CN101287760B

CN101287760B - Anti-IL13 human antibodies

Info

Publication number: CN101287760B
Application number: CN2006800374681A
Authority: CN
Inventors: E·M·坎贝尔; S·帕维恩; J·比希勒; G·瓦尔科尔斯
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2005-10-21
Filing date: 2006-10-19
Publication date: 2012-10-17
Anticipated expiration: 2026-10-19
Also published as: TNSN08175A1; CN101287760A; ZA200802541B; GB0521509D0; UA96426C2

Abstract

The present invention relates to human anti-IL-13 binding molecules, particularly antibodies, and to methods for using anti-IL-13 antibody molecules in diagnosis or treatment of IL-13 related disorders, such as asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease and Hodgkin's lymphoma.

Description

People's antibody and the therepic use of anti-IL13

Technical field

The present invention relates to specific binding members, especially the people anti--the IL-13 antibody molecule with particularly in and active those molecules of IL-13.Also relate to the method for in the diagnosis of IL-13 associated conditions or treatment, using the anti-il-13 antibody molecule, said illness is such as asthma, atopic dermatitis, rhinallergosis, fibrosis, inflammatory bowel and Hodgkin lymphoma.

Background of invention

Interleukin (IL)-13 is 114 amino acid whose cytokines, has molecular weight [McKenzie, the A.N. of the unmodified of about 12kDa; Deng people J Immunol, 1993.150 (12): p.5436-44, and Minty; A., wait people Nature, 1993.362 (6417): p.248-50.].IL-13 and IL-4 are the most closely related, on amino acid levels, have 30% sequence similarity with it.People IL-13 gene is positioned at karyomit(e) 5q31 and goes up adjacent with the IL-4 gene.The gene order of the cytokine (comprising GM-CSF and IL-5) in other Th2 lymphocytes source is contained in this zone of karyomit(e) 5q, and their level and IL-4 have been proved to be disease seriousness in the rodent model with asthma patient and allergic inflammation together relevant [Nakamura, Y. wait people Am J Respir Cell Mol Biol; 1996.15 (5): p.680-7, Robinson, D.S. waits people N Engl J Med; 1992.326 (5): p.298-304, Walker, C. waits people Am J Respir Crit Care Med; 1994.150 (4): p.1038-48, Humbert, M. waits people Am J Respir Crit Care Med; 1996,154 (5): p.1497-504, Corrigan; C.J.and A.B.Kay Int Arch Allergy Appl Immunol, 1991.94 (1-4): p.270-1, Bentley; A.M., wait people Am J Respir Cell Mol Biol, 1993.].

Although be accredited as the cytokine in Th2 CD4+ lymphocyte source at first, IL-13 also produces through Th1 CD4+T cell, CD8+T lymphocyte NK cell and acellular colony such as mastocyte, basophilic granulocyte, eosinophilic granulocyte, scavenger cell, monocyte and asm cell.

It is reported that IL-13 passes through its effect of receptor system mediation, said receptor system comprises IL-4 acceptor a chain (IL-4R α), but himself can combine IL-4 debond IL-13 and at least two kinds of other cell surface proteins: IL-13R α ₁With IL-13R α ₂[Murata, T. wait people Int J Hematol, 1999.69 (1): p.13-20, Andrews, A.L. waits people J Biol Chem, 2002.277 (48): p.46073-8.].IL-13R α ₁Can combine IL-13 with low-affinity, raise IL-4R α subsequently and form high-affinity functional receptor [Miloux, the B. that transmits signal; Deng people FEBS Lett; 1997.401 (2-3): p.163-6, Hilton, D.J.; Deng people Proc Natl Acad Sci U S A, 1996.93 (1): p.497-501].The Genbank DB has been listed IL-13R α with NP 001551 and Y10659 respectively ₁Aminoacid sequence and nucleotide sequence.Research in STAT6 (signal transducer and activator of transcription 6) deficient mice has disclosed IL-13 and has transmitted signal [Kuperman with the mode that is similar to IL-4 through utilizing the JAK-STAT6 approach; D., wait people J Exp Med, 1998.187 (6): p.939-48; Nelms; K., wait people Annu Rev Immunol, 1999.17:p.701-38.].IL-13R α ₂With IL-13R α ₁On amino acid levels, have 37% sequence identity and with high-affinity combine IL-13 [Zhang, J.G. wait people J Biol Chem, 1997.272 (14): p.9474-80, Caput, D. waits people J Biol Chem, 1996.271 (28): p.16921-6.].Yet, IL-13R α ₂Have short cytoplasmic tail, it lacks known signal motif.Express IL-13R α ₂Cell in addition in the presence of IL-4R α, do not reply yet IL-13 [Kawakami, K. wait people Blood, 2001.97 (9): p.2673-9].Therefore, infer IL-13R α ₂Still do not regulate the bait acceptor of IL-3 function as regulating IL-13.This obtains the α at IL-13R ₂The support of studying in the deficient mice, phenotype of said mouse and responsiveness consistent [Wood, N. to the increase of IL-13; Deng people J Exp Med, 2003.197 (6): p.703-709, Chiaramonte; M.G., wait people J Exp Med, 2003.197 (6): p.687-701].The Genbank DB is with IL-13R α ₂Aminoacid sequence and nucleotide sequence list with NP000631 and Y08768 respectively.

Summary of the invention

Embodiment of the present invention provide isolating people or humanized antibody or its function fragment, and it has the antigen binding domain special to target protein IL-13, and said antibody or its function fragment combination IL-13.In related embodiment, prevent that through cell surface IL-13 receptors bind inflammatory mediator release from measuring the combination of IL-13 at least.

In another embodiment, the invention provides the isolating antigen binding domain of antibody or its function fragment.In some embodiments, isolating antigen binding domain comprises H-CDR3 district and its conservative variant with the aminoacid sequence that is selected from SEQ ID NO:9-10.As described herein, conservative variant is included in the amino-acid residue in the aminoacid sequence of arbitrary evaluation.In related embodiment, isolating antigen binding domain is H-CDR2 district and its conservative variant with aminoacid sequence of SEQ ID NO:8.In another related embodiment, isolating antigen binding domain is H-CDR1 district and its conservative variant with the aminoacid sequence that is selected from SEQ IDNO:6-7.

In another embodiment, isolating antigen binding domain is L-CDR3 district and its conservative variant with the aminoacid sequence that is selected from SEQ ID NO:20-22.In another related embodiment, isolating antigen binding domain is L-CDR1 district and its conservative variant with the aminoacid sequence that is selected from SEQ ID NO:16-18.In a related embodiment again, isolating antigen binding domain is L-CDR2 district and its conservative variant with aminoacid sequence of SEQID NO:19.

In some embodiments, isolating antigen binding domain is variable light chain district and its conservative variant with the aminoacid sequence that is selected from SEQ ID 16-22.

In another embodiment, isolating antigen binding domain is the heavy chain that has one to three aminoacid sequence being selected from SEQ ID 6-10 and the sequence of at least 60,70,80,90 or 95% sequence identity is arranged in CDR district and the CDR district with SEQ ID NO:6-10.In related embodiment, isolating antigen binding domain is the light chain that has one to three aminoacid sequence being selected from SEQ ID NO:16-22 and the sequence of at least 60,70,80,90 or 95% sequence identity is arranged in CDR district and the CDR district with SEQ ID NO:16-22.

In some embodiments, isolated antibody is IgG.In another embodiment, isolated antibody is IgG1 and IgG4.

In a further embodiment, the invention provides isolating people or humanized antibody or its function fragment, it has the antigen binding domain special to the epi-position of IL-13, and the IL-13 surface receptor on said antibody or the function fragment combination cell.In related embodiment; The invention provides isolated antibody or humanized antibody or its function fragment; It has the antigen binding domain special to the epi-position of target IL-13, and said epi-position contains one or more amino-acid residues of the amino-acid residue 1-112 of target IL-13.In related embodiment, said epi-position is a conformational epitope.

In a further embodiment, said antibody or function fragment are Fab or scFv antibody fragment.In related embodiment, said isolated antibody is IgG.In another related embodiment, said isolated antibody is IgG1 or IgG4.

In a further embodiment, the invention provides pharmaceutical composition, it has at least a any above-mentioned antibody or function fragment or conservative variant and pharmaceutically acceptable carrier or vehicle.

In a further embodiment, the invention provides transgenic animal, it carries the gene of any above-mentioned antibody of coding or its function fragment.

In some embodiments, the invention provides the treatment illness relevant or the method for disease with the cell that has receptor targets with IL-13.This method relates to any aforementioned pharmaceutical compositions of its experimenter of needs being used significant quantity.In related embodiment, illness of being treated or disease are to breathe illness.

In another embodiment, the illness or the disease of being treated are bronchial asthma, and it is the common persistence inflammatory diseases of lung, and characteristic is the serum IgE level of respiratory tract hyperergy (AHR), the excessive generation of mucus, fibrosis and rising.People such as Li; Placard summary (the Abstract for poster submitted at The AmericanThoraics Society Annual Meeting that submits in American Thoracic Society's annual meeting of Seattle in 2003; 2003, the Seattle) effect of report neutrality anti-mouse IL-13 antibody in the chronic mouse model of asthma.

The illness or the disease of being treated in another embodiment, are chronic obstructive pulmonary disease (COPD).People J Clin Invest such as Zheng, 2000.106 (9): p.1081-93 illustrated the mucus that overexpression IL-13 in the mouse lung causes pulmonary emphysema, rising and produced and inflammation, reflected the aspect of people COPD.Shown and compared from patient's the lung sample of the lung disease that does not have report; The mRNA level of IL-13 higher (J.Elias, the world-of-mouth communication in American Thoracic Society's annual meeting in 2002 (Oral communication atAmerican Thoracic Society Annual Meeting 2002)) in autopsy tissue's sample of the experimenter with COPD medical history.In another research, from level [Wardlaw, A.J., Clin Med, 2001.1 (3): p.214-8.] of the rising of the immunohistochemistry proof IL-13 of COPD patient's periphery lung sections.

In another embodiment; The illness or the disease of being treated are selected from other inflammatories or OAD and situation; Like acute lung injury (ALI), acute/grow up respiratory distress syndrome (ARDS), expiratory dyspnea, allergy respiratory inflammation, respiratory tract tip disease, lung cancer, have acute chest complication and pulmonary hypertension among the patient of sickle shaped cell disease; And other drug treatment, especially the increasing the weight of of respiratory tract hyperergy after the pharmacological agent of other suctions.

In another embodiment; The illness or the disease of being treated are the bronchitis of any kind or origin; For example comprise acute bronchitis, arachidic bronchitis, catarrhal bronchitis, croupous bronchitis, chronic bronchitis or phthinoid bronchitis.

In another embodiment; The pneumoconiosis that illness of being treated or disease comprise any kind or origin (inflammatory of lung, common occupational disease; Often be accompanied by respiratory tract obstruction, chronic or acute, once in a while owing to suck dust repeatedly); For example comprise aluminosis, anthracosis, asbestosis, chalicosis, ptilosis, arc-welder's disease, silicosis, tabacosis and byssinosis.

The illness or the disease of being treated in another embodiment, are selected from atopic rhinitis's (pollinosis), allergic dermatitis (eczema) and chronic sinusitis.In human experimenter, measured the level of the rising of IL-13 with atopic rhinitis's (pollinosis), allergic dermatitis (eczema) and chronic sinusitis.For example, find to compare with the contrast experimenter, higher [Humbert, M. wait people JAllergy Clin Immunol to the level of IL-13 in from bronchial biopsy, phlegm and broncho-alveolar lavage (BAL) cell of asthma patient; 1997.99 (5): p.657-65, Kotsimbos, T.C., P.Ernst, and Q.A.Hamid; Proc Assoc Am Physicians, 1996.108 (5): p.368-73, Komai-Koma, M.; F.Y.Liew, and P.C.Wilkinson, J Immunol, 1995.155 (3): p.1110-6; Naseer, T. waits people Am J Respir Crit Care Med, 1997].

In another embodiment, other inflammatory situations that illness of being treated or disease are selected from skin, for example, psoriatic or lupus erythematosus.

In another embodiment, the illness or the disease of being treated are inflammatory bowels, like ulcerative colitis or crohn.People such as Heller (2002) Immunity, 17 (5): 629-38b report has alleviated the colonic inflammation in the mouse model of people's ulcerative colitis through using among the solubility IL-13Ra2 with IL-13.Accordingly, when when comparing, express higher from IL-13 in the rectum biopsy specimen of patients of ulcerative colitis.

In another embodiment, the illness or the disease of being treated are selected from other fibrotic conditions, like systemic sclerosis, pulmonary fibrosis, idiopathic pulmonary fibrosis or fibroid lung.Has the patients serum [Hasegawa of systemic sclerosis; M.; Deng people J Rheumatol, p.328-32] and receive the patient's of other forms of pulmonary fibrosis invasion and attack BAL sample [Hancock, A. 1997.24 (2):; Deng people Am J Respir Cell Mol Biol, 1998] measure the level of the rising of IL-13 in.

The illness or the disease of being treated in another embodiment, are hepatic fibrosis.Through using the special inhibition of solubility IL-13Ra2 or IL-13 gene disruption, do not produce and stoped the fiber in the liver to form that [Fallon, P.G. wait people J Immunol but do not eliminate IL-4 to IL-13; 2000.164 (5): p.2585-91, Chiaramonte, M.G.; Deng people J Clin Invest, 1999.104 (6): p.777-85, Chiaramonte; M.G., wait people Hepatology, 2001.34 (2): p.273-82.].

The illness or the disease of being treated in another embodiment, are Hodgkins.Hodgkin is uncommon in malignant tumour, because in the knurl property usually from the B cell-and Shi (Reed-Sternberg) cell only forms the small portion that can detect piece clinically.The clone in Hodgkin source and former Reed Sternberg cell express usually IL-13 and its acceptor [Skinnider, B.F. wait people Blood, 2001.97 (1): p.250-5].Because IL-13 promotes the propagation in cell survival and the normal B cell, so propose the growth factor that IL-13 can be used as Reed Sternberg cell.People such as Skinnider have illustrated the growth in vitro of the clone that neutralizing antibody to IL-13 can suppress the Hodgkin source, and [Kapp, U. wait people J Exp Med, 1999.189 (12): p.1939-46.].This finds that prompting Reed Sternberg cell maybe be through the survival of IL-13 autocrine and the enhancing of paracrine cell factor ring they self.Consistent with this hypothesis is when comparing with normal control, in some Hodgkin patients serums, to have detected elevated levels [Fiumara, P., F.Cabanillas, and A.Younes, Blood, 2001.98 (9): p.2877-8.] of IL-13.Therefore, the IL-13 suppressor factor can stop PD through suppressing pernicious Reed Sternberg cell propagation.

The illness or the disease of being treated in another embodiment, are tumor recurrence or transfer.The inhibition that has shown IL-13 strengthens the antiviral vaccine in the animal model and in treatment HIV and other infection, is useful [Ahlers, J.D. wait people Proc Natl Acad Sci U S A, 2002].Many human cancer cells express the special antigen of immunogenic cancer.Yet although many tumour spontaneous regressions, immunity system (immunosurveillance) is escaped in many immunity through the suppressor T cell mediation.People Nat Immunol such as Terabe, 2000.1 (6): p.515-20 illustrated IL-13 immunosuppressant effect in mouse model, wherein tumour disappears after initial growth, then recurrence.Avoid tumor recurrence with these mouse of the special inhibition of solubility IL-13Ra2 IL-13 protection.People such as Terabe continue to prove that IL-13 suppresses the differentiation of tumour-specific CD8+ cytotoxic lymphocyte, said cell mediated anti-tumor immune response.

In another embodiment, the illness or the disease of being treated are respiratory viral infectionses, and it has increased the weight of the potential chronic disease, like asthma, chronic bronchitis, COPD, otitis media and sinusitis paranasal sinusitis.The respiratory viral infections of being treated can be relevant with the Secondary cases infectation of bacteria, like otitis media, sinusitis paranasal sinusitis or pneumonia.

In another embodiment, the illness or the disease of being treated are selected from other diseases or illness, especially have the disease or the illness of inflammatory component; For example; The disease in bone and joint comprises rheumatoid arthritis, psoriatic arthritis and other diseases, like atherosclerosis, multiple sclerosis and acute and chronic allograft rejection; For example, the allograft rejection after heart, kidney, liver, lung or the bone marrow transplantation.

