CN104231083A

CN104231083A - Antibody for resisting to glucagon receptor and use thereof

Info

Publication number: CN104231083A
Application number: CN201410247918.6A
Authority: CN
Inventors: 张�成; 汪笑峰; 景书谦
Original assignee: Gmax Biopharm LLC
Current assignee: Gmax Biopharm LLC
Priority date: 2013-06-09
Filing date: 2014-06-06
Publication date: 2014-12-24

Abstract

The invention discloses an antibody for resisting to a glucagon receptor and use thereof, and more particularly relates to a monoclonal antibody capable of being specifically combined with a human glucagon receptor as well as use of the antibody for treating or preventing Diabetes mellitus II and related diseases in mammals. The invention provides the novel antibody for resisting to a glucagon receptor capable of being specifically combined with a glucagon receptor. The cAMP level in a cell is reduced by means of inhibiting combination of glucagon and the receptor.

Description

Antibody of anti-glucagon receptor and uses thereof

Technical field

The present invention relates to a kind of antibody, particularly a kind of antibody and uses thereof of anti-glucagon receptor.

Background technology

Porcine glucagon is the important hormone class material worked with Regular Insulin one, participates in the regulation and control of human body Glucose in Blood by Cyclic.Hyperglycemic-glycogenolytic factor and Regular Insulin are all peptide hormones.Hyperglycemic-glycogenolytic factor produces in the α islet cells of pancreas, and Regular Insulin produces in β islet cells.When glucose level reduces, hyperglycemic-glycogenolytic factor discharges glucose mainly through stimulating some target cells (mainly liver cell), hyperglycemic-glycogenolytic factor and Regular Insulin regulate the effect of blood sugar just in time contrary, and Regular Insulin irritation cell when blood sugar increasing absorbs and stores glucose, reduces blood sugar concentration.

Natural human hyperglycemic-glycogenolytic factor is by 29 amino-acid residues:

NH ₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH

Hyperglycemic-glycogenolytic factor is by activating downstream signaling pathway in conjunction with its acceptor, glucagon receptor (being called for short GCGR) belongs to seven transmembrane G protein coupled receptor family secretory element hypotypes, after ligand-receptor combines, functionating is carried out by activating second messenger's adenylate cyclase, cAMP level in elevate cellular, then de novo synthesis and the liver starch degradation pathway of glucose is started, blood sugar concentration is made to raise (Wakelam etc., Nature (1986) 323:68-71, Pittner and Fain, Biochem J (1991) 277:371-378).

Diabetes are common glucose metabolism diseases, it is characterized in that hyperglycemia, are divided into the type i diabetes of insulin-dependent and the type ii diabetes of Regular Insulin non-dependent.Type i diabetes patients goes out hyperglycemia and hypoinsulinemia, and to the conventional treatment measure of this disease for providing Regular Insulin.But in some I type or type ii diabetes, absolute or relatively high hyperglycemic-glycogenolytic factor causes hyperglycemia.In the animal or I type type ii diabetes animal pattern of health, glucose level decline (Brand etc., Diabetes (1996) 45:1076 can be made with the hyperglycemic-glycogenolytic factor in selectivity or specific cleaning antibody blood circulation; Jiang and Zhang, AJP Endo(2003) 284:E671).These researchs show, glucagon suppression or antagonism glucagon receptor can as the supplementary meanss of the conventional In Treatment of Hyperglycemia of diabetes.

By the antibody blocking hyperglycemic-glycogenolytic factor of target glucagon receptor and the combination of glucagon receptor, also as control or hypoglycemic a kind of means can be fallen, thus become the novel method (U.S.Pat. No.2008/036341 A2 and U.S.Pat. No.2012/0128679 A1) for the treatment of diabetes.Like this, the antagonist of hyperglycemic-glycogenolytic factor or glucagon receptor can be small molecules, also can be the biomacromolecule of polypeptide or antibody class.

Summary of the invention

The antibody of the anti-glucagon receptor that the object of the present invention is to provide a class new, can with glucagon receptor specific binding, by the combination of glucagon suppression and its acceptor, reduce intracellular cAMP levels, and the method for this kind of biomacromolecule of Synthesis and applications.

The technical solution adopted for the present invention to solve the technical problems is:

An antibody for anti-glucagon receptor, described antibody comprises the aminoacid sequence of one of following scheme:

A. the light chain CDR3 sequence of one of following sequence is selected from:

With the light chain CDR3 sequence being selected from L1-L6: SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50 one of them differ the light chain CDR3 sequence being altogether no more than three aminoacid addition, replacement and/or disappearance;

B. the heavy chain CDR3 sequence of one of following sequence is selected from:

With the heavy chain CDR3 sequence being selected from H1-H6: SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:45, SEQ ID NO:55 one of them differ the heavy chain CDR3 sequence being altogether no more than four aminoacid addition, replacement and/or disappearance;

C. the light chain CDR3 sequence of scheme a and the heavy chain CDR3 sequence of scheme b;

Described antibody and human glucagon receptor specific binding.

Described antibody also comprises the aminoacid sequence of the one or more combination of following scheme:

A. one of following light chain CDR1 sequence is selected from:

With the light chain CDR1 sequence being selected from L1-L6: SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:38, SEQ ID NO:48, SEQ ID NO:58 one of them differ the light chain CDR1 being no more than three aminoacid addition, replacement and/or disappearance;

B. one of following light chain CDR2 sequence is selected from:

With the light chain CDR2 sequence being selected from L1-L6: SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:39, SEQ ID NO:49 one of them differ the light chain CDR2 being no more than two aminoacid addition, replacement and/or disappearance;

C. one of following heavy chain CDR1 sequence is selected from:

With the heavy chain CDR1 sequence being selected from H1-H6: SEQ ID NO:5, SEQ ID NO:24, SEQ ID NO:33, SEQ ID NO:43, SEQ ID NO:53 one of them differ the heavy chain CDR1 being no more than two aminoacid addition, replacement and/or disappearance;

D. one of following heavy chain CDR2 sequence is selected from:

With the heavy chain CDR2 sequence being selected from H1-H6: SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:34, SEQ ID NO:44, SEQ ID NO:54 one of them differ the sequence of heavy chain being no more than three aminoacid addition, replacement and/or disappearance.

A. the light variable domains sequence of one of following scheme is selected from:

I. have and the light variable domains sequence being selected from L1-L6: SEQ ID NO:13, SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:41, one of them at least 80% identical aminoacid sequence of SEQ ID NO:51, SEQ ID NO:59;

Ii. there is the polynucleotide sequence of light variable domains sequence with coding L1-L6: the aminoacid sequence that SEQ ID NO:14, SEQ ID NO:23, SEQ ID NO:32, SEQ ID NO:42, one of them at least 80% identical polynucleotide sequence of SEQ ID NO:52, SEQ ID NO:60 are encoded;

B. the heavy-chain variable domains sequence of one of following scheme is selected from:

I. there is the heavy-chain variable domains sequence with H1-H6: SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:36, one of them at least 80% identical aminoacid sequence of SEQ ID NO:46, SEQ ID NO:56;

Ii. there is the polynucleotide sequence of heavy-chain variable domains sequence with coding H1-H6: the aminoacid sequence that SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:37, one of them at least 80% identical polynucleotide sequence of SEQ ID NO:47, SEQ ID NO:57 are encoded;

The light variable domains sequence of c.a and the heavy-chain variable domains sequence of b;

Described antibody and human glucagon receptor specific binding.

In scheme c, the combination of the light variable domains sequence of a and the heavy-chain variable domains sequence of b is selected from one of following: L1H1, L1H1, L2H2, L3H3, L4H4, L5H5 and L6H6.

Described antibody is selected from the one in mouse source antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibodies, antigen binding antibody fragment, single-chain antibody, double-chain antibody, three chain antibodies, four chain antibodies, Fab fragment, F (fa ') x fragment, domain antibodies, IgD antibody, IgE antibody, IgM antibody, IgGl antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody.

When described antibody and human glucagon receptor in conjunction with time:

A. with amino acid Gln27 to the Gln142 specific binding of human glucagon receptor;

B. hyperglycemic-glycogenolytic factor intracellular signaling is reduced with the IC50 value of≤100 nM;

C. in animal model, blood sugar is reduced;

D. both (a) and (b);

E. (a), (b) and (c).

Described animal model is ob/ob mouse model.

A nucleic acid for separation, it comprises the polynucleotide sequence of the light variable domains of antibody of coding claim 3, heavy-chain variable domains or light variable domains and heavy-chain variable domains.

A kind of recombinant expression vector, it comprises the nucleic acid of claim 8.

A kind of host cell, it comprises the carrier of claim 9.

Produce a method for the antibody of anti-glucagon receptor, under being included in the condition allowing to express described antibody, cultivate the host cell of claim 10.

A kind of medicinal compositions, it comprises antibody or the humanized antibody of the claim 3 mixed with pharmaceutical acceptable carrier.

Be used for the treatment of a test kit for type ii diabetes, it comprises the medicinal compositions of claim 12.

The invention has the beneficial effects as follows: the antibody of the anti-glucagon receptor providing a class new, can with glucagon receptor specific binding, by the combination of glucagon suppression and its acceptor, reduce intracellular cAMP levels.

Accompanying drawing explanation

Fig. 1 is monoclonal antibody A-1 and expresses the flow cytometer showed result having the CHO-DHFR cell-specific of hGCGR to combine, and grey peak and dotted line peak are negative control, and hybridoma supernatant has in conjunction with the solid line peak that CHO-DHFR-GCGR is corresponding and obviously moves to right.

Fig. 2 is monoclonal antibody A-2 and expresses the flow cytometer showed result having the CHO-DHFR cell-specific of hGCGR to combine, and grey peak and dotted line peak are negative control, and hybridoma supernatant has in conjunction with the solid line peak that CHO-DHFR-GCGR is corresponding and obviously moves to right.

Fig. 3 is monoclonal antibody A-3 and expresses the flow cytometer showed result having the CHO-DHFR cell-specific of hGCGR to combine, and grey peak and dotted line peak are negative control, and hybridoma supernatant has in conjunction with the solid line peak that CHO-DHFR-GCGR is corresponding and obviously moves to right.

Fig. 4 be HitHunter cAMP method detect purifying hybridoma supernatant in antibody suppression Glucagon to the activation figure of GCGR acceptor.

Embodiment

Below by specific embodiment, and by reference to the accompanying drawings, technical scheme of the present invention is described in further detail.

In the present invention, if not refer in particular to, the raw material adopted and equipment etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the ordinary method of this area.

The present invention relates to antibody such as can with the antibody of human glucagon receptor (GCGR) specific binding.These comprise and can suppress or block hyperglycemic-glycogenolytic factor and be combined with people GCGR and reduce the antigen-binding proteins of hyperglycemic-glycogenolytic factor through acceptor intracellular signaling.Provide the mouse source antibody of the reduced animal blood glucose comprising antagonistic antibodies in one embodiment.

The present invention further provides can specific binding to relevant composition, test kit and the method for the antibody of human glucagon receptor.Also provide nucleic acid molecule and derivative thereof and fragment, it comprises all or part of polynucleotide of the polypeptide be combined with glucagon receptor of encoding, the nucleic acid of such as encode all or part of anti-glucagon receptor antibodies, antibody fragment or antibody derivatives.The present invention further provides the carrier and plasmid that comprise such nucleic acid and the cell comprising such nucleic acid and/or carrier and plasmid and clone.Institute's supplying method comprises, and such as, the method for the antibody such as anti-GCGR antibody that preparation, qualification or separation are combined with people GCGR, measures the method whether this antibody is combined with GCGR and the method antibody be combined with GCGR being given animal model.

definition

The single-letter of use standard or trigram abbreviation show polynucleotide and peptide sequence.Except as otherwise noted, the aminoterminal of peptide sequence at left and their carboxyl terminal on the right side, 5 ' end of the upstream chain of single strand nucleotide sequence and double-strandednucleic acid sequence at left and their 3 ' end on the right side.The concrete part of polypeptide can be represented by numbering amino acid residues, such as amino acid 27 to 142, or represents such as Gln27 to Gln142 by the actual residue in this site.Also by explaining that the difference of itself and reference sequence describes concrete polypeptide or polynucleotide sequence.The polynucleotide of concrete light chain and heavy-chain variable domains and polypeptide can be expressed as L1(" light variable domains 1 "), H1(" heavy-chain variable domains 1 ").Comprise the antigen-binding proteins of light chain and heavy chain or antibody by representing in conjunction with the title of light chain and the title of heavy-chain variable domains.Such as, " L4H7 " represents the antibody comprising L4 light variable domains and H7 heavy-chain variable domains.

