CN103154032A - Antibodies to IL-1beta and IL-18, for treatment of disease - Google Patents

Abstract

The present invention relates to compositions and methods for treatment of disease. More particularly, the present invention relates to anti-IL-1beta and anti-IL-18 antibodies, including anti-IL-1beta and anti-IL-18 bispecific antibodies, and methods of treating disease using such antibodies.

Description

The antibody for IL-1 β and IL-18 for disease treatment

Related application

The rights and interests of the right of priority of the U.S. Provisional Application sequence number 61/373,760 of this non-provisional application requirement submission on August 13rd, 2010, be incorporated herein by reference with its integral body.

Invention field

The present invention relates generally to anti-il-i-beta and anti-IL-18 antibody, comprise anti-il-i-beta and anti-IL-18 bi-specific antibody and monoclonal antibody, and use the method for this antibody-like for disease treatment.

Background of invention

The interleukin 1 of cytokine (IL-1) and IL-18 family are correlated with by the mechanism that originates from, receptor structure and the signal transduction pathway that uses.These cytokines synthesize precursor molecule and before discharging from cell or cut by Caspase-1 in process.NALP-3 inflammation corpusculum (inflammasome) is most important (Cassel etc., 2009 in producing active Caspase-1; Ferrero-Miliani etc., 2007).IL-1 family is containing two kinds of agonists: IL-1 α and IL-1 β, a kind of specific inhibitor: IL-1 receptor antagonist (IL-1Ra) and two kinds of acceptors: the Type II IL-1R of activated type I L-1R and non-activity (Arend etc., 2008) biologically.IL-1RI and IL-33R all utilize the identical auxiliary protein (IL-1RAcP) of doing mutually.The disease aspect be equilibrated in the multiple organ of prevention between IL-1 and IL-1Ra is important, and has involved the excessive generation of IL-1 in the various human disease.IL-18 family also comprises specific inhibitor IL-18BP matter (IL-18BP), its in moving phase in conjunction with IL-18.IL-18 acceptor and IL-1 receptor complex are similar, comprise single ligand binding chain and the different auxiliary protein of doing mutually.IL-18 provides important connection between congenital and adaptive immune response.

May relate to the activation of inflammation corpusculum and IL-1 β/IL-18 processing and secretion in progression of disease.The correlative study of genome range has shown the effect of inflammation corpusculum in inflammatory bowel (IBD).It is reported that the patient who has polymorphism in inflammation corpusculum-compound N ALP-3 suffers from risk rising (Ferrero-Miliani etc., 2007 of regional ileitis; Villani etc., 2009).In addition, control Caspase-1 activation and the IL-1 β/autophagy component Atg16l1 of IL-18 processing and polymorphism (Baldassano etc., 2007 relevant to regional ileitis in IRGM have been reported; Cadwell etc., 2008; Kuballa etc., 2008; Saitoh etc., 2008).Independent studies has been reported IL-1 β and IL-18 serum level (Ludwiczek etc., 2005 that raise in the patient who suffers from IBD; Ludwiczek etc., 2004; Monteleone etc., 1999).Research in the people is further supported by preclinical study.It is reported that blocking-up IL-18 or IL-1 β cause improve (Ten Hove etc., 2001) of in the preclinical models of described disease clinical score.

In addition, be reported in the IL-1 β (Kowluru and Odenbach, 2004) that there is elevated levels in the patient's who suffers from diabetic retinopathy eye.

Summary of the invention

The invention provides anti-il-i-beta and anti-IL-18 antibody, comprise for example anti-il-i-beta and anti-IL-18 bi-specific antibody, and use the method for this antibody-like for disease treatment.In some embodiments, described anti-il-i-beta and anti-IL-18 antibody are monoclonal antibody, and simultaneously or to the patient, use for disease treatment continuously.In other embodiments, described anti-il-i-beta and anti-IL-18 are bi-specific antibody and use for disease treatment to the patient.In some embodiments, the disease that described disease is the mediation of inflammation corpusculum, the disease that for example the inflammation corpusculum is activated.The disease example comprises Immunological diseases and autoimmune disease, and comprises inflammatory bowel (IBD), age-related macular degeneration (AMD) and diabetes B (T2D).

In some embodiments, anti-il-i-beta of the present invention and anti-IL-18 antibody blocking or in and the activity of IL-1 β and/or IL-18, and/or in conjunction with IL-1 β and/or IL-18.In some embodiments, the blocking-up of described bi-specific antibody or in and the activity of IL-1 β and/or IL-18, and/or in conjunction with IL-1 β and/or IL-18.

On the one hand, provide in the patient method for the treatment of disease, described method comprises to described patient uses significant quantity:

A.IL-1 β/IL-18 bi-specific antibody; Or

B. in conjunction with the antibody of IL-1 β and IL-18; Or

C. in conjunction with the antibody of IL-1 β with in conjunction with the antibody of IL-18;

Wherein described antibody or a plurality of antibody of a, b or c part can neutralize or block the activity of IL-1 β and IL-18 in cell or tissue.

In some embodiments, the antibody/antibody used in described method is humanized.In some embodiments, described antibody is bifunctional antibody.

In some embodiments, described method is used the combination treatment that comprises anti-il-i-beta antibody and anti-IL-18 antibody.In one embodiment, at least one antibody is monoclonal.In some embodiments, each antibody is all monoclonal.In some embodiments, simultaneously or (c) antibody of part is provided continuously.In some embodiments, used described antibody in one hour.

In some embodiments, disease to be treated is immunological disease or autoimmune disease or inflammation or self inflammatory disease.In some embodiments, the disease that disease is the mediation of inflammation corpusculum.In some embodiments, disease is IL-1 ss related diseases or IL-18 relative disease or IL-1 β/IL-18 β disease.

In some embodiments, disease is age-related macular degeneration (AMD).In some embodiments, disease is diabetes B (T2D).In some embodiments, disease is inflammatory bowel (IBD).In some embodiments, disease is regional ileitis (CD).In some embodiments, disease is ulcerative colitis (UC).In some embodiments, disease is atherosclerosis.In some embodiments, disease is the heart metabolic trouble.In some embodiments, disease is fibrous stenosis regional ileitis (fibrostenosing Crohn ' s disease).

In some embodiments, the patient who accepts present method treatment also non-confrontational TNF therapy replys.

In some embodiments, in the patient, the method for the treatment of disease comprises to described patient and uses the monoclonal antibody in conjunction with IL-1 β of significant quantity and in conjunction with the monoclonal antibody of IL-18.

On the other hand, the method that neutralizes or block IL-1 β and/or IL-18 activity in cell or tissue is provided, described method comprises makes described cell or tissue contact with the monoclonal antibody in conjunction with IL-18 with the monoclonal antibody in conjunction with IL-1 β, and neutralizes thus or block described activity.In some embodiments, while or administration of antibodies continuously.In some embodiments, cell simultaneously or with the described monoclonal antibody in conjunction with IL-1 β, with the described monoclonal antibody in conjunction with IL-18, contact continuously.

The antibody of neutralization or blocking-up IL-1 β and IL-18 activity is provided on the other hand.In some embodiments, antibody is bi-specific antibody.In some embodiments, antibody is humanized.In some embodiments, antibodies IL-1 β and IL-18.

The accompanying drawing summary

Fig. 1 has described for IL-1 β with for the part of IL-18 and the example of acceptor.For example, signal can be initial by the linking of two receptor chains through IL-1 β or IL-18.Think that interior Toll-interleukin-1 receptor (Rexeptor) class (TIR) structural domain of born of the same parents causes the activation of transcription factor NF-kB and AP1; describedly transcribe the generation that tired sub-NF-kB and AP1 have increased cytokine conversely, finally cause protective immunity, self inflammatory condition or chronic inflammatory diseases.

Fig. 2 has described the example of the hypothetical modal of IL-1 β/IL-18 participation inflammatory bowel.The stimulation of the lamina propria scavenger cell caused by microorganism in intestines causes the autocatalysis activation of Caspase-1, and it is processed conversely and secretes IL-1 β and IL-18.IL-1 β and IL-18 work to the panimmunity cell and induce the tired son of proinflammatory cell in scavenger cell, make the T cell to Th1 and Th17 pathogenicity bo T polarization and destroy the epithelial cell barrier, make the more pathogenic agent can stimulating expression of macrophage.

Fig. 3 has described the example how genetics points out the effect of inflammation corpusculum activation in regional ileitis.Polymorphism in autophagy related gene ATG16L1 and IRGM and the tired NOD2 of inflammation corpusculum regulation and control base and NALP3 causes Caspase-1 activation of rising and the secretion of IL-1 β and IL-18.