In another embodiment; The illness or the disease of being treated are endotoxin shock, glomerulonephritis, brain and myocardial ischemia, Alzheimer, cystic fibrosis, virus infection and deterioration, AIDS (AIDS), multiple sclerosis (MS), helicobacter pylori (Helicobacter pylori) relevant gastritis and the cancer relevant with them, the especially growth of ovarian cancer.

In another embodiment; The illness or the disease of being treated are the symptoms that the philtrum virus infection causes, said virus infection is caused by ERC group virus, other enteroviruses, coronavirus, herpes virus, influenza virus, parainfluenza virus, respiratory syncytial virus or adenovirus.

According to treatment of the present invention can be symptomatic or preventative.

Material of the present invention is suppressing inflammatory diseases; For example the validity in inflammatory respiratory disease can be at the animal model of respiratory inflammation or other inflammatory diseasess; As illustrating in mouse, rat or the rabbit model, like people such as Wada, J.Exp.Med (1994) 180:1135-40; People such as Sekido, Nature (1993) 365:654-57; People such as Modelska, Am.J.Respir.Crit.Care.Med (1999) 160:1450-56; Said with people (1999) Am.J.Respir.Crit.Care Med.160:1443-49 such as Laffon.

In a further embodiment, the invention provides the method for identifying cell with IL-13 acceptor.This method relates to above-mentioned antibody of cell and any or antibody fragment is contacted, and said antibody or antibody fragment also have detectable mark.Said mark is radioactive, fluorescence, mark magnetic, paramagnetic or chemiluminescent.This method can also relate to imaging or separate any above-mentioned cell through mark.

In another embodiment, any above-mentioned people or humanized antibody or antibody fragment are synthetic.

In another embodiment, the invention provides pharmaceutical composition and extra therapeutical agent.

Said extra therapeutical agent can be selected from anti-inflammatory, bronchiectasis, antihistamine or antitussive medicine material; Be particularly useful for treating obstructive or inflammatory respiratory disease; As above-mentioned those; For example, as the toughener of the therapeutic activity of this type of medicine or as alleviating the required dosage of this type of medicine or the means of potential spinoff.Can with therapeutical agent of the present invention and other drug material in the fixed pharmaceutical composition, mix or it can be with said another kind of drug substance separate administration, before, use simultaneously or afterwards.Therefore, the present invention includes the combination like aforesaid material of the present invention and anti-inflammatory, tracheae amplification, antihistamine or antitussive medicine material, said material of the present invention and said drug substance are in identical or different pharmaceutical composition.

Suitable anti-inflammatory drug comprises steroid; Especially glucocorticosteroid; Like budesonide, betamethasone dipropionate, Fluticasone Propionate, ciclesonide or Mometasone Furoate or WO 02/88167; WO02/12266; WO 02/100879, WO 02/00679 (particularly embodiment 3,11,14,17,19,26,34,37,39,51,60,67,72,73,90,99 and 101), the steroid of describing among WO03/35668, WO 03/48181, WO 03/62259, WO 03/64445, WO 03/72592, WO 04/39827 and the WO 04/66920; The on-steroidal glucocorticoid receptor agonist, those as describing among DE 10261874, WO 00/00531, WO 02/10143, WO03/82280, WO 03/82787, WO 03/86294, WO 03/104195, WO03/101932, WO 04/05229, WO 04/18429, WO 04/19935 and the WO 04/26248; The LTB4 antagonist, those as describing among BIIL 284, CP-195543, DPC11870, LTB4 glycollic amide, LY 293111, LY 255283, CGS025019C, CP-195543, ONO-4057, SB 209247, SC-53228 and the US 5451700; The LTD4 antagonist comprises Singulair, pranlukast, Zafirlukast, Accolate, SR2640, Wy-48,252, ICI 198615, MK-571, LY-171883, Ro 24-5913 and L-648051; The PDE4 suppressor factor, as cilomilast (

GlaxoSmithKline), roflumilast (BykGulden), those that describe among V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-P1ough), LAS31025 (Almirall Prodesfarma), PD189659/PD168787 (Parke-Davis), AWD-12-281 (Asta Medica), CDC-801 (Celgene), SelCID (TM) CC-10004 (Celgene), VM554/UM565 (Vernalis), T-440 (Tanabe), KW-4490 (Kyowa Hakko Kogyo) and WO92/19594, WO 93/19749, WO 93/19750, WO 93/19751, WO 98/18796, WO 99/16766, WO 01/13953, WO 03/104204, WO 03/104205, WO03/39544, WO 04/000814, WO 04/000839, WO 04/005258, WO04/018450, WO 04/018451, WO 04/018457, WO 04/018465, WO04/018431, WO 04/018449, WO 04/018450, WO 04/018451, WO04/018457, WO 04/018465, WO 04/019944, WO 04/019945, WO04/045607 and the WO 04/037805; A _2AAgonist; As describing among EP 1052264, EP 1241176, EP 409595A2, WO 94/17090, WO 96/02543, WO96/02553, WO 98/28319, WO 99/24449, WO 99/24450, WO 99/24451, WO 99/38877, WO 99/41267, WO 99/67263, WO 99/67264, WO99/67265, WO 99/67266, WO 00/23457, WO 00/77018, WO00/78774, WO 01/23399, WO 01/27130, WO 01/27131, WO01/60835, WO 01/94368, WO 02/00676, WO 02/22630, WO 02/96462 and the WO 03/086408 those; And A _2BAntagonist, those as describing among the WO 02/42298.

Suitable bronchiectasis medicine comprises anticholinergic or muscarine antagonist; Especially Ipratropium Bromured, Oxitropium Bromide, tiotropium salts and CHF 4226 (Chiesi); And Glycopyrronium Bromide, and describe among EP424021, US 3714357, US 5171744, WO 01/04118, WO 02/00652, WO02/51841, WO 02/53564, WO 03/00840, WO 03/33495, WO 03/53966, WO 03/87094, WO 04/018422 and the WO 04/05285 those; With the beta-2-adrenoceptor agonist; Like salbutamol (salbutamol), orciprenaline sulfate, terbutaline, Salmeterol, Partusisten, procaterol; Particularly the formula I compound of formoterol, carmoterol and its pharmacologically acceptable salt and WO 00/75114 (free or salt or solvate form thereof) is introduced this paper as a reference with its file; The compound of preferred embodiment compound, particularly following formula

Promptly; (5-[(R)-2-(5; 6-diethylammonium-indane-2-base is amino)-1-hydroxyl-ethyl]-8-hydroxyl-1H-quinoline-2-one-) and its pharmacologically acceptable salt; And the formula I compound of WO 04/16601 (free or salt or solvate form thereof), and the compound of EP 1440966, JP 05025045, WO 93/18007, WO99/64035, US 2002/0055651, WO 01/42193, WO 01/83462, WO 02/66422, WO 02/70490, WO 02/76933, WO 03/24439, WO 03/42160, WO 03/42164, WO 03/72539, WO 03/91204, WO 03/99764, WO 04/16578, WO 04/22547, WO 04/32921, WO 04/33412, WO 04/37768, WO 04/37773, WO 04/37807, WO 04/39762, WO 04/39766, WO 04/45618 WO 04/46083, WO04/80964, EP1460064, WO 04/087142, WO 04/089892, EP 01477167, US 2004/0242622, US 2004/0229904, WO 04/108675, WO 04/108676, WO 05/033121, WO 05/040103 and WO 05/044787.

Suitable dual anti-inflammatory and bronchiectasis medicine comprise dual beta-adrenoceptor agonists/muscarine antagonist, like those disclosed among US 2004/0167167, WO 04/74246 and the WO 04/74812.

Suitable antihistamine drug substances comprises cetrizine hcl; Acetaminophen; Clemastine fumarate; Promethazine; LT; Desloratadine; Diphenhydramine and hydrochloric acid Fei Suonading; Activastine; Astemizole; Nitrogen department fourth; Ebastine; Epinastine; Mizolastine and tefenadine and JP 2004107299; Those disclosed among WO 03/099807 and the WO 04/026841.

Can also use the combination of therapeutical agent of the present invention and anticholinergic or muscarine antagonist, steroid, β-2 agonist, PDE4 suppressor factor, dopamine-receptor stimulant, LTD4 antagonist or LTB4 antagonist.Other useful combinations of medicament of the present invention and anti-inflammatory drug are other antagonists with Chemokine Receptors; Like CCR-1, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5; Especially CCR-5 antagonist, like Schering-Plough antagonist SC-351125, SCH-55700 and SCH-D, [[4-[[[6 for Takeda antagonist such as N-; 7-dihydro-2-(4-aminomethyl phenyl)-5H-benzocyclohepta alkene-8-yl] carbonyl] amino] phenyl]-methyl]-tetrahydrochysene-N; N-dimethyl--2H-pyrans-4-ammonium chloride (TAK-770), US 6166037 (especially claim 18 and 19), WO 0066558 (especially claim 8); WO 0066559 (especially claim 9), the CCR-5 antagonist of describing among WO 04/018425 and the WO 04/026873.

Extra therapeutical agent also can be selected from other cytokine binding molecules; Especially the antibody of other cytokines; Especially with the combination of anti--IL4 antibody; Described in PCT/EP2005/00836; Anti--IgE antibody, like

anti-IL31 antibody, anti-IL31R antibody, anti-TSLP antibody, anti-TSLP receptor antibody, anti-endothelium gp antibody, anti-IL1b antibody or another kind of anti-IL13 antibody, described in WO05/007699.

In some embodiments; The invention provides antibody with first aminoacid sequence and second aminoacid sequence; Said first aminoacid sequence is the heavy chain that is selected to three of SEQ ID NO:6-10 and the sequence of at least 60,70,80,90 or 95% sequence identity is arranged in CDR and the CDR district with SEQ ID NO:6-10, and said second aminoacid sequence is the light chain that is selected to three of SEQID NO:16-22 and the sequence of at least 60,70,80,90 or 95% sequence identity is arranged in CDR and CDR district with SEQ ID NO:16-22.

In a further embodiment, the invention provides immune connector, it is by being antibody or its segmental first component and second component preparation with second aminoacid sequence.For example, immune connector is a cytotoxin, and perhaps immune connector is the conjugated protein or antibody that the target that is different from IL-13 is had binding specificity.

In some embodiments, the invention provides bi-specific antibody.

In another embodiment, the invention provides medicine box with antibody or its antibody fragment.In some embodiments, said medicine box also contains pharmaceutically acceptable carrier or vehicle.In other related embodiment, the antibody in the said medicine box exists with unitary dose.In another related embodiment, said medicine box comprises the working instructions that are applied to the experimenter.

Detailed Description Of The Invention

The present invention relates to isolated antibody, people's antibody especially, its specific combination IL-13 and suppress the functional property of IL-13.In some embodiments, antibody of the present invention is from concrete heavy chain and sequence of light chain and/or comprise concrete constitutional features, as comprises the CDR district of concrete aminoacid sequence.The invention provides isolated antibody, prepare the method for this antibody-like, comprise immune connector and the bispecific molecule and the pharmaceutical composition that contains antibody of the present invention, immune connector or bispecific molecule of this antibody-like.The invention still further relates to and use this antibody-like to suppress illness or the disease relevant with the existence of cell receptor target IL-13, for example, in treatment inflammatory or allergic disease, the especially method of inflammatory or OAD.

In order more easily to understand the present invention, at first define some term.In detailed description, provide extra definition.

Only if context points out that on the contrary term " interleukin-13 " or " IL-13 " refer to people IL-13.The invention provides the antibody of anti-people IL-13, people's antibody especially, itself and non-human primates IL-13 comprise macaque and rhesus monkey IL-13 cross reaction.According to the variant of the antibody recognition IL-13 of embodiments more of the present invention, wherein the arginine residues of 130 of amino acid positions is substituted by Stimulina.In other respects with embodiment in, the invention provides to mouse IL-13, particularly the specific binding members of mouse IL-13.

Term " immunne response " for example refers to; The effect of the soluble large molecule (comprising antibody, cytokine and complement) that lymphocyte, antigen presenting cell, phagocytic cell, granulocyte and above-mentioned cell or liver produce; It causes the selectivity infringement, destroys or removes invasive pathogenic agent, the cell or tissue that receives pathogenic infection, cancerous cells, perhaps (under the situation of autoimmunization or pathology inflammation) normal cell or tissue from human body.

" signal transduction pathway " refers to the biological chemistry relation between the multiple signal transducers, and said molecule works signal is delivered to another part of cell from the part of cell.As used herein, phrase " cell surface receptor " for example comprises, molecule or molecular complex, and it can received signal and can be with the plasma membrane transmission of sort signal through cell.The instance of " cell surface receptor " of the present invention is an IL-13 protein molecule bonded IL-13 acceptor.

The term that this paper relates to " antibody " comprises complete antibody and its any Fab (that is, " antigen-binding portion thereof ") or strand.Naturally occurring " antibody " is gp, and it comprises at least two weights (H) chain and two light (L) chains that connect through disulfide linkage.Every heavy chain comprises variable region of heavy chain and (is abbreviated as V among this paper _H) and CH.CH comprises three structural domain: CH1, CH2 and CH3.Every light chain comprises variable region of light chain and (is abbreviated as V among this paper _L) and constant region of light chain.Constant region of light chain comprises a domain C _LCan be with V _HAnd V _LFurther be subdivided into the hypervariable region, be called complementary determining region (CDR), it is studded with the more conservative zone that is called framework region (FR).Each V _HAnd V _LForm by three CDR and four DR, they from the N-terminal to the C-terminal with following series arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The binding domains with AI is contained in the variable region of heavy chain and light chain.The constant region of antibody can mediate combining of Tegeline and the host tissue or the factor, and the said tissue or the factor comprise first component (Clq) of immune various kinds of cell (for example, effector cell) and classical complement system.

As used herein, " antigen-binding portion thereof " of term antibody (or " antigen part ") simply refers to one or more fragments of antibody, and it keeps specific combination antigen (for example, IL-13) ability.The antigen combined function that has shown antibody can be carried out through the fragment of full length antibody.The instance of the binding fragment that comprises in " antigen-binding portion thereof " of term antibody comprises the Fab fragment, by V _L, V _H, C _LUnit price fragment with CH1 structural domain composition; F (ab) ₂Fragment, comprise the segmental divalence fragment of two Fab that connects at hinge area through disulfide linkage; By V _HFd fragment with CH1 structural domain composition; V by the single armed of antibody _LAnd V _HThe Fv fragment that structural domain is formed; By V _HThe dAb fragment that structural domain is formed people such as (, 1989 Nature 341:544-546) Ward; With isolating complementary determining region (CDR).

In addition, although Fv is segmental two structural domain V _LAnd V _HBy independent genes encoding, but they can connect through synthetic linker with recombination method, and said joint makes them become protein chain, wherein a V _LAnd V _HDistrict's pairing forms monovalent molecule and (is called strand Fv (scFv); For example see people such as Bird, 1988 Science 242:423-426; With people such as Huston, 1988 Proc.Natl.Acad.Sci.85:5879-5883).This type of single-chain antibody is intended to comprised by " antigen-binding portion thereof " of term antibody.Use routine techniques well known by persons skilled in the art to obtain these antibody fragments, and screen said segmental effectiveness with the mode identical with complete antibody.

" isolated antibody " used like this paper refers to antibody, and it does not have other antibody (for example, the isolated antibody of specific combination IL-13 has basically no the antigenic antibody that specific combination is different from IL-13) of different antigen-specifiies basically.Yet, the isolated antibody of specific combination IL-13 can with other antigen, have cross reactivity like IL-13 molecule from other materials.In addition, isolated antibody can have basically no other cellular materials and/or chemical.

Term as used herein " monoclonal antibody " or " monoclonal antibody combination " refer to the preparation of single molecular antibody molecule.Monoclonal antibody combination demonstrates single binding specificity and the affinity to defined epitope.

The term " people's antibody " that uses like this paper is intended to comprise the antibody with variable region, wherein framework region and the CDR district sequence of all originating from the people.In addition, if antibody contains constant region, this constant region is also from this type of human sequence so, and for example, ethnic group is a sequence, and perhaps ethnic group is the mutant form of sequence.People's antibody of the present invention can comprise can't help human sequence's amino acids coding residue (for example, through at random external or site-specific mutagenesis or the sudden change that imports through somatic mutation in the body).Yet term as used herein " people's antibody " is not intended to and comprises such antibody, wherein from the CDR sequence of the kind of another mammalian species such as mouse system by grafting to people's frame sequence.