Unless definition herein in addition, should have to relevant Science and Technology term the implication that those of ordinary skill in the art understand herein.Further, unless the context otherwise requires, singular references should comprise plural number and plural reference term should comprise odd number implication.Usually, to cell and tissue culture described herein, molecular biology, immunology, microbiology, genetics and protein nucleic acid chemistry and to hybridize relevant nomenclature and technology be well known and frequent use.Method and Technology of the present invention usually according to ordinary method known in the art and this specification sheets quote and discuss various common and the carrying out more specifically described by reference, unless otherwise indicated.See, such as, Sambrook etc., Molecular Cloning:A Laboratory Manual, 2nd edition., Cold Spring Harbor Laboratory Press (1989) and Ausubel etc., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies:A Laboratory Manual Cold Spring Harbor Laboratory Press (1990), all with reference form and in herein.Enzyme reaction and purification technique carry out according to operation instructions, as this area completes or described herein usually.The wooden language relevant with medicinal chemistry to analytical chemistry described herein, synthetic organic chemistry and medical science and experimental implementation and technology are well known and general user.Standard technique can be used for the treatment of chemosynthesis, chemical analysis, medicine preparation, preparation and administration and patient.

Following term, except as otherwise noted, be interpreted as that there is following implication: term " molecule of separation " (wherein said molecule is such as polypeptide, polynucleotide or antibody) for regard to its source or derive (1) with regard to source and be separated with the natural related component accompanied with it under native state, (2) basic and of the same race other molecule free (3) is by cell expressing not of the same race or (4) not natural existence.Therefore, chemosynthesis or the molecule of being expressed by the cell system different from the cell of its natural origin " will be separated " with natural related component.Also purification technique known in the art can be used by separation, the basic related component natural with it of molecule to be dissociated.Such as, polyacrylamide gel electrophoresis can be used to measure the purity of polypeptide sample and use technology well known in the art by gel-colored to observe this polypeptide.For some object, HPLC or other purification process well known in the art can be used to provide higher resolving power.

Term " peptide ", " polypeptide " and " albumen " all refer to comprise two or morely is good for interconnective amino acid whose molecule by peptide.These terms contain such as natural and artificial protein, protein fragments and protein sequence polypeptide analog (such as mutain, varient and fusion rotein) and after transcribing or otherwise albumen for covalently or non-covalently modifying.Peptide, polypeptide or albumen can be monomer or polymer.

" varient " of polypeptide (such as antibody) comprises the aminoacid sequence inserting, lack and/or substituted for one or more amino-acid residue relative to another peptide sequence in aminoacid sequence.Varient of the present invention comprises fusion rotein.

" derivative " of polypeptide is the polypeptide (such as, antibody) through chemically modified, such as by combining with other chemical part such as polyoxyethylene glycol, albumin (such as human serum albumin), phosphorylation and glycosylation.Unless otherwise indicated, term " antibody " comprises its derivative except comprising the antibody of two total length heavy chains and two full-length light chains, and varient, fragment and mutain, the example sees below.

" antibody " is for comprising with antigen-binding portion thereof and optionally promoting the support of conformation that this antibody is combined with this antigen or the albumen of frame part for allowing antigen-binding portion thereof to take.The example of antibody comprises antibody, antibody fragment (antigen-binding portion thereof of such as antibody), antibody derivatives and antibody analog.This antibody can comprise such as selectable albumen support or have the man-made support transplanting CDRs or CDRs derivative.This support include but not limited to comprise be introduced into such as derive support with the antibody of the three-dimensional structure of this antigen-binding proteins of stabilization and comprise the polymeric complete synthesis support of biological example consistency.See, such as, Korndorfer etc., 2003, Proteins:Structure, Function, and Bioinformatics, Volume 53, Issue l:121-129; Roque etc., 2004, Biotechnol. Prog. 20:639-654.In addition, can use simulating peptide antibody (" PAMs ") and the support based on analog antibody, it utilizes fibronectin as support.

Antibody can have the structure of such as native immunoglobulin." immunoglobulin (Ig) " is tetrameric molecule.In natural immunoglobulin (Ig), each tetramer by two identical polypeptide chains to forming, each to having one " gently " (about 25 kDa) and " weight " chain (about 50-70kDa).The aminoterminal of each chain comprises about 100 to 110 or more amino acid whose variable domains, main relevant to antigen recognition.The carboxy terminal half of each chain determines the main constant region relevant to effector effect.The light chain of people is divided into κ and lambda light chain.Heavy chain is divided into μ, δ, α or ε, and determines the isotype of antigen, such as, be respectively IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain, to be variablely connected by about 12 or more amino acid whose " J " district with constant region, heavy chain also comprises about individual amino acid whose " D " district more than 10.See, Fundamental Immunology Ch.7 (Paul writes, the 2nd edition .Raven Press (1989)) (its complete content with reference to form and in herein for any object).The right variable region of each light/heavy chain forms antibody combining site, and so complete immunoglobulin (Ig) has two basic change site.

Native immunoglobulin chain demonstrates the same principle construction in the relatively conservative skeleton district (FR) connected by three hypervariable regions, is also referred to as complementary determining region or CDRs.Hold C to hold from N, light and heavy chain all comprises structural domain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.The distribution of each domain amino acid and Kabat etc. are at Sequences of Proteins of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, definition in NIH, NIH Publication no. 91--3242.1991 is consistent.

Except as otherwise noted, " antibody " refer to complete immunoglobulin (Ig) or its can compete the antigen-binding portion thereof of specific binding with complete antibody.Can by recombinant DNA technology or by enzyme or chemical cracking complete antibody production antigen-binding portion thereof.Antigen-binding portion thereof comprises, especially, Fab, Fab ', F (ab ') ₂, Fv, domain antibodies (dAbs), comprise the fragment of complementary determining region (CDRs), single-chain antibody (scFv), chimeric antibody, double-chain antibody (diabodies), three chain antibodies (triabodies), four chain antibodies (tetrabodies) and at least comprise the polypeptide being enough to a part of giving the immunoglobulin (Ig) that polypeptide antigen combines.

Fab fragment is for having V _l, V _h, C _land C _hthe monovalent fragment of 1 structural domain; F (ab ') ₂fragment is the bivalent fragment with the Fab fragment that two are connected by disulfide linkage in hinge area; Fd fragment has V _hor V _lstructural domain; DAb fragment has V _hstructural domain, V _lstructural domain, or V _hor V _lfab (the U.S. Patent number 6846634,6696245 of structural domain, U.S. Patent Application Publication No. 05/0202512,04/0202995,04/0038291,04/0009507,03/0039958, Ward etc., 1989, Nature 341:544-546).

Single-chain antibody (scFv) is V wherein _lbutton V _hdistrict by joint (such as, the amino acid residue sequence of synthesis) connect with the antibody forming continuous protein, wherein this joint sufficiently long with allow this protein chain to fold back self also to be formed monovalent antigen binding site (see, such as, Bird etc., 1988, Science 242:423-26 and Huston etc., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).Double-chain antibody is the bivalent antibody comprising two polypeptide chains, and wherein each polypeptide chain comprises the V connected by joint _hand V ^lstructural domain, this joint is very short so that do not allow two pairings of structural domain in same chain, therefore allow the complementary domain on each structural domain and another polypeptide chain match (see, such as, Holliger etc., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, with Poljak etc., 1994, Structure 2:1121-23).If two of double-chain antibody polypeptide chains are identical, the double-chain antibody so obtained by their pairings will have identical antigen binding site.There is not homotactic polypeptide chain and can be used for preparing the double-chain antibody with different antigen binding site.Similarly, three chain antibodies and four chain antibodies are respectively the antibody comprising three and four polypeptide chains and also form three and four antigen binding sites respectively, and it may be the same or different.

Kabat etc. can be used at Sequences of Proteins of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, the method described in 1991 identifies complementary determining region (CDRs) and skeleton district (FR) of given antibody.Can covalently or non-covalently be incorporated to one or more CDRs in molecule and become antibody.Antibody can be incorporated to CDR (s) by larger polypeptide chain.CDR (s) can be covalently attached to another township's peptide chain, or non-covalently be incorporated to CDR (s).CDRs allows antibody to be combined with concrete correlation antigen specific.

Antibody can have one or more binding site.If more than one binding site, this binding site can be identical or different with another.Such as, natural human immunoglobulin has two identical binding sites usually, and " dual specific " or " difunctional ' ' antibody has two different binding sites.

Term " mouse source antibody " comprises having and one or morely derives from the variable region of mouse immunoglobulin sequences and all antibody of constant region.

" antigen-binding domains ", " antigen binding domain " or " antigen binding site " are for comprising with the amino-acid residue of AI (or other parts) and contributing to the part of antibody to the specificity of antigen and the antibody of avidity.For the antibody be combined with its antigen-specific, this will comprise at least one at least part of its CDR structural domain.

" epi-position " is the molecular moiety with antibodies.Epi-position can comprise molecule discontinuous part (such as, in polypeptide, in the primary sequence of polypeptide discontinuous amino-acid residue in three grades of this polypeptide and quaternary structure mutually enough close to so that by an antibodies).

" same percentage " of two polynucleotides or two peptide sequences is by use GAP computer program (GCG Wisconsin Package; A part of version 10.3 (Accelrys, San Diego, CA)) use its default parameters comparative sequences to measure.

Term " polynucleotide ", " oligonucleotide " and " nucleic acid " can be used alternatingly in the text and comprise DNA molecular (such as, cDNA or genomic dna), RNA molecule (such as mRNA), use nucleotide analog (such as, peptide nucleic acid(PNA) and non-natural nucleoside acid-like substance) DNA or the RNA analogue that generates and crossbred thereof.Nucleic acid molecule can be strand or double-strand.In one embodiment, nucleic acid molecule of the present invention comprises code book invention antibody or its fragment, derivative, mutain or varient continuous print open reading frame.

" carrier " is for can be used for the nucleic acid another coupled nucleic acid being introduced cell.One type of carrier is " plasmid ", and it refers to connect the linear of additional nucleic acid section or circular double stranded DNA molecule.The another type of carrier is virus vector (such as, replication defective retrovirus, adenovirus and adeno-associated virus), wherein additional region of DNA section can be introduced viral genome.Self-replicating (such as, comprising bacteria carrier and the episomal mammalian vectors of bacterial origin of replication) in the host cell that some carrier can be introduced at them.Other carrier (such as, non-free type mammalian vector) is integrated in the genome of host cell when introducing host cell and also therefore copies together with host genome.The bearer type that " expression vector " expresses for bootable selected polynucleotide.

If the expression (such as, expression level, time or site) that regulating and controlling sequence affects nucleotide sequence so nucleotide sequence " is operationally connected " with regulating and controlling sequence." regulating and controlling sequence " is for affecting the nucleic acid of the expression (such as, expression level, time or site) that can operate the nucleic acid be connected with it.Regulatory gene, such as, directly to play a role to modulated nucleic acid or by the effect of other molecule one or more (polynucleotide such as, be combined with regulating and controlling sequence and/or nucleic acid).The example of regulating and controlling sequence comprises promotor, enhanser and other expression controlling elements (such as, polyadenylation signal).The further example of regulating and controlling sequence is described in such as Goeddel, 1990, Gene Expression Technology:Methods in Enzymology 185, Academic Press and Baron etc., 1995, Nucleic Acids Res. 23:3605-06.

" host cell " is the cell for express nucleic acid such as nucleic acid of the present invention.Host cell can be prokaryotic organism, such as intestinal bacteria, or it can be eukaryote, such as unicellular eukaryote (such as, yeast or other fungi), vegetable cell (such as tobacco or tomato plant cell), zooblast (such as, people's cell, MC, hamster cell, rat cell, mouse cell or insect cell) or hybridoma.Usually, host cell is the culturing cell of the conversion of available polypeptide encoding nucleic acid or transfection, and it can then express in host cell.Phrase " recombinant host cell " can be used for the nuclear transformation of statement expection expression or the host cell of transfection.But host cell also can be and comprises this nucleic acid not to expect the cell of horizontal expression, be operationally connected unless introduced such its of regulating and controlling sequence to this host cell with nucleic acid.Be understood that term host cell not only refers to that concrete subject cell also refers to the filial generation of this cell or possible filial generation.Modify because such as sudden change or environmental influence subsequent generation there will be some, but this filial generation in fact differently from mother cell may still belong to term scope used herein.

glucagon receptor

Glucagon receptor (GCGR) belongs to the B subfamily of 7-transmembrane receptor family, it is by heterotrimer guanine-nucleotide-binding protein (G-protein) and one or more intracellular signaling pathways coupling (Jelinek etc., Science 259:1614-1616 (1993), Segre etc., Trends Endocrinol. Metab. 4:309-314 (1993)." glucagon receptor " as used herein and " GCGR " can be used alternatingly.

