200 μg of the test bispecific antibody protein was dissolved in 1 ml of 1×PBS (pH 7.4) and stored in an incubator at 37°C; samples were taken on days 0 and 14, respectively, and intact heavy chain retention was determined by LC-MS on an Agilent 6530 Q-TOF. The results are shown in Table 14 below. Each of the mutated GLP-1 peptide bispecific proteins has a significantly higher stability compared to the bispecific protein containing GLP-1A (SEQ ID NO: 91) and hu1803-9B, hu1803-9D and hu1803 -9G show better stability.

Test Case 5: GCGR Binding Blocking Assay in Cells

The principle and steps of the experiment are the same as in Test Case 2.

Samples tested:

1) antibodies to GCGR (hu1803-9, hu1810-12)

2) Bispecific proteins: hu1803-9B and hu1803-9D.

In FIG. 2 and Table 15 show that both hu1803-9B and hu1803-9D can completely inhibit GCGR antagonistic activity and have comparable potency and IC50 to an anti-GCGR monoclonal antibody, indicating that both hu1803-9B and hu1803 -9D retains the full biological activity of the anti-GCGR antibody portion. In addition to the confirmed antagonistic activity of hu1803-9 and the bispecific protein containing hu1803-9 against inhibition of human GCGR, both hu1803-9 and hu1810-12 were further shown (see Table 16) to have antagonistic activity against mouse and cynomolgus monkey GCGR inhibition.

Test Case 6: GLP-1R Activation Assay in Cells

1. Purpose:

The aim is to evaluate the activating activity of the GLP-1 portion of the GLP-1 bispecific protein/anti-GCGR antibody against GLP-1R.

2. The principle of the test:

Expression of the luciferase gene downstream of the CRE can be induced by cAMP binding to the CRE. The change in the fluorescence signal emitted by the binding of a luciferase to its substrate may reflect the effectiveness of the inhibition. CRE was cloned upstream of the luciferase gene and co-transfected into CHO-K1 cells with a plasmid containing the GLP-1R gene. Selected monoclonal cells, at a high level expressing both CRE and GLP-1R. Both the GLP-1 bispecific protein/anti-GCGR antibody and dulaglutide (positive control) can bind to GLP-1R, activate signaling downstream of GLP-1R, and stimulate downstream cAMP expression. The agonist activities of the GLP-1 bispecific proteins/anti-GCGR antibodies against GLP-1R can be assessed by measuring changes in the fluorescence signal.

3. Samples tested:

1) Positive control: Dulaglutide

2) hu1803-9B, hu1803-9D.

4. Experimental technique

a. The cell suspension was prepared with fresh cell culture medium and added to a 96-well cell culture plate with 90 μl culture system, 25,000 cells/well, and incubated at 5% CO2, 37° C. for 16 hours.

b. 10 µl of each of the prepared test proteins is added to each well and incubated at 5% CO2, 37°C for 5 hours.

With. 100 µl of ONE Glo detection solution (Promega) is added to each well and placed in the dark at room temperature for 7 minutes.

d. Fluorescence is determined on a Victor3 microplate reader and EC50 values of GLP-1 bispecific proteins/anti-GCGR antibodies are calculated for activation of GLP-1R by binding.

In FIG. 3 and Table 17 show that both hu1803-9B and hu1803-9D can fully activate GLP-1R and have comparable potency and EC50 to the positive control (dulaglutide), indicating that both hu1803-9B and hu1803-9D retain the full biological activity of the GLP-1 portion.

Table 18 shows that all of the ch1805-D, ch1808-D, and ch1817-D chimeric antibodies coupled to the GLP1 peptide can fully activate GLP-1 R and have comparable potency and EC50 (concentration required to activate 50% of maximum activity). ) with a positive control (dulaglutide), indicating that the bispecific proteins formed by binding various anti-GCGR antibodies to the GLP1 peptide in the manner disclosed herein do not affect the biological activity of the GLP1 peptide.

Pharmacokinetic evaluation

Test Example 7: Determination of in vivo pharmacokinetics in C57 mice

Four C57 laboratory mice, females, were kept under a 12/12 hour day/night cycle, a constant temperature of 24±3°C, a humidity of 50-60%, and unlimited access to water and food. The mice were purchased from Jiesijie Laboratory Animal Co., Ltd. On the day of the test, equimolar hu1803-9D and the positive control drug dulaglutide were injected into the tail vein of C57 mice at a dose of 6 mg/kg and 2.35 mg/kg, respectively, and an injection volume of 10 ml/kg.

Since both the GLP-1 bispecific protein/anti-GCGR antibody of the present invention and the positive control dulaglutide have cross-activity with mice, the drug's metabolism time is shorter in mice. The selected time points for blood collection were: 0 h, 1 h, 24 h (day 2) on day 1 after administration and day 3. Blood was collected from a mouse retinal vein 150 μl at a time (1.5 μl of DPP-4 inhibitor was added to the blood collection tube before blood collection). The collected blood sample was placed at 4°C for half an hour before coagulation, and then centrifuged at 14000×g for 5 minutes at 4°C. The supernatant (about 80 µl) was collected and immediately stored at minus 80°C.

The determination process is described as follows:

a. 1 μg/ml anti-GLP1 antibody (Novus, NBP1-05180) was plated at 4°C overnight.

b. Washed 4 times with 250 μl 1×PBST, 200 μl PBS containing 5% milk was added and blocked at 37°C for 3 hours.

With. Washed 4 times with 250 μl of 1×PBST, 100 μl of test drug gradient diluted with empty mouse serum was added and incubated at 37° C. for 2 hours.

d. Washed 3 times with 250 μl 1×PBST.

e. 100 μl of horseradish peroxidase-labeled anti-human IgG Fc secondary antibody was added to each well and incubated at 37° C. for 1 hour.

f. Washed 3 times with 250 μl 1×PBST.

g. 100 μl TMB was added to each well, incubated at room temperature for 10 minutes; and then 100 µl of 1M H ₂ SO ₄ was added to stop the reaction.

h. Absorbance was measured at 450 nm on a microplate reader; and data was analyzed using Graphpad Prism 5.