Term " human monoclonal antibodies " refers to demonstrate unijunction and closes specific antibody, and it has the variable region, and wherein framework region and CDR district are all from the human sequence.In one embodiment, produce human monoclonal antibodies through hybridoma, hybridoma comprises from transgenic nonhuman animal, the B cell of transgenic mice for example, and it has and comprises people's heavy chain transgenic and the genetically modified genome of light chain that merges with immortality cell.

Term as used herein " recombinant human antibody " comprises through recombinant means preparation, expression, generation or isolating everyone antibody; As from for the human immunoglobulin gene be transgenic or transfection chromosome animal (for example; Mouse) or from the hybridoma isolated antibody of its preparation; From being transformed and the host cell isolated antibody of expressing human antibody, for example, from the transfectoma isolated antibody; From the combination people antibody library isolated antibody of reorganization, with through relating to any other means preparation, expression, generation or the isolated antibody that all or part of montage of human immunoglobulin gene's sequence is become other dna sequence dnas.This type of recombinant human antibody has the variable region, and wherein framework and CDR district are immunoglobulin sequences from ethnic group.Yet in some embodiments, this type of recombinant human antibody can carry out vitro mutagenesis (perhaps, when the genetically modified animal of end user Ig sequence, body endosome cell mutation), thus and the V of recombinant antibodies _HAnd V _LThe aminoacid sequence in district is such sequence, although its from and to relate to ethnic group be V _HAnd V _LSequence, but can not naturally be present in people's antibody kind in vivo is repertoire.

As used herein, " isotype " refers to the antibody classification (for example, IgM, IgE, IgG such as IgG1 or IgG4) of weight chain constant area gene coding.

Phrase " discern antigenic antibody " and " to the antibody of antigen-specific " in this article with the interchangeable use of term " the antigenic antibody of specific combination ".

As used herein, the antibody of " specific combination people IL-13 " means with 5 * 10 ^-9M or littler K _DAntibody in conjunction with people IL-13.The antibody of " with the antigenic cross-reaction that is different from people IL-13 " means with 5 * 10 ^-9M or littler K _DIn conjunction with this antigenic antibody.The antibody of " not with the specific antigen cross reaction " means with 1.5 * 10 ^-8M or bigger, or 5-10 * 10 ^-8M or 1 * 10 ^-7M or bigger K _DIn conjunction with this antigenic antibody.In some embodiments, not with this antibody-like of said antigenic cross-reaction in standard combines to measure, demonstrate for these proteinic detect basically less than combination.

As used herein, the antibody of " suppressing IL-13 and IL-13 receptors bind " refers to suppress with 5nM or littler KD the antibody of IL-13 and receptors bind.

As used herein, the antibody of " release of inflammation-inhibiting medium " means with less than 10nM, 5nM, 2.5nM, 1.0nM, 0.5nM, or littler IC ₅₀Suppress the antibody that IL-13 inductive eotaxin (eotaxin) discharges from people's lung fibroblast.

As used herein, term " K _Assoc" or " K _a" mean the association rate of antibodies specific-AI, and as used herein, term " K _Dis" or " K _D, " mean the dissociation rate of antibodies specific-AI.As used herein, term " K _D" mean dissociation constant, it is from K _dWith K _aRatio (be K _d/ K _a) obtain and be expressed as volumetric molar concentration (M).Can use the sophisticated method in this area to measure the K of antibody _DValue.Be used to measure antibody K _DMethod be through using surface plasma body resonant vibration, perhaps use bio-sensor system, as

System.

As used herein, " high-affinity " of term IgG antibody refers to that target antigen is had 10 ^-8M or littler, 10 ^-9M or littler or 10 ^-10M or littler K _DAntibody.

As used herein, term " experimenter " comprises anyone or non-human animal.Term " non-human animal " comprises all vertebratess, for example, Mammals and nonmammalian, like non-human primate, sheep, dog, cat, horse, milk cow, chicken, Amphibians, Reptilia, or the like.

Chapters and sections below describe in further detail many aspects of the present invention.

Being used to assess antibody is known in the art to the standard test method of the binding ability of multiple species IL-13, for example comprises ELISA, western blotting and RIA.Suitable assay method details in an embodiment.Also can be through standard test method known in the art, like binding kinetics (for example, binding affinity) through Biacore analysis and evaluation antibody.Being used to assess antibody describes in further detail the assay method of the influence of the functional property of IL-13 in an embodiment.

Therefore; Like " inhibition " these IL-13 functional propertys of measuring through method known in the art and as herein described (for example; Biological chemistry, immunochemistry, cell, physiological or other biological is active or the like) one or more antibody will be understood that to relate to when not having said antibody (for example, perhaps when having uncorrelated specific control antibodies) concrete active statistics and significantly reduce.Suppress the active antibody of IL-13 and realize that this statistics of measured parameter significantly is reduced by at least 10%, at least 50%, 80% or 90%, in some embodiments, antibody of the present invention suppresses the IL-13 functionally active greater than 95%, 98% or 99%.

Monoclonal antibody

Antibody of the present invention is the human monoclonal antibodies like described isolating and structural characterization of embodiment 1-5.The V of said antibody _HAminoacid sequence shows in SEQ ID NO:6-10 respectively.The V of said antibody _LAminoacid sequence shows in SEQ ID NO:16-22 respectively.Other antibody of the present invention comprise and being suddenlyd change, however in the CDR district with above-mentioned sequence in the CDR district described amino acid with at least 60,70,80,90 or 95% identity.

Because each of these antibody can combine IL-13, so, can be with V _HAnd V _LSequence " is mixed and coupling " to produce other anti-il-13 binding molecules of the present invention.(for example, ELISA) testing this type of IL-13 that " mixes and mate " antibody combines can to use the binding assay described in above-mentioned and the embodiment.Work as V _HAnd V _LChain mixed with when coupling, from concrete V _H/ V _LPaired V _HSequence should be with V similar on the structure _HThe sequence displacement.Equally, from concrete V _H/ V _LPaired V _LSequence should be with V similar on the structure _LThe sequence displacement.The V of antibody of the present invention _HAnd V _LSequence is especially obeyed and is mixed and displacement because these antibody use from mutually of the same race be the V of sequence _HAnd V _LSequence, thus and demonstrate structural similarity.

On the other hand, the invention provides antibody, it comprises heavy chain and light chain CDR1, CDR2 and the CDR3 of antibody, or its combination.The V of antibody _HThe aminoacid sequence of CDR1 shows in SEQ ID NO:6-7.The V of antibody _HThe aminoacid sequence of CDR2 shows through SEQ ID NO:8.The V of antibody _HThe aminoacid sequence of CDR3 shows in SEQ ID NO:9-10.The V of antibody _LThe aminoacid sequence of CDR1 shows in SEQ ID NO:16-18.The V of antibody _LThe aminoacid sequence of CDR2 shows in SEQ ID NO:19.The V of antibody _LThe aminoacid sequence of CDR3 shows in SEQ IDNO:20-22.(Kabat, E.A. wait the people to use Kabat system description CDR district; 1991Sequences of Proteins of Immunological Interest; The 5th edition, U.S.Departmentof Health and Human Services, NIH Publication No.91-3242).

Each that consider these antibody can combine IL-13 and antibody binding specificity mainly to provide through CDR1,2 and 3 districts, can be with V _HCDR1,2 and 3 sequence and V _LCDR1,2 and 3 sequences " mix and coupling " (that is, and can mixed and coupling from the CDR of different antibodies, although each antibody must contain V _HCDR1,2 and 3 and V _LCDR1,2 and 3) to produce other anti-il-13 binding molecules of the present invention.(for example, ELISA) testing this type of IL-13 that " mixes and mate " antibody combines can to use the binding assay of describing among preceding text and the embodiment.When mixing and coupling V _HDuring the CDR sequence, from concrete V _HThe CDR1 of sequence, CDR2 and/or CDR3 sequence should be with CDR sequence displacements similar on the structure.Equally, when mixing and coupling V _LDuring the CDR sequence, from concrete V _LThe CDR1 of sequence, CDR2 and/or CDR3 sequence should be with CDR sequence displacements similar on the structure.The technician will it is obvious that easily, for monoclonal antibody of the present invention, through using the one or more V of sequence replacing similar on the structure from the sequence of CDR shown in this paper _HAnd/or V _LThe CDR sequence can produce new V _HAnd V _LSequence.

Isolating monoclonal antibody, perhaps its antigen-binding portion thereof has: the variable region of heavy chain CDR1 that comprises the aminoacid sequence that is selected from SEQ ID NO:6-7; The variable region of heavy chain CDR2 that comprises the aminoacid sequence of SEQ ID NO:8; The variable region of heavy chain CDR3 that comprises the aminoacid sequence that is selected from SEQ ID NO:9-10; The variable region of light chain CDR1 that comprises the aminoacid sequence that is selected from SEQ ID NO:16-18; The variable region of light chain CDR2 that comprises the aminoacid sequence of SEQ ID NO:19; The variable region of light chain CDR3 that comprises the aminoacid sequence that is selected from SEQ IDNO:20-22; This antibody specific combination IL-13 wherein.

In some embodiments, antibody consists of: the variable region of heavy chain CDR1 that comprises SEQ ID NO:6; The variable region of heavy chain CDR2 that comprises SEQ ID NO:8; The variable region of heavy chain CDR3 that comprises SEQ ID NO:9; The variable region of light chain CDR1 that comprises SEQ ID NO:16; The variable region of light chain CDR2 that comprises SEQID NO:19; With the variable region of light chain CDR3 that comprises SEQ ID NO:20.

In another embodiment, antibody consists of: the variable region of heavy chain CDR1 that comprises SEQ ID NO:7; The variable region of heavy chain CDR2 that comprises SEQ ID NO:8; The variable region of heavy chain CDR3 that comprises SEQ ID NO:10; The variable region of light chain CDR1 that comprises SEQ ID NO:17; The variable region of light chain CDR2 that comprises SEQID NO:19; With the variable region of light chain CDR3 that comprises SEQ ID NO:21.

In a further embodiment, antibody consists of: the variable region of heavy chain CDR1 that comprises SEQ ID NO:7; The variable region of heavy chain CDR2 that comprises SEQ ID NO:8; The variable region of heavy chain CDR3 that comprises SEQ ID NO:10; The variable region of light chain CDR1 that comprises SEQ ID NO:18; The variable region of light chain CDR2 that comprises SEQID NO:19; With the variable region of light chain CDR3 that comprises SEQ ID NO:22.

As used herein, people's antibody comprises heavy chain or variable region of light chain, if the variable region of this antibody obtains from the system that uses the racial immunity globulin gene, so said variable region be specific kind be sequence " product " or " from " this specific kind is sequence.This type systematic comprises the human immunoglobulin gene library of perhaps showing on phage with the purpose antigen selection with the transgenic mice of purpose antigen immune carrier immunoglobulin gene." product " of behaviour racial immunity sphaeroprotein sequence perhaps " from " people's antibody of said sequence can identify as follows: the aminoacid sequence that aminoacid sequence and the ethnic group through people's antibody relatively is Tegeline also is selected from sequence and the sequence of people's antibody is an immunoglobulin sequences near the ethnic group of (that is the % identity of maximum)." product " of behaviour racial immunity sphaeroprotein sequence perhaps " from " people's antibody of said sequence can or have a mind to introduce site-directed mutagenesis and be that sequence is compared and contained amino acid difference with kind owing to for example naturally occurring somatic mutation.Yet; Selected people's antibody usually and ethnic group be that the immunoglobulin gene amino acid sequence coded is at least 90% same and contain such amino-acid residue on aminoacid sequence; When with the racial immunity sphaeroprotein aminoacid sequence of other species (for example; The mouse kind is a sequence) when comparing, said amino-acid residue identifies said people's antibody into the people's.In some cases, people's antibody can be on aminoacid sequence and racial immunity globulin gene amino acid sequence coded at least 60%, 70%, 80%, 90% or at least 95% or even at least 96%, 97%, 98% or 99% same.Usually, from concrete ethnic group be people's antibody of sequence will demonstrate with ethnic group be that the immunoglobulin gene amino acid sequence coded is no more than 10 amino acid differences.In some cases, people's antibody can demonstrate with racial immunity globulin gene amino acid sequence coded and be no more than 5, or does not even surpass 4,3,2 or 1 amino acid differences.

Homologous antibody

In a further embodiment; Antibody of the present invention has heavy chain and variable region of light chain; It has the aminoacid sequence with the amino acid sequence homologous of antibody described herein, and wherein said antibody keeps the desirable functional property of anti-il-13 antibody of the present invention.

For example, the invention provides isolating monoclonal antibody, or its antigen-binding portion thereof, it comprises variable region of heavy chain and variable region of light chain, and wherein: variable region of heavy chain comprises and the aminoacid sequence 80% homologous aminoacid sequence that is selected from SEQ ID NO:6-10 at least; Variable region of light chain comprises and the aminoacid sequence 80% homologous aminoacid sequence that is selected from SEQID NO:16-22 at least; Said antibody specific combination IL-13; And this antibody demonstrates at least a following functional property: antibody inhibition IL-13 albumen combines or this antibody inhibition IL-13 receptors bind with the IL-13 acceptor; Prevent or alleviate inflammatory or allergy situation, especially inflammatory or OAD, perhaps this antibody suppresses the IL-13 receptors bind; Prevent or alleviate asthma or this antibody to suppress the IL-13 receptors bind, prevent or alleviate COPD.

In multiple embodiments, one or more of the functional property of discussing above antibody can demonstrate, two or more, or three kinds.This antibody for example can be, people's antibody, humanized antibody or chimeric antibody.

In other embodiments, V _HAnd/or V _LAminoacid sequence can with top sequence that provides 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology.Has respectively V with SEQ ID NO:6-10 and 16-22 _HAnd V _LThe V of Qu Yougao (for example, 80% or higher) homology _HAnd V _LThe antibody in district can obtain as follows: mutagenesis (for example; Site-directed mutagenesis or PCR mediated mutagenesis) nucleic acid molecule of coding SEQ ID NO:6-10 and/or 16-22; Then use functional examination method that this paper discusses to coded through changing the function that antibody test keeps (that is, preceding text provide function).

As used herein, the percent homology between two aminoacid sequences equals two identity per-cents between the sequence.Identity per-cent between two sequences be the total same position number of these two sequences function (promptly; % homology=same position number/total number of positions * 100); Consider the length of breach number, each breach, they are introduced into to carry out the best comparison of two sequences.Can use mathematical algorithm described in following limiting examples to accomplish confirming of identity percentage ratio between comparison and two sequences of sequence.

Can use E.Meyers and W.Miller (Comput.Appl.Biosci.; 4:11-17; 1988) algorithm; Use PAM120 weight residue table, notch length point penalty 12 and breach point penalty 4, confirm two identity percentage ratios between the aminoacid sequence, this algorithm has been incorporated in the ALIGN program (version 2 .0).In addition, can use be incorporated into the GCG software package (can Http:// www.gcg.comObtain) the Needleman and the Wunsch (J.Mol of GAP program; Biol.48:444-453; 1970) algorithm; Use Blossom 62 matrixes or PAM250 matrix, and breach weight 16,14,12,10,8,6 or 4 and the identity percentage ratio confirmed between two aminoacid sequences of length weight 1,2,3,4,5 or 6.

Extra or alternatively, protein sequence of the present invention can also further be used as " search sequence " public database is searched for for example to identify relevant sequence.Can use Altschul, wait the people, the XBLAST program of 1990 J.Mol.Biol.215:403-10 (version 2 .0) is carried out this type of search.Can use the XBLAST program, score=50, word length=3 are carried out the BLAST protein search and are obtained and antibody molecule homologous aminoacid sequence of the present invention.In order to obtain being used for the breach comparison of comparison purpose, can use like people such as Altschul 1997 Nucleic Acids Res.25 (17): the breach BLAST that describes among the 3389-3402.When utilizing BLAST and breach BLAST, can use the default parameters of program (for example XBLAST and NBLAST) separately.See http://www.ncbi.nlm.nih.gov.