In one embodiment, antibodies of the present invention can be selected to be expressed in film on cell in conjunction with glucagon receptor suppressed by glucagon receptor or block hyperglycemic-glycogenolytic factor intracellular signaling.In one embodiment, antibody of the present invention and human glucagon receptor specific binding.In further embodiment, the antibody be combined with human glucagon receptor also can be combined with the glucagon receptor of other species, such as rat.Embodiment hereinafter provides the mouse source antibody generating and combine in conjunction with glucagon receptor with people's cytolemma, is combined in further embodiment with the glucagon receptor of other species.

HGCGR(human glucagon receptor, glucagon receptor) comprise 477 amino-acid residues.The amino acid whose sequence of hGCGR is shown in SEQ ID NO:2, and its nucleotide coding sequence is shown in SEQ ID NO:1.The amino acid whose sequence of GCGR of mouse is shown in SEQ ID NO:3, and its nucleotide coding sequence is shown in SEQ ID NO:4.

Antibody

On the one hand, the invention provides and the antibody of human glucagon receptor specific binding (such as, antibody, antibody fragment, antibody derivatives, antibody mutation albumen and antibody variants why).This antibody is mouse source antibody in one embodiment.

Antibody according to the present invention comprises the antibody tying merga pass glucagon receptor glucagon suppression intracellular signaling with human glucagon receptor.In one embodiment, the IC of this antibody ₅₀value is 100 nM or lower.On the other hand, this antibody and glucagon receptor specific binding, suppress intracellular signaling and demonstrate therapeutic biological effect, such as, reducing the blood sugar of animal model.In one embodiment, this antibody is with glucagon receptor specific binding and is suppressed the mouse source antibody of intracellular signaling by glucagon receptor.

In one embodiment, this antibody comprises the sequence respectively differing 5,4,3,2,1 or 0 monamino acids with the CDR sequence of A1-A6 in following table and add, replace and/or lack.As used herein, be no more than at most such as four aminoacid addition with the CDR sequence shown in following table, blind change with or the CDR sequence that lacks refer to have compared with the sequence in following table the sequence that 4,3,2,1 or 0 monamino acids add, replace and/or lack.

In another embodiment, this antibody comprises one or more hereafter shown CDR concensus sequence (consensus sequences).Provide the concensus sequence of light chain CDR1, CDR2, CDR3 and heavy chain CDR1, CDR2 and CDR3.

The light chain CDRs of antigen-binding proteins (antibody) Al-A6 and the heavy chain CDRs of Exemplary antigens associated proteins (antibody) Al-A6 sees the following form.A-l to A-6 is corresponding to Ll to L6 hereafter and H1 to H6 hereafter.Also show the polynucleotide sequence of coding CDRs aminoacid sequence.

On the other hand, the invention provides the variable region of light chain comprising and be selected from L1-L6 or be selected from the variable region of heavy chain of H1-H6 and the antibody of fragment, derivative, mutain or varient thereof.Available name " LxHy " represents this antibody-like, wherein " x " corresponding to variable region of light chain and " y " corresponding to variable region of heavy chain.Antibody of the present invention comprises such as comprising and is selected from the combination light chain of LIH1, L2H2, L3H3, L4H4, L5H5 and L6H6 and the combination of heavy-chain variable domains.In one embodiment, this antibody is mouse source antibody.

Light chain L1-L6

Heavy chain L1-L6

The specific embodiments of antibody of the present invention comprises the one or more aminoacid sequences identical with the aminoacid sequence of one or more listed CDRs and/or FRs (skeleton district) above.In one embodiment, this antibody comprises listed light chain CDR1 sequence above.In another embodiment, this antibody comprises listed light chain CDR2 sequence above.In another embodiment, this antibody comprises listed light chain CDR3 sequence above.In another embodiment, this antibody comprises listed heavy chain CDR1 sequence above.In another embodiment, this antibody comprises listed heavy chain CDR2 sequence above.In another embodiment, this antibody comprises listed heavy chain CDR3 sequence above.

In another embodiment, the CDR3 sequence of at least one antibody is no more than at most 6,5,4,3,2,1 or 0 monamino acids with the difference from the CDR3 sequence of A1-A6 and adds, replaces and/or lack, as described above.In another embodiment, the difference of the light chain CDR3 sequence of antibody and the light chain CDR3 sequence of above-mentioned Al-A6 must not add, replace and/or lack more than 6,5,4,3,2,1 or 0 monamino acids and the difference of the heavy chain CDR3 sequence of the heavy chain CDR3 sequence of antibody and Al-A6 mentioned above is no more than at most 6,5,4,3,2,1 or 0 monamino acids adds, replaces and/or lack.In another embodiment, antibody comprises 1,2,3,4 or 5 CDR sequence further, and each sequence is no more than at most 6,5,4,3,2,1 or 0 single amino acid differences with the difference of the CDR sequence of Al-A6 alone.In another embodiment, antibody comprises the listed CDRs of variable region of light chain and the CDRs of variable region of heavy chain above.In another embodiment, antibody comprises 1,2,3,4,5 and/or 6 consistent CDR sequence mentioned above.In further embodiment, this antibody comprises CDRs one of any in L1H1, L2H2, L3H3, L4H4, L5H5 and L6H6.In one embodiment, this antibody is mouse source antibody.

In one embodiment, this antibody (such as antibody or antibody fragment) comprises light variable domains, it comprises the aminoacid sequence that there are 15,14,13,12,11,10,9,8,7,6,5,4,3,2,1 or 0 amino acid differences with the light variable domains sequence being selected from L1-L6, and wherein respectively the difference of this sequence is independently the disappearance of an amino-acid residue, insertion or replacement.In another embodiment, this light variable domains comprise with the light variable domains at least 70% being selected from L1-L6,75%, 80%, 85%, 90%, 95%, 97% or 99% identical aminoacid sequence.In another embodiment, this light variable domains comprise with L1-L6 polynucleotide sequence listed hereinafter at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identical nucleotide sequence coded aminoacid sequence.In another embodiment, this light variable domains is included under moderate condition and is selected from the aminoacid sequence of the polymerized nucleoside acid encoding of the polynucleotide complementary sequence hybridization of the light variable domains of L1-L6 with coding.In another side's of enforcement Chinese bush cherry, this light variable domains comprises and is selected from the aminoacid sequence of the polymerized nucleoside acid encoding of the complementary sequence hybridization of the polynucleotide of the light variable domains of L1-L6 with coding under strict conditions.

In another embodiment, the invention provides the antibody comprising heavy-chain variable domains, this variable domains comprises the heavy-chain variable domains sequence being selected from H1-H6 and there is the aminoacid sequence that there are differences of 15,14,13,12,11,10,9,8,7,6,5,4,3,2,1 or 0 residues, and wherein respectively this sequence difference is independently amino acid whose disappearance, insertion or a replacement.In another embodiment, this heavy-chain variable domains comprise with the heavy-chain variable domains sequence at least 70% being selected from H1-H6,75%, 80%, 85%, 90%, 95%, 97% or 99% identical aminoacid sequence.In another embodiment, this heavy-chain variable domains is included under medium stringency condition and is selected from the aminoacid sequence of the polymerized nucleoside acid encoding of the polynucleotide complementary sequence hybridization of the heavy-chain variable domains of H1-H6 with coding.In one embodiment, this heavy-chain variable domains comprise be selected from coding under strict conditions the polynucleotide complementary sequence hybridization of the heavy-chain variable domains of H1-H6 the aminoacid sequence of polymerized nucleoside acid encoding.

Additional embodiment comprises the antibody comprising combination L1H1, L2H2, L3H3, L4H4, L5H5 and L6H6.

Antibody of the present invention comprises and such as comprises combination L1H1, L2H2, L3H3, L4H4, L5H5 and L6H6 and have expectation phenotype (such as, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE and IgD) and itself Fab or F (ab') ₂those of fragment.

Antibody of the present invention preferably described herein based on the assay method of cell in and/or regulate the combination of one of antibody described in hyperglycemic-glycogenolytic factor intracellular signaling and/or cross-blocks the application in vivoassay method described herein and/or through the combined GCGR cross-blocks of one of antibody described in the application.Therefore assay method described herein can be used to identify such combination.

In some embodiments, by first identify described herein based on cell and/or in vivoassay method with the Cell binding of process LAN GCGR and/or in and and/or the antibody of the combined GCGR cross-blocks of one of the antibody that describes of cross-blocks the application and/or the antibody that describes through the application to generate antibody.

What produce mouse monoclonal antibody can system of selection be peritoneal cavity hybridoma being injected homologous gene mouse, and such as treated (such as pristane) promotes to form the mouse comprising the ascites of monoclonal antibody.By the multiple technology separation established and monoclonal antibody purification.Such isolation technique comprise use the affinity chromatography of Protein A-agarose, size exclusive chromatography and height exchange chromatography (see, such as, Coligan 2.7.1-2.7.12 page and the

2.9.1-2.9.3 page; Baines etc., " Purification of Immunoglobulin G (IgG), " Methods in Molecular Biology, the 10th volume, 79-104 page (The Humana Press, Inc., 1992).Suitable aglucon that the special property (such as, heavy chain or light chain isotype, binding specificity etc.) based on antibody is screened can be used by affinity chromatography monoclonal antibody purification.The example being immobilized onto the suitable aglucon of solid carrier comprises albumin A, Protein G, anti-constant region (light chain or heavy chain) antibody, antiidiotypic antibody and TGF-p associated proteins or its fragment or varient.

The molecular evolution of the complementary determining region (CDRs) of antibody combining site central authorities can be used to be separated the antibody of affinity increase, such as, to the antibody that c-erbB-2 affinity increases, as Schier etc., described in 1996, J.Mol. Biol. 263:551.Therefore, such technology can be used for the antibody preparing human glucagon receptor.

Such as whether can there is in the external of glucagon receptor or in vivoassay method in detection the antibody used for human glucagon receptor.

Also by any conventional art Dispersal risk.Such as, (such as, can being purified from the hybridoma producing antibody) can be purified from the cell of natural these antibody of expression or use any technique known in the art to produce recombinant expression system.See, such as, Monoclonal Antibodies, Hybridomas:A New Dimension in Biological Analyses, Kennet etc. write, Plenum Press (1980); Write with Antibodies:A Laboratory Manual, Harlow and Land, Cold Spring Harbor Laboratory Press (1988).This is in nucleic acid moiety discussion hereafter.

Screen desirable properties by any known technology Dispersal risk.Some technology relate to the nucleic acid of the polypeptide chain (or its part) being separated coding associated antibodies (such as, anti-glucagon receptor antibodies), and operate nucleic acid by recombinant DNA technology.This nucleic acid can merge with another associated nucleic acid or modified (such as by mutagenesis or other conventional art) to add, to lack or to replace one or more amino-acid residue.

When needing raising to comprise the affinity of the antibody of one or more above-mentioned CDRs according to the present invention, comprise by multiple Affinity maturation scheme and maintain CDRs (Yang etc., 1995, J.Mol. Biol., 254:392-403), (Marks etc. replaced by chain, 1992, Bio/Technology, 10:779-783), use colibacillary mutant strain (Low etc., 1996, J.Mol. Biol., 250:350-368) DNA resets (Patten etc., 1997, Curr. Opin. Biotechnol., 8:724-733), phage display (Thompson etc., 1996, J.Mol. Biol., 256:7-88) and other round pcr (Crameri etc., 1998, Nature, 391:288-291).All these affinity maturation methods are discussed at Vaughan etc., and 1998, Nature Biotechnology, in 16:535-539.

antibody fragment

The invention provides the anti-glucagon receptor fragment of the present invention on the other hand.This fragment can be made up of antibody derived sequence completely maybe can comprise appended sequence.The example of Fab comprises Fab, F (ab ') 2, single-chain antibody, double-chain antibody, three chain antibodies, four chain antibodies and domain antibodies, and other example is provided in Lunde etc., 2002, Biochem, Soc. Trans. 30:500-06.