The results of the PK analysis demonstrate that the half-life of the hu1803-9D bispecific protein molecule of the present invention in mice is about 23.4 hours, which is twice that of the positive control.

Assessment of biological activity in vivo

Test Example 8: In Vivo Drug Efficacy Test on ob/ob Mice 1. The strain of mice used in this test were diabetic ob/ob mice and wild-type mice of the same age (Institute of Animal Model, Nanjing University). The aim is to observe the effect of GLP-1/anti-GCGR bispecific protein treatment on diabetes-related parameters such as blood glucose, glycosylated hemoglobin, body weight and food intake after continuous administration to ob/ob mice.

Before the test, the model animals were divided into 6 groups according to random body weight and fasting body weight, random blood glucose level and fasting blood glucose level on the day of the test, respectively:

Model control group, anti-GCGR monoclonal antibody (hu1803-9) 2.84 mg/kg and 1.42 mg/kg groups, positive control group 1.16 mg/kg (dulaglutide) and 3 mg/kg and 1.5 mg /kg hu1803-9D. Mice in the control group were injected subcutaneously (sc) with phosphate buffer, and mice in each group were injected subcutaneously once a week (9:00 am), 4 times in total (Table 20).

2. Experimental technique

a. Fasting body weight and random body weight were measured once a week, and food and water intake was measured once a day.

b. The blood glucose level is measured randomly before the first administration and on days 1, 2, 3 and 7 after the administration, and thereafter the blood glucose level is measured once a week. The blood glucose level after 6 hours of fasting is measured before the first administration and on days 3 and 7 after the administration, and then the blood glucose level is measured once a week.

With. On the 26th day of administration, after the animals are not fed for 6 hours (8:00-14:00), a single dose of glucose solution of 2 g/kg is administered intraperitoneally, and the time of administration of glucose is recorded as 0. Animals are checked for glucose levels in blood 0 min before glucose administration and 15, 30, 60, 90 and 120 minutes after glucose administration. A glucose tolerance curve was plotted from blood glucose versus time data and the area under the curve (AUC) was calculated.

d. After the experiment, mice are not fed for 6 hours (8:00-14:00) and then sacrificed. Blood samples were taken from the heart, whole blood was divided into two parts, one part of about 30 µl was added to a centrifuge tube with anticoagulant previously added and stored to determine glycosylated hemoglobin, and the other part was left alone on the table and then centrifuged to obtain serum to determine the levels of TG (triglycerides), FFA (free fatty acids), Cholesterol (cholesterol), HDL (high density lipoprotein) and LDL (low density lipoprotein).

e. Data are analyzed with Graphpad Prism 6 software and Student's t-test is used for statistical data analysis.

3. Results of the experiment:

1) Effect of long-term administration on random blood glucose concentrations in ob/ob mice:

As shown in FIG. 4, random blood glucose is maintained at a high level in the model control group of ob/ob mice throughout the experiment. Random blood glucose levels in each of the administration groups decreased to varying degrees following subcutaneous injection of each agent at different doses once a week, demonstrating a favorable dose efficacy that was significantly lower than that of the model control group. Administration of 2.84mg/kg hu1803-9 and 3mg/kg hu1803-9D showed a significantly improved blood glucose lowering effect compared to other test groups, while administration of 3mg/kg hu1803-9D by 3, 6, 14 and day 30 shows the best effect in terms of reducing random blood glucose levels.

2) Effect of long-term administration on fasting blood glucose in ob/ob mice:

As shown in FIG. 5, random blood glucose was maintained at a high level in the model control group of ob/ob mice throughout the experiment. Fasting blood glucose levels in each of the administration groups decreased to varying degrees following subcutaneous injection of each agent at different doses once a week, demonstrating a favorable dose efficacy that is significantly lower than that of the model control group. Similar to the random blood glucose test, administration of 2.84mg/kg hu1803-9 and 3mg/kg hu1803-9D showed a significantly improved blood glucose lowering effect compared to other test groups, while administration of 3mg/kg hu1803-9D at days 3, 6, 13 and 30 shows the best effect in terms of lowering fasting blood glucose.

3) Effect of long-term administration on glycosylated hemoglobin (HbA1c) in ob/ob mice:

The results are shown in Table 21. Glycosylated hemoglobin levels in each of the administration groups decreased to varying degrees 30 days after weekly subcutaneous injection of each agent at different doses, and was significantly lower than in the model control group (P<0.05). Glycosylated 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, indicating a significant dose-response ratio; among them, the 3mg/kg hu1803-9D group has a lower level than the equimolar 1.16mg/kg dulaglutide positive control group and 2.84mg/kg hu1803-9 anti-GCGR monoclonal antibody (P<0.05).

Test Example 9: Anti-GCGR Antibody Competitive ELISA

GCGR epitopes associated with anti-GCGR antibodies were classified by ELISA, which determined the competitive binding of biotin-labeled antibodies with various concentrations of standard antibody to GCGR-overexpressing CHO cells. The production method of the cell plate was the same as in Test Example 1. The following specific procedures were:

The antibody was labeled according to the instructions of the biotin labeling kit (Dojindo Molecular Technologies, Inc. LK03). The unlabeled test antibody was diluted to a range of concentrations with sample dilution solution in a 96-well cell plate, 50 μl/well, and incubated at 37° C. for 2 hours. After completion of the incubation, the plate was washed 3 times with PBST, 50 μl/well of biotin-labeled antibody diluted with sample dilution solution was added at a concentration of 0.1 μg/ml, incubated at 37°C for 2 hours, the plate was washed 3 times with PBST, added HRP-labeled secondary goat anti-human antibody (JacksoW ImmuWo Research cat. no. 109-035-003) and incubated at 37° C. for 1 hour. The plate was washed 3 times with PBST, 50 µl/well TMB chromogenic substrate (KPL, Cat #52-00-03) was added, incubated at room temperature for 10 minutes, and 50 µl/well 1M H ₂ SO ₄ was added to stop reactions. The absorbance value was read with a microplate reader (Thermo scientific Multiskan MK3) at a wavelength of 450 nm, and the data was analyzed using a GraphPad Prism 5.