The data that Fig. 4 (A) raises in the biopsy of colon from CrohnShi and UC patient for the expression that shows IL-1 β and IL-18mRNA.The relative intensity of numerical value based on hybridization signal on Agilent gene platform.(B) data for showing that IL-1 β and IL-18 raise in the serum from regional ileitis and UC patient.

Fig. 5 is the data that are presented at IL-1 β and IL-18 differential expression in the colon of inflammation.Immunohistochemistry from the slices across of the patient's who suffers from UC biopsy of colon.Use antibody for people IL-1 β and IL-18 to section statining.Although IL-1 β mainly is found in the scavenger cell that is arranged in wall inflammation position, IL-18 mainly is found in the dendritic cell that are arranged in lymphoid follicle.In both cases, only in areas of inflammation, observe dyeing.

Fig. 6 shows to come in the colon of the mouse of having accepted 3.5%DSS in comfortable 5 days in random drinking-water, the data of the secretion rising of IL-1 β and IL-18.

Fig. 7 is in the colon of demonstration from the mouse of the CD4+CD45RBhi T cell of having accepted the transfer of adopting property, the data of the IL-1 β of rising and the secretion of IL-18.

In the colon that Fig. 8 is the IL-10KO mouse that shows the piroxicam processing of use by oneself, the data of the IL-1 β of rising and the secretion of IL-18.

Fig. 9 demonstrates the data of the significantly reduced seriousness of colitis that DSS induces for showing IL-1R1 and ASC KO mouse.The colon scoring is from the mouse that lacks IL-1R1, IL-18Ra and ASC.

Figure 10 shows that IL-1R1 lacks the remarkable reduction of IL-1 β, IL-18, IL-17 and TNF-α in the colitis that causes DSS to induce.

Figure 11 lacks the significantly reduced data of level of IL-1 β and IL-12p40 in the colitis that causes DSS to induce for showing IL-18R.

Figure 12 lacks the significantly reduced data of level of IL-1 β, IL-18, IL-12p40 and IL-17 in the colitis that causes DSS to induce for showing ASC.

Figure 13 is the general introduction available from the Isolated colon culture of different mouse IBD models, the exemplary cells factor is replied.

The data of Figure 14 for showing that IL-1 β expresses in the vitreum of AMD patient subgroups.Vitreum is collected the patient that self diagnosis is moist AMD, geotype atrophy (geographic atrophy, GA) or is collected from the patient who suffers from macular pucker or macular hole.Measure cytokine levels with ELISA.

The data that Figure 15 expresses for being presented at the IL-1 β that raises in the eye continued after exposure (light exposure) and Caspase-1.(A) small mouse is exposed to constant light (1800Lux) 10 days, takes out subsequently eye; (B) in, from the retina separating mRNA, and measure IL-1 β mRNA level by PCR in real time; (C) in, in lysis buffer, full eye is homogenized, and by cell extract separation, trace use the antibody for muroid Caspase-1 to be dyeed on sds gel.

Figure 16 is the data that are presented at the expression of IL-1 β former (pro-IL-1 β) and Caspase-1 in the eye that IL-1 β infects.Adeno associated virus (AAV) subretinal injection of ripe muroid IL-1 β will be expressed.After three weeks, mouse is exposed to high light (5000Lux) (ILE; Heavy exposure) lower 6 hours.Process eye after 1 day for the IL-1 β that describes as Figure 15 and the Western engram analysis of Caspase-1.

Figure 17 is presented at IL1 β in little rathole to cross the data of expressing the rear inflammation raise and neovascularity generation.(A), in, the albefaction mouse is accepted the empty AAV virus of subretinal injection or expresses the virus of IL-1 β.After three weeks, scan their eye with FITC injection of solution mouse and by FA.Sword fingers shows the have choroidal neovascularization zone of (CNV).(B), in, eye is extracted, fixes and processes for paraffin embedding and section.Use the antibody for CD45 section statining to be infiltrated to immunocyte (seeing illustration) to develop.There is no inflammation in the mouse with empty AAV carrier subretinal injection.

Figure 18 has the former AAV eye of IL-1 β to demonstrate the data of the inflammation that does not rely on Caspase-1 activity for showing to infect.Use as described in Figure 17 AAV-IL-1 β former subretinal injection Caspase-1 wild-type or ko mouse.After three weeks, as described in extraction eye processing.Inflammation does not rely on Caspase-1 activity.

Figure 19 significantly reduces for showing AAV-IL1 β and AAV-IL-18 the data that dark adatpation ERG replys.With the mouse with the empty carrier injection, compare, the Electro Retino Grams (ERGs) of the mouse of processing with AAV-IL-1 β and AAV-IL-18 demonstrates remarkable reduction in " a " and " b " ripple is replied.

Figure 20 is IL-1 β and the biological general introduction of IL-18 in AMD, described IL-1 β and IL-18 for example, reach the anti-il-i-beta of the clinical reagent that is used as people's research and the neutralizing antibody of anti-IL-18 for the preclinical study in non-human animal (, mouse) for exploitation.

Figure 21 is for showing the data of the method example of using the technology screening anti-il-i-beta neutralizing antibody based on ELISA.

Figure 22 measures for the ELISA of the neutralization activity of the anti-mouse IL-1 of demonstration hamster β hybridoma hypotype.

Figure 23 is the table of the IC50 value of the anti-mouse anti-il-i-beta of explanation hamster antibody blocking activity.

Figure 24 is for showing for measuring the data of people and muroid IL-1 β/IL-18 and active clone.

Figure 25 is the data of the dose response of NF-kB receptor active in the NIH3T3 clone that has shown (A) and process at the muroid IL-1 β by rising concentration; (B) contain in IL-1 β and the data of the blocking-up activity of the hybridoma supernatant liquor of Abs.

The general introduction that Figure 26 is the exemplary antibodies that produces by phage technology.Screened at variable region of heavy chain (V _h) or heavy chain and variable region of light chain (V _hv _l) in there is multifarious multiple phage display library.Synthetic library (YSGX) and peptide library have also been screened, described synthetic library is to produce the simplification genetic code sublibrary (Fellouse etc. of randomization CDR with the codon that is rich in tyrosine, Serine and glycine, J Mol Biol, 373,924-940), described peptide library is the antibody library that is designed for possibility binding specificity peptide sequence.

The data that Figure 27 is the locking activity of multiple phage antibody in the neutralization mensuration be presented at based on ELISA.

Figure 28 is for describing the cartoon of exemplary event sequence, and described exemplary event sequence causes pancreatic beta cell to be withered away and disturbs the potential level of anti-il-i-beta/IL-18 neutralizing antibody.

The sketch plan that Figure 29 is the experimental program of use in embodiment 4.

Figure 30 is illustrated in (A) histology colon appraisal result that anti-il-i-beta in piroxicam IL-10KO mouse IBD model and/or anti-IL18 process and (B) figure of vision colon appraisal result.The result that has also shown the TNF-α-block.Anti-il-i-beta and anti-IL18 combination therapy and TNF blocking-up equivalence.

Detailed Description Of The Invention

The invention provides anti-il-i-beta and anti-IL-18 antibody, comprise for example anti-il-i-beta and anti-IL-18 bi-specific antibody, and use the method for this antibody-like for disease treatment.In some embodiments, anti-il-i-beta and anti-IL-18 antibody are monoclonal antibody, and simultaneously or to the patient, use for disease treatment continuously.In other embodiments, anti-il-i-beta and anti-IL-18 are bi-specific antibody and use for disease treatment to the patient.The disease example comprises Immunological diseases and autoimmune disease, and comprises inflammatory bowel (IBD), age-related macular degeneration (AMD) and diabetes B (T2D).

In some embodiments, anti-il-i-beta of the present invention and anti-IL-18 antibody blocking or in and the activity of IL-1 β and/or IL-18, and/or in conjunction with IL-1 β and/or IL-18.

All references cited herein comprise that patent, application and scientific literature all are incorporated herein by reference with its integral body.

General technology

Technology and the method for description or reference are generally fully understood by those skilled in the art and often use by ordinary method herein, for example, at Sambrook etc., the Molecular Cloning:A Laboratory Manual third edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M.Ausubel, wait editor, (2003)); Series Methods in Enzymology (Academic Press, Inc.): PCR2:A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor edit (1995)), Harlow and Lane, editor (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I.Freshney, editor (1987)); Oligonucleotide Synthesis (M.J.Gait, editor, 1984); Methods in Molecular Biology, Humana Press; Cell Biology:A Laboratory Notebook (J.E.Cellis, editor, 1998) Academic Press; Animal Cell Culture (R.I.Freshney), editor, 1987); Introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell, editor, 1993-8) J.wiley and Sons; Handbook of Experimental Immunology (D.M.Weir and C.C.Blackwell, editor); Gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos, editor, 1987); PCR:The Polymerase Chain Reaction, (Mullis etc., editor, 1994); Current Protocols in Immunology (editor such as J.E.Coligan, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A.Janeway and P.TraVers, 1997); Antibodies (P.Finch, 1997); Antibodies:A Practical Approach (D.Catty., editor, IRL Press, 1988-1989); Monoclonal Antibodies:A Practical Approach (P.Shepherd and C.Dean, editor, Oxford University Press, 2000); Using Antibo dies:A Laboratory Manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zanetti and J.D.Capra, editor, Harvood Academic Publishers, 1995); With the widely used methodology of describing in Cancer:Principles and Practice of Oneology editors such as (, J.B.Lippincott Company, 1993) V.T.DeVita.