Heavy chain can be connected through amino acid bridge (short peptide linkers) and light variable domains (Fv district) forms single-chain antibody, thus obtain single polypeptide chain.By the DNA of encoded peptide joint is merged at coding two varied texture domain polypeptide (V _land V _h) DNAs between prepare this strand FVs (scFvs).Gained polypeptide self forms antigen for collapsible time in conjunction with monomer, or they can form polymer (such as, dimer, tripolymer or the tetramer), depend on the length (Kortt etc. of the flexible joint between two variable domains, 1997, Prot.Eng. 10:423; Kortt etc. 2001, Biomol. Eng. 18:95-108).The different V of polypeptide are comprised by combination _land V _h, the many bodies scFvs (Kriangkum etc., 2001, Biomol. Eng. 18:31-40) combined from different phenotype can be formed.The technology for the production of single-chain antibody researched and developed comprises U.S. Patent number 4946778; Bird, 1988, Science 242:423; Huston etc., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward etc., 1989, Nature 334:544, de Graaf etc., 2002, Methods Mol Biol. 178:379-87 Shens describe those.The single-chain antibody deriving from antibody provided herein includes but not limited to the scFvs comprising variable domains combination L1H1, L2H2, L3H3, L4H4, L5H5 and L6H6, is all covered by the present invention.

Also the proteolyzing by antibody such as obtains according to traditional method stomach en-or the complete antibody of papain digestion the Fab deriving from antibody.For example, available stomach en-enzymatic lysis antibody provides the SS fragment being called F (ab ') 2 to produce antibody fragment.This fragment of the further cracking of SH-group reductant can be used to produce 3.5S Fab ' monovalent fragment.Optionally, thiol protecting group can be used to carry out cracking that this scission reaction obtains disulfide linkage.As selectable, the enzymatic lysis of papoid is used directly to produce two monovalent Fab fragment and a Fc fragment.These methods are described in such as Goldenberg, U.S. Patent number 4,331,647, Nisonoff etc., Arch. Biochem. Biophys. 89:230,1960; Porter, Biochem.J. 73:119,1959; Edelman etc., Methods in Enzymology l:422 (Academic Press, 1967); And Andrews and Titus, Current Protocols in Immunology (Coligan etc. write), John Wiley & Sons (2003), 2.8.1-2.8.10 page and 2.10A.1-2.10A.5 page.The method of other cracking antibody, unit price is heavy, light chain segments (Fd) to be formed such as to prepare heavy chain, further crack fragment or also can use other enzyme, chemistry or gene engineering, as long as fragment with can be combined by the antigen of this complete antibody identification.

The antibody fragment of another kind of form is for comprising the peptide of one or more complementary antibody determining area (CDRs).Polypeptide by building the relevant CDR of coding obtains CDRs.Such as by use the mRNA of polymerase chain reaction antibody-producting cell as templated synthesis variable region prepare such polypeptide (see, such as, Larrick etc., Methods:A Companion to Methods in Enzymology 2:106,1991; Courtenay-Luck, " Genetic Manipulation of Monoclonal Antibodies; " Monoclonal Antibodies:Production, Engineering and Clinical Application, Ritter etc. write, 166 pages (Cambridge University Press, 1995); With Ward etc., " Genetic Manipulation and Expression of Antibodies, " Monoclonal Antibodies:Principles and Applications, Birch etc. write, 137 pages (Wiley-Liss, Inc, 1995).This antibody fragment can comprise at least one variable domains of antibody described herein further.Therefore, such as, V region domain can be monomer and is V _hor V _lstructural domain, it can at least equal 1 x 10 as mentioned below ^-7the affinity of M or lower is independent to be combined with glucagon receptor.

This Variable domain can be any natural variable structural domain or its engineered forms.Engineered forms refers to the Variable domain using recombinant DNA engineering to produce.This engineered forms comprises such as by inserting, lacking or changing from the generation of specific antibody variable region to the aminoacid sequence of specific antibody.Specific examples comprises the genetically engineered Variable domain comprised only containing a CDR and optional one or more framework amino acid from an antibody and the Variable domain remainder from another antibody.

Variable domain can with at least one other antibody domain or its fragment covalently bound at C terminal amino acid.Therefore, for example, the V of Variable domain is present in _hstructural domain can be connected with immunoglobulin (Ig) CH1 structural domain or its fragment.Similarly, V _lstructural domain can with C _kstructural domain or its fragment are connected.By this way, such as, this antibody can be Fab fragment, wherein antigen-binding domains comprise they C end respectively with CH1 and C _kthe associating V that structural domain is covalently bound _hand V _lstructural domain.Other amino acid available extends C _h1structural domain, such as, to provide hinge area or such as, as the part hinge structural domain in Fab ' fragment or provide other structural domain, antibody CH2 and CH3 structural domain.

the derivative of antibody and varient

Such as change nucleotide sequence L1-L6 with H1-H6 of coding corresponding to aminoacid sequence A1-A6 to produce the polynucleotide through changing comprising one or more concrete Nucleotide and replace, lack or insert compared with non-Variant polynucleotides by random mutagenesis or by site-directed mutagenesis (site-directed mutagenesis of such as oligonucleotide induction).The technical examples changed for generation of such is described in Walder etc., 1986, Gene 42:133; Bauer etc., 1985, Gene 37:73; Craik, BioTechniques, January 1985,12-19; Smith etc., 1981, Genetic Engineering:Principles and Methods, Plenum Press; And U.S. Patent number 4518584 and 4737462.These and other method can be used for producing and such as has compared with underivatized antibody that desirable properties such as affinity, avidity or the specificity to glucagon receptor strengthen, in body or external activity or stability strengthens or the derivative of the anti-glucagon receptor antibodies that side effect reduces in body.

Other anti-glucagon receptor antibodies derivative in field of the present invention comprises the covalency of anti-glucagon receptor antibodies or its fragment and its albumen or polypeptide or assembles binding substances, such as, to hold with the N of anti-glucagon receptor antibodies polypeptide or C holds the recombination fusion protein of the heterologous polypeptide merged by expressing to comprise.Such as, this binding peptide can be Heterologous signal (or guiding) polypeptide, the such as peptide of yeast alpha-factor leader or such as epitope tag.The antibody comprising fusion rotein can comprise the peptide (such as polyhistidine) be added with the purifying of auxiliary antibody or qualification.Antibody also can be connected with FLAG peptide, as Hopp etc., described in 1988, Bio/Technology 6:1204 and United States Patent (USP) 5011912.FLAG peptide has high antigenic and provides by the epi-position of specific monoclonal antibody (mAb) Reversible binding, allows to express the rapid detection of recombinant protein and facilitates purifying.Business can buy (Sigma, St. Louis, MO) for the preparation of the reagent of the wherein fusion rotein of FLAG peptide and given peptide fusion, in another embodiment, the oligomer comprising one or more antibody can be used as glucagon receptor antagonist or with more senior oligomer.Oligomer can be covalently bound or the dimer of non-covalent linking, tripolymer or higher oligomeric forms.The widow comprising two or more antigen-binding proteins can be used concrete, and one of them example is homodimer.Other oligomer comprises heterodimer, homotrimer, heterotrimer, the homotype tetramer, the assorted tetramer etc.

Should be understood that antibody of the present invention can have at least one amino acid and replace, as long as this antibody remains binding specificity.Therefore, the modification of antibody structure is contained in scope.These can comprise the amino acid not destroying antibody glucagon receptor binding ability and replace, and it can be conservative or nonconservative.Conserved amino acid is replaced can comprise Unnatural amino acid residues, and it is integrated instead of biosystem synthesis through chemical peptide symthesis usually.These comprise intends peptide and other amino acid moiety that is reverse or reversing form.Conserved amino acid is replaced also can relate to and is replaced native amino acid residues like this to the polarity of this site amino acid residues or charge effect is very little or not effect with non-natural residues.The member that non-conservative replacement can relate to class of amino acid or amino acid analogue exchanges with the another kind of amino acid whose member with different physical properties (such as, volume, polarity, hydrophobicity, electric charge).

And those skilled in the art can be created on the varient to be measured each expectation amino-acid residue comprising amino acid replacement.Activation measurement well known by persons skilled in the art can be used to screen such varient.Such varient can be used for collecting the information about suitable varient.For example, if find that certain amino acid residue can cause active destruction, unexpected reduction or active improperly, the varient with such change can be avoided.In other words, based on the information of collecting from these routine tests, those skilled in the art easily can determine to avoid the amino acid of further replacement (separately or with other mutation combination).

Technician can use known technology to determine the suitable varient of polypeptide as set forth herein.In some embodiments, those skilled in the art can not destroy active molecule appropriate area for activity unessential region qualification by target after changing.In some embodiments, residue conservative in similar polypeptide or molecular moiety can be identified.In some embodiments, even can guard and replace for biological activity or the important region of structure and do not destroy biological activity or adversely act on polypeptide structure.In addition, those skilled in the art can investigate structure. and functional study qualification is to the residue in active or that structure is important similar polypeptide.In view of this contrast, the measurable importance corresponded to the Amino Acids in Proteins residue of active or that structure is important amino-acid residue in similar protein.Those skilled in the art can be these through predicting that important amino-acid residue selects chemical Similar amino acids to replace.

Those skilled in the art also can analyze the three-dimensional structure relevant to the structure of similar polypeptide and aminoacid sequence.In view of this category information, the amino-acid residue comparison of the measurable antibody with regard to three-dimensional structure of those skilled in the art.In some embodiments, those skilled in the art can select significantly not change at the amino-acid residue of protein surface, because such residue may participate in the important interaction with other molecule through prediction.Many scientific publications are devoted to the prediction of secondary structure.See Moult, Curr. Op. Biotech., 7:422-427 (1996); Chou etc., Biochemistry, 13:222-245 (1974); Chou etc., Biochemistry, 113:211-222 (1974); Chou etc., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou etc., Ann. Rev. Biochem., 47:251-276 and Chou etc., Biophys.J., 26:367-384 (1979).In addition, computer program can be used at present to assist Secondary structure predietion.For example, sequence iden be greater than 30% or similarity be greater than 40% two polypeptide or protein there is similar topology usually.The growth of recent Protein Structural Databank (PDB) enhances the predictability of secondary structure, comprises foldable number potential in polypeptide or protein structure.See Holm etc., Nucl. Acid. Res., 27:244-247 (1999).Show Brenner etc., Curr.Op. Struct. Biol., 7:369-376 (1997) in given polypeptide or protein, there is the folding of limited quantity and once determine the structure of critical mass, structure prediction will become significantly more accurate.

Those skilled in the art can determine when needing such to replace that the amino acid expected is replaced (conservative or non-conservative).In some embodiments, amino acid replaces the important residue or increase that can be used for identifier's glucagon receptor antibodies or the avidity reducing human glucagon receptor antibody described herein.

According to some embodiments, preferred amino acid replaces with following: (1) reduces proteolysis susceptibility, (2) oxidation sensitive is reduced, (3) change and form the binding affinity of protein complex, (4) change binding affinity and/or (4) and give or other physical chemistry of modifying on such polypeptide or functional property.According to some embodiments, single or multiple amino acid replacement (in some embodiments for conserved amino acid is replaced) can be carried out at natural deposit in the sequence (polypeptide portion in some embodiments, outside the structural domain forming intermolecular contacts).In some embodiments, conserved amino acid replaces the structural performance (such as, replace amino acid and should not crack other type secondary structure being present in spiral in parental sequences or interference characteristic parental sequences) that usually can not change parental sequences in essence.This area team can polypeptide secondary and the example of tertiary structure be described in Proteins, (Creighton writes Structures and Molecular Principles, W.H.Freeman and Company (1984); (C. Branden and J.Tooze writes Introduction to Protein Structure, Garland Publishing (1991); And Thornton etc., Nature 354:105 (1991), it is with reference form and in herein.