The lower the concentration of biotin labeled antibody bound to the competition cell plate, the lower the optical density (OD) value, and vice versa. The effectiveness of competition was calculated by the formula:

результаты приведены ниже в таблице 22.

the results are shown in Table 22 below.

The results demonstrate that ch1817 and ch1822 have very similar epitopes; ch1808, hu1803-9 and hu1810-12 have very similar epitopes; while the epitopes associated with ch1808, hu1803-9 and hu1810-12 are expected to be in the range of epitopes associated with ch1805.

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SEQUENCE LIST

<110> JIANGSU HENGRUI MEDSIN CO., LTD.,

SHANGHAI HENGUI PHARMACEUTICAL CO., LTD.

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Ala Ile Leu Gly Gly Leu Ser Lys Leu His Cys Thr Arg Asn Ala Ile

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His Ala Asn Leu Phe Ala Ser Phe Val Leu Lys Ala Ser Ser Val Leu

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Val Ile Asp Gly Leu Leu Arg Thr Arg Tyr Ser Gln Lys Ile Gly Asp

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Asp Leu Ser Val Ser Thr Trp Leu Ser Asp Gly Ala Val Ala Gly Cys

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Arg Val Ala Ala Val Phe Met Gln Tyr Gly Ile Val Ala Asn Tyr Cys

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Gly Ala Pro Met Leu Phe Val Val Pro Trp Ala Val Val Lys Cys Leu

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Phe Glu Asn Val Gln Cys Trp Thr Ser Asn Asp Asn Met Gly Phe Trp

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Trp Ile Leu Arg Phe Pro Val Phe Leu Ala Ile Leu Ile Asn Phe Phe

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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

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Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Phe Pro Trp

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Gly Glu Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Lys Thr Met Tyr

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Met Glu Leu Arg Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys

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Ala Ser Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr

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Trp Gly Gln Gly Ser Pro Leu Thr Val Ser Ser

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<212> PRT

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

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Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Thr Thr Ala Asp Tyr Phe Tyr Gly Ile Tyr Phe Ala Leu

100 105 110

Asp Ala Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 11

<211> 111

<212> PRT

<213> Gray rat

<220>

<221> DOMAIN

<223> rat1817 light chain variable region

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Gln Phe Thr Leu Thr Gln Pro Lys Ser Val Ser Gly Ser Leu Arg Ser

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Thr Ile Thr Ile Pro Cys Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser

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Tyr Val Asn Trp Tyr Gln Gln His Leu Gly Arg Pro Pro Leu Asn Val

35 40 45

Ile Tyr Ala Asp Val Gln Arg Pro Ser Glu Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Thr Asn

65 70 75 80

Leu Gln Met Asp Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Thr

85 90 95

Asn Ile Asp Ile Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 12

<211> 125

<212> PRT

<213> Gray rat

<220>

<221> DOMAIN

<223> rat1822 heavy chain variable region

<400> 12

Glu Val Arg Leu Val Glu Ser Gly Gly Asp Phe Val Gln Pro Gly Arg

1 5 10 15

Ser Val Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Gly Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Thr Thr Pro Asp Tyr His Tyr Gly Ile Tyr Phe Ala Met

100 105 110

Asp Ala Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 13

<211> 111

<212> PRT

<213> Gray rat

<220>

<221> DOMAIN

<223> rat1822 light chain variable region

<400> 13

Gln Val Thr Leu Thr Gln Pro Lys Ser Val Ser Gly Ser Leu Arg Ser

1 5 10 15

Thr Ile Thr Ile Pro Cys Glu Arg Ser Ser Gly Asp Ile Gly Glu Ser

20 25 30

Tyr Val Asn Trp Tyr Gln Gln His Leu Gly Arg Pro Pro Ile Asn Val

35 40 45

Ile Tyr Ala Asp Asp Gln Arg Pro Ser Glu Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Thr Asn