Definition

In order to explain the purpose of this specification sheets, by use to give a definition and any suitable in, the term used with odd number also will comprise plural number and vice versa.In any definition of illustrating hereinafter and the afoul situation of any file that herein is incorporated herein by reference, with the definition of set forth hereinafter, be as the criterion.

For example, for the molecule (molecule of antibody and antibodies) of reference herein, " activity " or " activity " refer to biology, immunology and/or the functionally active of this quasi-molecule.For example, in some embodiments, therefore anti-il-i-beta of the present invention and anti-IL-18 antibody for also having in conjunction with active bi-specific antibody in conjunction with IL-1 β and IL-18.In another embodiment, anti-il-i-beta of the present invention and anti-IL-18 antibody have neutralization or blocking-up is active, and this antibody-like can neutralize or block the activity of IL-1 β and/or IL-18.

" affibody " refers to be connected to by peptide bond the purposes of the protein in Fc district, wherein with described protein, as support, for target molecule, provides mating surface.Can change mating surface by mutagenesis can be in conjunction with the protein library of other epi-positions on other target molecules or identical target molecule to produce.Initiation protein is generally the natural protein that exists, as SP or IgG in conjunction with the B structural domain or by it derivative Z protein (consult (1987) such as Nilsson, Prot Eng1,107-133, with U.S. Patent number 5,143,844) or its fragment or derivative.For example, can from the Z protein variant with binding affinity that target molecule is changed, produce affibody, wherein by random mutagenesis to the section of Z protein carried out sudden change with produce can binding target molecule the variant library.The example of affibody comprises U.S. Patent number 6,534,628, Nord K etc., Prot Eng8:601-608 (1995) and Nord K etc., Nat Biotech15:772-777 (1997) .Biotechnol Appl Biochem.2008Jun; 50 (Pt2): 97-112.

Term herein " antibody " is used with broad sense and refers to no matter natural any immunoglobulin (Ig) (Ig) molecule that exists or transform, with its any fragment, mutant, variant or derivative, for example, as long as it shows required biologic activity (epi-position is in conjunction with activity).The example of antibody includes but not limited to monoclonal antibody, polyclonal antibody, multi-specificity antibody, antibody fragment, single domain antibody, octopus antibody and DVD antibody.In one embodiment, antibody of the present invention comprises at least one variable domains.In another embodiment, antibody of the present invention is bi-specific antibody.

Generally speaking, according to the aminoacid sequence of heavy chain constant domain, immunoglobulin (Ig) can be divided into different classes of.The immunoglobulin (Ig) of five primary categories has been described: IgA, IgD, IgE, IgG and IgM.These can be further divided into subclass (isotype), such as IgG-1, IgG-2, IgA-1, IgA-2 etc.For IgA, D, E, G and M, corresponding to the heavy chain constant domain of each immunoglobulin class, can be defined as respectively α, δ, ε, γ and μ.The subunit structure of different classes of immunoglobulin (Ig) and 3-d modelling be known and general description in, such as Abbas etc., in 2000, Cellular and Mol.Immunology the 4th edition.Antibody can be the part of larger fusion molecule, and described fusion molecule covalently or non-covalently is connected and forms with one or more other protein or peptide by antibody.

In one embodiment, antibody of the present invention has minimizing (still less) disulfide linkage.In one embodiment, antibody of the present invention comprises the hinge area that wherein makes at least one cysteine residues can not form disulfide linkage, wherein disulfide linkage be preferably intermolecular, preferably between two heavy chains.Can make hinge cysteine can not form disulfide linkage with multiple suitable method known in the art (described the some of them method herein, include but not limited to lack cysteine residues or with another kind of amino acid replacement halfcystine).

Based on context, phrase " anti-il-i-beta antibody and/or anti-IL-18 antibody/a plurality of antibody " refers to the combination (that is, two kinds of antibody) of (1) anti-il-i-beta antibody or (2) anti-IL-18 antibody or (3) anti-il-i-beta antibody and anti-IL-18 antibody or (4) antibody in conjunction with IL-1 β and IL-18.

" affinity maturation " antibody refers to the parental antibody that does not have those changes and compares to have the antibody of the one or more changes that make antibody be improved to antigen avidity in its one or more CDR.The antibody of preferred affinity maturation can have nmole level or the avidity of picomole quantities even to target antigen.Produce the antibody of affinity maturation by known method.For example, consult Marks etc., 1992, Biotechnology10:779-783, it has been described by variable heavy chain (V _h) and variable light chain (V _l) affinity maturation of structural domain reorganization.For example, the random mutagenesis of CDR and/or framework residue is described in: for example, and Barbas, etc., 1994, Proc.Nat.Acad.Sci, USA91:3809-3813; Shier etc., 1995, Gene169:147-155; Yelton etc., 1995, J.Immunol.155:1994-2004; Jackson etc., 1995, J.Immunol.154 (7): 3310-9; With Hawkins etc., 1992, J.Mol.Biol.226:889-896.

" agonist antibody " or " agonistic antibody " refers to the antibody of combination active antigen (as acceptor).Usually, the receptor activation ability of agonist antibody can be with the receptor activation ability of the natural agonist ligand of acceptor at least at similar (and may be quantitatively substantially similar) qualitatively.

The antibody of the part (being preferably antigen binding domain or the variable region of complete antibody) that " antibody fragment " refers to comprise complete antibody.The example of antibody fragment comprises Fab, Fab,, F (ab ') 2 and Fv fragment; Binary (Db); The series connection binary (taDb), (consult U.S. Patent number 5,641,870, embodiment 2 for linear antibody; Zapata etc., Protein Eng.8 (10): 1057-1062 (1995)); Single armed antibody, corpusculum (minibody), single-chain antibody molecule; For example, with the multi-specificity antibody formed from antibody fragment (, including but not limited to Db-Fc, taDb-Fc, taDb-CH3 and (scFV) 4-Fc).

In some embodiments, antibody fragment only comprises the part of complete antibody, and wherein this part retains at least one of normal correlation function when described part is present in complete antibody, and may retain most function or all functions.In another embodiment, the abundant part that antibody fragment of the present invention comprises constant region for example, with the heavy chain dimerization (or multimerization) of the disulfide linkage ability that allows to have reduction (as herein describe and changed at least one hinge cysteine that wherein normally participates in disulfide linkage between heavy chain).In one embodiment, therefore the antigen binding site that antibody fragment comprises complete antibody or variable domain also retain the ability of conjugated antigen.In another embodiment, antibody fragment, the antibody fragment that for example comprises the Fc district, retain Liao Dang Fc district and be present at least one biological function that in complete antibody, Shi Yu Fc district normally is correlated with, as FcRn in conjunction with adjusting, antibody half life, ADCC function and/or complement be for example, in conjunction with (, wherein antibody has ADCC function or complement in conjunction with essential glycosylation spectrum).The example of antibody fragment includes but not limited to linear antibody; The single-chain antibody molecule; With the multi-specificity antibody formed from antibody fragment.

The reaction of " the cell-mediated cytotoxicity that antibody relies on " and the mediation of " ADCC " phalangeal cell, wherein express the non-specific cell toxic cell (as natural killer (NK) cell, neutrophilic granulocyte and scavenger cell) of FcR and identify the binding antibody on target cell and cause subsequently the target cell cracking.The main cell NK cell of mediation ADCC is only expressed Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.On hematopoietic cell, the expression of FcR is summarized in Ravetch etc., the table 3 of the 464th page of 1991, Annu.Rev.Immunol9:457-92.For the ADCC activity of assessment objective molecule, can carry out as U.S. Patent number 5,500, the external ADCC described in 362 or 5,821,337 measures.Measure useful effector cell for this type of and comprise peripheral blood lymphocytes (PBMC) and natural killer (NK) cell.Perhaps, or in addition, can be in vivo such as Clynes etc., the ADCC activity of assessment objective molecule in disclosed animal model in 1998, PNAS (USA) 95:652-656.