In addition, one skilled in the art will recognize that suitable bonding agent comprises the part of these antibody, such as one or more heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2 and CDR3, as specifically disclosed herein.At least one heavy chain CDR1, CDR2, CDR3, CDR1, CDR2 and CDR3 district has at least one amino acid and replaces, as long as this antibody remains the binding specificity of non-replaced CDR.The non-CDR part of this antibody can be non-protein molecule, wherein this bonding agent cross-blocks antibody disclosed herein and people GCGR combination and/or suppress through this receptor hyperglycemic-glycogenolytic factor intracellular signaling.The non-CDR part of this antibody can be non-proteinaceous molecule, and wherein this antibody demonstrates shown similar at least one of antibody A l-A6 with bond type that is people GCGR in competition binding assay method, and/or in and the activity of hyperglycemic-glycogenolytic factor.The non-CDR part of antibody can be made up of amino acid, and wherein this antibody is recombinant binding protein or synthetic peptide, and the combination of this recombinant binding protein cross-blocks antibody disclosed herein and people GCGR and/or in and body is interior or external GLA.The non-CDR part of antibody can be made up of amino acid, wherein this antibody is recombinant antibodies, and this recombinant antibodies demonstrates shown similar at least one of antibody A 1-A6 with bond type that is people GCGR peptide in competition binding assay method, and/or in and hyperglycemic-glycogenolytic factor intracellular signaling.

nucleic acid

On the one hand, the invention provides the nucleic acid molecule of separation.This nucleic acid molecule comprises the polynucleotide of all or part of antigen-binding proteins of such as encoding, a chain of such as antibody of the present invention or two chains, or its fragment, derivative, mutain or varient; Be enough to the polynucleotide being used as hybridization probe; PCR primer or for the identification of, analyze, the sequencing primer of polynucleotide of sudden change or amplification coding polypeptide; The antisense nucleic acid of expressing for suppressing polynucleotide and its complementary sequence.This nucleic acid can be any length.Such as their length can be 5,10,15,20,25,30,35,40,45,50,75,100,125,150,175,200,250,300,350,400,450,500,750,1000,1500,3000,5000 or more Nucleotide, and/or comprise one or more appended sequence, such as regulating and controlling sequence, and/or the part being larger nucleic acid such as carrier.This nucleic acid can be strand or double-strand and comprises RNA and/or DNA Nucleotide and its artificial variant (such as, peptide nucleic acid(PNA)).

The nucleic acid of encode antibody polypeptides (such as, heavy chain or light chain, only variable domains or total length) can be separated from the mouse B cell through GCGR antigen immune.This nucleic acid is separated by ordinary method such as polymerase chain reaction (PCR).

The nucleotide sequence of encoding heavy chain and variable region of light chain as indicated above.Those skilled in the art can understand the degeneracy due to genetic code, and each peptide sequence disclosed herein can by other nucleic acid sequence encodings of greater amt.The invention provides each degenerate core nucleotide sequence of code book invention antigen-binding proteins.

In nucleic acid, introduce change by sudden change, cause the change of its polypeptide of encoding (such as, antigen knot house albumen) aminoacid sequence by this.Any technology known in the art can be used to introduce sudden change.In one embodiment, such as site directed mutagenesis protocol is used to change one or more concrete amino-acid residue.In another embodiment, such as random mutagenesis scheme is used to change the residue of one or more Stochastic choice.No matter how it generates, and can express mutant polypeptide and screen desirable properties.

Sudden change can be introduced nucleic acid and significantly do not change the biologic activity of its coded polypeptide.Such as, the Nucleotide that can carry out causing non-essential amino acid residues place amino acid to replace is replaced.In one embodiment, the nucleotide sequence that sudden change provides for L-1 to L-6 and H-1 to H-6 herein or its fragment, varient or derivative like this its encoded packets, containing one or more disappearance of the amino-acid residue of L-1 to L-6 and H-1 to H-6 shown in herein or replacement, become the residue that two or more sequence is different.In another embodiment, mutagenesis inserts an amino acid near one or more amino-acid residues of L-1 to L-6 and H-1 to H-6 shown in this paper becomes the different residue of two or more sequence.Or, one or more sudden change can be introduced nucleic acid with the biologic activity of its coded polypeptide of selectively changing (such as, being combined with GCGR).Such as, this sudden change quantitatively or in nature can change biologic activity.The example of quantitative change comprises increase, reduces or eliminates this activity.The example of qualitative change comprises the antigen-specific changing antigen-binding proteins.

On the other hand, the invention provides the nucleic acid molecule being suitable for the hybridization probe being used as primer or detecting nucleotide sequence of the present invention.Nucleic acid molecule of the present invention only can comprise a part for the nucleotide sequence of code book invention full-length polypeptide, such as, can be used as the fragment of the fragment (such as, GCGR bound fraction) of probe or primer or code book invention polypeptide active part.

Probe based on nucleotide sequence of the present invention can be used for detecting this nucleic acid or analogous nucleic acid, such as the transcript of code book invention polypeptide.This probe can comprise labelling groups, such as radio isotope, fluorescent chemicals, enzyme or enzyme cofactor.Such probe can be used for identifying the cell of expressing this polypeptide.

The invention provides the carrier of the nucleic acid comprising code book invention polypeptide or its part on the other hand.The example of carrier includes, but are not limited to plasmid, virus vector, non-free gene mammalian vector and expression vector, such as recombinant expression vector.

Recombinant expression vector of the present invention can comprise the nucleic acid of the present invention being suitable for the form that this nucleic acid is expressed in host cell.This recombinant expression vector comprises one or more regulating and controlling sequence, screens based on the host cell for expressing, and its nucleotide sequence operability expressed in advance with this is connected.Regulating and controlling sequence comprise guiding nucleus nucleotide sequence in multiple kind host cell constitutive expression (such as, SV40 early gene toughener, Rous sarcoma virus promotor and cytomegalovirus promoter), guide only some host cell nucleotide sequence expression (such as, organizing specific regulating and controlling sequence, see Voss etc., 1986, Trends Biochem. Sci.11:287, Maniatis etc., 1987, Science 236:1237, its complete content is with reference to form and in herein) and the concrete process of guiding nucleus nucleotide sequence response or condition inducible expression (such as, tetracycline reaction (tet-sesponsive) promotor in both metal metalothionine promoter in mammalian cell and protokaryon and eukaryotic system and/or Streptomycin sulphate reaction promotor (ditto)).It will be understood by those skilled in the art that the factor such as selection, desirable proteins expression level of host cell such as transforming is depended in the design of expression vector.Expression vector of the present invention can introduce host cell, produces by this by the albumen of nucleic acid encoding described herein or peptide, comprises fusion rotein or peptide.

On the other hand, the invention provides the host cell can introducing expression vector of the present invention.Host cell can be any protokaryon or eukaryotic cell.Prokaryotic host cell comprises Gram-negative or gram-positive organism, such as intestinal bacteria or bacillus.More senior eukaryotic cell comprises the establishment clone of insect cell, yeast cell and mammalian source.The example of suitable mammalian host cell line comprises Chinese hamster ovary (CHO) cell or their derivative such as Veggie CHO and the relevant cell system that grows in serum free medium (see Rasmussen etc., 1998, Cytotechnology 28:31) or CHO strain DXB-11, DHFR is (see Urlaub etc. for its disappearance, 1980, Proc. Natl. Acad, Sci. USA 77:4216-20).Other Chinese hamster ovary celI system comprises CHO-K1 (ATCC# CCL-61), EM9 (ATCC# CRL-1861), with UV20 (ATCC# CRL-1862), other host cell comprises the COS-7 system (ATCC# CRL-1651) (see Gluzman etc., 1981, Cell 23:175) of monkey-kidney cells, L cell, C127 cell, 3T3 cell (ATCC# CCL-163), AM-1/D cell (being described in U.S. Patents Serial numbers 6210924), HeLa cell, BHK (ATCC# CRL-10) clone, derive from the CV1/EBNA clone (ATCC# CCL-70) (see McMahan etc., 1991, EMBO J.10:2821) of African green monkey kidney cell line CV1, HEKC such as 293,293 EBNA or MSR 293, people's epithelium A431 cell, people C010205 cell, other is through transforming primate cell system, normal diploid cell, derive from the cell strain of prior structure's vitro culture thing, first transplant, HL-60, U937, HaK or Jurkat cell.Suitable cloning and expressing carrier for bacterium, fungi, yeast and mammalian cell host is described in Pouwels etc., Cloning Vectors:A Laboratory Manual, Elsevier (1985).

By Conventional reformat or rotaring dyeing technology, carrier DNA is introduced in protokaryon or eukaryotic cell.For stable mammalian transfection, depend on expression vector and the rotaring dyeing technology of use, known only have sub-fraction cell foreign DNA chelating can be entered in their genome.In order to identify and screen these integrons, usually the gene of selection markers of encoding (such as antibiotics resistance) is introduced host cell together with paid close attention to gene.Preferred selection markers comprises those that can give medicine (as G418, Totomycin and Rheumatrex) resistance.Differentiate to comprise the stable transfected cells (such as, the cell incorporating screening-gene can be survived, and other cells die) being introduced into nucleic acid by drug screening in other method.

Transformant can be cultivated under the condition improving expression of polypeptides, reclaim polypeptide by standard protein purification method.This purification process a kind of is described in Examples below.Comprise the anti-glucagon receptor antibodies polypeptide of recombinant mammalian of basic homology in advance for polypeptide herein, it is not substantially containing contaminative endogenous material.

the activity of antibody

On the one hand, the invention provides can with the mouse source antibody of human glucagon receptor specific binding.This antibody-like comprise can reduce or in and the antagonism of hyperglycemic-glycogenolytic factor intracellular signaling or neutralizing antibody.In one embodiment, antibody, the such as IC of murine antibody of the present invention ₅₀value is 100 nM or lower; In another embodiment, IC ₅₀value is 80nM or lower; In another embodiment, 70nM or lower; In another embodiment, 60nM or lower; In another embodiment, 50nM or lower; In another embodiment, 40nM or lower; In another embodiment, 30nM or lower; In another embodiment, 25nM or lower.In another embodiment, antibody such as murine antibody of the present invention can with human glucagon receptor specific binding, and its IC ₅₀value and reference antibody basic simlarity.In another embodiment, the antibody assay method (the similar assay method of army) had described by following embodiment detect with Kb(or Kd of reference antibody basic simlarity).In this article, term " basic simlarity " means the IC with reference antibody ₅₀or Kb(or Kd) comparable or about 100%, 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 50%.Reference antibody comprises, and such as, has the antibody of heavy chain and light chain combination L1H1, L2H2, L3H3, L4H4, L5H5 and L6H6.In one embodiment, reference antibody comprises A-1, A-2, A-3, A-4, A-5 and A-6.In another embodiment, antibody such as murine antibody of the present invention can with human glucagon receptor specific binding, and the blood sugar of animal model can be reduced.In one embodiment, blood sugar reduces by 2% compared with untreated animal; In another embodiment, blood sugar reduces by 5% compared with untreated animal; In another embodiment, blood sugar reduces by 10% compared with untreated animal; In another embodiment, blood sugar reduces by 15% compared with untreated animal, is 20% in another embodiment, is 25% or more in another embodiment.Blood sugar reducing amount is by dosage control, and for animal or human patient, treatment effective dose is the dosage for blood sugar being reduced to normal range.

indication

Diabetes especially diabetes B and complication thereof have become the day by day serious problem in the whole world.Usually, this disease generates impaired causing by pancreatic beta cell Regular Insulin.In diabetes B (the modal form of this disease), it is believed that h and E factors in combination causes beta cell failure, make in human body, cause insulin secretion and activity is impaired and insulin resistant.It is generally acknowledged that obesity impels the illness that in the even children of being grown up, diabetes B increases.

It is also believed that hyperlipemia, or abnormal HDL(high-density lipoprotein (HDL)) with LDL(low-density lipoprotein) all relevant to diabetes B.The feature of diabetes B is the Regular Insulin that muscle and other organ can not respond normal circulation concentration.Pancreatic beta cell excreting insulin increases subsequently, i.e. the illness of hyperinsulinemia.Final β cell cannot compensate again, causes that glucose tolerance reduces, impaired, the chronic hyperglycemia of fasting glucose concentration and tissue injury.In addition, it is believed that and early send out diabetes B and hyperlipemia, or abnormal HDL(high-density lipoprotein (HDL)) and LDL(low-density lipoprotein) relevant.Metabolic syndrome patients shows hyperlipemia and hyperglycemia.