65 70 75 80

Leu Gln Val Asp Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Ser

85 90 95

Ser Ile Asp Ile Phe Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 14

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR1 m1803

<400> 14

Asp Tyr Trp Ile Glu

fifteen

<210> 15

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 m1803

<400> 15

Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

<210> 16

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 m1803

<400> 16

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr

1 5 10

<210> 17

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 m1803

<400> 17

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 m1803

<400> 18

Tyr Ser Ser Thr Leu His Ser

fifteen

<210> 19

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 m1803

<400> 19

Gln Gln Thr Asn Ile Phe Pro Trp Thr

fifteen

<210> 20

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR1 m1805

<400> 20

Asp Tyr Asn Met Asp

fifteen

<210> 21

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 m1805

<400> 21

Ser Ile Asp Pro Asp Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys

1 5 10 15

gly

<210> 22

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 m1805

<400> 22

Asp Tyr Tyr Gly Ser Ser Ser Trp Phe Ala Tyr

1 5 10

<210> 23

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 m1805

<400> 23

Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His

1 5 10

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 m1805

<400> 24

Tyr Ala Ser Gln Ser Ile Ser

fifteen

<210> 25

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 m1805

<400> 25

Gln Asn Gly His Ser Phe Pro Tyr Thr

fifteen

<210> 26

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR1 m1808

<400> 26

Thr Asn Gly Ile Ser

fifteen

<210> 27

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 m1808

<400> 27

Glu Ile Tyr Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Asn Phe Lys

1 5 10 15

gly

<210> 28

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 m1808

<400> 28

Ser Ile Thr Ser Val Ile Gly Ala Asp Tyr Phe Asp Tyr

1 5 10

<210> 29

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 m1808

<400> 29

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 30

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 m1808

<400> 30

Tyr Ser Ser Thr Leu His Ser

fifteen

<210> 31

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 m1808

<400> 31

Gln Gln Gly Asn Thr Phe Pro Trp Thr

fifteen

<210> 32

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR1 m1810

<400> 32

Asn Tyr Ala Ile Ser

fifteen

<210> 33

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 m1810

<400> 33

Glu Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

<210> 34

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 m1810

<400> 34

Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr

1 5 10

<210> 35

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 m1810

<400> 35

Arg Ala Ser Leu Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 36

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 m1810

<400> 36

Tyr Thr Ser Thr Leu His Ser

fifteen

<210> 37

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 m1810

<400> 37

Gln Gln Gly Asn Met Val Pro Tyr Thr

fifteen

<210> 38

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR1 rat1817

<400> 38

Asn Tyr Tyr Met Ala

fifteen

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 rat1817

<400> 39

Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val Lys

1 5 10 15

gly

<210> 40

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 rat1817

<400> 40

His Thr Thr Ala Asp Tyr Phe Tyr Gly Ile Tyr Phe Ala Leu Asp Ala

1 5 10 15

<210> 41

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 rat1817

<400> 41

Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser Tyr Val Asn

1 5 10

<210> 42

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 rat1817

<400> 42

Ala Asp Val Gln Arg Pro Ser

fifteen

<210> 43

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 rat1817

<400> 43

Gln Ser Tyr Asp Thr Asn Ile Asp Ile Ile

1 5 10

<210> 44

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 rat1822

<400> 44

His Thr Thr Pro Asp Tyr His Tyr Gly Ile Tyr Phe Ala Met Asp Ala

1 5 10 15

<210> 45

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 rat1822

<400> 45

Glu Arg Ser Ser Gly Asp Ile Gly Glu Ser Tyr Val Asn

1 5 10

<210> 46

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 rat1822

<400> 46

Ala Asp Asp Gln Arg Pro Ser

fifteen

<210> 47

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 rat1822

<400> 47

Gln Ser Tyr Asp Ser Ser Ile Asp Ile Phe

1 5 10

<210> 48

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula of HCDR1 mouse antibody formula

<220>

<221> DOMAIN

<222> (1)..(3)

<223> Xaa-Xaa-Xaa is selected from Asp-Tyr-Trp, Thr-Asn-Gly, or Asn-Tyr-Ala

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa selected from Glu or Ser

<400> 48

Xaa Xaa Xaa Ile Xaa

fifteen

<210> 49

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula of HCDR2 mouse antibody

<220>

<221> DOMAIN

<222> (3)..(3)

<223> Xaa is selected from Leu or Tyr.

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Gly, Arg, or Thr.

<220>

<221> DOMAIN

<222>(7)..(7)

<223> Xaa is selected from Gly or Thr.

<220>

<221> DOMAIN

<222>(8)..(8)

<223> Xaa selected from Tyr or Asn.

<220>

<221> DOMAIN

<222> (10)..(10)

<223> Xaa selected from Asn or Tyr.

<400> 49

Glu Ile Xaa Pro Xaa Ser Xaa Xaa Thr Xaa Tyr Asn Glu Xaa Phe Lys

1 5 10 15

gly

<210> 50

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula of HCDR3 mouse antibody

<220>

<221> DOMAIN

<222> (1)..(1)

<223> Xaa is selected from Gly or Ser.

<220>

<221> DOMAIN

<222> (2)..(2)

<223> Xaa is selected from Ile, Leu, or Val-Ile.

<220>

<221> DOMAIN

<222> (3)..(3)

<223> Xaa selected from Ser or Thr

<220>

<221> DOMAIN

<222> (4)..(4)

<223> Xaa selected from Ser or Thr

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Leu or Val.

<220>

<221> DOMAIN

<222>(6)..(6)

<223> Xaa is selected from Met, Val, or Ile.

<220>

<221> DOMAIN

<222>(7)..(7)

<223> Xaa is selected from Ser, Gly, or Ala.

<220>

<221> DOMAIN

<222>(8)..(8)

<223> Xaa is selected from Thr, Ala, or Val.

<400> 50

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Phe Asp Tyr

1 5 10

<210> 51

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula LCDR1 mouse antibody

<220>

<221> DOMAIN

<222> (4)..(4)

<223> Xaa is selected from Leu or Gln.

<400> 51

Arg Ala Ser Xaa Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 52

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula LCDR2 mouse antibody

<220>

<221> DOMAIN

<222> (2)..(2)

<223> Xaa selected from Ser or Thr.

<400> 52

Tyr Xaa Ser Thr Leu His Ser

fifteen

<210> 53

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula LCDR3 mouse antibody

<220>

<221> DOMAIN

<222> (3)..(3)

<223> Xaa is selected from Thr or Gly.

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa is selected from Met, Thr, or Ile.

<220>

<221> DOMAIN

<222>(6)..(6)

<223> Xaa is selected from Phe or Val.

<220>

<221> DOMAIN

<222>(8)..(8)

<223> Xaa selected from Trp or Tyr.

<400> 53

Gln Gln Xaa Asn Xaa Xaa Pro Xaa Thr

fifteen

<210> 54

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula of HCDR3 rat antibody

<220>

<221> DOMAIN

<222> (4)..(4)

<223> Xaa selected from Pro or Ala.

<220>

<221> DOMAIN

<222>(7)..(7)

<223> Xaa is selected from Phe or His.

<220>

<221> DOMAIN

<222> (14)..(14)

<223> Xaa is selected from Leu or Met.

<400> 54

His Thr Thr Xaa Asp Tyr Xaa Tyr Gly Ile Tyr Phe Ala Xaa Asp Ala

1 5 10 15

<210> 55

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula LCDR1 rat antibody

<220>

<221> DOMAIN

<222>(9)..(9)

<223> Xaa is selected from Glu or Asp.

<400> 55

Glu Arg Ser Ser Gly Asp Ile Gly Xaa Ser Tyr Val Asn

1 5 10

<210> 56

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula LCDR2 rat antibody

<220>

<221> DOMAIN

<222> (3)..(3)

<223> Xaa is selected from Asp or Val.