Has the conservative antibody of modifying

In some embodiments; Antibody of the present invention has variable region of heavy chain of being made up of CDR1, CDR2 and CDR3 sequence and the variable region of light chain of being made up of CDR1, CDR2 and CDR3 sequence; Wherein one or more specific amino acids sequence or its conservative modifications that have based on antibody described herein of these CDR sequences, and wherein said antibody keeps the required function character of anti-il-13 antibody of the present invention.Therefore, the invention provides isolated antibody or its antigen-binding portion thereof, it consists of; Variable region of heavy chain of forming by CDR1, CDR2 and CDR3 sequence and the variable region of light chain of forming by CDR1, CDR2 and CDR3 sequence, wherein: the variable region of heavy chain of CDR1 is the aminoacid sequence and its conservative sequence of forming of modifying that is selected from the aminoacid sequence of SEQ IDNO:6-7; The variable region of heavy chain of CDR2 is aminoacid sequence and its conservative sequence of forming of modifying of SEQ ID NO:8; The variable region of heavy chain of CDR3 is aminoacid sequence and its conservative sequence of forming of modifying that is selected from the aminoacid sequence of SEQ ID NO:9-10; The variable region of light chain of CDR1 is aminoacid sequence and its conservative sequence of forming of modifying that is selected from the aminoacid sequence of SEQ ID NO:16-18; The variable region of light chain of CDR2 is aminoacid sequence and its conservative sequence of forming of modifying of SEQ ID NO:19; The variable region of light chain of CDR3 is aminoacid sequence and its conservative sequence of forming of modifying that is selected from the aminoacid sequence of SEQ ID NO:20-22; This antibody specific combination IL-13; And this antibody suppresses the IL-13 receptors bind, prevents inflammatory mediator release.

In multiple embodiments, antibody can demonstrate one or more, two or more, three kinds or multiple above the functional property listed.This antibody-like can be for example people's antibody, humanized antibody or chimeric antibody.

As used herein, term " conserved sequence modification " means amino acid modified, and its not remarkably influenced or change contain the combination characteristic of the antibody of this aminoacid sequence.This type of conservative modification comprises amino acid replacement, interpolation and disappearance.Can in antibody of the present invention, introduce modification like site-directed mutagenesis and PCR mediated mutagenesis through standard technique known in the art.

It is wherein with amino-acid residue substituting with the radical amino acid replacement with similar side chain that conserved amino acid substitutes.Defined amino-acid residue family in the art with similar side chain.These families comprise have basic side chain amino acid (for example; Methionin, l-arginine, Histidine), acid side-chain amino acid (for example; Aspartic acid, L-glutamic acid), the uncharged polar side chain amino acid (for example; Glycocoll, l-asparagine, Stimulina, Serine, Threonine, tyrosine, halfcystine, tryptophane), non-polar sidechain amino acid (for example; L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine(Phe), methionine(Met)), β branch side chain amino acid (for example, Threonine, Xie Ansuan, Isoleucine) and aromatic series side chain amino acid (for example, tyrosine, phenylalanine(Phe), tryptophane, Histidine).Thereby the one or more amino-acid residues in the CDR district of antibody of the present invention can be used other radical amino acid replacements from same side chain family, and can use the function that antibody kept of functional examination method test as herein described through changing.

In conjunction with the antibody of the identical epi-position of anti-il-13 antibody of the present invention

In another embodiment, the invention provides the antibody that combines to combine identical epi-position with the of the present invention multiple anti-il-13 antibody that this paper provides.This type of extra antibody can be identified with other antibody cross competitions of the present invention (that is, combining with the remarkable mode competitive inhibition of statistics) in standard I L-13 binding assay based on them.Being tried antibody suppresses antibody of the present invention and shows that with people IL-13 bonded ability this is tried antibody and combines people IL-13 with antibody competition of the present invention; According to non-limiting theory, this antibody can with identical or relevant (for example, approaching on the similar or space on the structure) epi-position on the antibodies people IL-13 that it is competed.In some embodiments, with antibodies people IL-13 of the present invention on the antibody of identical epi-position be human monoclonal antibodies.This type of human monoclonal antibodies can be as be shown in the examples preparation with separate.

The antibody of transforming and modifying

Can also use and have the one or more V shown in this paper _HAnd/or V _LFurther preparation antibody of the present invention is to transform the antibody of modifying as starting substance for the antibody of sequence, and the antibody of this modification can have the form that changes from initial antibody.Through modifying one or two variable region (that is V, _HAnd/or V _L), for example, in one or more CDR district and/or the variable region in one or more framework region can engineered antibody.Extra or alternatively, through modifying the residue in the constant region, for example, come engineered antibody with the effector function that changes antibody.

One type variable region transformation can carrying out is the CDR grafting.Antibody mainly interacts through amino-acid residue and the target antigen that is arranged in six heavy chains and light chain complementary determining region (CDR).For this reason, the sequence more variation outer of the aminoacid sequence in the CDR between each antibody than CDR.Because the CDR sequence is responsible for most antibody-AIs; So possibly pass through the construction of expression vector expressing recombinant antibody, it simulates the character of specific naturally occurring antibody, said expression vector comprises the CDR sequence from specific naturally occurring antibody; Its grafting is to from (for example seeing on the framework region with different antibodies of different nature; Riechmann, people such as L., 1998 Nature 332:323-327; Jones, people such as P., 1986 Nature 321:522-525; Queen, people such as C., 1989 Proc.Natl.Acad.See.U.S.A.86:10029-10033; People's such as the U.S. Patent number 5,225,539 of winter and Queen U.S. Patent number 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Therefore, another embodiment of the present invention relates to isolating monoclonal antibody, or its antigen-binding portion thereof, and it comprises variable region of heavy chain and variable region of light chain, and variable region of heavy chain comprises the CDR1 sequence that has the aminoacid sequence that is selected from SEQ ID NO:6-7 respectively; CDR2 sequence with aminoacid sequence of SEQ ID NO:8; CDR3 sequence with the aminoacid sequence that is selected from SEQ ID NO:9-10, variable region of light chain have the CDR1 sequence of the aminoacid sequence that is selected from SEQ ID NO:16-18 respectively; CDR2 sequence with aminoacid sequence of SEQ ID NO:18; The CDR3 sequence of forming by the aminoacid sequence of the aminoacid sequence that is selected from SEQ IDNO:20-22.Thereby this antibody-like contains the V of monoclonal antibody _HAnd V _LThe CDR sequence, and can contain different frame sequences from these antibody.

This type of frame sequence can obtain from the reference of public DNA DB or announcement, and it comprises that planting is the antibody gene sequence.For example, the kind of people's heavy chain and chain variable region gene is that dna sequence dna can be sequence library (can obtain at internet network address www.mrc-cpe.cam.ac.uk/vbase) in " VBase " ethnic group, and at Kabat; E.A.; Deng the people, 1991 Sequences ofProteins of Immunological Interest, the 5th edition; U.S.Department of Healthand Human Services, NIH Publication No.91-3242; Tomlinson, I.M. waits the people, 1992 J.fol.Biol.227:776-798; And Cox, people such as J.P.L. find among 1994 Eur.JImmunol.24:827-836, and they are all clearly introduced this paper as a reference.

The instance that is used for the framework region of antibody of the present invention is the frame sequence that uses with selected antibody of the present invention, for example, and those similar frame sequences on consensus sequence that monoclonal antibody of the present invention is used and/or the frame sequence structure.Can be with V _HCDR1,2 and 3 sequences, and V _LCDR1,2 and 3 graftings are on framework region; This framework region has the identical sequence of finding in the racial immunity globulin gene of originating with this frame sequence; Perhaps can be on framework region with the grafting of CDR sequence, this framework region and kind are that sequence is compared and had one or more sudden changes.For example, have been found that in some cases, the residue in useful the is sudden change framework region with the antigen binding capacity that keeps or strengthen antibody (see, for example, people's such as Queen U.S. Patent number 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

It is sudden change V that the variable region of another type is modified _HAnd/or V _LThereby the amino-acid residue in CDR1, CDR2 and/or the CDR3 district improves one or more combination character (for example, avidity) of purpose antibody, is called " affinity maturation ".Can carry out site-directed mutagenesis or PCR mediated mutagenesis with importing sudden change, and can combine or the influence of other purpose functional propertys with assay method assessment antagonist in the external or body that provides among as described herein and the embodiment.Can introduce conservative modify (like the preceding text discussion).Sudden change can be amino acid replacement, interpolation or disappearance.In addition, change usually and be no more than one, two, three, four or five residues in the CDR district.

Therefore; In another embodiment; The invention provides isolating anti-il-13 monoclonal antibody; Or its antigen-binding portion thereof, it is made up of variable region of heavy chain, and this variable region of heavy chain has: compare the V that aminoacid sequence with 1,2,3,4 or 5 amino acid replacement, disappearance or interpolation is formed by the aminoacid sequence that is selected from SEQ ID NO:6-7 or with SEQ ID NO:6-7 _HThe CDR1 district; Aminoacid sequence with SEQ ID NO:8 is perhaps compared the V of the aminoacid sequence with 1,2,3,4 or 5 amino acid replacement, disappearance or interpolation with SEQ ID NO:8 _HThe CDR2 district; Have the aminoacid sequence that is selected from SEQ ID NO:9-10 or compare the V of aminoacid sequence with SEQ ID NO:9-10 with 1,2,3,4 or 5 amino acid replacement, disappearance or interpolation _HThe CDR3 district; Have the aminoacid sequence that is selected from SEQ ID NO:16-18 or compare the V of aminoacid sequence with SEQ ID NO:16-18 with 1,2,3,4 or 5 amino acid replacement, disappearance or interpolation _LThe CDR1 district; Aminoacid sequence with SEQID NO:19 is perhaps compared the V of the aminoacid sequence with 1,2,3,4 or 5 amino acid replacement, disappearance or interpolation with SEQ ID NO:19 _LThe CDR2 district; And have the aminoacid sequence that is selected from SEQ ID NO:20-22 or compare the V of aminoacid sequence with SEQ ID NO:20-22 with 1,2,3,4 or 5 amino acid replacement, disappearance or interpolation _LThe CDR3 district.

Antibody through transformation of the present invention comprises such antibody, wherein to V _HAnd/or V _LInterior framework residue is modified for example to improve the character of antibody.Usually, carrying out this type of framework modifies to reduce the immunogenicity of antibody.For example, a kind of method is that the corresponding kind of " reverse mutation " one or more framework residues one-tenth is a sequence.More particularly, it is the different framework residue of sequence that the antibody that has experienced somatic mutation can contain the kind of originating with this antibody.The kind of originating through comparison antibody frame sequence and this antibody is that sequence can be identified this type of residue.For the kind that makes the framework region sequence be returned to them is a configuration, can for example through site-directed mutagenesis or PCR mediated mutagenesis somatic mutation " reverse mutation " become to plant be sequence.This type of " reverse mutation " antibody also is intended to the present invention includes.

The framework of another type is modified and is related in the sudden change framework region, perhaps even the one or more residues in one or more CDR district, removing t cell epitope, thus the potential immunogenicity of reduction antibody.This method is also referred to as " going immunity " and in people's such as Carr U.S. Patent Publication numbers 20030153043, describes in further detail.

Extra or alternatively as in framework or CDR district, modifying; Can transform antibody of the present invention to comprise the modification in the Fc district; Usually change one or more functional propertys of antibody, like serum half life, complement fixation(CF), Fc receptors bind, and/or the antigen dependent cellular cytotoxicity.In addition, antibody of the present invention can change its glycosylation by chemically modified (for example, one or more chemical parts can be connected to antibody) or through modifying, and changes one or more functional propertys of this antibody once more.Each of these embodiments all is discussed in more detail below.Residue in the Fc district is numbered the EU index number of Kabat.

In one embodiment, the hinge area of modifying CH1 makes and changes, and for example, increases or reduce the number of cysteine residues in the hinge area.This method further describes in people's such as Bodmer U.S. Patent number 5,677,425.Cysteine residues number in the hinge area of change CH1 is with assembling or increase that for example makes things convenient for light chain and heavy chain or the stability that reduces antibody.

In another embodiment, the Fc hinge area with antibody suddenlys change to reduce the biology half life of this antibody.More specifically, one or more amino acid mutations are imported the segmental CH2-CH3 structural domain of Fc-hinge interface region, make this antibody combine to have impaired staphylococcal protein A,SPA (SpA) and combine with respect to natural Fc hinge arrangement territory SpA.This method describes in further detail in people's such as Ward U.S. Patent number 6,165,745.

In another embodiment, modified antibodies is to increase its biology half life.Several different methods is possible.For example, can import one or more following sudden changes: T252L, T254S, T256F, like the U.S. Patent number 6,277 of Ward, 375 is said.Alternatively, in order to prolong the biology half life, can in the CH1 of antibody or CL district, change to contain and remedy the receptors bind epi-position from what two rings of the CH2 structural domain in the Fc district of IgG obtained; U.S. Patent number 5,869,046 and 6 like people such as Presta; Describe in 121,022.

In other embodiments, through at least one amino-acid residue is changed the Fc district with the displacement of different amino acid residue with the effector function that changes antibody.But for example, one or more amino acid can be used different amino acid residue displacement, make this antibody have the avidity of change for the effect part keep the antigen binding capacity of parental antibody.The effect part that changes avidity for example can be, the C1 component of Fc acceptor or complement.This method is at people's such as Winter U.S. Patent number 5,624,821 and 5,648, further details in 260.

In another embodiment, be selected from the CDC (CDC) that one or more amino acid of amino-acid residue can combine and/or reduce or eliminate with the C1q that different aminoacids residue displacement make this antibody have change.This method further details in people's such as Idusogie U.S. Patent number 6,194,551.

In another embodiment, change one or more amino-acid residues to change the ability of this antibody complement-fixing.This method is announced among the WO 94/29351 at people's such as Bodmer PCT and is further described.

In a further embodiment, through modifying one or more amino acid modified Fc district to increase this antibody-mediated ADCC (ADCC) and/or to increase the avidity of antibody to Fc γ acceptor.This method further describes in the PCT of Presta publication No. WO 00/42072.In addition, for the human IgG1 to the mapping of the binding site of Fc γ RI, Fc γ RII, Fc γ RIII and FcRn and described bonded variant (seeing Shields, people such as R.L., 2001 J.Biol.Chen.276:6591-6604) with raising.

In a further embodiment, the glycosylation of modified antibodies.For example, can prepare sugar based antibody (that is, lacking glycosylated antibody).Can change glycosylation with for example, increase the avidity of antibody " antigen ".This type of carbohydrate is modified can be through for example changing one or more glycosylation sites completion in the antibody sequence.For example, can carry out one or more amino acid replacements, it causes removing one or more variable regions framework glycosylation site, thereby removes the glycosylation in this site.This type of sugar basedization can increase antibody to antigenic avidity.This method is at people's such as Co U.S. Patent number 5,714,350 and 6,350, describes in further detail in 861.

Extra or alternatively, can prepare and have the glycosylated antibody that changes type, as have low fucosylation antibody or the antibody of the fucosyl residues of reduction with five equilibrium GlcNac structure of increase.Shown that the glycosylation pattern of this type of change increases the ADCC ability of antibody.Through for example this type of carbohydrate modification of expressing antibodies completion in the host cell of glycosylation machine with change.Cell with glycosylation machine of change is described in this article and can be used as host cell, thereby expresses the glycosylated antibody that recombinant antibodies generation of the present invention has change therein.For example, people's such as Hang EP 1,176,195 has described the clone with the ruined FUT8 gene of function, said FUT8 genes encoding fucosyltransferase, and it is low fucosylated to make the antibody of in this clone, expressing demonstrate.The PCT of Presta announces that it is the Lecl3 cell that WO 03/035835 has described the variant Chinese hamster ovary celI; Its ability that Fucose is connected to the carbohydrate of Asn (297)-connection reduces; The low fucosylated of antibody that also causes in this host cell, expressing (also sees Shields; R.L. wait the people, 2002 J.Biol.Chem.277:26733-26740).People's such as Umana PCT announces that WO 99/54342 has described clone; Its fucosyltransferase of being expressed modified glucoprotein by transformation (for example; β (1,4)-N acetyl glucosamine based transferase III (GnTIII) makes the antibody of in this engineered cells system, expressing demonstrate the five equilibrium GlcNac structure of increase; The ADCC active (also seeing people such as Umana, 1999 Nat.Biotech.17:176-180) that it causes antibody to increase.