The invention provides antibody, especially can with glucagon receptor Binding in vivo, the human antibodies reducing animal model blood sugar concentration.Antibody also can improve glucose tolerance.In one embodiment, the invention provides the complete human antibodies having and tire in body.Blood sugar can be reduced, for hyperglycemia, diabetes B and associated conditions provide effective, lasting treatment in a couple of days after injection to the effect of ob/ob mouse single injection antibody.Single injection antibody also can improve the glucose clearance (improving glucose tolerance) of blood sample in the glucose tolerance test (GTT) carried out on hereinafter described cynomolgus monkey.Antigen-binding proteins of the present invention, especially human antibodies can be used for reducing blood or plasma glucose, improve glucose tolerance and reduce, improve fasting glucose level and improve hyperlipemia.Like this, antigen-binding proteins of the present invention, especially human antibodies can be used for treatment hyperglycemia, diabetes B, metabolic syndrome and comprises other related symptoms of hyperlipemia.In addition, shown reduce blood sugar can in some cases for the cancer such as colorectal carcinoma that prevention and therapy is concrete, as Richardson etc., Nature ClinPract Oncol 2:48-53 (2005); Giovannucci etc., Gastroenterology 132:2208-2225 (2007); Krone etc., Integrative Cancer Ther 4:25-31 (2005); Chang etc., Diabetologia 46:595-607 (2003); Described in Jee etc., Yonsei Med J 46:449-55 (2005).

concrete enforcement:

1, immunity cell antigen stablizes the structure of strain

Inoculation CHO-DHFR ^-to in 6 orifice plates, transfection after cultivation 24h, changes nutrient solution before transfection, carries out transfection according to the transfection conditions of Invitrogen business recommendations Lipofectamine 2000.After 48 hours, then be changed to the perfect medium containing 10 nM MTX, change liquid every 3 days, treat about two weeks, occur the clone of stable growth, digestion cell dispersion colony, in time growing to 50% healing grade, the concentration improving MTX gradually carries out pressurization screening, until the concentration of MTX is 10 uM.The stable cell line built carries out FACS detection respectively, utilizes the cell colony after the homemade Identification of the antibodies pressurization of GCGR, the CHO-DHFR after 10 uM MTX screen ^-on hGCGR cytolemma, hGCGR has great expression, eventually passes subclone screening, qualification acquisition GCGR high expressing cell stablizes strain.

2, the preparation of antibody

By complete for the CHO-DHFR-hGCGR of emulsification in freund's adjuvant cell, with the injected sc BALB/c mouse of 2x10^6 cell/mouse (6-8 age in week).After 2 weeks, cannot be used up full freund's adjuvant emulsified immunogen, and booster immunization mouse, after this strengthens weekly once.Taken a blood sample by the mode of cutting tail, centrifugation serum, carries out FACS and detects serum titer.Until when reaching applicable antibody titers, disconnected neck puts to death mouse, obtains spleen cell under sterile state.Collect the SP2/0 cell that growth conditions is in logarithmic phase, 2000rpm, 3min are centrifugal, and precipitation serum-free culture is to resuspended, and recentrifuge-resuspended, counts.Mixing spleen cell and SP2/0 cell, ensure SP2/0: spleen cell >=1:1, is total to " washing-centrifugal " 3 times.Bullet fall apart centrifugal for the last time after precipitation, dropwise add in the pre-warm PEG-1350(30s of 1mL and drip off), upper and lower pressure-vaccum 1 minute, slowly add the serum free medium of 30mL preheating to stop the fusion of PEG, 1500rpm, 5min are centrifugal, bullet falls apart cell precipitation, add fusion substratum, be laid in 96 orifice plates by 20000, every hole splenocyte and 5000 feeder layer cells, every hole 100uL.Hybridoma after fusion is cultivated together with feeder layer cells in 96 orifice plates, and carries out HAT(xanthoglobulin, methotrexate and thymidine) screening, to remove nonfused cell.The hybridoma supernatant collected after 10 days in culture plate carries out ELISA detection.

3, full cell ELISA screening

CHO-DHFR-hGCGR process LAN hGCGR cell and the CHO-DHFR-blanc cell of not expressing hGCGR are seeded to 96 orifice plates respectively, grow to 90% healing grade.Removing cells and supernatant, PBS washes twice, adds 100ul 100% methyl alcohol 4 DEG C of fixing 10min.Add the freshly prepared 0.6% H2O2-PBS of 100 ul again, room temperature treatment 20min, PBS wash 2 times.After PBS-1%BSA closes, add hybridoma supernatant 4 DEG C and hatch 90 min.Repeatedly after washing, the GxM-HRP-Fc bis-that every hole adds 100ul dilution resists, and hatches 0.5h for 37 DEG C.After washing 5 times, every hole adds 100ul TMB chromogenic substrate, 37 DEG C of reaction 15min, 2M H ₂sO ₄stop, read OD450 value.Positive control is the serum of immune mouse; Negative control is cell culture medium supernatant.Choose the hybridoma cell strains secreting anti-hGCGR antibody, carry out cloning to obtain the cell strain of energy stably excreting for hGCGR.The antibody supernatant finally choosing hybridoma secretion carries out FACS checking.

4, positive hybridoma cell supernatant flow cytometer showed qualification (FACS)

Digest with the PBS containing 10mM EDTA, collect 10 ⁵individual CHO-DHFR-hGCGR cell, adds 1.5ml EP respectively and manages, abandon supernatant after centrifugal, and negative control sample is resuspended with streaming sample-loading buffer (PBS, 2% FBS).Positive treatment group cell often pipe adds 200ul antibody supernatant, incubated at room; 1500rpm is centrifugal, abandons supernatant, washes once with streaming sample-loading buffer, more centrifugal, resuspended, and add the sheep anti mouse fluorescence two anti-(BD, 200ul/ hole) of the FITC mark of 1:50 dilution, room temperature lucifuge hatches 30 min; Centrifugal, abandon supernatant, then wash once with streaming sample-loading buffer, centrifugal, finally use streaming sample-loading buffer resuspended, upper machine testing.Hybridoma supernatant and expression have the CHO-DHFR cell of GCGR to have specific binding, and grey peak and dotted line peak are negative control, and the solid line peak that hybridoma supernatant is corresponding has obviously move to right (Fig. 1,2,3).

5, the clone of antibody variable gene and antibody transient expression

Collect the hybridoma of secretory antibody, according to the mRNA extraction agent box working specification of QIAGEN, extract the mRNA of hybridoma.Then the mRNA reverse transcription after extraction is become cDNA, reverse transcriptase primer is that mouse is light, the Auele Specific Primer of CH, heavy chain reverse transcriptase primer is (5 '-TTTGGRGGGAAGATGAAGAC-3 ', SEQ ID NO:61), light chain reverse transcriptase primer be (5 '-TTAACACTCTCCCCTGTTGAA-3 ', SEQ ID NO:62) and (5 '-TTAACACTCATTCCTGTTGAA-3 ', SEQ ID NO:63).The TE of the cDNA 0.1mM of reverse transcription is diluted, by ultra-filtration centrifuge tube purifying.Get the cDNA after the purifying of 10ul as template, add the 5xtailing buffer of 4 ul, the dATP(1mM of 4 ul) and the terminal enzyme (DNA) (Promega) of 10U mix afterwards, hatch 65 DEG C of 5min after 5min for 37 DEG C; Then to add that the cDNA of PolyA tail is for template, light, the heavy chain variable region gene of pcr amplification antibody.Upstream primer is OligodT, heavy chain downstream primer be (5 '-TGGACAGGGATCCAGAGTTCC-3 ', SEQ ID NO:64) and (5 '-TGGACAGGGCTCCATAGTTCC-3 ', SEQ ID NO:65), light chain downstream primer is (5 '-ACTCGTCCTTGGTCAACGTG-3 ', SEQ ID NO:66).PCR reacts amplification, and PCR primer checks order after being connected to PMD 18-T carrier.

Based on the DNA sequence dna design PCR primer of the antibody obtained that checks order, thus Whole light chains, heavy chain signal peptide and variable domain and mouse IgG 1 constant region are connected with expression vector pTM5.

6, antigen-binding proteins is recombinant expressed

The suspension HEK293 express cell of inoculation 40ml logarithmic phase is in T125 rolling bottle, get 4ml fresh medium configuration PEI-DNA transfection composite, in PEI:DNA(0.5ug/ml substratum) ratio of=3:1 mixing PEI and DNA, room temperature all adds in Tissue Culture Flask after placing 15min.After 24 hours, add Tryptones as expressing the nitrogenous source needed, then continue cultivation more than 96 hours, collecting cell supernatant directly carries out functional selection or the separation and purification for antigen binding proteins.

7, HitHunter cAMP experiment screening functional antibodies

CHO-DHFR-cell to the 384 porocyte culture plate of hGCGR is expressed, 37 DEG C of overnight incubation with the inoculation of 10000, every hole.After second day removing substratum supernatant, add the antibody after 10ul purifying, hatch 15 minutes for 37 DEG C, then add the Glucagon of 5ul serum free medium dilution, hatch 30 minutes for 37 DEG C.Then adopt the HitHunter cAMP test kit of DiscoverX to detect the cAMP generated, namely add 5ul cAMP antibody, the mixture of 20ul ED reagent, lysis buffer and luminous substrate, incubated at room 1h, then add 20ul EA reagent room temperature and continue to hatch 1h.Finally in the SpectraMax L microplate reader of Molecular Devices, read relative intensity of fluorescence.With not having the contrast of inhibitor to compare, A-1, A-2, A-3 antibody supernatant obviously suppresses 0.5nM hyperglycemic-glycogenolytic factor to the activation (Fig. 4) of GCGR acceptor.

8, Reporter Gene Experiments screening function antibody

With CHO-DHFR-cell to the 96 porocyte culture plate of 20000, every hole inoculation coexpression hGCGR-CRE-Luciferase, 37 DEG C of overnight incubation.Second day removing substratum supernatant, with serum free medium cleaning cell surface twice, sucks raffinate, and add antibody incubation after 50ul purifying 30 minutes, then add the Glucagon of 50ul serum free medium dilution, 37 DEG C are continued to hatch 4 hours.Stimulate after terminating, add the Bright Glo chemical luminous substrate lysing cell of 100ulPromega, finally cell lysate is transferred to white 96 orifice plates, the SpectraMax L microplate reader of Molecular Devices reads relative intensity of fluorescence.

Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.

SEQUENCE LISTING

<110> Hangzhou Gmax Biopharm Engineering Co., Ltd.

Antibody of the anti-glucagon receptor of <120> and uses thereof

<130> 2013

<160> 66

<170> PatentIn version 3.5

<210> 1

<211> 1434

<212> DNA

<213> people

<400> 1

atgcccccct gccagccaca gcgacccctg ctgctgttgc tgctgctgct ggcctgccag 60

ccacaggtcc cctccgctca ggtgatggac ttcctgtttg agaagtggaa gctctacggt 120

gaccagtgtc accacaacct gagcctgctg ccccctccca cggagctggt gtgcaacaga 180

accttcgaca agtattcctg ctggccggac acccccgcca ataccacggc caacatctcc 240

tgcccctggt acctgccttg gcaccacaaa gtgcaacacc gcttcgtgtt caagagatgc 300

gggcccgacg gtcagtgggt gcgtggaccc cgggggcagc cttggcgtga tgcctcccag 360

tgccagatgg atggcgagga gattgaggtc cagaaggagg tggccaagat gtacagcagc 420

ttccaggtga tgtacacagt gggctacagc ctgtccctgg gggccctgct cctcgccttg 480

gccatcctgg ggggcctcag caagctgcac tgcacccgca atgccatcca cgcgaatctg 540

tttgcgtcct tcgtgctgaa agccagctcc gtgctggtca ttgatgggct gctcaggacc 600

cgctacagcc agaaaattgg cgacgacctc agtgtcagca cctggctcag tgatggagcg 660

gtggctggct gccgtgtggc cgcggtgttc atgcaatatg gcatcgtggc caactactgc 720

tggctgctgg tggagggcct gtacctgcac aacctgctgg gcctggccac cctccccgag 780

aggagcttct tcagcctcta cctgggcatc ggctggggtg cccccatgct gttcgtcgtc 840

ccctgggcag tggtcaagtg tctgttcgag aacgtccagt gctggaccag caatgacaac 900

atgggcttct ggtggatcct gcggttcccc gtcttcctgg ccatcctgat caacttcttc 960

atcttcgtcc gcatcgttca gctgctcgtg gccaagctgc gggcacggca gatgcaccac 1020

acagactaca agttccggct ggccaagtcc acgctgaccc tcatccctct gctgggcgtc 1080

cacgaagtgg tctttgcctt cgtgacggac gagcacgccc agggcaccct gcgctccgcc 1140

aagctcttct tcgacctctt cctcagctcc ttccagggcc tgctggtggc tgtcctctac 1200

tgcttcctca acaaggaggt gcagtcggag ctgcggcggc gttggcaccg ctggcgcctg 1260

ggcaaagtgc tatgggagga gcggaacacc agcaaccaca gggcctcatc ttcgcccggc 1320

cacggccctc ccagcaagga gctgcagttt gggaggggtg gtggcagcca ggattcatct 1380

gcggagaccc ccttggctgg tggcctccct agattggctg agagcccctt ctga 1434

<210> 2

<211> 477

<212> PRT

<213> people

<400> 2

Met Pro Pro Cys Gln Pro Gln Arg Pro Leu Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Ala Cys Gln Pro Gln Val Pro Ser Ala Gln Val Met Asp Phe Leu