<400> 56

Ala Asp Xaa Gln Arg Pro Ser

fifteen

<210> 57

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> general formula LCDR3 rat antibody

<220>

<221> DOMAIN

<222> (5)..(5)

<223> Xaa selected from Ser or Thr.

<220>

<221> DOMAIN

<222>(6)..(6)

<223> Xaa selected from Ser or Asn.

<220>

<221> DOMAIN

<222> (10)..(10)

<223> Xaa is selected from Phe or Ile.

<400> 57

Gln Ser Tyr Asp Xaa Xaa Ile Asp Ile Xaa

1 5 10

<210> 58

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu1803_VL.1

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ser Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Ile Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 59

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu1803_VL.1A

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Ser Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Asn Ile Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu1803_VL.1B

<400> 60

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Ser Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Ile Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu1803_VH.1

<400> 61

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 62

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu1803_VH.1A

<400> 62

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 63

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu1803_VH.1B

<400> 63

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 64

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> hu1803_VH.1C

<400> 64

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Ala Asp Pro Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 65

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu1810_VL.1

<400> 65

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Met Val Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 66

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu1810_VL.1A

<400> 66

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Met Val Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 67

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu1810_VL.1B

<400> 67

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Leu Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Met Val Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 68

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu1810_VH.1

<400> 68

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 69

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu1810_VH.1A

<400> 69

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Arg Ser Thr Ser Thr Met Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 70

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu1810_VH.1B

<400> 70

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Arg Ser Thr Ser Thr Met Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 71

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu1810_VH.1C

<400> 71

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Ala Ile Ser Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Thr Ser Thr Met Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 72

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IgG4-AA heavy chain constant region

<400> 72

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 73

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> antibody light chain constant region (kappa chain)

<400> 73

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 74

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain ch1803

<400> 74

Gln Phe Gln Leu His Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Thr Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Ala Glu Pro Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ser Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 75

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain ch1803

<400> 75

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Asn Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Ser Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser His Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Thr Asn Ile Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 76

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain hu1803-1

<400> 76

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 77

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain hu1803-1

<400> 77

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ser Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Ile Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 78

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain hu1803-9

<400> 78

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 79

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain hu1803-9

<400> 79

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Ser Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Ile Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 80

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain ch1805

<400> 80

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Pro Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met Asp Trp Val Lys Gln Ser His Gly Arg Ser Leu Glu Trp Ile

35 40 45

Gly Ser Ile Asp Pro Asp Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Asp Tyr Tyr Gly Ser Ser Ser Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 81

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain ch1805

<400> 81

Asp Val Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 82

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain ch1808

<400> 82

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Asp Thr Phe Thr Thr Asn

20 25 30

Gly Ile Ser Trp Val Lys Gln Arg Ile Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Ile Thr Ser Val Ile Gly Ala Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 83

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain ch1808

<400> 83

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Lys Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Ser Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 84

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain hu1810-12

<400> 84

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Ala Ile Ser Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Thr Ser Thr Met Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

210 215 220

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440 445

Gly Lys

450

<210> 85

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain hu1810-12

<400> 85

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Leu Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Met Val Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 86

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain ch1817

<400> 86

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Thr Thr Ala Asp Tyr Phe Tyr Gly Ile Tyr Phe Ala Leu

100 105 110

Asp Ala Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Lys

450

<210> 87

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain ch1817

<400> 87

Gln Phe Thr Leu Thr Gln Pro Lys Ser Val Ser Gly Ser Leu Arg Ser

1 5 10 15

Thr Ile Thr Ile Pro Cys Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser

20 25 30

Tyr Val Asn Trp Tyr Gln Gln His Leu Gly Arg Pro Pro Leu Asn Val

35 40 45

Ile Tyr Ala Asp Val Gln Arg Pro Ser Glu Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Thr Asn

65 70 75 80

Leu Gln Met Asp Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Thr

85 90 95

Asn Ile Asp Ile Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 88

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> heavy chain ch1822

<400> 88

Glu Val Arg Leu Val Glu Ser Gly Gly Asp Phe Val Gln Pro Gly Arg

1 5 10 15

Ser Val Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Gly Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Thr Thr Pro Asp Tyr His Tyr Gly Ile Tyr Phe Ala Met

100 105 110

Asp Ala Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Lys

450

<210> 89

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> light chain ch1822

<400> 89

Gln Val Thr Leu Thr Gln Pro Lys Ser Val Ser Gly Ser Leu Arg Ser

1 5 10 15

Thr Ile Thr Ile Pro Cys Glu Arg Ser Ser Gly Asp Ile Gly Glu Ser

20 25 30

Tyr Val Asn Trp Tyr Gln Gln His Leu Gly Arg Pro Pro Ile Asn Val

35 40 45

Ile Tyr Ala Asp Asp Gln Arg Pro Ser Glu Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Thr Asn

65 70 75 80

Leu Gln Val Asp Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Ser

85 90 95

Ser Ile Asp Ile Phe Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 90

<211> 275

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Dulaglutide

<400> 90

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu

35 40 45

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

50 55 60

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

65 70 75 80

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

85 90 95

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

100 105 110

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

115 120 125

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

130 135 140

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

145 150 155 160

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

165 170 175

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

180 185 190

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

195 200 205

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

210 215 220

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

225 230 235 240

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

245 250 255

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

260 265 270

Ser Leu Gly

275

<210> 91

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1A

<400> 91

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly

20 25 30

<210> 92

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1B

<400> 92

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly

20 25 30

<210> 93

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1C

<400> 93

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210> 94

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1D

<400> 94

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Gly Gly

20 25 30

<210> 95

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1E

<400> 95

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210> 96

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1F

<400> 96

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly

20 25 30

<210> 97

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1G

<400> 97

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Arg Gly Gly Gly

20 25 30

<210> 98

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1H

<400> 98

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

20 25 30

<210> 99

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> GLP-1J

<400> 99

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Arg Gly Arg Gly

20 25 30

<210> 100

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9A containing part of the heavy chain