Term " anti-il-i-beta antibody " and " in conjunction with the antibody of IL-1 β " refer to such antibody, thereby described antibody can make antibody be used as diagnostic reagent and/or therapeutical agent that target is determined IL-1 β in conjunction with IL-1 β with enough avidity.In one embodiment, for example by radioimmunoassay (RIA), measure, anti-il-i-beta antibody to the combination degree of incoherent non-IL-1 beta protein lower than antibody to approximately 10% of IL-1 β combination.In certain embodiments, the epi-position of anti-il-i-beta antibodies conservative IL-1 β between the IL-1 β from different plant species.

Term " anti-IL-18 " antibody and " in conjunction with the antibody of IL-18 " refer to such antibody, thereby described antibody can make antibody be used as diagnostic reagent and/or therapeutical agent that target is determined IL-18 in conjunction with IL-18 with enough avidity.In one embodiment, for example by radioimmunoassay (RIA), measure, anti-IL-18 antibody to the combination degree of incoherent non-IL-18 protein lower than antibody to approximately 10% of IL-18 combination.In certain embodiments, the epi-position of anti-IL-18 antibodies conservative IL-18 between the IL-18 from different plant species.

" autoimmune disease " used herein is for being derived from and pointing to nonmalignant disease or the illness of individual autologous tissue.Autoimmune disease described herein is clearly got rid of pernicious or Cancerous disease or situation, especially gets rid of B cell lymphoma, acute lymphoblastic leukemia (ALL), lymphocytic leukemia (CLL), hairy cell leukemia and chronic myeloblastic leukemia.The example of autoimmune disease or illness includes but not limited to that age-related macular degeneration (AMD), inflammatory response for example, as comprised the inflammatory skin disease of psoriatic and dermatitis (atopic dermatitis); Systemic scleroderma and sclerosis; To relevant the replying of inflammatory bowel (as regional ileitis and ulcerative colitis); Respiratory distress syndrome (comprises adult respiratory distress syndrome; ARDS); Dermatitis; Meningitis; Encephalitis; Uveitis; Colitis; Glomerulonephritis; The supersensitivity situation is as eczema and asthma and relate to the T cellular infiltration and other situations that chronic inflammatory diseases is replied; Atherosclerosis; Leukocyte adhesion deficiency; Rheumatoid arthritis; Systemic lupus erythematous (SLE); Lupus nephritis (LN); Diabetes (for example type i diabetes or insulin-dependent diabetes mellitus); Multiple sclerosis; The Reynaud Cotard; Autoimmune thyroiditis; Allergic encephalitis; The Sjorgen Cotard; Juvenile onset diabetes; And the immunne response relevant to acute and delayed hypersensitivity (DH) (cytokine in being typically found at tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis and T cell mediated); Pernicious anemia (Addison disease); The disease that relates to leukocyte infiltration; Central nervous system (CNS) inflammatory conditions; Multiple organ injury's syndrome; Hemolytic anemia (including but not limited to the positive anaemia of cryoglobinemia or Coombs); Myasthenia gravis; The disease of immune complex mediation; The anti-GBM disease; Antiphospholipid syndrome; The supersensitivity neuritis; Graves disease; The Lambert-Eaton myasthenic syndrome; The pemphigoid bleb; BP; Autoimmune polyendocrinopathy; RD; Unyielding man's syndrome; Behcet disease; Giant cell arteritis; Immune complex nephritis; IgA nephropathy; The IgM polyneuropathy; Immunologic thrombocytopenic purpura (ITP) or AT etc.

AMD is the leading reason (for example consulting Bressler (2004) JAMA291:1900-01) of serious irreversible visual loss in the elderly.Clinical and the pathology that it is characterized in that wide spectrum find, comprise that the breaking of the pale macula lutea that is known as drusen, retinal pigment epithelium (RPE), choroidal neovascularization form (CNV) and disciform degeneration of macula.The performance of this disease can be divided into two kinds of forms: non-exudative (dry) and exudative (moist or new vessel).Recently, several therapies that the are used for the treatment of moist AMD Visudyne that goes through-use photodynamic therapy; VEGF is in conjunction with fit, pegaptantib

with the VEGF antibody fragment, Lucentis (ranibizumab)

" self inflammatory disease " used herein refers to total similar features, is specially the illness of one group of rare inherited immunity mediation of heating.Self inflammatory disease is characterized as the recidivity nonirritant inflammation in autoantibody, infection or the antigen-specific T lymphocyte situation that does not have high titre.Exemplary self inflammatory disease includes but not limited to familial Mediterranean fever (FMF); Tumour necrosis factor (TNF) the hot syndrome of acceptor associated period (TRAPS); Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS); The whole body juvenile primary sacroiliitis (Chauffard-Still disease) of falling ill; Cryopyrin associated period syndrome (CAPS); Family's flu self inflammatory syndromes (familial cold autoinflammatory syndrome); Hereditary familial urticaria syndrome; Interleukin-1 receptor antagonist lacks (DIRA); With newborn infant fall ill multisystem inflammatory disease (NOMID)/chronic baby's neuroscience skin and joint (CINCA) syndrome.

Autoimmunization and self inflammatory disease have common feature, describedly are characterised in that two groups of illnesss are by due to immune system attack health autologous tissue, and also cause the inflammation increased.Distinguishing characteristics between the two is to lack (high titre) autoantibody in self inflammatory disease.

" biologic activity " or " functional " immunoglobulin (Ig) is to bring into play the immunoglobulin (Ig) of one or more its natural radioactivities in structure, regulation and control, biological chemistry or biophysics event.For example, the antibody of biologic activity can have the ability of specific combination antigen and this combination and can induce or change cytology or molecular events as signal transduction or enzymic activity.The antibody of biologic activity also acceptor capable of blocking ligand activation or with the agonist antibody effect.The ability that antibody is brought into play one or more its natural radioactivities depends on several tired elements, comprises the folding and assembling that polypeptide chain is correct.

" binding affinity " refers generally to the intensity of for example, for example, between the single binding site of molecule (, antibody) and its binding partners (, the antigen) summation of noncovalent interaction.Molecule X generally represents with dissociation constant (Kd) the avidity of its mating partner Y.Can be by usual way well known in the art, comprise that those methods described herein measure avidity.Low-affinity antibody is conjugated antigen be tending towards easily dissociating faintly, and the high-affinity antibody conjugated antigen is more tight and keep in conjunction with more permanent.

" biomolecules " refers to nucleic acid, protein, carbohydrate, lipid and composition thereof.In one embodiment, biomolecules is present in occurring in nature.

" blocking-up " antibody or " antagonist " antibody are a kind of antibody of the biologic activity of inhibition or the antigen that reduces its combination.This type of blocking-up can occur by any method, for example, by disturbing: the phosphorylation of Tyrosylprotein kinase residue or by the Tyrosylprotein kinase residue of receptor phosphorylation in the tyrosine kinase activity of tyrosine kinase receptor and/or acceptor in the combination of part and acceptor, receptor complex formation, receptor complex.Preferred blocking antibody or antagonist antibodies suppress the biologic activity of antigen basically or fully.

Term " cancer " and " cancer " refer to or describe the physiological situation in Mammals, and the characteristic feature of described physiological situation is not modulated Growth of Cells.The example of cancer includes but not limited to cancer, lymphoma, blastoma, sarcoma and leukemia.The more specifically example of this type of cancer comprises squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer, lung's gland cancer, lung's squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, carcinoma of the pancreas, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, mammary cancer, colorectal carcinoma, colorectal carcinoma, uterine endometrium or uterus carcinoma, salivary-gland carcinoma, kidney, liver cancer, prostate cancer, carcinoma vulvae, thyroid carcinoma, liver cancer and polytype H&N cancer.

Term " chimeric " antibody refers to the fragment (as long as they show required biologic activity) of such antibody and this antibody-like, heavy and/or the light chain of part or homology identical with corresponding sequence from specific species or antibody that belong to specific antibodies class or subclass in described antibody, and the rest part of described chain is identical with corresponding sequence from other species or antibody that belong to other antibody class or subclass or homology (for example is shown in U.S. Patent number 4,816,567 and Morrison etc., 1984, Proc.Natl.Acad.Sci.USA81:6851-6855).

As used herein, the statement " cell ", " clone " and " cell culture " is used interchangeably and all this type of indications include the offspring.Therefore, word " transformant " and " transformant " comprise primary individual cells and the irrelevant culture with shifting number therefrom produced.Will also be understood that due to have a mind to or sudden change unintentionally, all offsprings may can not be accurately consistent on DNA content.Comprised and there is the function identical with function as for original transformant was screened or biologic activity or the mutant offspring of biologic activity.When the clear and definite indication of needs, from the context can be clearly definite.

Statement " control sequence " refers to express in the specific host biology the essential DNA sequence dna of encoding sequence effectively connected.Be suitable for procaryotic control sequence and for example comprise promotor, optional operon sequence and ribosome bind site.The known genuine karyocyte utilizes promotor, polyadenylation signal and enhanser.