It is Pegylation that the another kind of this paper antibody of the present invention's imagination is modified.Can be with the antibody Pegylation, for example, with biology (for example, the serum) half life that prolongs this antibody.For Pegylation antibody, usually with this antibody or its fragment and polyoxyethylene glycol (PEG), as the active ester of PEG or aldehyde derivatives condition under react, under the described conditions, one or more PEG groups become and are attached to antibody or antibody fragment.Through carrying out acylation reaction or alkylated reaction, can carry out Pegylation with reactive PEG molecule (or its similar reaction water-soluble polymkeric substance).As used herein, term " polyoxyethylene glycol " is intended to comprise the PEG of any form, and it has been used to other protein of deriving, like list (C1-C10) alkoxyl group-or aryloxy poly glycol or polyoxyethylene glycol-maleimide.In some embodiments, the antibody of treating Pegylation is the antibody of sugar basedization.The method of pegylated protein is known in the art, and can be applied to antibody of the present invention.For example see people's such as people's such as Nishimura EP 0 154 316 and Ishikawa EP 0,401 384.

The method of engineered antibody

Like top discussion, has the V shown in this paper _HAnd V _LThe anti-il-13 antibody of sequence can be used for through modifying V _HAnd/or V _LThe constant region of sequence or its connection and produce new anti-il-13 antibody.Thereby; In another aspect of this invention; The constitutional features of anti-il-13 antibody of the present invention is used to produce the anti-il-13 antibody of structurally associated; It keeps at least a functional property of antibody of the present invention, like one or more functional propertys (for example, the inhibition of receptors bind, medium release) that combine people IL-13 and suppress IL-13.

For example, one or more CDR district of antibody of the present invention or its sudden change can with known framework region and/or other CDR reorganization combination, to produce the anti-il-13 antibody of extra modified recombinant as discussed above.The modification of other types comprises those modifications of describing in the previous section.The parent material that is used for remodeling method is one or more V that this paper provides _HAnd/or V _LSequence, perhaps its one or more CDR district.In order to produce the antibody of transformation, needn't in fact prepare (that is, as protein expression) and have one or more V that this paper provides _HAnd/or V _LThe antibody in sequence or its one or more CDR district.On the contrary, information contained in the sequence is produced " s-generation " sequence from initial sequence as parent material, prepare " s-generation " sequence then and be expressed as protein.

Therefore; In another embodiment; The invention provides the method for preparing anti-il-13 antibody; Said anti-il-13 antibody is made up of variable fragments of heavy chain sequence and variable region of light chain antibody sequence, and said variable fragments of heavy chain sequence has the CDR2 sequence of the CDR1 sequence that is selected from SEQ ID NO:6-7, SEQ IDNO:8 and/or is selected from the CDR3 sequence of SEQ ID NO:9-10; Said variable region of light chain antibody sequence has the CDR2 sequence of the CDR1 sequence that is selected from SEQ ID NO:16-18, SEQ ID NO:19 and/or is selected from the CDR3 sequence of SEQ ID NO:20-22; Change at least one interior amino-acid residue of said variable fragments of heavy chain sequence and/or variable region of light chain antibody sequence to produce the antibody sequence of at least one change; And the antibody sequence that will change is expressed as protein.

Can and express the antibody sequence that changes with the standard molecular biological technique preparation.The antibody of the antibody sequence that changed coding is the antibody that keeps a kind of, the some or all of functional propertys of anti-il-13 antibody as herein described, and its functional property includes, but not limited to specific combination IL-13; With the antibody that demonstrates the functional property below at least a: this antibody suppresses combining of IL-13 protein and IL-13 acceptor; Perhaps this antibody suppresses the IL-13 receptors bind, and prevent or alleviate inflammatory, fibrosis or allergy situation, especially inflammatory or OAD, perhaps this antibody suppresses the IL-13 receptors bind, thereby prevents or alleviate asthma.

The antibody that is changed can demonstrate one or more, two or more, three kinds or multiple above the functional property discussed.

Can use standard test method that can get in this area and/or as herein described, like those (for example, ELISA) functional propertys of the antibody that changes of assessment that provide among the embodiment.

In some embodiments of the method for transforming antibody of the present invention; Can along the anti-il-13 antibody encoding sequence all or part of at random or selectivity import sudden change, and can be to the anti-il-13 antibody screening that obtains active and/or other functional propertys of combination as described herein through modifying.Mutation method is described in this area.For example, the PCT of Short announces that WO 02/092780 has described use saturation mutagenesis, synthetic being linked and packed, perhaps the method for its combination results and screening antibody mutation.Alternatively, people's such as Lazar PCT announces that WO 03/074679 has described the method that the screening method that uses a computer is optimized the physico-chemical property of antibody.

The nucleic acid molecule of code book invention antibody

Another aspect of the present invention relates to the nucleic acid molecule of code book invention antibody.Nucleic acid may reside in intact cell, the cell lysate, perhaps can be partial purification or pure basically form.When through the standard technique purification of nucleic acid to remove other cellular components or other pollutents; For example; When other nucleus or protein; Nucleic acid is " isolating " or " making pure basically ", and said standard technique comprises that alkali/SDS handles, CsCl divides band (binding), column chromatography, agarose gel electrophoresis and other technologies well known in the art.See F.Ausubel, wait the people, write, 1987 Current Protocolsin Molecular Biology, Greene Publishing and Wiley Interscience, NewYork.Nucleic acid of the present invention can be for example DNA or RNA, and can contain or not contain intron sequences.In one embodiment, nucleic acid is the cDNA molecule.Nucleic acid may reside in carrier, like Vector for Phage Display, perhaps in the recombinant plasmid vector.

Can use standard molecular biological technique to obtain nucleic acid of the present invention.For hybridoma (for example; The antibody of the hybridoma of the transgenic mice preparation of carrier's immunoglobulin gene as described further below) expressing, coding can obtain through standard pcr amplification or cDNA clone technology through the heavy chain of the antibody of hybridoma preparation and the cDNA of light chain.Antibody for from the immunoglobulin gene library (for example, the use display technique of bacteriophage) obtains can reclaim nucleic acid encoding said antibody from the multiple phage clone as the library member.

In case obtain the V that encodes _HAnd V _LThe dna fragmentation of section just can further be operated these dna fragmentations through characterizing recombinant DNA technology, for example, and variable region gene is transformed into full length antibody chain gene, Fab fragment gene, perhaps scFv gene.In these operations, coding V _L-or V _HDna fragmentation effectively be connected to another dna molecular, another proteinic fragment of perhaps encoding is like antibody constant region or flexible joint.The term that uses in this context " effectively connection " means two dna fragmentations and connects with functional mode, for example, makes these two dna fragmentation amino acid sequence coded keep meeting frame, perhaps makes this protein under desirable promotor control, express.

Can be with coding V _HThe separated DNA in district is transformed into the total length heavy chain gene, through with V _HCoding DNA effectively is connected to another dna molecular of encoding heavy chain constant region (CH1, CH2 and CH3) and realizes said transformation.The sequence of people's weight chain constant area gene is known in the artly (for example to see; Kabat, E.A. waits the people; 1991 Sequences of Proteins of Immunological Interest; The 5th edition, U.S.Department of Health and Human Services, NIH Publication No.91-3242) and can obtain comprising these regional dna fragmentations through the standard pcr amplification.CH can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.For Fab fragment heavy chain gene, can be with coding V _HDNA effectively be connected to only another dna molecular of heavy chain CH1 constant region of encoding.

Through with V _LCoding DNA effectively is connected to another dna molecular of coding constant region of light chain CL, can be with separated coding V _LThe DNA in district is transformed into full-length light chains gene (and Fab light chain gene).The sequence of people's constant region of light chain gene is known in the artly (for example to see; Kabat, E.A. waits the people; 1991 Sequences of Proteins of Immunological Interest; The 5th edition, U.S.Department of Health and Human Services, NIH Publication No.91-3242) and the dna fragmentation that comprises these districts can obtain through the standard DNA amplification.Constant region of light chain can be κ or λ constant region.

In order to produce the scFv gene, can be with coding V _HAnd V _LDna fragmentation effectively be connected to the coding flexible joint, for example, encoding amino acid sequence (Gly4-Ser) ₃Another fragment, make V _HAnd V _LSequence can be used as successive single chain protein matter and expresses, and connects V through flexible joint _LAnd V _HThe district (see, for example, people such as Bird, 1988 Science 242:423-426; People such as Huston, 1988 Proc.Natl.Acad.Sci.USA 85:5879-5883; People such as McCafferty, 1990 Nature348:552-554).

Produce monoclonal antibody of the present invention

Can produce monoclonal antibody (mAb) through multiple technologies, said technology comprises conventional monoclonal anti body method, for example, and Kohler and Milstein, the standard body hybridoma technique of 1975 Nature 256:495.Can use the multiple technology that is used to produce monoclonal antibody, for example, the virus of bone-marrow-derived lymphocyte or oncogene transform.

The animal system that is used to prepare hybridoma is the mouse system.It is sophisticated method that hybridoma in mouse produces.It is known in the art being used to separate immunization protocol and the technology through the immune splenocyte that are used to merge.Fusion partner (for example, rat bone marrow tumour cell) and fusion steps also are known.

Can based on as the sequence of the mouse monoclonal antibody of above-mentioned preparation prepare chimeric or humanized antibody of the present invention.The DNA of encoding heavy chain and light chain Tegeline can obtain and use standard molecular biological technique to be transformed into containing non-mouse (for example people) immunoglobulin sequences from the purpose murine hybridoma.For example, in order to produce chimeric antibody, can use methods known in the art that the mouse variable region is connected to human constant region (see, for example, people's such as Cabilly U.S. Patent number 4,816,567).In order to produce humanized antibody, can use methods known in the art that mouse CDR district is inserted into people's framework region and (for example see people's such as the U.S. Patent number 5,225,539 of Winter and Queen. U.S. Patent number 5,530,101; 5,585,089; 5,693,762 and 6; 180; 370).

In some embodiments, antibody of the present invention is human monoclonal antibodies.Can use the transgenic or the transchromosomic mice that carry groups of people's immunity system rather than mouse immune system to produce this type of human monoclonal antibodies to IL-13.These transgenics and transchromosomic mice comprise the mouse that is called HuMAb mouse and KM mouse in this article respectively, and they are referred to as " people Ig mouse " in this article.

HuMAb mouse

(Medarex; Inc.) contain human immunoglobulin gene's minigene seat; People's heavy chain (μ and γ) and к light chain immunoglobulin sequences that its coding is not reset; And orthomutation, endogenous μ of its inactivation and к chain gene seat (are for example seen Lonberg; Deng the people, 1994 Nature 368 (6474): 856-859).Therefore; Mouse demonstrates the expression of the reduction of mouse IgM or к, and when replying immunity, people's heavy chain that is imported and the classification conversion of light chain transgenic experience and somatic mutation are to produce high-affinity human IgG к monoclonal antibody (Lonberg; N. wait the people, 1994 preceding text; Lonberg, N., 1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D., 1995 Intern.Rev.Immunol.13:65-93, and Harding, F.andLonberg, N. summarizes among 1995 Ann.N.Y.Acad.Sci.764:536-546).The genomic modification that preparation of HuMAb mouse and purposes and this type of mouse are carried is at Taylor, people such as L., 1992Nucleic Acids Research 20:6287-6295; Chen, people such as J., 1993 InternationalImmunology 5:647-656; People such as Tuaillon, 1993 Proc.Natl.Acad.Sci.USA94:3720-3724; People such as Choi, 1993 Nature Genetics 4:117-123; Chen, people such as J., 1993 EMBO are J.12:821-830; People such as Tuaillon, 1994 J.Immunol.152:2912-2920; Taylor, people such as L., 1994 International Immunology 579-591; And Fishwild, people such as D. further describe among the 1996 Nature Biotechnology 14:845-851, with they all complete introducing this paper of content as a reference.Also see the U.S. Patent number 5,545,806 of Lonberg and Kay; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; With 5,770,429; People's such as Surani U.S. Patent number 5,545,807; PCT publication No. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO 98/24884 and WO 99/45962 with Lonberg and Kay; PCT publication No. WO 01/14424 with people such as Korman.

In another embodiment, use the mouse of carrier's immunoglobulin sequences on transgenic or transfection chromosome, produce people's antibody of the present invention like the mouse of carrier's heavy chain transgenic and people's light chain transfection chromosome.This type of mouse is called " KM mouse " in this article, in people's such as Ishida PCT publication No. WO 02/43478, describes in detail.

In addition, the alternative transgenic animal system of expressing human immunoglobulin gene can obtain in the art and can be used to produce anti-il-13 antibody of the present invention.For example, can use and be called Xenomouse (Abgenix, alternative transgenic system Inc.); This type of mouse is at people's such as for example Kucherlapati U.S. Patent number 5,939,598; 6,075,181; 6,114,598; Describe in 6,150,584 and 6,162,963.

In addition, the alternative trans-chromosome animal system of expressing human immunoglobulin gene can obtain in the art and can be used to produce anti-il-13 antibody of the present invention.For example, can use the carrier's heavy chain transfection chromosome that is called " TC mouse " and the mouse of people's light chain transfection chromosome; This type of mouse is described among the 2000 Proc.Natl.Acad.Sci.USA 97:722-727 people such as Tomizuka.In addition, the milk cow of carrier's heavy chain and light chain transfection chromosome people such as (, 2002 Nature Biotechnology 20:889-894) Kuroiwa described in this area and can be used to produce of the present invention anti--IL-13 antibody.

Also can use the phage display method in screening human immunoglobulin gene library to prepare human monoclonal antibodies of the present invention.This type of phage display method that is used for isolating human antibodies is set up in the art.For example see: people's such as Ladner U.S. Patent number 5,223,409; 5,403,484; With 5,571,698; People's such as Dower U.S. Patent number 5,427,908 and 5,580,717; People's such as McCafferty U.S. Patent number 5,969,108 and 6,172,197; U.S. Patent number 5,885,793 with people such as Griffiths; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

Also can use the SCID mouse of wherein having rebuild people's immunocyte to prepare human monoclonal antibodies of the present invention, thereby when immunity, can produce people's antibody response.This type of mouse is at people's such as for example Wilson U.S. Patent number 5,476,996 and 5,698, describes in 767.

Produce human monoclonal antibodies to IL-13

Purified recombinant people (hr) IL-13 that is conjugated to the auxiliary epi-position (PADRE) of Pan DR T is as antigen.Use the HCo7 strain of the HuMab transgenic mice of expressing human antibody gene to prepare complete human monoclonal antibodies to IL-13.In this mouse species; Can be like people such as Chen, 1993EMBO describes in J.12:811-820 destroys endogenous mouse κ light chain gene with isozygotying and can announce the destruction endogenous mouse heavy chain gene described in the embodiment 1 of WO 01109187 like PCT with isozygotying.This mouse species carries like people such as Fishwild, the human kappa light chain transgenic of describing among the 1996 Nature Biotechnology14:845-851 and like U.S. Patent number 5,545,806; 5,625,825; With 5,545, the HCo7 people's heavy chain transgenic described in 807.

In order to produce the complete human monoclonal antibodies to IL-13 of the present invention; The HuMab mouse is used the mixture immunity from the purified recombinant IL-13 of HEK-EBNA/PADRE conjugate (42ug/ mouse) and Quil A (15ug/ mouse, Accurate Chemical).The general immunization protocol of HuMab mouse is at Lonberg, people such as N., 1994 Nature 368 (6474): 856-859; Fishwild, people such as D., 1996 Nature Biotechnology 14:845-851 and PCT announce among the WO98/24884 and describe.Intravenously between 1-71 days (IV) or subcutaneous (SC) immune transgenic mouse.Strengthen mouse with antigen (no adjuvant) intravenously, put to death and remove spleen after 2 days.Use Nucleospin RNA II separating kit (BD Biosciences/Clontech) from the spleen isolation of RNA.RNA is used for producing the H of random assignment and the phage display library of L chain variable domains with the Fab Vector for Phage Display, like USP 6,794, described in 132.In the solution described in this patent balances each other association schemes, use biotinylated hrIL-13, phage display library is carried out five take turns selection.Preceding four-wheel is selected to use 10 ^-8The hrIL-13 of M, last is taken turns and selects to use 10 ^-9The hrIL-13 of M.For this library, confirm that through the pfu that the pfu counting that has recover at antigen is reclaimed when not having this antigen last SNR is 37, show the antibody that combines hrIL-13 greater than 90% selected phage expression.Then this phage display library subclone is used to express soluble Fab in plasmid vector, like USP 6,794,132 is said.