<400> 100

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Ile

65 70 75 80

Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu

85 90 95

Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

130 135 140

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 101

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9B containing part of the heavy chain

<400> 101

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Ile

65 70 75 80

Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu

85 90 95

Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

130 135 140

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 102

<211> 496

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9C containing part of the heavy chain

<400> 102

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly

20 25 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

35 40 45

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

50 55 60

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp

65 70 75 80

Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

85 90 95

Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys

100 105 110

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met

115 120 125

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

130 135 140

Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly

145 150 155 160

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

165 170 175

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

180 185 190

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

195 200 205

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

210 215 220

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

225 230 235 240

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

245 250 255

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

260 265 270

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

275 280 285

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

290 295 300

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

305 310 315 320

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

325 330 335

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

340 345 350

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

355 360 365

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

370 375 380

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

385 390 395 400

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

405 410 415

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

420 425 430

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

435 440 445

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

450 455 460

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

465 470 475 480

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 103

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9D containing part of the heavy chain

<400> 103

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Ile

65 70 75 80

Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu

85 90 95

Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

130 135 140

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 104

<211> 496

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9E containing part of the heavy chain

<400> 104

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Arg Gly Gly

20 25 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

35 40 45

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

50 55 60

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp

65 70 75 80

Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

85 90 95

Glu Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys

100 105 110

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met

115 120 125

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

130 135 140

Arg Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly

145 150 155 160

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

165 170 175

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

180 185 190

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

195 200 205

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

210 215 220

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

225 230 235 240

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

245 250 255

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

260 265 270

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

275 280 285

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

290 295 300

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

305 310 315 320

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

325 330 335

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

340 345 350

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

355 360 365

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

370 375 380

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

385 390 395 400

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

405 410 415

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

420 425 430

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

435 440 445

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

450 455 460

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

465 470 475 480

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 105

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9F containing part of the heavy chain

<400> 105

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Ile

65 70 75 80

Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu

85 90 95

Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

130 135 140

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 106

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9G containing part of the heavy chain

<400> 106

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Arg Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Ile

65 70 75 80

Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu

85 90 95

Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

130 135 140

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 107

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9H containing part of the heavy chain

<400> 107

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Ile

65 70 75 80

Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu

85 90 95

Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

130 135 140

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 108

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1803-9J containing part of the heavy chain

<400> 108

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Arg Gly Arg Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Ile

65 70 75 80

Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu

85 90 95

Ile Leu Pro Gly Ser Thr Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

130 135 140

Gly Leu Ser Thr Leu Met Ala Val Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 109

<211> 493

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> ch1805-D containing part of the heavy chain

<400> 109

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val

50 55 60

Lys Ile Pro Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met

65 70 75 80

Asp Trp Val Lys Gln Ser His Gly Arg Ser Leu Glu Trp Ile Gly Ser

85 90 95

Ile Asp Pro Asp Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly

100 105 110

Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu

115 120 125

Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg

130 135 140

Asp Tyr Tyr Gly Ser Ser Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

145 150 155 160

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

165 170 175

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

180 185 190

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

195 200 205

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

210 215 220

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

225 230 235 240

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

245 250 255

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

420 425 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490

<210> 110

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> ch1808-D containing part of the heavy chain

<400> 110

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

35 40 45

Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val

50 55 60

Lys Leu Ser Cys Lys Ala Ser Gly Asp Thr Phe Thr Thr Asn Gly Ile

65 70 75 80

Ser Trp Val Lys Gln Arg Ile Gly Gln Gly Leu Glu Trp Ile Gly Glu

85 90 95

Ile Tyr Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Asn Phe Lys Gly

100 105 110

Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr Met Glu

115 120 125

Leu Arg Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

130 135 140

Ser Ile Thr Ser Val Ile Gly Ala Asp Tyr Phe Asp Tyr Trp Gly Gln

145 150 155 160

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

165 170 175

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

180 185 190

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

195 200 205

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

210 215 220

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

225 230 235 240

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

245 250 255

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 111

<211> 496

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> hu1810-12D containing part of the heavy chain

<400> 111

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

35 40 45

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val

50 55 60

Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr Ala Ile

65 70 75 80

Ser Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Glu

85 90 95

Ile Tyr Pro Thr Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly

100 105 110

Lys Ala Thr Leu Thr Ala Asp Arg Ser Thr Ser Thr Met Tyr Met Glu

115 120 125

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser

130 135 140

Gly Val Ile Thr Thr Val Val Ser Thr Asp Tyr Phe Asp Tyr Trp Gly

145 150 155 160

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

165 170 175

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

180 185 190

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

195 200 205

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

210 215 220

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

225 230 235 240

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

245 250 255

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

260 265 270

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

275 280 285

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

290 295 300

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

305 310 315 320

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

325 330 335

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

340 345 350

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

355 360 365

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

370 375 380

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

385 390 395 400

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

405 410 415

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

420 425 430

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

435 440 445

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

450 455 460

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

465 470 475 480

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

485 490 495

<210> 112

<211> 498

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> ch1817-D containing part of the heavy chain

<400> 112

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg Ser Met

50 55 60

Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Tyr Met

65 70 75 80

Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Ser

85 90 95

Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val Lys Gly

100 105 110

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr Leu Gln

115 120 125

Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys Ala Arg

130 135 140

His Thr Thr Ala Asp Tyr Phe Tyr Gly Ile Tyr Phe Ala Leu Asp Ala

145 150 155 160

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly

165 170 175

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

180 185 190

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

195 200 205

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

210 215 220

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

225 230 235 240

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

245 250 255

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

260 265 270

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

275 280 285

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

290 295 300

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

305 310 315 320

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

325 330 335

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

340 345 350

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

355 360 365

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

370 375 380

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

385 390 395 400

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

405 410 415

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

420 425 430

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

435 440 445

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

450 455 460

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

465 470 475 480

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

485 490 495

Gly Lys

<210> 113

<211> 498

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> ch1822-D containing part of the heavy chain

<400> 113

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Gly Gly Gly Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