" diabetes " used herein are the chronic diseases that affects carbohydrate, fat and protein metabolism in animal.Diabetes are leading reasons of blind in the grownup, renal failure and lower extremity amputation and are the major risk factors of cardiovascular disorder and apoplexy.Type i diabetes (or insulin-dependent diabetes mellitus (" IDDM ") or juvenile onset diabetes) accounts for approximately 10% in all diabetes situations.This genius morbi is the carrying out property forfeiture of the insulin secretion function of pancreatic beta cell.This feature also is that by its origin non-primary or " secondary " diabetes in the pancreas disease are common.Type i diabetes is relevant to following clinical sign or symptom, blood sugar concentration or hyperglycemia that for example persistence raises; Diuresis; Polydipsia and/or hyperalimentation; Chronic microvascular complication is as retinopathy, ephrosis and neuropathy; And macrovascular complications is as blind in caused, hyperlipidaemia and the hypertension of end stage kidney disease, amputation and myocardial infarction.

Type ii diabetes (non-insulin-dependent diabetes mellitus (NIDDM) or NIDDM) relates to the metabolic disorder of glucose metabolism imbalance and impaired insulin sensitivity.Type ii diabetes generally occurs and can't to utilize or produce enough Regular Insulin relevant to health in adulthood.Except the insulin resistant of observing in target is organized surely, the patient who suffers from type ii diabetes has relative insulin deficit---, for given blood sugar concentration patient, there is the insulin level lower than expection.Type ii diabetes is characterised in that following clinical sign or symptom, blood sugar concentration or hyperglycemia that for example persistence raises; Diuresis; Polydipsia and/or hyperalimentation; Chronic microvascular complication is as retinopathy, ephrosis and neuropathy; And macrovascular complications is as blind in caused, hyperlipidaemia and the hypertension of end stage kidney disease, amputation and myocardial infarction.X syndrome, also referred to as insulin resistance syndrome (IRS), metabolism syndrome or metabolism syndrome X, approximately recognized in 2% the crown catheterization of diagnosis.Although be not often, but it has represented syndrome or the risk factors that type ii diabetes and cardiovascular disorder occur, comprise that for example impaired glucose tolerance (IGT), fasting glucose amount reduce (IFG), hyperinsulinemia, insulin resistant, abnormal lipidemia (for example high triglyceride, low HDL), hypertension and obesity.

" illness " is can be from any situation with benefiting therapeutic antibodies or a plurality of Antybody therapy.This comprises chronic and acute disease or disease, and described illness or disease comprise those pathological conditions that make Mammals be easy to produce the illness of discussing.In some embodiments, described illness is cancer, inflammation, immunity, self inflammation or autoimmune disease.

" extracellular domain " is defined as herein the cross-film polypeptide in the zone of outside, as FcR.

Term " Fc acceptor " or " FcR " are for the acceptor in the Fc district of describing binding antibody.

Term " Fc district ", for defining the C-terminal district of heavy chain immunoglobulin, comprises native sequences Fc district and variant Fc district herein.In one embodiment, the Fc district comprises CH2 structural domain and/or CH3 structural domain.Although the border in the Fc district of heavy chain immunoglobulin may change, human IgG heavy chain Fc district is commonly defined as from Cys226 or from the amino-acid residue of Pro230 position to its carboxyl terminal one section.For example antibody produce or purge process in, or carry out by the nucleic acid to the encoding antibody heavy chain C-terminal Methionin (being residue 447 according to the EU numbering system) that modified recombinant is removed the Fc district.Tired this, the composition of complete antibody can comprise the antibody population of removing all K447 residues, not remove the antibody population of K447 residue and have the antibody population that contains or do not contain the mixtures of antibodies of K447 residue." Fc mixture " used herein refers to two CH2 structural domains and/or two CH3 structural domains, and wherein CH2 structural domain and/or CH3 structural domain connect together by the interaction of non-peptide bond.

" framework region " (FR) refers to those variable domain residues except the CDR residue.Each IgG variable domain has four FR usually, is accredited as FR1, FR2, FR3 and FR4.If according to Kabat definition CDR, light chain FR residue is positioned at about residue 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4), and heavy chain FR residue is arranged in about residue 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and the 103-113 (HCFR4) of heavy chain residue.If CDR comprises the amino-acid residue from the hypermutation ring, light chain FR residue is arranged in about residue 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and the 97-107 (LCFR4) of light chain, and heavy chain FR residue is arranged in about residue 1-25 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3) and the 102-113 (HCFR4) of heavy chain residue.In some cases, when CDR comprises to come the amino acid of the CDR of the definition of Kabat freely and hypermutation ring, the FR residue is by corresponding adjustment.For example, when CDRH1 comprises amino acid H26-H35, heavy chain FR1 residue in the 1-25 position and the FR2 residue in the 36-49 position.

" functional Fc district " has " effector function " in native sequences Fc district.Exemplary " effector function " comprises the C1q combination; Complement dependent cytotoxicity; The Fc receptors bind; The cytotoxicity (ADCC) of antibody-dependant cell mediation; Phagolysis; Cell surface receptor is (as B-cell receptor; BCR) downward etc.This type of effector function generally needs the Fc district and combines in conjunction with territory (as antibody variable domains) and can use the assessment of many measure method, for example assay method disclosed herein." native sequences Fc district " comprises the aminoacid sequence identical with the aminoacid sequence in the Fc district found in nature.Native sequences people Fc district comprises native sequences human IgG1 Fc district (non-A and A allotype); Native sequences human IgG2 Fc district; Native sequences human IgG 3Fc district; With native sequences human IgG 4Fc district with and the variant of natural generation." variant Fc district " comprise due at least one place of definition herein " amino acid modified,, and the aminoacid sequence different from native sequences Fc district.Variant Fc district compares and can have at least one place amino acid substitution with the Fc district of native sequences Fc district or parental antibody, and can for example in the Fc district of native sequences Fc district or parental antibody, have from one to about ten place's amino acid substitutions or from approximately one to about five place's amino acid substitutions.Variant Fc district can have with the Fc district of native sequences Fc district and/or parental antibody at least about 80% identity, and can have with it identity at least about 90%, or has the identity at least about 95% with it.

Term " full length antibody ", " complete antibody " and " all antibody " is Alternate herein, is used in reference to its antibody that complete form exists basically, rather than the antibody fragment of definition hereinafter.This term especially refers to have the antibody in heavy chain and Fc district.Antibody variants of the present invention can be for example full length antibody.And full length antibody can be for example the people, humanized, chimeric and/or affinity maturation.

" hinge area " and version thereof used herein comprise connotation known in the art, it is illustrated in such as Janeway etc., 1999, Immuno Biology:The hmmune System in Health and Disease, Elsevier Science Ltd., NY. the 4th edition; Bloom etc., 1997, Protein Science, 6:407-415; Humphreys etc., 1997, J.Immunol.Methods, 209:193-202.

" homology " is defined as in aligned sequences and introduces room (as needs, to obtain maximum per-cent homology) afterwards, the per-cent of identical residue in the aminoacid sequence variant.Method and computer program for comparison is being known in the art.This type of computer program is " Align2; " the author is Genentech, Inc. and together be filed in (the United States Copyright Office of American National Copyright Bureau that is positioned at Washington DC on December 10th, 1991 with user file, Washington, D.C.20559).

As used herein term " host cell " (or " recombinant host cell ") refer to by introduce exogenous polynucleotide as recombinant plasmid or carrier by hereditary change or can be by the cell of hereditary change.Be to be understood that this type of term is intended to not only refer to specific individual cells, also refers to the offspring of this cell.Because due to sudden change or environmental influence and some modification may occur in the offspring, so this type of offspring in fact may be incomplete same with parental cell, but still within being included in the scope of term used herein " host cell ".

" people has framework " is the human normal immunoglobulin V selected _lor V _hthe framework of the amino-acid residue that in frame sequence, representative the most often occurs.Generally speaking, select human normal immunoglobulin V from the subgroup of variable domain sequence _lor V _hsequence.Generally speaking, the sequence subgroup is as the subgroup in Kabat.In one embodiment, for V _l, subgroup is as the subgroup kappa I in Kabat.In one embodiment, for V _h, subgroup is as the subgroup III in Kabat.

During the residue in mentioning variable domain (about residue 1-107 of light chain and the residue 1-113 of heavy chain), the general Kabat numbering system of using is (such as Kabat etc., the 5th edition .Public Health Service of Sequences of Immunological Interest., National Institutes of Health, Bethesda, Md. (1991)).During residue in mentioning immunoglobulin heavy chain constant region, general use " EU numbering system " or " EU index " is (such as the EU index be reported in Kabat etc., above)." as the EU index in Kabat " refers to the residue numbering of human IgG1 EU antibody.Unless referred else herein, in antibody variable domains, the reference of residue numbering refers to the residue numbering of Kabat numbering system.Unless referred else herein, in the antibody constant domain, reference of residue numbering refers to the residue numbering (for example consulting U.S. Provisional Application number 60/640,323, the figure that EU numbers) of EU numbering system.