Produce the preparation of the transfectoma of monoclonal antibody

Can also in the host cell transfectoma, produce antibody of the present invention, for example use the combination of recombinant DNA technology well known in the art and gene transfection method (for example, Morrison, S. (1985) Science 229:1202).

For example; For expressing antibodies, perhaps its antibody fragment can (for example pass through standard molecular biological technique; Pcr amplification or cDNA clone; Use the hybridoma of expressing purpose antibody) can obtain the DNA of encoding part or full-length light chains and heavy chain, and can DNA be inserted in the expression vector, make gene effectively be connected to and transcribe and translate control sequence.In this context, term " effectively connection " means antibody gene and is connected in the carrier, thereby carries the intravital expectation function that their adjusting antibody genes of control sequence performance are transcribed and translated of transcribing and translate.Select expression vector and expression control sequenc with compatible with used expression host cell.Can light chain of antibody gene and heavy chain of antibody gene be inserted into independent carrier, perhaps more typically, can two kinds of genes be inserted in the identical expression vector.In expression vector, insert antibody gene (for example, on antibody gene fragment or carrier, connect complementary restriction site,, be flat terminal the connection so) through standard method if perhaps there is not restriction site.The light chain of antibody described herein and variable region of heavy chain can be used to produce the full length antibody gene of any antibody isotype, through said gene is inserted into encode hope in the expression vector of CH and constant region of light chain of isotype, make V _HCH section and V in the effective connection carrier of section _LCL section in the effective connection carrier of section can be realized said generation.Extra or alternatively, recombinant expression vector can the coded signal peptide, and it makes things convenient for the secretion of antibody chain from host cell.Can make signal peptide meet frame ground with the N-terminal of antibody chain gene in the carrier antibody chain gene clone be connected.Signal peptide can be Tegeline signal peptide or allos signal peptide (that is, from the proteinic signal peptide of NIg).

Except the antibody chain gene, recombinant expression vector of the present invention can also carry the adjusting sequence that control antibody chain gene is expressed in host cell.Term " adjusting sequence " is intended to comprise promotor, enhanser and other expression controlling elementss (for example, polyadenylation signal), its control antibody chain gene transcription or translation.This type of regulate sequence description in for example Goeddel (Gene ExpressionTechnology.Methods in Enzymology 185, Academic Press, San Diego, CA1990).It will be appreciated by one of skill in the art that the design of expression vector, comprise the selection of regulating sequence, can depend on this type of factor, like the selection of host cell to be transformed, desirable protein expression level, or the like.The adjusting sequence that mammalian host cell is expressed comprises the viral element that instructs high-level protein expression in the mammalian cell; Like promotor and/or enhanser; It from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (for example; And polyoma adenovirus major late promoter (AdMLP)).Alternatively, can use non-virus to regulate sequence, like ubiquitin promotor or P globin promotor.In addition; By the regulatory element that the sequence of different sources is formed, like the SRa promoter systems, it contains the terminal repetition sequence (Takebe of length from the sequence of SV40 early promoter and human T-cell leukemia virus's 1 type; Y. wait the people, 1988 Mol.Cell.Biol.8:466-472).

Except antibody chain gene and adjusting sequence, recombinant expression vector of the present invention can carry extra sequence, as regulating sequence (like replication origin) and the selectable marker gene that carrier duplicates in the host cell.Selectable marker gene is convenient to have imported the selection (see, for example, people's such as Axel U.S. Patent number 4,399,216,4,634,665 and 5,179,017) of the host cell of carrier.For example, usually, selectable marker gene is given the resistance to medicine (like G418, Totomycin or methotrexate) to the host cell that imports carrier.Selectable marker gene comprises Tetrahydrofolate dehydrogenase (DHFR) gene (be used for the dhfr-host cell, use methotrexate selection/amplification) and neo gene (being used for G418 selects).

For the expression of light chain and heavy chain, host cell is arrived in the expression vector transfection of encoding heavy chain and light chain through standard technique.The various ways of term " transfection " is intended to comprise the multiple technologies that are usually used in protokaryon or eukaryotic host cell, importing foreign DNA, for example, and electroporation, calcium phosphate precipitation, the transfection of DEAE-VISOSE or the like.Possibly in protokaryon or eukaryotic host cell, express antibody of the present invention in theory.Antibody has been discussed at eukaryotic cell, the especially expression in the mammalian host cell, because this type of eukaryotic cell, especially mammalian cell more possibly assemble and secrete correct fold and immunoreactivity antibody than prokaryotic cell prokaryocyte.The prokaryotic expression of having reported antibody gene is invalid (Boss, M.A. and Wood, C.R., 1985 Immunology Today6:12-13) for the active antibody that produces high yield.

The mammalian host cell that is used to express recombinant antibodies of the present invention comprises that Chinese hamster ovary (CHO) cell (comprises the dhfr-CHO cell; Be described in Urlaub and Chasin; 1980 Proc.Natl.Acad.Sci.USA 77:4216-4220; Use DH FR selective marker,, describe among 1982 Mol.Biol.159:601-621 like R.J.Kaufman and P.A.Sharp), NSO myeloma cell, COS cell and SP2 cell.When the recombinant expression vector of encoding antibody gene is imported mammalian host cell, through cultivate that host cell enough allows time that antibody expresses in host cell or substratum that antibody-secreting is grown to host cell in produce antibody.Can use the standard protein purification process to reclaim antibody from substratum.

The immunity connector

On the other hand, the invention describes the anti-il-13 antibody that is conjugated to the treatment part, or its fragment, said treatment part is like cytotoxin, medicine (for example, immunosuppressor) or radiotoxin.This type of conjugate is known as " immune connector " in this article.Comprise that one or more cytotoxic immune connectors are known as " immunotoxin ".Cytotoxin or cytotoxic agent comprise any material of pair cell harmful (for example, killing).Instance comprises taxon, cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, MTC, VP, teniposide, vincristine(VCR), vinealeucoblastine(VLB), NST-757, Zorubicin, daunorubicin, dihydroxyl anthracin diketone, mitoxantrone, Plicamycin, dactinomycin, 1-boldenone, glucocorticoids, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte and tetracycline and its analogue or homologue.Therapeutical agent for example also comprises; Metabolic antagonist (for example; Methotrexate, Ismipur, 6-Tioguanine, cytosine arabinoside, 5 FU 5 fluorouracil, decarbazine), etching reagent (for example, mustargen, thio-tepa (thioepa), chloraxnbucil, melphalan, carmustine (BSNU) and CCNU (CCNU), endoxan, busulfan, mitobronitol, U-9889, ametycin; With suitable-dichloro diamines platinum (II) (DDP), cis-platinum, anthracene nucleus class (for example; Daunorubicin (daunomycin in the past) and Zorubicin), microbiotic (for example, gengshengmeisu (NSC-3053 in the past), bleomycin, Plicamycin and anthramycin (AMC)); And antimitotic agent (for example, vincristine(VCR) and vinealeucoblastine(VLB)).

Cytotoxic other instances of therapeutic that can be conjugated to antibody of the present invention comprise many card Mi Xing, calicheamicin, maytenin and auristatins and its verivate.The instance of calicheamicin antibody conjugates can obtain (MylotargTm through commercial sources; Wyeth-Ayerst).

Use obtainable joint technology in this area, can cytotoxin be conjugated to antibody of the present invention.Be used for the instance that cytotoxin is conjugated to the joint categories of antibody is included, but not limited to hydrazone class, thioether, ester, disulphide and contains the joint of peptide.For example can select responsively or to protease-sensitive joint to the low pH cutting in the lysosome compartment, said proteolytic enzyme is such as preferential expressed proteins enzyme in tumor tissues, like kathepsins (for example, cathepsin B, C, D).

About the cytotoxin type, be used for therapeutical agent is conjugated to the joint of antibody and the further discussion of method, also see people such as Saito, G., 2003 Adv.Drug Deliv.Rev.55:199-215; Trail, people such as P.A., 2003 Cancer Immunol.Immunother.52:328-337; Payne, G., 2003 Cancer Cell 3:207-212; Allen, T.M., 2002 Nat.Rev.Cancer 2:750-763; Pastan, I. and Kreitman, R.J., 2002 Curr.Opin.Investig.Drugs3:1089-1091; Senter, P.D. and Springer, C.J., 2001 Adv.Drug Deliv.Rev.53:247-264.

Can also antibody of the present invention be conjugated to ri to produce radioactive cytotoxic drugs, be also referred to as the radioimmunity connector.The radioisotopic instance that can be conjugated to the antibody of diagnosis or treatment application includes, but not limited to iodine ¹³¹, indium ¹¹¹, yttrium ⁹⁰And lutetium ¹⁷⁷The method for preparing the radioimmunity connector is sophisticated in this area.The instance of radioimmunoassay connector can obtain through commercial sources, comprises Zevalin ^TM(DEC Pharmaceuticals) and Bexxar ^TMAnd can use similar method (CorixaPharmaceuticals), with Antibody Preparation radioimmunoassay connector of the present invention.

Antibody conjugates of the present invention can be used to modify given biological response, and drug moiety will not be understood that to be confined to typical chemotherapeutic.For example, drug moiety can be to have desirable bioactive protein or polypeptide.This proteinoid can comprise, for example, the enzymatic activity toxin, perhaps its active fragments is like toxalbumin, ricin A, PE or diphtheria toxin; Protein is like tumour necrosis factor or IFN-; Or biological response modifier; Like lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), rHuGM-CSF (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF "), or other growth factors.

The technology that is used for this type of treatment part is conjugated to antibody is well known in the art; For example see people such as Amon, " Monoclonal Antibodies For Immunotargeting Of Drugs InCancer Therapy "; Monoclonal Antibodies And Cancer Therapy; People such as Reisfeld (eds.), and pp.243-56 (Alan R.Liss, Inc.1985); Hellstrom et at., " Antibodies For Drug Delivery ", Controlled Drug Delivery (2nd Ed.), people such as Robinson (eds.), pp.623-53 (Marcel Dekker, Inc.1987); Thorpe; " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review "; Monoclonal Antibodies ' 84:Biological And Clinical Applications, people such as Pinchera (eds.), pp.475-506 (1985); " Analysis; Results, And FutureProspective Of The Therapeutic Use Of Radiolabeled Antibody In CancerTherapy ", Monoclonal Antibodies For Cancer Detection And Therapy; People such as Baldwin (eds.); People such as pp.303-16 (Academic Press 1985) and Thorpe, " The Preparation And Cytotoxic Properties Of Antibody-ToxinConjugates "; Inmunol.Rev., 62:119-58 (1982).

Bispecific molecule

On the other hand, the invention describes bispecific molecule, it comprises of the present invention resisting-IL-13 antibody or its fragment.Antibody of the present invention or its antigen-binding portion thereof can be derived or be connected to another functional molecular; For example; Another peptide or protein (for example, the part of another antibody or acceptor) are to produce bispecific molecule, and it combines at least two different binding sites or target molecule.In fact can antibody of the present invention be derived or is connected to the more than one function molecule and combine the binding sites different more than two and/or the polyspecific molecule of target molecule to produce; This type of polyspecific molecule also is intended to comprised by the term " bispecific molecule " that this paper uses.In order to produce bispecific molecule of the present invention; Can antibody function of the present invention (for example be connected; Through chemical coupling, heredity fusion, non-covalent combination or additive method) to one or more other binding molecules; Like another antibody, antibody fragment, peptide or combination stand-in, thereby produce bispecific molecule.

Therefore, the present invention includes bispecific molecule, it comprises at least to first kind of binding specificity of IL-13 with to second kind of binding specificity of second kind of target epi-position.For example, second kind of target epi-position is the Fc acceptor, for example, and people Fc γ RI (CD64) or people Fc α acceptor (CD89).Therefore, the present invention includes the bispecific molecule that can combine to express Fc γ R, Fc α R or Fc ε R effector cell (for example, monocyte, scavenger cell or polymorphonuclear cell (PMNs)) and combine to express the target cell of IL-13.Cell-targeting effector cell and Fc that these bispecific molecules will be expressed IL-13 cause receptor-mediated effector cell's activity; Like the phagolysis of IL-13 express cell, cytotoxicity (ADCC), the cytokine release of antibody dependent cellular mediation, the perhaps generation of superoxide anion.

In addition, be the present invention of polyspecific for bispecific molecule, molecule can also comprise the third binding specificity except anti--Fc binding specificity and anti-il-13 binding specificity.For example, the third binding specificity can be anti-enhancement factor (EF) part, for example, thereby increases the molecule to the immunne response of target cell in conjunction with the surface protein of participating in cellular cytoxicity activity." anti-enhancement factor part " can be antibody, function antibody fragment or part, and it combines given molecule, for example, antigen or acceptor, thus cause combining the enhancing of determinant to Fc acceptor or the antigenic effect of target cell.

" anti-enhancement factor part " can combine Fc acceptor or target cell antigen.Alternatively, the anti-acceptor portion that strengthens can be incorporated into and first kind and second kind of different entity of binding specificity bonded entity.For example, anti-enhancement factor partly can be incorporated into cytotoxic T cell (for example, through CD2, CD3, CD8, CD28, CD4, CD44, ICAM-1 or other immunocytes, it causes the enhanced immunne response to target cell).

In one embodiment, bispecific molecule of the present invention comprises at least a antibody as binding specificity, or its antibody fragment, comprises Fab, Fab ', F (ab ') ₂, Fv or strand Fv.Antibody can also be light chain or heavy chain homodimer, and perhaps its any least part like Fv or the light chain construct of describing in people's such as Ladner the U.S. Patent number 4,946,778, is clearly introduced this paper as a reference with its content.

In one embodiment, the binding specificity of Fc γ acceptor is provided through monoclonal antibody, its combination is not blocked by immunoglobulin G while (IgG).As used herein, term " IgG acceptor " refers to be positioned at any of No. 18 γ chain genes on the karyomit(e).These genes encodings totally 12 kinds stride film or soluble receptors isotype, they are divided into 3 F γ acceptor classification: Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD 16).In another embodiment, Fc γ acceptor is people's high-affinity Fc γ RI.People Fc γ RI is the 72kDa molecule, and it demonstrates high-affinity (10 to monomer I gG ⁸-10 ⁹M ^-1).

The generation of some anti-Fc γ monoclonal antibody and characterize by people such as Fanger and announce WO88/00052 and at U.S. Patent number 4,954 at PCT is described in 617, and it is instructed complete introducing this paper as a reference.The different site of Fc γ binding site epi-position and acceptor of these antibodies Fc γ RI, Fc γ RII or Fc γ RIII, thus their combination is not blocked by the physiological level of IgG basically.Can be used for special anti-Fc γ RI antibody of the present invention is mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197.The hybridoma that produces mAb 32 can obtain with ATCC preserving number HB9469 from American type culture collection.In other embodiments, the anti-Fc γ receptor antibody monoclonal antibody 22 (H22) that is the humanization form.The H22 production of antibodies be characterized in people such as Graziano, R.F., 1995 J.Immunol 155 (10): 4996-5002 and PCT announce description among the WO 94/10332.The clone that produces 1122 antibody is deposited in American type culture collection with sign HA022CL1 preservation, and has preserving number CRL 11177.

In other embodiments, through combining people IgA acceptor, for example, (antibody of Fc α RI (CD89) provides the binding specificity to the Fc acceptor to Fc-α acceptor, and the combination of said acceptor needn't be passed through human immunoglobulin A (IgA) blocking-up.Term " IgA acceptor " is intended to comprise the gene product that is positioned at No. 19 a genes (Fc α RI) on the karyomit(e).Several kinds of alternative splicings of known this genes encoding 55 to 110kDa stride the film isotype.Fc α RI (CD89) constitutive expression on monocyte/macrophage, eosinophilic granulocyte and neutrophilic granulocyte, but in non-effector cell colony, do not express.Fc α RI has medium avidity (5 * 10 to IgA1 and IgA2 ⁷M ^-1), it increases (Morton, people such as H.C., 1996 Critical Reviews inImmunology 116:423-440) when being exposed to cytokine such as G-CSF or GM-CSF.Described four kinds of Fc α RI-monoclonal antibody specifics that combine the outer Fc α RI of IgA ligand binding domains, they are accredited as A3, A59, A62 and A77 (Monteiro, people such as R.C., 1992 J.Immunol.148:1764).