35 40 45

Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg Ser Met

50 55 60

Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Tyr Met

65 70 75 80

Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala Ser

85 90 95

Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val Lys Gly

100 105 110

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr Leu Gln

115 120 125

Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys Ala Arg

130 135 140

His Thr Thr Ala Asp Tyr Phe Tyr Gly Ile Tyr Phe Ala Leu Asp Ala

145 150 155 160

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly

165 170 175

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

180 185 190

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

195 200 205

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

210 215 220

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

225 230 235 240

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

245 250 255

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

260 265 270

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

275 280 285

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

290 295 300

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

305 310 315 320

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

325 330 335

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

340 345 350

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

355 360 365

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

370 375 380

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

385 390 395 400

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

405 410 415

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

420 425 430

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

435 440 445

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

450 455 460

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

465 470 475 480

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

485 490 495

Gly Lys

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Claims (85)

1. An anti-GCGR (glucagon receptor) monoclonal antibody or antigen-binding fragment thereof, comprising a combination of a heavy chain variable region and a light chain variable region selected from the group consisting of: i) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOS: 14, 15 and 16, respectively, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID NOs: 17, 18, and 19, respectively; ii) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 20, 21 and 22, respectively, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOs: 23, 24 and 25, respectively; iii) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 26, 27 and 28, respectively, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOs: 29, 30 and 31, respectively; iv) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 32, 33 and 34, respectively, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID NOs: 35, 36, and 37, respectively; v) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOS: 38, 39 and 40, respectively, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 regions shown in SEQ ID NOs: 41, 42, and 43, respectively; and vi) a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOS: 38, 39 and 44, respectively, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOs: 45, 46 and 47, respectively. 2. An anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is a mouse antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof. 3. An anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody contains a framework region derived from a human antibody, or a variant of this framework region, where: the framework region variant has no more than 10 amino acid reverse mutations in the framework region of the light chain and/or the framework region of the heavy chain of a human antibody, respectively. 4. An anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the reverse amino acid mutation is selected from: aa) one or more backmutations in 42G, 44V, 71Y and 87F contained in the light chain variable region, and/or one or more backmutations in 38K, 48I, 67A, 69F, 71A, 73P, 78A and 93S contained in the variable region of the heavy chain; or ab) one or more backmutations in 38L, 44V, 59S, 70E and 71Y contained in the light chain variable region, and/or one or more backmutations at 38K, 48I, 66K, 67A, 69L, 73R, 78M and 94S contained in the heavy chain variable region. 5. An anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to claim 2, comprising a combination of a light chain variable region and a heavy chain variable region selected from the group consisting of: c) a heavy chain variable region whose sequence is as shown in any of SEQ ID NOs: 2, 61, 62, 63 and 64 or has at least 90% sequence identity with any of SEQ ID NOs: 2, 61 , 62, 63 and 64; and a light chain variable region whose sequence is as shown in any of SEQ ID NOs: 3, 58, 59 and 60 or has at least 90% sequence identity with any of SEQ ID NOs: 3, 58, 59 and 60 ; d) a heavy chain variable region whose sequence is as shown in SEQ ID NO: 4 or has at least 90% sequence identity with SEQ ID NO: 4; and a light chain variable region whose sequence is as shown in SEQ ID NO: 5 or has at least 90% sequence identity with SEQ ID NO: 5; e) a heavy chain variable region whose sequence is as shown in SEQ ID NO: 6 or has at least 90% sequence identity with SEQ ID NO: 6; and a light chain variable region whose sequence is as shown in SEQ ID NO: 7 or has at least 90% sequence identity with SEQ ID NO: 7; f) a heavy chain variable region whose sequence is as shown in any of SEQ ID NOs: 8, 68, 69, 70 and 71 or has at least 90% sequence identity with any of SEQ ID NOs: 8, 68 , 69, 70 and 71; and a light chain variable region whose sequence is as shown in any of SEQ ID NOs: 9, 65, 66 and 67 or has at least 90% sequence identity with any of SEQ ID NOs: 9, 65, 66 and 67 ; g) a heavy chain variable region whose sequence is as shown in SEQ ID NO: 10 or has at least 90% sequence identity with SEQ ID NO: 10; and a light chain variable region whose sequence is as shown in SEQ ID NO: 11 or has at least 90% sequence identity with SEQ ID NO: 11; and h) a heavy chain variable region whose sequence is as shown in SEQ ID NO: 12 or has at least 90% sequence identity with SEQ ID NO: 12; and a light chain variable region whose sequence is as shown in SEQ ID NO: 13 or has at least 90% sequence identity with SEQ ID NO: 13. 6. Monoclonal antibody to GCGR or antigennegative fragment according to any one of paragraphs. 1-5, where the antibody is a full-length antibody, additionally containing the constant region (regions) of the antibody, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from the group consisting of human antibody κ- and λ-chain constant regions and conventional variants thereof . 7. Monoclonal antibody to GCGR or antigennegative fragment according to any one of paragraphs. 1-5, wherein the antibody is a full length antibody further comprising the human antibody heavy chain constant region set forth in SEQ ID NO: 72 and the human antibody light chain constant region set forth in SEQ ID NO: 73. 