20 25 30

Phe Glu Lys Trp Lys Leu Tyr Gly Asp Gln Cys His His Asn Leu Ser

35 40 45

Leu Leu Pro Pro Pro Thr Glu Leu Val Cys Asn Arg Thr Phe Asp Lys

50 55 60

Tyr Ser Cys Trp Pro Asp Thr Pro Ala Asn Thr Thr Ala Asn Ile Ser

65 70 75 80

Cys Pro Trp Tyr Leu Pro Trp His His Lys Val Gln His Arg Phe Val

85 90 95

Phe Lys Arg Cys Gly Pro Asp Gly Gln Trp Val Arg Gly Pro Arg Gly

100 105 110

Gln Pro Trp Arg Asp Ala Ser Gln Cys Gln Met Asp Gly Glu Glu Ile

115 120 125

Glu Val Gln Lys Glu Val Ala Lys Met Tyr Ser Ser Phe Gln Val Met

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Trp Leu Leu Val Glu Gly Leu Tyr Leu His Asn Leu Leu Gly Leu Ala

245 250 255

Thr Leu Pro Glu Arg Ser Phe Phe Ser Leu Tyr Leu Gly Ile Gly Trp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ile Phe Val Arg Ile Val Gln Leu Leu Val Ala Lys Leu Arg Ala Arg

325 330 335

Gln Met His His Thr Asp Tyr Lys Phe Arg Leu Ala Lys Ser Thr Leu

340 345 350

Thr Leu Ile Pro Leu Leu Gly Val His Glu Val Val Phe Ala Phe Val

355 360 365

Thr Asp Glu His Ala Gln Gly Thr Leu Arg Ser Ala Lys Leu Phe Phe

370 375 380

Asp Leu Phe Leu Ser Ser Phe Gln Gly Leu Leu Val Ala Val Leu Tyr

385 390 395 400

Cys Phe Leu Asn Lys Glu Val Gln Ser Glu Leu Arg Arg Arg Trp His

405 410 415

Arg Trp Arg Leu Gly Lys Val Leu Trp Glu Glu Arg Asn Thr Ser Asn

420 425 430

His Arg Ala Ser Ser Ser Pro Gly His Gly Pro Pro Ser Lys Glu Leu

435 440 445

Gln Phe Gly Arg Gly Gly Gly Ser Gln Asp Ser Ser Ala Glu Thr Pro

450 455 460

Leu Ala Gly Gly Leu Pro Arg Leu Ala Glu Ser Pro Phe

465 470 475

<210> 3

<211> 485

<212> PRT

<213> mouse

<400> 3

Met Pro Leu Thr Gln Leu His Cys Pro His Leu Leu Leu Leu Leu Leu

1 5 10 15

Val Leu Ser Cys Leu Pro Glu Ala Pro Ser Ala Gln Val Met Asp Phe

20 25 30

Leu Phe Glu Lys Trp Lys Leu Tyr Ser Asp Gln Cys His His Asn Leu

35 40 45

Ser Leu Leu Pro Pro Pro Thr Glu Leu Val Cys Asn Arg Thr Phe Asp

50 55 60

Lys Tyr Ser Cys Trp Pro Asp Thr Pro Pro Asn Thr Thr Ala Asn Ile

65 70 75 80

Ser Cys Pro Trp Tyr Leu Pro Trp Tyr His Lys Val Gln His Arg Leu

85 90 95

Val Phe Lys Arg Cys Gly Pro Asp Gly Gln Trp Val Arg Gly Pro Arg

100 105 110

Gly Gln Pro Trp Arg Asn Ala Ser Gln Cys Gln Leu Asp Asp Glu Glu

115 120 125

Ile Glu Val Gln Lys Gly Val Ala Lys Met Tyr Ser Ser Gln Gln Val

130 135 140

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

145 150 155 160

Leu Val Ile Leu Leu Gly Leu Arg Lys Leu His Cys Thr Arg Asn Tyr

165 170 175

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

180 185 190

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

195 200 205

Asp Asp Leu Ser Val Ser Val Trp Leu Ser Asp Gly Ala Met Ala Gly

210 215 220

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

225 230 235 240

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

245 250 255

Ala Thr Phe Ser Glu Arg Ser Phe Phe Ser Leu Tyr Leu Gly Ile Gly

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Phe Ile Phe Val His Ile Ile His Leu Leu Val Ala Lys Leu Arg Ala

325 330 335

His Gln Met His Tyr Ala Asp Tyr Lys Phe Arg Leu Ala Arg Ser Thr

340 345 350

Leu Thr Leu Ile Pro Leu Leu Gly Val His Glu Val Val Phe Ala Phe

355 360 365

Val Thr Asp Glu His Ala Gln Gly Thr Leu Arg Ser Thr Lys Leu Phe

370 375 380

Phe Asp Leu Phe Leu Ser Ser Phe Gln Gly Leu Leu Val Ala Val Leu

385 390 395 400

Tyr Cys Phe Leu Asn Lys Glu Val Gln Ala Glu Leu Met Arg Arg Trp

405 410 415

Arg Gln Trp Gln Glu Gly Lys Ala Leu Gln Glu Glu Arg Leu Ala Ser

420 425 430

Ser His Gly Ser His Met Ala Pro Ala Gly Pro Cys His Gly Asp Pro

435 440 445

Cys Glu Lys Leu Gln Leu Met Ser Ala Gly Ser Ser Ser Gly Thr Gly

450 455 460

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

465 470 475 480

Ala Asp Ser Pro Thr

485

<210> 4

<211> 1000

<212> DNA

<213> mouse

<400> 4

atgcccctca cccagctcca ctgtccccac ctgctgctgc tgctgttggt gctgtcatgt 60

ctgccagagg caccctctgc ccaggtaatg gactttttgt ttgagaagtg gaagctctat 120

agtgaccaat gtcaccacaa cctaagcctg ctgcccccac ctactgagct ggtctgtaac 180

agaaccttcg acaactactc ctgctggcct gacacccctc ccaacaccac tgccaacatt 240

tcctgcccct ggtacctacc ttggtgccac aaagtgcagc accgcctagt gttcaagagg 300

tgtgggcccg atgggcagtg ggttcgaggg ccacgggggc agccgtggcg caacgcctcc 360

caatgtcagt tggatgatga agagatcgag gtccagaagg gggtggccaa gatgtatagc 420

agccagcagg tgatgtacac cgtgggctac agtctgtccc tgggggcctt gctccttgcg 480

ctggtcatcc tgctgggcct caggaagctg cactgcaccc gaaactacat ccatgggaac 540

ctgtttgcgt cctttgtgct caaggctggc tctgtgttgg tcatcgattg gctgctgaag 600

acacggtaca gccagaagat tggcgatgac ctcagtgtga gcgtctggct cagtgacggg 660

gcgatggccg gctgcagagt ggccacagtg atcatgcagt acggcatcat acccaactat 720

tgctggttgc tggtagaggg cgtgtacctg tacagcctgc tgagccttgc caccttctct 780

gagaggagct tcttttccct ctacctgggc attggctggg gtgcgcccct gctgtttgtc 840

atcccctggg tggtggtcaa gtgtctgttt gagaatgttc agtgctggac cagcaatgac 900

aacatgggat tctggtggat cctgcgtatt cctgtcttcc tggccttact gatcaatttt 960

ttcatctttg tccacatcat tcaacttctt gtggccaagc 1000

<210> 5

<211> 12

<212> PRT

<213> mouse

<400> 5

Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val

1 5 10

<210> 6

<211> 10

<212> PRT

<213> mouse

<400> 6

Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr

1 5 10

<210> 7

<211> 4

<212> PRT

<213> mouse

<400> 7

Ala Asn Leu Tyr

1

<210> 8

<211> 111

<212> PRT

<213> mouse

<400> 8

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

1 5 10 15

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

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Val Glu Glu Phe

50 55 60

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

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

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

100 105 110

<210> 9

<211> 333

<212> DNA

<213> mouse

<400> 9

cagatacagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cttctgggta taccttcaca aactatggaa tgaactgggt gaaacaggct 120

ccaggaaagg gtttaaagtg gatgggctgg ataaacacca acactggaga gccaacatat 180

gttgaagagt tcaagggacg gtttgccttc tctttggaaa cctctgacag cactgcctat 240

ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aaacctttac 300

tggggccaag gcaccactct cacagtctcc tca 333

<210> 10

<211> 12

<212> PRT

<213> mouse

<400> 10

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

1 5 10

<210> 11

<211> 10

<212> PRT

<213> mouse

<400> 11

Lys Val Ser Asn Arg Phe Ser Gly Val Pro

1 5 10

<210> 12

<211> 9

<212> PRT

<213> mouse

<400> 12

Phe Gln Gly Ser His Val Pro Trp Thr

1 5

<210> 13

<211> 112

<212> PRT

<213> mouse

<400> 13

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Leu His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Phe Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 14

<211> 336

<212> DNA

<213> mouse

<400> 14

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gaacatttta catagtaatg ggaacaccta tttagaatgg 120

tacctgcaga aaccaggcca gtctccaaag ctcctgatct tcaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

tggacgttcg gtggaggcac caagctggaa atcaaa 336

<210> 15

<211> 10

<212> PRT

<213> mouse

<400> 15

Ile Asn Thr Tyr Asn Gly Glu Pro Thr Tyr

1 5 10

<210> 16

<211> 14

<212> PRT

<213> mouse

<400> 16

Thr Arg Glu Tyr Tyr Ile Ser Asn Tyr Gly Ala Met Asp Tyr

1 5 10

<210> 17

<211> 121

<212> PRT

<213> mouse

<400> 17

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

1 5 10 15

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

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

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

50 55 60

Lys Gly Arg Cys Ala Phe Phe Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Thr Arg Glu Tyr Tyr Ile Ser Asn Tyr Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 18

<211> 363

<212> DNA

<213> mouse

<400> 18

cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cttctggata taccttcaca aactatggaa tgaactgggt gaagcaggct 120

ccaggaaagg gtttaaagtg gatgggctgg attaacacct acaatggaga gccaacatat 180

gctgatgact tcaagggacg gtgtgccttt tttttggaaa cctctgccag cactgcctat 240

ctgcaactca acaacctcaa aaatgaggac acggctacct atttctgtac aagagaatat 300

tacattagta attacggggc tatggactac tggggtcaag gaacctcagt caccgtctcc 360

tca 363

<210> 19

<211> 12

<212> PRT

<213> mouse

<400> 19

Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln

1 5 10

<210> 20

<211> 10

<212> PRT

<213> mouse

<400> 20

Tyr Thr Ser Arg Leu His Ser Gly Val Pro

1 5 10

<210> 21

<211> 9

<212> PRT

<213> mouse

<400> 21

Gln Gln Gly Ile Leu Phe Pro Leu Thr

1 5

<210> 22

<211> 107

<212> PRT

<213> mouse

<400> 22

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 Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ile Leu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 23

<211> 321

<212> DNA

<213> mouse

<400> 23

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctttgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctattac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggagcagat tattctctca ccattgccaa cctggatcaa 240

gaagatattg ccacttactt ttgccagcag ggtattctgt ttccgctcac gttcggtgct 300

gggaccaagc tggagctgaa a 321

<210> 24

<211> 12

<212> PRT

<213> mouse

<400> 24

Gly Phe Pro Phe Ser Asp Tyr Gly Leu Ala Trp Phe

1 5 10

<210> 25

<211> 10

<212> PRT

<213> mouse

<400> 25

Ile Ser Asn Leu Ala Tyr Ser Thr Tyr Tyr

1 5 10

<210> 26

<211> 9

<212> PRT

<213> mouse

<400> 26

Ala Arg Asp Arg Gly Leu Phe Asp Tyr

1 5

<210> 27

<211> 116

<212> PRT

<213> mouse

<400> 27

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

1 5 10 15

Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Asp Tyr

20 25 30

Gly Leu Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ala Phe Ile Ser Asn Leu Ala Tyr Ser Thr Tyr Tyr Ala Asp Thr Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 28