Fc α RI and Fc γ RI are the initiation acceptors that is used for of the present invention pair of special molecular, because they are mainly at immune effector cell, as expressing on monocyte, PMN, scavenger cell and the dendritic cell; With high level expression (for example, each cell 5,000-100,000); Cellular cytoxicity activity (for example, ADCC, phagolysis) amboceptor; The mediation target is decided the enhanced antigen presentation of their antigen (comprising autoantigen).

Other antibody that can be used for bispecific molecule of the present invention are mouse, chimeric and humanized monoclonal antibody.

Use the known method of this paper, through puting together the component binding specificity, for example, anti-FcR and anti-il-13 binding specificity can prepare bispecific molecule of the present invention.For example, can produce every kind of binding specificity of bispecific molecule separately, then they interconnected.When binding specificity was protein or peptide, multiple coupling or linking agent can be used for covalently bound.The instance of linking agent comprises albumin A, carbodiimide, N-succinimido-S-ethanoyl-thioacetate (SATA), 5; 5 '-dithio two (2-nitrobenzoic acid) (DTNB), adjacent phenylenedimaleimide (oPDM), N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP); With thiosuccimide base 4-(N-maleimide methyl) Trimetylene-1-carboxylicesters (sulfo--SMCC) (see for example people such as Karpovsky, 1984 J.Exp.Med.160:1686; Liu, people such as MA, 1985 Proc.Natl.Acad.Sci.USA 82:8648).Additive method comprises Paulus, 1985 Behring Ins.Mitt.No.78,118-132; People such as Brennan, 1985 Science 229:81-83) and people such as Glennie, those that describe 1987 J.Immunol.139:2367-2375).Puting together agent is SATA and sulfo-SMCC, and they can (Rockford IL) obtains from Pierce Chemical Co..

When binding specificity was antibody, the sulfydryl bonding that they can pass through the terminal hinge area of C-of two heavy chains combined.In specific embodiments, modify hinge area before puting together, to contain the odd number sulfydryl, for example, a sulfydryl.

Alternatively, can in same vehicle, encode two kinds of binding specificities and in identical host cell, expressing and assembling.When bispecific molecule is mAb x mAb, mAb x Fab, Fab x F (ab ') ₂Or during part x Fab fusion rotein, this method is particularly useful.Bispecific molecule of the present invention can be a single chain molecule, and it comprises a single-chain antibody and combines determinant, perhaps comprises two strand bispecific molecules that combine determinant.Bispecific molecule can comprise at least two single chain molecules.The method for preparing bispecific molecule is at for example U.S. Patent number 5,260,203; U.S. Patent number 5,455,030; U.S. Patent number 4,881,175; U.S. Patent number 5,132,405; U.S. Patent number 5,091,513; U.S. Patent number 5,476,786; U.S. Patent number 5,013,653; U.S. Patent number 5,258,498; With U.S. Patent number 5,482, describe in 858.

Bispecific molecule with they particular target combine can be through for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), facs analysis, bioassay method (for example, growth-inhibiting), perhaps the western blotting assay method confirms.Each of these assay methods is usually through using the labelled reagent (for example, antibody) special to the purpose complex body to detect the existence of specific purposes protein-antibody complex body.For example, can use the antibody or the antibody fragment that connect like enzyme to detect FcR antibody complex body, said antibody or antibody fragment identification and specific combination antibody-FcR complex body.Alternatively, can use any detection complex body of multiple other immunoassays (RIA).For example, can and be used for radioimmunoassay (RIA) with radioactive antibody property mark and (for example see Weintraub; B., Principles of Radioimmunoassays, Seventh Training Course onRadioligand Assay Techniques, The Endocrine Society, March, 1986, be introduced into this paper as a reference).Can pass through such as using gamma counter or scintillometer perhaps through radioautograph detection of radioactive isotropic substance.

Pharmaceutical composition

On the other hand, the invention provides compsn, for example, pharmaceutical composition, it contains one of monoclonal antibody of the present invention or its Fab or combination, prepares with pharmaceutically acceptable carrier.This based composition can comprise antibody of the present invention or one of immune connector or bispecific molecule or combination (for example, the combination of two or more different antibodies).For example, pharmaceutical composition of the present invention can comprise the combination that combines different epi-positions on the target antigen or have the antibody (perhaps immune connector or bispecific molecule) of complementary activity.

Also can be with combination treatment, promptly use pharmaceutical composition of the present invention with other medicaments are combined.For example, combination treatment can comprise the of the present invention anti--IL-13 antibody that makes up with at least a other anti-inflammatory agenies.The instance that can be used for the therapeutical agent of combination treatment is described about the chapters and sections of antibody purposes of the present invention below in more detail.

As used herein, " pharmaceutically acceptable carrier " comprise any of physical compatibility and all solvents, dispersion medium, dressing, antibacterium and anti-mycotic agent, etc. blend the absorption delay agent, or the like.Carrier should be suitable for intravenously, intramuscular, subcutaneous, parenteral, backbone or epidermis and use (for example, through injection or inculcate).Depend on route of administration, active compound, i.e. antibody, immune connector, or bispecific molecule can avoid the effect of acid and other natural condition that can this compound of inactivation with the material dressing with the protection compound.

Medical compounds of the present invention can comprise one or more pharmacologically acceptable salts.The salt that " pharmacologically acceptable salt " refers to keep the desirable biological activity of parent compound and do not give any undesirable toxicological effect (for example see, Berge, S.M. waits the people, 1977 J.Pharm.Sci.66:1-19).The instance of this type of salt comprises acid salt and base addition salt.Acid salt comprises the salt from nontoxic mineral acid; The salt of example hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, Hydrogen bromide, hydroiodic acid HI, phosphorous acid; Or the like; And from the sour salt of non-toxic organic, said non-toxic organic acid is like aliphatics one or dicarboxylicacid, the substituted paraffinic acid of phenyl, hydroxyl alkane acid, aromatic acid, aliphatics and aromatic sulfonic acid, or the like.Base addition salt comprises those salt from earth alkali metal such as sodium, potassium, magnesium, calcium or the like; And from the salt of non-toxic organic amine; Said non-toxic organic amine is like N; N '-diphenyl-methyl quadrol, N-NMG, chloroprocaine, choline, diethylolamine, quadrol, PROCAINE HCL, PHARMA GRADE, or the like.

Pharmaceutical composition of the present invention can also comprise pharmaceutically acceptable inhibitor.The instance of pharmaceutically acceptable inhibitor comprises: water soluble antioxidant, like xitix, cysteine hydrochloride, sodium pyrosulfate, sodium metabisulfite, S-WAT or the like; The soluble inhibitor of oil, like ascorbyl palmitate, butylated hydroxyanisol (BHA), Yoshinox BHT (BHT), Yelkin TTS, Tenox PG, alpha-tocopherol, or the like; And metal chelator, like Hydrocerol A, YD 30 (EDTA), sorbyl alcohol, tartrate, phosphoric acid, or the like.

The instance that can be used for suitable water-based and the non-aqueous carrier of pharmaceutical composition of the present invention comprises water, ethanol, polyvalent alcohol (like glycerine, Ucar 35, polyoxyethylene glycol, or the like) and its suitable mixture; Vegetables oil; Like sweet oil and injectable organic ester, like OE.For example,,, pass through the particle size that keeps required and pass through the use tensio-active agent, can keep suitable flowability for the situation of dispersion-s like Yelkin TTS through using coating material.

These compsns can also contain adjuvant, like sanitas, wetting agent, emulsifying agent and dispersion agent.Mikrobe exists, and prevent can be through as above disinfectant program with through comprising that multiple antibacterium and anti-mycotic agent guarantee that said antibacterium and anti-mycotic agent are for example p-Hydroxybenzoate, chlorobutanol, phenol, Sorbic Acid or the like.Also hope in compsn, to comprise isotonic agent, like sugar, sodium-chlor or the like.In addition, through comprising the reagent that postpones absorption, can cause that like aluminum monostearate and gelatin the prolongation of injectable drug form absorbs.

Pharmaceutically acceptable carrier comprises aseptic aqueous solution or dispersion-s and sterile powder, and it is used for preparing aseptic parenteral solution or dispersion-s temporarily.This type of medium and the reagent purposes in pharmaceutically active substances is known in the art.Except with inconsistent conventional media of active compound or reagent, expect that it is used for pharmaceutical composition of the present invention.Can also the complementarity active compound be mixed in the compsn.

Therapeutic compsn must be usually produce and preservation condition under be aseptic with stable.Can compsn be mixed with solution, microemulsion, liposome or be suitable for other ordered structures of high drug level.Carrier can be solvent or dispersion medium, for example contains water, ethanol, polyvalent alcohol (for example, glycerine, Ucar 35 and liquid macrogol, or the like) and its suitable mixture.Can keep adequate liquidity, for example,,, pass through the particle size that keeps required and pass through to use tensio-active agent for the situation of dispersion-s like Yelkin TTS through using dressing.In many cases, can in compsn, comprise isotonic agent, for example, sugar, polyvalent alcohol, like N.F,USP MANNITOL, sorbyl alcohol, perhaps sodium-chlor.The prolongation of Injectable composition absorbs and can realize like Monostearate and gelatin through in compsn, comprising the material that postpones absorption.

Through active compound is admixed with one of above-named composition or combination in suitable solvent with required amount, if desired, follow aseptic microfiltration and prepare aseptic injectable solution.Usually, prepare dispersion-s through active compound is mixed sterile carrier, said carrier contains basic dispersion medium and from other required compositions of above-named those compositions.For the situation of the sterile powder that is used to prepare aseptic injectable solution, the preparation method is vacuum-drying and lyophilize (freeze-drying), and it produces activeconstituents and adds the powder from any extra required composition of sterile filtration solution before it.

Can will depend on the experimenter who is treated, concrete method of application with the amount of the activeconstituents that produces single formulation with carrier substance combination and become.Can will normally produce the amount of the compsn of result of treatment with carrier substance combination with the amount of the activeconstituents that produces single formulation.Usually, in per-cent, this amount will for about 0.01% to about 99% activeconstituents, about 0.1% to about 70%, or about 1% make up to about 30% activeconstituents and pharmaceutically acceptable carrier.

Regulate dosage and reply (for example, treatment is replied) so that best purpose to be provided.For example, can use single bolus infusion, can use several broken doses in time, perhaps can reduce or increase dosage pro rata according to the urgency of treatment situation.Particularly advantageous is unified to use easily with dosage with dosage unit form preparation parenteral composition.Dosage unit form as used herein refers to suitable dispersive unit physically as the single dose of waiting to treat the experimenter; Each unit contains the activeconstituents of predetermined amount, and said amount is through calculating to combine to produce desirable result of treatment with required pharmaceutical carrier.The specification of dosage unit form of the present invention by as make decision and directly depend on: preparation is used for treating the inherent limitation of this active compound of individuality susceptibility in the specific characteristic of active compound and concrete result of treatment to be achieved and this area.

Use for antibody, dosage is about 0.0001 to 100mg/kg, and more generally 0.01 to 5mg/kg host's body weight.For example, dosage can scope for 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight or 10mg/kg body weight or 1-10mg/kg in.The exemplary treatment scheme need use weekly once, whenever biweekly, per three weeks once, every around once, every month once, per March once or per 3 to June once.Of the present invention anti--dosage of IL-13 antibody can comprise through intravenously or subcutaneous administration 1mg/kg body weight or 3mg/kg body weight, with one of following dosage regimen administration of antibodies: for example, every 6 dosage of applied once all around, per then March once; Per 3 weeks once; The 3mg/kg body weight is once followed per 3 all 1mg/kg body weight.

In certain methods, use two or more monoclonal antibodies simultaneously with different binding specificities, the dosage of every kind of using in this case antibody falls in the pointed scope.Usually in administration of antibodies on a plurality of opportunity.Interval between the single dose can be for example weekly, every month, per March or every year.Also can be irregular at interval, indicate through the blood level that is directed against the antibody of target antigen in the measuring patient.In certain methods, regulate dosage and serve as about 1-1000 μ g/ml and serve as about 25-300 μ g/ml in certain methods to realize plasma antibody concentration.

Alternatively, can be used as the extended release preparation administration of antibodies, in this situation, need frequent lower using.Dosage and frequency depend on antibody in the patient half life and become.Usually, people's antibody demonstrates the longest half life, secondly is humanized antibody, chimeric antibody, and the non-human antibody.Dosage and frequency of administration can be preventative or curative and become along with treatment.In prophylactic application, use low relatively dosage with relative not frequent interval in during long-time.Some patients continue to receive treatment throughout one's life.In treatment is used, need use relative high dosage with relatively short interval sometimes and slow down or stops perhaps demonstrating partially or completely alleviating of disease symptoms up to the patient up to the progress of disease.Afterwards, can use prevention scheme to the patient.

The actual dose level of activeconstituents can change in the pharmaceutical composition of the present invention, as long as the amount of activeconstituents realizes that for concrete patient, compsn and method of application desirable treatment replys, and to this patient's nontoxicity.Selected dosage level will depend on multiple pharmacokinetics factor; The activity that comprises of the present invention concrete compsn or its ester, salt or the acid amides of use, route of administration, time of application; The discharge rate of the particular compound of using; The treatment time length, known other factors in other drug, compound and/or the material that uses with used concrete combination of components, the patient's age of being treated, sex, body weight, situation, general health and former medical history and the medical field.

The present invention is anti--and " the treatment effective dose " of IL-13 antibody can cause reduction, the frequency of no disease symptoms phase and the increase of time length of disease symptoms seriousness, perhaps because disease torments the disease damage that causes or the prevention of deformity.

Can use one or more methods known in the art, use compsn of the present invention through one or more route of administration.To understand like the technician, route of administration and/or mode will depend on desirable result and become.The route of administration of antibody of the present invention comprises intravenously, intramuscular, intracutaneous, intraperitoneal, subcutaneous, other parenteral administration approach of backbone, for example, and through injection or perfusion.The phrase " parenteral administration " that uses like this paper refers to be different from the method for application through intestines and topical application; Usually through injection; And include but not limited to, in the intravenously, intramuscular, intra-arterial, sheath, in the capsule, interior, intracardiac, the intradermal of socket of the eye, intraperitoneal, under tracheae, subcutaneous, epidermis, under the intraarticular, tunicle, in the subarachnoid space, backbone, epidural and intrastemal injection and pour into.

Alternatively, antibody of the present invention can be used through the outer approach of parenteral, like local, epidermis or mucosal administration approach, for example, in the nose, oral cavity, vagina, rectum, hypogloeeis or part.

The preparing carriers active compound that can avoid snap-out release with the protection active compound like controlled release preparation, comprises implant, through skin patch and microencapsulation delivery system.Can use biodegradable biocompatible polymkeric substance, like ethylene vinyl acetate, polyanhydride, Sodium bromoacetate homopolymer, SRU, collagen, poe and POLYACTIC ACID.The many methods that prepare this type of preparation are applied for a patent or well known to a person skilled in the art.For example see Sustained and Controlled Release Drug DeliverySystems, J.R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Can use medical apparatus administering therapeutic compsn known in the art.For example, in one embodiment, can use therapeutic compsn of the present invention with the hypodermic injection unit of needle-less, like U.S. Patent number 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596, the device shown in 556.The instance that can be used for known implant of the present invention or assembly comprises: U.S. Patent number 4,487,603, and it has explained the implantable microinfusion pump that is used for distributing with controllable rate medicine; U.S. Patent number 4,486,194, it has explained the therapeutic system that is used for through the dermal administration medicine; U.S. Patent number 4,447,233, it has explained the medicinal liquid pump that is used for accurate infusion rates delivering drugs; U.S. Patent number 4,447,224, it has been explained and has been used for the implantable transfusion device of variable flow that continuous medicine is sent; U.S. Patent number 4,439,196, it has explained the osmotic drug delivery system with a plurality of chambers; With U.S. Patent number 4,475,196, it has explained the osmotic drug delivery system.These patents are introduced this paper as a reference.Many other these type of implants, delivery system and assembly are well known by persons skilled in the art.