8. An anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to claim 7, comprising a combination of a heavy chain and a light chain selected from any of the group consisting of: j) a heavy chain shown in SEQ ID NO: 74, 76 or 78 or having at least 85% sequence identity with it, and a light chain shown in SEQ ID NO: 75, 77 or 79 or having at least 85% sequence identity with it; k) a heavy chain shown in SEQ ID NO: 80 or having at least 85% sequence identity with it, and a light chain shown in SEQ ID NO: 81 or having at least 85% sequence identity with it; l) a heavy chain shown in SEQ ID NO: 82 or having at least 85% sequence identity with it, and a light chain shown in SEQ ID NO: 83 or having at least 85% sequence identity with it; m) a heavy chain shown in SEQ ID NO: 84 or having at least 85% sequence identity with it, and a light chain shown in SEQ ID NO: 85 or having at least 85% sequence identity with it; n) a heavy chain shown in SEQ ID NO: 86 or having at least 85% sequence identity with it, and a light chain shown in SEQ ID NO: 87 or having at least 85% sequence identity with it; and o) a heavy chain shown in SEQ ID NO: 88 or having at least 85% sequence identity with it, and a light chain shown in SEQ ID NO: 89 or having at least 85% sequence identity with it. 9. Monoclonal antibody to GCGR or antigennegative fragment according to any one of paragraphs. 1-5, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single chain antibody, dimerized V region (diabody), and disulfide stabilized V region (dsFv). 10. A bispecific protein containing a GLP-1 peptide (glucagon-like peptide-1) and an antibody to GCGR or its antigen-binding fragment, where: the GLP-1 peptide is covalently linked to the anti-GCGR antibody or antigen-binding fragment thereof via a peptide bond or a linker; and the anti-GCGR antibody or antigen-binding fragment thereof is a monoclonal anti-GCGR antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-9, wherein said bispecific protein is capable of binding to GCGR and GLP-1R (glucagon-like peptide-1 receptor). 11. Bispecific protein according to claim 10, where: the carboxy terminus of the GLP-1 peptide is linked to the amino terminus of the heavy chain variable region of an anti-GCGR monoclonal antibody or antigen-binding fragment thereof via a peptide bond or a linker; or the carboxy terminus of the GLP-1 peptide is linked to the amino terminus of the light chain variable region of an anti-GCGR antibody or antigen-binding fragment thereof via a peptide bond or a linker. 12. A bispecific protein according to claim 10 or 11, where: the GLP-1 peptide is the GLP-1 peptide shown in SEQ ID NO: 91, or a variant thereof, the GLP-1 peptide variant contains one or more amino acid mutations from Q17E, I23V, K28R and G30R relative to the GLP-1 peptide shown in SEQ ID NO: 91. 13. A bispecific protein according to claim 10 or 11, wherein the GLP-1 peptide variant contains a Q17E mutation or contains Q17E and I23V mutations. 14. The bispecific protein of claim 12, wherein the sequence of the GLP-1 variant peptide is as shown in SEQ ID NOs: 92, 93, 94, 95, 96, 97, 98, or 99. 15. Bispecific protein according to claim 14, containing the first polypeptide chain and the second polypeptide chain, and the first polypeptide chain contains a heavy chain of a monoclonal antibody to GCGR according to any one of paragraphs. 1-9 and the second polypeptide chain contains a light chain of a monoclonal antibody to GCGR according to any one of paragraphs. 1-9; where: (ai) the first polypeptide chain contains a polypeptide selected from any of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107 and 108, and the second polypeptide chain contains the polypeptide shown in SEQ ID NO: 79; (aj) the first polypeptide chain contains the polypeptide shown in SEQ ID NO: 109, and the second polypeptide chain contains the polypeptide shown in SEQ ID NO: 81; (ak) the first polypeptide chain contains the polypeptide shown in SEQ ID NO: 110 and the second polypeptide chain contains the polypeptide shown in SEQ ID NO: 83; (al) the first polypeptide chain contains the polypeptide shown in SEQ ID NO: 111 and the second polypeptide chain contains the polypeptide shown in SEQ ID NO: 85; (am) the first polypeptide chain contains the polypeptide shown in SEQ ID NO: 112 and the second polypeptide chain contains the polypeptide shown in SEQ ID NO: 87; or (an) the first polypeptide chain contains the polypeptide shown in SEQ ID NO: 113 and the second polypeptide chain contains the polypeptide shown in SEQ ID NO: 89. 16. Pharmaceutical composition for lowering the concentration of glucose in the blood of a subject, containing: a therapeutically effective amount of an anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-9 or bispecific protein according to any one of paragraphs. 10-15 and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. 17. Pharmaceutical composition for the treatment of metabolic disorders, containing: a therapeutically effective amount of an anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-9 or bispecific protein according to any one of paragraphs. 10-15 and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. 18. An isolated nucleic acid molecule encoding a monoclonal antibody to GCGR or its antigen-binding fragment according to any one of paragraphs. 1-9. 19. A recombinant vector for the expression of a monoclonal antibody to GCGR or its antigen-binding fragment, containing the isolated nucleic acid molecule according to claim 18. 20. A host cell for expressing an anti-GCGR monoclonal antibody or an antigen-binding fragment thereof, wherein said host cell is transformed with the recombinant vector of claim 19, selected from the group consisting of mammalian cells or insect cells. 21. The method of obtaining a monoclonal antibody to GCGR or its antigen-binding fragment according to any one of paragraphs. 1-9, including: culturing the host cell according to claim 20 to obtain a monoclonal antibody to GCGR or its antigen-binding fragment according to any one of paragraphs. 1-9 and isolating the monoclonal antibody or its antigen-binding fragment from the culture. 22. Reagent for detecting human GCGR in a sample, containing a monoclonal antibody to GCGR or antigennegative fragment according to any one of paragraphs. 1-9. 23. A method for lowering a blood glucose concentration in a subject, comprising: administering to said subject a therapeutically effective amount of an anti-GCGR monoclonal antibody or antigen-binding fragment thereof according to any one of claims. 1-9, or a bispecific protein according to any one of paragraphs. 10-15, or a pharmaceutical composition according to claim 16, where the specified therapeutically effective amount is from 0.1 to 3000 mg of a monoclonal antibody to GCGR or its antigen-binding fragment according to any one of paragraphs. 1-9 or bispecific protein according to any one of paragraphs. 10-15 in a single dose. 24. A method for treating a metabolic disorder, comprising: administering to a subject a therapeutically effective amount of an anti-GCGR monoclonal antibody, or antigen-binding fragment thereof, according to any one of paragraphs. 1-9, or a bispecific protein according to any one of paragraphs. 10-15, or a pharmaceutical composition according to claim 17; where the metabolic disorder is selected from the group consisting of obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperinsulinemia and insulin resistance syndrome.

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