(IgM antibody is comprised of different tetramer unit, 5 bases and the extra polypeptide that is called the J chain the different four glycan albumen that the basic 4-chain antibody unit of natural generation is comprised of two identical light (L) chains and two identical weights (H) chain, and contain thus 10 antigen binding sites, and the multivalence assemblage that the IgA antibody of secretion can multimerization contains 2-5 basic 4-chain unit and J chain with formation).In the situation of IgG, the 4-chain unit generally is about 150,000 dalton.Each L chain is connected with the H chain by a covalent disulfide bonds, and two H chains are connected to each other by one or more disulfide linkage according to H chain isotype.Each H and L chain also have the intrachain disulfide bond at regular interval.Each H chain has variable domain (V at N-terminal _h), there are thereafter three constant domain (C for each α and γ chain _h), and there are thereafter four C for μ and ε isotype _hstructural domain.Each L chain has variable domain (V at N-terminal _l), there is constant domain (C at its another end subsequently _l).V _lwith V _halignment, and C _lfirst constant domain (C with heavy chain _h1) alignment.Believe that particular amino acid residue forms the interface between light chain and heavy chain variable domain.V _hwith V _lpairing formed together single antigen binding site.For structure and the character of dissimilar antibody, for example consult Basic and Clinical Immunology, the 8th edition, Daniel P.Stites, Abba I.Terr and Tristram G.Parslow (editor), Appleton& Lange, Norwalk, CT, 1994, the 71 pages and the 6th chapter.

L chain from any invertebrate species can be classified as to two kinds of being called K and λ according to the aminoacid sequence of its constant domain complete one of dissimilar.According to its heavy chain (C _h) aminoacid sequence of constant domain, immunoglobulin (Ig) can be classified as to different kinds or isotype.There are five immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, there is respectively the heavy chain that is called α, δ, γ, ε and μ.Based on C _hsequence and relative fine difference on function, can be further divided into subclass by γ and α class, and for example the people expresses following subclass: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The white corpuscle that " people's effector cell " refers to express one or more FcR and exercise effector function.In some embodiments, cell expressing Fc γ RIII exercise the ADCC effector function at least.The example of the human leukocyte of mediation ADCC comprises peripheral blood lymphocytes (PBMC), natural killer (NK) cell, monocyte, cytotoxin T cell and neutrophilic granulocyte.Can be as described from natural origin separate effect daughter cell from blood or PBMC (peripheral blood lymphocytes) for example herein.

" humanized " form of inhuman (for example, muroid) antibody is the chimeric antibody contained from the non-human immunoglobulin minmal sequence.For most of parts, humanized antibody is human normal immunoglobulin (receptor antibody), and the residue as the hypervariable region of mouse, rat, rabbit or non-human primates with specificity, avidity and ability of wanting is substituted by inhuman species (donor antibody) wherein to carry out the residue of hypervariable region of autoreceptor.In some cases, Fv framework region (FR) residue of human normal immunoglobulin is substituted by corresponding inhuman residue.In addition, humanized antibody can be included in receptor antibody or undiscovered residue in donor antibody.Carrying out these modifies further to improve the antibody performance.Generally speaking, humanized antibody can comprise all parts basically of at least one and common two variable domains, wherein all or basically all hypermutation ring corresponding to those hypermutation rings of non-human immunoglobulin and all or those FR districts of all FR district behaviour immunoglobulin sequences basically.Humanized antibody optionally also can comprise at least part of constant region for immunoglobulin (Fc), is generally the constant region of human normal immunoglobulin.Other details are consulted Jones etc., Nature321:522-525 (1986); Riechmann etc., Nature332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

" people's antibody " is such antibody, and described antibody has corresponding to the aminoacid sequence of the antibody produced by the people and/or used any technology for generation of people's antibody disclosed herein to prepare.This definition clear-cut has been got rid of the humanized antibody in conjunction with residue containing non-human antigen.

As used herein, term " hyperglycemia illness " refers to the diabetes of form of ownership and the illness caused by insulin resistant, as I type and type ii diabetes, and serious insulin resistant, hyperinsulinemia and hyperlipidaemia, as obese subjects, and insulin-resistant diabetes, as Mendenhall Cotard, Werner syndrome, donohue's syndrome, lipoatrophic diabetes and other lipoatrophy.Specific hyperglycemia illness disclosed herein is diabetes, especially I type and type ii diabetes." diabetes " reference own and Regular Insulin produce or utilize the PD of insufficient carbohydrate metabolism and be characterised in that hyperglycemia and glycosuria.

When using herein, term " hypervariable region ", " HVR " or " HV " refer to hypermutation on sequence and/or zone that form the antibody variable domains of ring definite on structure.Generally speaking, antibody comprises six HVR; Three at V _hin (H1, H2, H3), three at V _lin (L1, L2, L3).In natural antibody, H3 and L3 show the diversity of the maximum of six HVR, and especially think H3 give on the meticulous specificity of antibody, play a part unique.Such as consulting Xu etc., Immunity13:37-45 (2000); Johnson and wu, in Methods in Molecular Biology248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003).Yet, exist manyly only to there is heavy chain and lack the example of function antibody of the naturally occurring of light chain and transformation.Such as consulting Hamers-Casterman etc., Nature363:446-448 (1993); Sheriff etc., Nature Struct.Biol.3:733-736 (1996).

Many HVR describe just in use and are contained in this herein.Kabat complementary determining region (CDR) is based on sequence variability and be the most frequently used (Kabat etc., Sequences of Proteins of Immunological Interest, the 5th edition .Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).Chothia refers to the position (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)) of structure ring.AbM HVR has represented trading off between Kabat HVR and Chothia structure ring, and is used by Oxford Molecular ' s AbM antibody modeling software." contact ( contact) " analysis of HVR based on to obtainable compound crystal structure.From each the residue in these HVR, be presented at hereinafter.

HVR generally comprise from the hypermutation ring and/or from " complementary determining region " amino-acid residue (CDR), and the latter has the highest sequence variability and/or participate in antigen recognition.Exemplary hypermutation ring appears at the amino-acid residue of 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) position.(Chothia and Lesk, J.Mol.Biol.196:901-917 (1987).Exemplary CDR (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) appears at the amino-acid residue of the 95-102 position of the 50-65 of 31-35B, H2 of 89-97, H1 of 50-56, L3 of 24-34, L2 of L1 and H3.(Kabat etc., Sequences of Proteins of Immunological Interest, the 5th edition .Publie Health Service, National Institutes of Health, Bethesda, MD (1991).Except V _hin CDRl, CDR generally comprises the amino-acid residue that forms the hypermutation ring.CDR also comprises " specificity decision residue " or " SDR ", and it is the residue of contact antigen.Within SDR is contained in the CDR district that is called as the CDR that shortens or a-CDR.Exemplary a-CDR (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2 and a-CDR-H3) appears at the amino-acid residue of the 95-102 position of the 50-58 of 31-35B, H2 of 89-96, H1 of 50-55, L3 of 31-34, L2 of L1 and H3.(consult Almagro and Fransson, Front.Biosci.13:1619-1633 (2008).) IgG HVR can comprise " HVR of expansion ": V as follows _lin 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) and V _hin 26-35 (H1), 50-65 or 49-65 (H2) and 93-102,94-102 or 95-102 (H3).Unless otherwise stated, other residues in HVR residue and variable domain (as the FR residue) are numbered according to the article (seeing above) of Kabat etc. herein.

Can produce that have can be in conjunction with the V of two or more epi-positions _h/ V _lthe antibody of unit (Bostrom etc. (2009) Science323:1610-1614; WO2008/027236 (being incorporated herein by reference).This type of multi-specificity antibody being called to " two-in-one " antibody or " double-acting Fab " or " DAF " herein can be in conjunction with being positioned at least two epi-positions on same target molecule or being positioned at two epi-positions on different target molecules for the single arm (aka the VH/VL unit) that antibody is described.On the one hand, these DAF antibody can be by the V of the antibody in connection with the first epi-position _h/ V _lthe V of unit _lstructural domain is suddenlyd change, and selection can be in conjunction with the mutant V of described the first epi-position and the second epi-position _h/ V _lunit and preparing.For example, in one embodiment, the V in screening for the sudden change in conjunction with the second epi-position _h/ V _lbefore unit, can substitute at random or selectively the amino-acid residue that the one or more solvents in light chain CDR expose with one or more other amino-acid residues.