In some embodiments, can prepare human monoclonal antibodies of the present invention to guarantee appropriate allocation in vivo.For example, hemato encephalic barrier (BBB) is got rid of many high-hydrophilic compounds.Pass BBB (if hope) in order to ensure treatment compound of the present invention, can be with them with for example liposome formulation.About producing the method for liposome, for example see USP 4,522,811; 5,374,548; With 5,399,331.Liposome can comprise one or more parts, and its being selected property is transported in specific cells or the organ, thus the enhancing directed drug delivery (see, for example, V.V.Ranade, 1989 J.Cline Pharmacol.29:685).Exemplary orientation partly comprises folic acid or vitamin H (see, for example, people's such as Low USP 5,416,016); Seminose glycoside (people such as Umezawa, 1988 Biochem.Biophys.Res.Commun.153:1038); Antibody (people such as P.G.Bloeman, 1995 FEBS Lett.357:140; People such as M.Owais, 1995 Antimicrob.Agents Chernother.39:180); Surfactant A protein receptor (people such as Briscoe, 1995 Am.J.Physiol.1233:134); P120 (people such as Schreier, 1994 J.Biol.Chem.269:9090); Also see K.Keinanen; M.L.Laukkanen, 1994 FEBSLett.346:123; J.J.Killion; I.J.Fidler, 1994 Immnunomethods4:273.

Purposes of the present invention and method

Antibody of the present invention (with immune connector and bispecific molecule) has external and in-vivo diagnostic and treatment effectiveness.For example, can these molecules be applied to for example external or intravital culturing cell, perhaps be applied to experimenter's (for example, in body) with treatment, prevention or diagnosis various disease conditions.Term as used herein " experimenter " is intended to comprise people and non-human animal.The non-human animal comprises all vertebratess, and for example, Mammals and nonmammalian are like non-human primates, sheep, dog, cat, milk cow, horse, chicken, Amphibians and reptiles.Said method is particularly suited for treating has the human patients of expressing relevant illness with unusual IL-13.When using IL-13 antibody and another promoting agent together, can be with two kinds with arbitrary order or use simultaneously.

In one embodiment, antibody of the present invention (with immune connector and bispecific molecule) can be used to detect the level of IL-13, perhaps contains the cell levels of IL-13.This can be for example, through sample (like vitro samples) and control sample and anti--IL-3 antibody are contacted allowing to form between antibody and the IL-13 under the condition of complex body.Any complex body that forms between detection antibody and the IL-13 also compares in sample and contrast.For example, can use compsn of the present invention to carry out standard detecting method well known in the art, like ELISA and flow cytometry assay.

Therefore; On the one hand; The present invention also provides in the test sample IL-13 (for example; People IL-13 antigen) existence, or measure the method for the amount of IL-13, it comprises the antigen-binding portion thereof of sample, control sample and antibody of the present invention or its specific combination IL-13 is contacted under the condition that allows formation complex body between said antibody or its part and the IL-13.Detect the formation of complex body then, wherein sample is compared difference that complex body forms and is illustrated in this sample and has IL-13 with control sample.

Also comprise test kit in the scope of the present invention, it is made up of compsn of the present invention (for example, antibody, people's antibody, immune connector and bispecific molecule) and working instructions.Test kit can also contain at least a additional agents, perhaps one or more extra antibody of the present invention (for example, have the antibody of complementary activity, its with first kind of antibodies target antigen on different epi-positions).Test kit generally includes label, points out the desired use of this test kit inclusion.Term tag is included in and provides on the test kit or test kit provides or any written or recording materials of subsidiary this test kit.

Described the present invention fully, further illustrated the present invention through following embodiment and claim, said embodiment is illustrative and is not intended to further qualification.Those skilled in the art will recognize that perhaps only to use normal experiment to confirm many equivalents of concrete steps described herein.This type of equivalent is in the scope of the present invention and claim.With all reference that the application quotes in full, comprise that the content of patented claim of patent and the announcement of publication is all introduced this paper as a reference.

Embodiment

Embodiment 1: produce people IL-13 specific antibody from the spleen library of immunity

RNA from spleen is used to be created in like USP 6,794, the phage display library of the H of random assignment and L chain variable domains in the Fab Vector for Phage Display described in 132.The solution of in like this patent, describing balances each other in the association schemes, uses biotinylated hrIL-13 that phage display library is carried out five and takes turns selection.Preceding four-wheel is selected to use 10 ^-8The hrIL-13 of M, last is taken turns and selects to use 10 ^-9The hrIL-13 of M.Final SNR through calculating this library that pfu that the pfu that has a recover at antigen reclaims when not having this antigen confirms is 37, shows the antibody that combines hrIL-13 greater than 90% selected phage expression.Then this phage display library subclone is used to express solubility Fab in plasmid vector, like USP 6,794, described in 132.The library of subclone comprises the plasmid vector in the intestinal bacteria, each plasmid-encoded mono-clonal Fab fragment.With subclone library plating and select the representative single clone bacterium colony be used to inoculate 96 orifice plates.After the overnight growth, plate culture is used for setting up clone's frozen cell storehouse and also being used for inoculation at 96 orifice plates duplicating 96 orifice plates, it is by the abduction delivering monoclonal antibody.Second day, these 96 orifice plate cultures are carried out stain remover extract and the antibody of purifying with the recovery milligram quantities.The antibody of processing purified is to remove intracellular toxin and last filtration sterilization.The biotinylation rhIL-13 that use encapsulates on the avidin flat board carries out the ELISA assay method and contains the function positive antibody to identify which hole.Decide the sandwich assay method of antibody constant region with target and measure the AC in the different holes.Dull and stereotyped and the determination data assessment biological activity with 96 holes of containing antibody.Then the purpose cloning and sequencing in the frozen cell storehouse, 96 hole is expressed the clone of unique antibody with evaluation.Subsequently, inoculate shake-flask culture thing and overnight growth on a small scale with these unique clones' frozen cell storehouse.Use overnight culture inoculation large scale culturing bottle and abduction delivering antibody.Second day, with the antibody of machinery homogenate of culturing bottle inoculum and purifying generation milligram quantities.The Fab of processing purified is used to remove extracellular toxin and carries out last filtration sterilization.Through ELISA, use the biotinylated rhIL-13 that on the avidin flat board, encapsulates to illustrate the functionally active of these antibody.Through the absorbance measurement AC under the 280nm.Use external combination and the activity of assessing the Fab of purifying based on the assay method of cell.

Embodiment 2: the quantitative analysis of binding affinity: confirm anti-people IL-13Fab material standed for

Use optical biosensor BIAcore 2000 to carry out the surface plasmon resonance measurement amount, the interaction of its quantitative anti-il-13 Fab and some hrIL-13.IL-13 can measure through following the tracks of the accumulation of part on acceptor with the specific combination that is fixed on the IL-13Fab separately on the BIAcore chip.Microcosmic combines (k _On) and the speed (k that dissociates _Off) can be directly quality accumulation rate from the chip obtain and express with reacton (RU).Through secondary Anti-Human L κ antibody (JacksonImmunochemicals) anti-il-13 Fab is fixed on the chip surface.Use " amine coupling reagent kit " (' Amine coupling kit ') (BIAcore, Cat.No.BR-1000-50) as manufacturer's scheme recommend with this capture antibody covalent attachment.Inject the hrIL-13 of 250 μ l different concns and write down the kinetics trace with 20 μ l/ minutes flow velocity.Through two acid elution steps, use 100mMHCl and inject 10 μ l regeneration chip surface with 20 μ l/ minutes flow velocity.This processing is owing to reversible acid sex change causes dissociating of Fab IL-13 complex body.When for subsequently operation once more during injection of antibodies, do not observe and combine active remarkable loss.Use BIAcore software, application 1: 1 Langmuir combination model assessment kinetics trace.

The affine data that shown the people IL-13 that sums up in the table 1 here.

Table 1

Fab	KD [pM] people IL-13
		01471/G6	100±2
03161/H2	197±12
		01951/G12	480±68
01771/E10	343±54

Embodiment 3: change into the IgG form

Use QIAprep minipreps (Qiagen Inc.) from 3ml culture antibody purification dna sequencing template.Use Applied Biosystems 3100 Avant Genetic Analyzer template to be checked order according to manufacturer's specification sheets.Selected clone's heavy chain and κ chain variable region increase respectively through PCR from sequencing template, through the agarose gel electrophoresis purifying, from gel excision and purifying.V will encode _HAnd V _LPlasmid clone to human kappa light chain and human IgG ₁In the expression cassette of heavy chain.With two kinds of carrier transfection Sp2/0 parental cell lines, a kind of carrier is the light chain carrier, a kind of chain carrier of attaching most importance to.Select and the amplification cells transfected with G418 and methotrexate respectively, cause occurring resistance, expanded cells storehouse, it produces the antibody that 5mg/L tires to 30mg/L.Use the dilution clone then, cause separating 127 survival clones from 6 96 orifice plates.Distribute the shaking table formal testing yield-power of clone newly to occur with feed supplement then.Also in 90 day time limit, carried out stability test with the stable integration of confirmation expression vector and the sane expression of product.The RNA trace demonstrates single full-length RNA, has equal band strength, shows that heavy chain and light chain have similar expression level.

Embodiment 4: anti--IL-13 full length antibody is based on the vitro characterization in the assay method of cell

IL-13 is effective inductor that the eotaxin discharges from people's lung fibroblast.Discharge in the assessment of end user's lung fibroblast in the assay method antibody the bioactive ability with IL-13 IL-13 inductive eotaxin.In brief, with cell (every hole 2 * 10 ⁴Individual cell, volume 100 μ l) be seeded in every hole of 96 hole tissue culturing plates.With the II-13 irritation cell of giving 80% maximum eotaxin's release concentration, said concentration uses the typical curve of 0-100ng/mlIL-13 to confirm in advance for the cell of each batch.With the antibody common application of different concns in cell.Allow cell at 37 ℃, 5%CO ₂Following incubation 24 hours, results substratum and-20 ℃ of preservations up to needs.Measure the eotaxin's concentration in the substratum through specific ELISA (R&D system), wherein the sensitivity of assay method is 15-1000pg/ml.

Thereby as the EC of above-mentioned analysis anti-il-13 Fab ₅₀And in table 2, show.

Table 2

Antibody	EC ₅₀[nM] people IL-13
		01471/G6	1.23±0.4
03161/H2	0.95±0.2
		01951/G12	0.33±0.1
01771/E10	1.71±0.5

Embodiment 5: the sequential analysis of anti-il-13 antibody

Measure the heavy chain and the variable region of light chain (V of all antibody _HAnd V _L) nucleotide sequence.List in the aminoacid sequence of complementary determining region (CDR) table 3 and the table 4 here.The CDR that defines according to Kabat (people such as E.Kabat, 1991, Sequences of Proteinsof Immunological Interest in table 3a and 4a, have been listed; The 5th edition, Public Health Service, National Instituteof Health; Bethesda, MD).

Table 3

Antibody

HCDR1

SEQ?ID No. HCDR1

?HCDR2

SEQ?ID No. HCDR2

?HCDR3

SEQ?ID No. HCDR3

01471/G6

GFTFSNYG

1

?IWYDGSN

3

?VKGSGDIP

4

03161/H2

GFTFSNYG

1

?IWYDGSN

3

?VKGSGDIP

4

01951/G12

GFTFSSYG

2

?IWYDGSN

3

?ARLWFGDLD

5

01771/E10

GFTFSSYG

2

?IWYDGSN

3

?ARLWFGDLD

5

Table 3a

Antibody

HCDR1

SEQ?ID No. HCDR1

?HCDR2

SEQ?ID No. HCDR2

HCDR3

SEQ?ID No. HCDR3

01471/G6

NYGMH

6

?IIWYDGSNKYYADSVKG

8

GSGDIPFDY

9

03161/H2

NYGMH

6

?IIWYDGSNKYYADSVKG

8

GSGDIPFDY

9

01951/G12

SYGMH

7

?IIWYDGSNKYYADSVKG

8

LWFGDLDAFDI

10

01771/E10

SYGMH

7

?IIWYDGSNKYYADSVKG

8

LWFGDLDAFDI

10

Table 4

Antibody

LCDR1

SEQ ID?No. LCDR1

LCDR2

SEQ?ID No. LCDR2

LCDR3

SEQ ID?No. LCDR3

01471/G6

QSVSSY

11

DA

12

HQRSHWPPI

13

03161/H2

QSVSSY

11

DA

12

HQRSHWPPI

13

01951/G12

QSVSSY

11

DA

12

QQRSSWPPV

14

01771/E10

QSVSSY

11

DA

12

HQRSSWPPI

15

Table 4a

Antibody

LCDR1

SEQ?ID?No. LCDR1

?LCDR2

SEQ?ID?No. LCDR2

LCDR3

SEQ?ID No. LCDR3

01471/G6

RASQSVSSYLA

16

?DASNRAT

19

HQRSHWPPIFT

20

03161/H2

RASQSVSSYLA

16

?DASNRAT

19

HQRSHWPPIFT

20

01951/G12

RAGQSVSSYLV

17

?DASNRAT

19

QQRSSWPPVYT

21

01771/E10

RASQSVSSYLA

18

?DASNRAT

19

HQRSSWPPIFT

22

Show the antibody sequence of previous table below, comprised framework region.Also show complete IgG1 light chain of antibody and CH below, mixed the variable region (runic) of antibody 01951/G12 as an example.

The 01471/G6 antibody sequence

(i) HC variable region

The HC variable amino acid sequence of 01471/G6 shows in SEQ ID NO:23 and nucleotide sequence coded by among the SEQ ID NO:24

(ii) LC variable region

The LC variable amino acid sequence of 01471/G6 shows in SEQ ID NO:25 and nucleotide sequence coded by shown in the SEQID NO:26

03161/H2 antibody

(i) HC variable region

The HC variable amino acid sequence of 03161/H2 shows in SEQ ID NO:27 and nucleotide sequence coded by shown in the SEQ ID No.28

(ii) LC variable region

The LC variable amino acid sequence of 03161/H2 shows in SEQ ID NO:29 and by nucleotide sequence coded shown in the SEQ ID NO:30

The 01951/G12 antibody sequence

(i) HC variable region

The HC variable amino acid sequence of 01951/G12 shows in SEQ ID NO:31 and nucleotide sequence coded by shown in the SEQ ID NO:32

(ii) LC variable region

The LC variable amino acid sequence of 01951/G12 shows in SEQ ID NO:33 and nucleotide sequence coded by shown in the SEQ ID NO:34

The 01771/E10 antibody sequence

(i) HC variable region

The HC variable amino acid sequence of 01771/E10 shows in SEQ ID NO:35 and nucleotide sequence coded by shown in the SEQ ID NO:36

(ii) LC variable region

The LC variable amino acid sequence of 01771/E10 shows in SEQ ID NO:37 and nucleotide sequence coded by shown in the SEQ ID NO:38

Complete antibody IgG1 sequence of light chain, the variable region (runic) of mixing antibody A ntibody 01951/G12

The LC aminoacid sequence shows in SEQ ID NO:39 and nucleotide sequence coded by SEQ ID NO:40

Mix the complete antibody IgG1 sequence of heavy chain of the variable region (runic) of antibody 01951/G12

The HC aminoacid sequence shows in SEQ ID NO:41 and nucleotide sequence coded by SEQ ID NO:42

Claims

1. isolating people or humanization IL13 antibody, its antigen binding domain comprise the L-CDR3 shown in L-CDR2 shown in the L-CDR1 shown in the H-CDR3 shown in the H-CDR2 shown in the H-CDR1 shown in the SEQ ID NO:7, the SEQ ID NO:8, the SEQ ID NO:10, the SEQ ID NO:17, the SEQ ID NO:19 and the SEQ ID NO:21.

2. according to the antibody of claim 1, it has according to the HC variable region of SEQ ID NO:31 with according to the LC variable region of SEQ ID NO:33.

3. according to each antibody of claim 1-2, it is IgG1 or IgG4.

4. pharmaceutical composition, it comprises each antibody or its function fragment and its pharmaceutically acceptable carrier or vehicle according to claim 1-3.

5. according to the purposes of the pharmaceutical composition of claim 4, be used for the production for treating illness relevant or the medicine of disease with the existence of cell receptor target IL-13.

6. according to the purposes of claim 5, wherein said illness or disease are asthma.