<211> 348

<212> DNA

<213> mouse

<400> 28

gaggtgaaac tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc 60

tcctgtgcag cctctggatt ccctttcagt gactacggat tggcgtggtt tcgacaggct 120

ccagggaagg ggcctgaatg ggtagcgttc attagtaatt tggcatatag tacctactat 180

gcagacactg tgacgggccg attcaccatc tctagagaga atgccaagaa caccctgttc 240

ctggaaatga gcagtctgag gtctgaggac acagccatgt attactgtgc aagggatagg 300

gggctgtttg actactgggg ccaaggcacc actctcacag tctcctca 348

<210> 29

<211> 12

<212> PRT

<213> mouse

<400> 29

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

1 5 10

<210> 30

<211> 10

<212> PRT

<213> mouse

<400> 30

Ser Gln Ser Thr His Val Pro Pro Tyr Thr

1 5 10

<210> 31

<211> 114

<212> PRT

<213> mouse

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg

<210> 32

<211> 342

<212> DNA

<213> mouse

<400> 32

gatgttgtga tgacccaaat tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttcct 300

ccgtacacgt tcggaggggg gaccaagctg gaaataaaac gg 342

<210> 33

<211> 12

<212> PRT

<213> mouse

<400> 33

Gly Phe Thr Phe Ser Ser Tyr Asp Met Ser Trp Val

1 5 10

<210> 34

<211> 10

<212> PRT

<213> mouse

<400> 34

Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro

1 5 10

<210> 35

<211> 15

<212> PRT

<213> mouse

<400> 35

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

1 5 10 15

<210> 36

<211> 121

<212> PRT

<213> mouse

<400> 36

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

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

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

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

50 55 60

Gly Arg Phe Ile Ile Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu

65 70 75 80

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

85 90 95

Ser Gly Gly Phe Tyr Tyr Gly Ser Thr Tyr Asn Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 37

<211> 363

<212> DNA

<213> mouse

<400> 37

gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt agctatgaca tgtcttgggt gcgccagact 120

ccagagaaga ggctggagtg ggtcgcatcc ataagtagtg gtggtagtac gtactatcca 180

gacagtgtga agggccgatt catcatttcc agagataatg tcaggaatat cctgtacctg 240

caaatgagca gtctgaggtc tgaggacacg gccatgtatt actgtgcaag tggagggttt 300

tactacggta gtacctacaa cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360

tca 363

<210> 38

<211> 12

<212> PRT

<213> mouse

<400> 38

Gln Asp Ile Ser Gly Tyr Leu Ser Trp Phe Gln Gln

1 5 10

<210> 39

<211> 10

<212> PRT

<213> mouse

<400> 39

Arg Ala Asn Arg Leu Ile His Gly Val Pro

1 5 10

<210> 40

<211> 9

<212> PRT

<213> mouse

<400> 40

Leu Gln Tyr Asp Glu Phe Pro Phe Thr

1 5

<210> 41

<211> 107

<212> PRT

<213> mouse

<400> 41

Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Ile His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 42

<211> 321

<212> DNA

<213> mouse

<400> 42

gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60

atcacttgca aggcgagtca ggacattagt ggctatttaa gctggttcca gcagaaacca 120

ggcaaatctc ctaagaccct gatctatcgt gcaaacagat tgatacatgg ggtcccatca 180

aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240

gaagatatgg gaatttatta ttgtctacag tatgatgagt ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 43

<211> 12

<212> PRT

<213> mouse

<400> 43

Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val

1 5 10

<210> 44

<211> 10

<212> PRT

<213> mouse

<400> 44

Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr

1 5 10

<210> 45

<211> 15

<212> PRT

<213> mouse

<400> 45

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

1 5 10 15

<210> 46

<211> 122

<212> PRT

<213> mouse

<400> 46

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

1 5 10 15

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

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Ser Gly Gly Phe Tyr Tyr Gly Ser Thr Tyr Asn Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 47

<211> 363

<212> DNA

<213> mouse

<400> 47

gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt agctatgaca tgtcttgggt gcgccagact 120

ccagagaaga ggctggagtg ggtcgcatcc ataagtagtg gtggtagtac gtactatcca 180

gacagtgtga agggccgatt catcatttcc agagataatg tcaggaatat cctgtacctg 240

caaatgagca gtctgaggtc tgaggacacg gccatgtatt actgtgcaag tggagggttt 300

tactacggta gtacctacaa cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360

tca 363

<210> 48

<211> 12

<212> PRT

<213> mouse

<400> 48

Gln Gly Thr Ser Ile Asn Leu Asn Trp Phe Gln Gln

1 5 10

<210> 49

<211> 10

<212> PRT

<213> mouse

<400> 49

Gly Ala Ser Asn Leu Glu Asp Gly Val Pro

1 5 10

<210> 50

<211> 6

<212> PRT

<213> mouse

<400> 50

Leu Gln His Ser Tyr Leu

1 5

<210> 51

<211> 94

<212> PRT

<213> mouse

<400> 51

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

1 5 10 15

Asp Ile Val Thr Met Thr Cys Gln Ala Ser Gln Gly Thr Ser Ile Asn

20 25 30

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

35 40 45

Tyr Gly Ala Ser Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Asp

65 70 75 80

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

85 90

<210> 52

<211> 282

<212> DNA

<213> mouse

<400> 52

gatgtccaga tgattcagtc tccatcctcc ctgtctgcat ctttgggaga catagtcacc 60

atgacttgcc aggcaagtca gggcactagc attaatttaa actggtttca gcaaaaacca 120

gggaaagctc ctaagctcct gatctatggt gcaagcaact tggaagatgg ggtcccatca 180

aggttcagtg gcagtagata tgggacagat ttcactctca ccatcagcag cctggaggat 240

gaagatatgg caacttattt ctgtctacag catagttatc tc 282

<210> 53

<211> 12

<212> PRT

<213> mouse

<400> 53

Gly Phe Thr Phe Ser Gly Ser Gly Met His Trp Val

1 5 10

<210> 54

<211> 10

<212> PRT

<213> mouse

<400> 54

Ile Ser Ser Asp Ser Ser Thr Ile Tyr Tyr

1 5 10

<210> 55

<211> 13

<212> PRT

<213> mouse

<400> 55

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

1 5 10

<210> 56

<211> 120

<212> PRT

<213> mouse

<400> 56

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

1 5 10 15

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

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Asp Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 57

<211> 360

<212> PRT

<213> mouse

<400> 57

Gly Ala Thr Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly

1 5 10 15

Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr

20 25 30

Ala Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly

35 40 45

Thr Cys Cys Cys Gly Gly Ala Ala Ala Cys Thr Cys Thr Cys Cys Thr

50 55 60

Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr

65 70 75 80

Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Gly Cys Thr Cys Thr

85 90 95

Gly Gly Ala Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr Thr Cys

100 105 110

Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Gly Ala Ala

115 120 125

Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Cys

130 135 140

Gly Cys Ala Thr Ala Cys Ala Thr Thr Ala Gly Thr Ala Gly Thr Gly

145 150 155 160

Ala Cys Ala Gly Thr Ala Gly Thr Ala Cys Cys Ala Thr Cys Thr Ala

165 170 175

Cys Thr Ala Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Gly Thr Gly

180 185 190

Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala

195 200 205

Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Cys Cys

210 215 220

Cys Ala Ala Gly Ala Ala Cys Ala Cys Cys Cys Thr Gly Thr Thr Cys

225 230 235 240

Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Cys Cys Ala Gly Thr Cys

245 250 255

Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys

260 265 270

Gly Gly Cys Cys Ala Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr

275 280 285

Gly Cys Ala Ala Gly Ala Cys Cys Cys Thr Ala Thr Thr Ala Cys Thr

290 295 300

Thr Cys Gly Gly Thr Ala Gly Thr Ala Cys Thr Thr Cys Cys Thr Thr

305 310 315 320

Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Ala

325 330 335

Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys Ala Cys Ala Gly

340 345 350

Thr Cys Thr Cys Cys Thr Cys Ala

355 360

<210> 58

<211> 12

<212> PRT

<213> mouse

<400> 58

Gln Asp Ile Asn Gly Tyr Leu Ser Trp Phe Gln Gln

1 5 10

<210> 59

<211> 107

<212> PRT

<213> mouse

<400> 59

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

1 5 10 15

Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Gly Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Ile Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 60

<211> 321

<212> DNA

<213> mouse

<400> 60

gacatccaga tgacccaatc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60

atcacttgca aggcgagtca ggacattaat ggctatttaa gctggttcca gcagaaacca 120

gggaaatctc ctaagaccct gatctatcgt gcaaacagat tgatagatgg ggtcccatca 180

aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcggcag cctggaatat 240

gaagatatgg gaatttatta ttgtctacag tatgatgagt ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 61

<211> 20

<212> DNA

<213> artificial sequence

<400> 61

tttggrggga agatgaagac 20

<210> 62

<211> 21

<212> DNA

<213> artificial sequence

<400> 62

ttaacactct cccctgttga a 21

<210> 63

<211> 21

<212> DNA

<213> artificial sequence

<400> 63

ttaacactca ttcctgttga a 21

<210> 64

<211> 21

<212> DNA

<213> artificial sequence

<400> 64

tggacaggga tccagagttc c 21

<210> 65

<211> 21

<212> DNA

<213> artificial sequence

<400> 65

tggacagggc tccatagttc c 21

<210> 66

<211> 20

<212> DNA

<213> artificial sequence

<400> 66

actcgtcctt ggtcaacgtg 20

Claims

1. an antibody for anti-glucagon receptor, is characterized in that: described antibody comprises the aminoacid sequence of one of following scheme:

A. the light chain CDR3 sequence of one of following sequence is selected from:

B. the heavy chain CDR3 sequence of one of following sequence is selected from:

Described antibody and human glucagon receptor specific binding.

2. antibody according to claim 1, is characterized in that: described antibody also comprises the aminoacid sequence of the one or more combination of following scheme:

A. one of following light chain CDR1 sequence is selected from:

B. one of following light chain CDR2 sequence is selected from:

C. one of following heavy chain CDR1 sequence is selected from:

D. one of following heavy chain CDR2 sequence is selected from:

3. an antibody for anti-glucagon receptor, is characterized in that: described antibody comprises the aminoacid sequence of one of following scheme:

Described antibody and human glucagon receptor specific binding.

4. antibody according to claim 3, is characterized in that: in scheme c, the combination of the light variable domains sequence of a and the heavy-chain variable domains sequence of b is selected from one of following: L1H1, L1H1, L2H2, L3H3, L4H4, L5H5 and L6H6.

5. antibody according to claim 3, is characterized in that: described antibody is selected from the one in mouse source antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibodies, antigen binding antibody fragment, single-chain antibody, double-chain antibody, three chain antibodies, four chain antibodies, Fab fragment, F (fa ') x fragment, domain antibodies, IgD antibody, IgE antibody, IgM antibody, IgGl antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody.

6. antibody according to claim 3, is characterized in that: when described antibody and human glucagon receptor in conjunction with time:

C. in animal model, blood sugar is reduced;

D. both (a) and (b);

E. (a), (b) and (c).

7. antibody according to claim 6, is characterized in that: described animal model is ob/ob mouse model.

8. the nucleic acid be separated, is characterized in that: it comprises the polynucleotide sequence of the light variable domains of antibody of coding claim 3, heavy-chain variable domains or light variable domains and heavy-chain variable domains.

9. a recombinant expression vector, is characterized in that: it comprises the nucleic acid of claim 8.

10. a host cell, is characterized in that: it comprises the carrier of claim 9.

11. 1 kinds of methods of producing the antibody of anti-glucagon receptor, is characterized in that: the host cell cultivating claim 10 under being included in the condition allowing to express described antibody.

12. 1 kinds of medicinal compositionss, is characterized in that: it comprises antibody or the humanized antibody of the claim 3 mixed with pharmaceutical acceptable carrier.

13. be used for the treatment of a test kit for type ii diabetes, it is characterized in that: it comprises the medicinal compositions of claim 12.