" inflammation corpusculum mediation disease " refers to IL-1 β wherein and/or the IL-18 any disease raise to some extent of organizing with respect to normal not inflammation.Generally speaking, in the disease of inflammation corpusculum mediation, with respect to the compared with control cells of not inducing, it has related to processing and/or activation or the rising of Caspase-1.Can use commercially available mensuration test kit as Caspase 1 fluorometric assay test kit ((catalog number (Cat.No.) ab394120; AbCam, Cambridge, MA), Caspase 1 colorimetric estimation (catalog number (Cat.No.) BF14100; R& D Systems) etc. measure the activity of Caspase-1.

Generally speaking, if, if disease or situation raise relevant or take from the cell or tissue IL-1 β that the generation level raises in cultivation in health to IL-1 β level in body fluid or tissue, can think that this disease or situation are IL-1 ss related diseases or situation.Equally, if, if disease or situation raise relevant or take from the cell or tissue IL-18 that the generation level raises in cultivation in health to IL-18 level in body fluid or tissue, can think that this disease or situation are IL-18 relative disease or situation.Therefore, IL-1 were β/if IL-18 relative disease or situation would raise relevant or take from two kinds of cytokines of the cell or tissue generation level rising in cultivation in health to the level of IL-1 β in body fluid or tissue and IL-18, could be IL-1 β/IL-18 relative disease or situations.

Immunity and inflammatory disease comprise: chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, osteoarthritis, childhood chronic arthritis, spondyloarthropathy, Sjogren's syndrome disease (scleroderma), primary inflammatory myopathy (dermatomyositis), SV, sarcoidosis, autoimmune hemolytic anemia (immune cytopenia, paroxysmal nocturnal hemoglobinuria), AT (idiopathic thrombocytopenic purpura, immune-mediated thrombopenia), thyroiditis (Grave disease, Hashimoto thyroiditis, teenager's lymphocytic thyroiditis, atrophic thyroiditis) the autoimmunization inflammatory diseases is (as allergic encephalitis, multiple sclerosis, insulin-dependent diabetes mellitus, the autoimmunization uveal tract retinitis (autoimmune uveoretinitis), thyrotoxicosis, autoimmune thyroid disease, pernicious anemia, autograft rejection, diabetes, with immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis)), maincenter and peripheral nervous system demyelinating diseases are as multiple sclerosis, primary demyelination polyneuropathy or Guillain-Barr é syndrome and chronic inflammatory demyelinating polyneuropathy, the hepatic duct disease is as infectious hepatitis (first, second, third, fourth, hepatitis E and other non-hepatitis venereal disease poison), ACAH, primary biliary cirrhosis, granulomatous hepatitis and sclerosing cholangitis, gluten susceptibility enteropathy and Whipple disease, autoimmunization or immune-mediated tetter comprise bleb tetter, erythema multiforme and contact dermatitis, psoriatic, allergic disease as asthma, rhinallergosis, atopic dermatitis, vernal conjunctivitis, eczema, food hypersensitivity and urticaria, lung's Immunological diseases as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplant relative disease and comprise transplant rejection and graft versus host disease (GVH disease).

Immune interrelation and inflammatory diseases are very complex, normally phenomenon or the consequence of the biological pathway of a plurality of interconnection, described biological pathway under normal physiological conditions for the reparation of response wound or damage, initial wound or damage and to start the congenital and acquired defence for exogenous organisms be all vital.When these normal physiological routes cause extra wound or when damage, disease or symptom or with directly to reply intensity relevant or extremely to regulate and control or the result of overstimulation or to occur for id reaction or with the combination of these forms.

The example of IL-1 ss related diseases is acute pancreatitis; ALS; Emaciation/anorexia, comprise the emaciation that AIDS induces; Asthma and other pulmonary disorders; The autoimmunization vasculitis; CIAS1 associated period syndrome (CAPS); Adolescent primary sacroiliitis, Still disease, hereditary familial urticaria syndrome, the chronic tired syndrome of the multisystem inflammatory diseases (NOMID/CINCA) of newborn infant's morbidity, whole body morbidity; Shuttle mattress (Clostridium) relative disease, comprise the relevant diarrhoea of clostridium; Coronary disease and indication, comprise congestive heart failure, crown restenosis, myocardial infarction, myocardial dysfunction (as relevant to Sepsis) and coronary artery bypass graft; Cancer, as (as AML and CML) and other leukemia in multiple myeloma and marrow, and tumor metastasis; Diabetes (as the Regular Insulin diabetes); Endometriosis; Family's flu self inflammatory syndromes (FCAS); Familial Mediterranean fever (FMF); Heating; Fibromyalgia; Glomerulonephritis; Graft versus host disease/transplant rejection; Hemohorragic shock; Hyperpathia; Inflammatory bowel; The arthritis disease, comprise psoriatic arthritis (and osteoarthritis and rheumatoid arthritis); The inflammatory eye disease, as can be relevant to for example corneal transplantation; Ischemic, comprise cerebral ischemia (as wound, epilepsy, the brain injury that hemorrhage or apoplexy (these each all may cause neurodegeneration) causes); River abnormal (family name) disease; The study damage; Lung disease (as ARDS); Myopathy (as the especially mytolin metabolism in Sepsis); Neurotoxicity (as induced by HIV); Osteoporosis; Pain, comprise that cancer is ache related; Parkinson's disease; Periodontopathy; Preterm labor; Psoriatic; Reperfusion injury; Radiocurable side effect; Sleep disordered; Temporomandibular arthrosis; The periodic fever syndrome (TRAPS) that Tumor Necrosis Factor Receptors is relevant; Uveitis; Or nervous, sprain, inflammatory diseases that cartilage injury, wound, orthopedics, infection or other diseases process cause.

Interleukin 18 plays vital effect in the pathology of the various diseases that relates to immunity and inflammatory molecule.These diseases include but not limited to rheumatoid arthritis, osteoarthritis, childhood chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, lupus (as systemic lupus erythematous and systemic lupus erythematosus), regional ileitis, ulcerative colitis, inflammatory bowel, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic disease, psoriatic, 1 psoriasis pustulosa, 2 psoriasis pustulosas, the dermatitis scleroderma, graft versus host disease (GVH disease), the organ-graft refection, acute or the chronic immune disease relevant to organ transplantation, sarcoidosis, atherosclerosis, disseminated inravascular coagulation, kawasaki disease, the GraveShi disease, nephrotic syndrome, chronic tired syndrome, Wegner granulomatosis, Henoch-Schoenlein purpurea, kidney capillary blood vessel inflammation, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, emaciation, transmissible disease, parasitosis, acute transverse myelitis, Huntington Chorea, Parkinson's disease, alzheimer's disease, apoplexy, primary biliary cirrhosis, hemolytic anemia, malignant tumour, in heart failure, myocardial infarction, Addison disease, sporadic I type polyadenous lacks and II type polyadenous lacks, Schmidt syndrome, adult respiratory distress syndrome, alopecia, alopecia greata, seronegative arthopathy, joint disease, the Josef Reiter disease, psoriatic arthropathy, the ulcerative colitis joint disease, the enteropathy synovitis, chlamydozoan, the joint disease that Yersinia is relevant with Salmonellas, arthritis vertebralis (spondyloarthopathy), Atheromatosis 1 arteriosclerosis, atopic allergology, the autoimmunization bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, the positive hemolytic anemia of Coombs, acquired pernicious anemia, pernicious anemia,juvenile, myalgia encephalitis/Royal Free Disease.Chronic mucocutaneous candidiasis, giant cell arteritis, primary hardening hepatitis, hidden systemic autoimmune hepatitis, acquired immune deficiency syndrome (AIDS), the acquired immunodeficiency relative disease, hepatitis C, common various immune deficiency, common variable hypogammaglobulinemia, dilated cardiomyopathy, female infertility, ovarian function failure, Premature Ovarian Failure, , pulmonary fibrosis disease, hidden source fibrosis pulmonary alveolitis, interstitial lung disease after inflammation, interstitial pneumonia, the interstitial lung disease that connective tissue disease (CTD) is relevant, the tuberculosis that mixed connective tissue disease is relevant, the interstitial lung disease that systemic sclerosis is relevant, the interstitial lung disease that rheumatoid arthritis is relevant, the tuberculosis that systemic lupus erythematous is relevant, the tuberculosis that dermatomyositis/polymyositis is relevant,

the tuberculosis of disease-related, the tuberculosis that ankylosing spondylitis is relevant, vasculitis diffusivity lung disease, the tuberculosis that haemosiderosis is relevant, drug-induced interstitial lung disease, radioactive fibrosis, bronchitis obliterans, the chronic eosinophilic pneumonia, lymphocytic infiltration tuberculosis, interstitial lung disease after infecting, urarthritis, autoimmune hepatitis, 1 type autoimmune hepatitis, typical case's autoimmunity or lupoid hepatitis, 2 type autoimmune hepatitis, anti-LKM antibody hepatitis, the hypoglycemia of autoimmunization mediation, follow the Type B insulin resistant of acanthosis nigricans, hypoparathyroidism, the acute Immunological diseases relevant to organ transplantation, the chronic immune disease relevant to organ transplantation, osteoarthropathy, primary sclerosing cholangitis, primary leucopaenia, the autoimmunity neutropenia, nephropathy NOS, glomerulonephritis, the microscopic vastulitis of kidney, Lyme disease, discoid lupus erythematosus, primary male infertility or NOS, Sperm autoimmunity, all hypotypes of multiple sclerosis, sympathetic ophthalmia, be secondary to the pulmonary hypertension of connective tissue disease (CTD), Goodpastures syndrome, the pulmonary of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Chauffard-Still disease, Sjogren's syndrome disease,

syndrome, Takayasu ' s disease/arteritis, the autoimmunity thrombopenia, the primary thrombopenia, autoimmune thyroid disease, hyperthyroidism, thyrocele autoimmunity hypothyroidism or Hashimoto's disease, atrophic autoimmunity hypothyroidism, the primary solid edema, crystalline body source uveitis (phacogenic uveitis), primary angiitis, vitiligo, acute hepatopathy, chronic hepatopathy, allergy and asthma, mental disorder, dysthymia disorders, schizophrenia, the disease of Th2 type and the mediation of Th1 type, chronic obstructive pulmonary disease (COPD), inflammation, autoimmune disorder and osteopathia.

Antibody of the present invention and fragment also can be used for treatment or prevention IL-1 β is relevant or that IL-18 is correlated with or self inflammation or autoimmunity or inflammatory or Immunological diseases.

Although these diseases be usually directed to multistage approach and multiple different biosystem/approach, the intervention in the stagnation point in one or more these approach has and improves or therapeutic action.Can realize that treatment gets involved by the antagonism to harmful process/approach or to the stimulation of useful process/approach.

T lymphocyte (T cell) is the important component that mammalian immune is replied.The relevant antigen of self molecule of T cell recognition and genes encoding in ajor histocompatibility mixture (MHC).Antigen can be showed on the surface of antigen presenting cell, virus infected cell, cancer cells, graft etc. together with the MHC molecule.The T cell system is removed these that host animal is produced to health threat the cell changed has been occurred.The T cell comprises helper cell and cytotoxic T cell.Helper cell is bred identify antigen-MHC mixture on antigen presenting cell after in a large number.Helper cell is also secreted as lymphokine tired sub in interior various kinds of cell, and it plays a major role in the reactivation process of multiple other cells of B cell, cytotoxic T cell and participation immunne response.

In many immunne responses, inflammatory cell infiltration damage or sites of infection.As measured by the histological examination to affected tissue, migrating cell can be neutrophilic, oxyphilous, monocyte or lymphocyte.For example consult Current Protocols in Immunology, John E.Coligan edits, 1994, John Wiley& Sons, Inc.Many immune correlated diseases are known and have carried out broad research.This type of disease comprises inflammatory diseases, infectious diseases, immunodeficient disease, tumorigenesis and the transplant rejection etc. of immune-mediated inflammatory diseases (as rheumatoid arthritis, immune-mediated ephrosis, hepatic duct disease, inflammatory bowel (IBD), psoriatic and asthma), nonimmune mediation.In field of immunology, identify that target is used for the treatment of inflammation and inflammatory conditions.In field of immunology, identified that target is used for the treatment of inflammation and inflammatory conditions herein.In one case, can reply to treat immune correlated disease by Immunosuppression.The neutralizing antibody that uses inhibition to have the molecule of immunostimulatory activity will contribute to treat immune-mediated disease and inflammatory diseases.The molecule that can utilize (protein directly or by using antibody agonist) Immunosuppression to reply comes Immunosuppression to reply and so improves immune correlated disease.

As used herein, term " immunoadhesin " has referred to combine the molecule of the effector function of the binding specificity of heterologous protein (" adhesin ") and immunoglobulin (Ig) constant domain.Structurally, immunoadhesin comprises aminoacid sequence with binding specificity of wanting and the fusion in Fc district, described aminoacid sequence is not antigen recognition and the binding site (that is, being " allos ") of antibody, and described Fc district is for example CH2 and/or the CH3 sequence of IgG.Exemplary conglutnin sequence comprises the continuous amino acid sequence of the part that contains part acceptor (for example, extracellular domain) or binding purposes protein.The conglutnin sequence can also be the sequence of binding purposes protein, but is not acceptor or ligand sequence (for example, the conglutnin sequence in peptide body (peptibody)).This type of peptide sequence can be selected or identify by several different methods, and described method comprises display technique of bacteriophage and high-throughput sorting method.Can be from any immunoglobulin (Ig) as the immunoglobulin (Ig) constant domain sequence in adaptive immune adhesin IgG-1, IgG-2, IgG-3 or IgG-4 hypotype, IgA (comprising IgA-1 and IgA-2), IgE, IgD or IgM.Example molecule is to be described in Berg etc., 1991, PNAS (USA) 88:4723 and Chamow etc., the dual specific CD4-IgG molecule in 1994, J.Immunol.153:4268.

" separation " antibody is the antibody of from the component of its natural surroundings, identifying and separating and/or reclaim.The contaminant component of its natural surroundings is to disturb the diagnosis of antibody or the material of therepic use, and may comprise solute enzyme, hormone and other protein or nonprotein.In some embodiments, can be by antibody purification (1) to as what measure by the Lowry method, with the antibody weighing scale, surpassing 95%, and most preferably be by weight and surpass 99%, (2) to the degree of at least 15 residues that use the rotating cup sequence analyser to be enough to obtain N-terminal or inner aminoacid sequence, or (3) to by SDS-PAGE, under reduction or non-reduced condition, use Coomassie blue or, preferably silver dyes and is accredited as homogeneity.Due at least one component of the natural surroundings that will not have antibody, so the antibody separated is included in the original position antibody in reconstitution cell.Yet usually, will be prepared by least one step purification step by the antibody of separation.

" separation " nucleic acid molecule is the nucleic acid molecule of identifying and separating from least one contaminated nucleic acid molecule, and described contaminated nucleic acid molecule accompanies with antibody nucleic acid usually in the natural origin of antibody nucleic acid.In form or environment that the nucleic acid molecule separated is not found in occurring in nature.Therefore the nucleic acid molecule separated is distinguishing with the nucleic acid molecule be present in n cell.Yet the nucleic acid molecule of separation is included in the nucleic acid molecule contained in the cell of common expression antibody, for example described nucleic acid molecule is positioned on the chromosome position that is different from n cell amplifying nucleic acid molecule.

Technology as the term " pestle mortar structure (knob-into-hole) " herein mentioned or " KnH " refer to, in vitro or in body by projection (pestle shape structure) (pertuberance (knob)) is introduced to a polypeptide on their interactional interfaces and by chamber (mortar shape structure (hole)) thus introducing another polypeptide guides two polypeptide selective matchings.For example, KnH is incorporated into to Fc:Fc bonding interface, the C of antibody _l: C _h1 interface or V _h/ V _linterface (for example, (1997) Protein Science6:781-788 such as US20007/0178552, WO96/027011, WO98/050431 and Zhu).This is for driving two different heavy chains together to match and be particularly useful in preparing the multi-specificity antibody process.For example, the multi-specificity antibody that has KnH in Qi Fc district can also comprise the single variable domain be connected with each Fc district, or also comprises the different heavy chain variable domain matched from similar or different light chain variables territory.In fact, the KnH technology can be used for matching two different receptor extracellular domain or contain different target recognition sequences any other peptide sequence of (for example, comprising that affibody, peptide body and other Fc merge).

Statement " linear antibody " refers generally to be described in Zapata etc., the antibody in Protein Eng.8 (10): 1057-1062 (1995).Briefly, these antibody comprise pair of series Fd section (V _h-C _h1-V _h-C _h1), itself and complementary light chain polypeptide together form antigen binding domain pair.Linear antibody can be dual specific or monospecific.

Term " Mammals " comprises and is categorized as mammiferous any animal, comprises people, ox, horse, dog and cat.In one embodiment, Mammals is the people.

Term " monoclonal antibody " refers to the antibody obtained from homogeneous antibody group basically as used herein, that is, each antibody that forms the group is except may be being identical possible natural the undergoing mutation existed on a small quantity.Monoclonal antibody is high special for single antigen site.In addition, from routine (polyclone) the antibody preparation difference that generally includes the different determinants of sensing (epi-position), each monoclonal antibody is pointed to the single determinant on antigen.Qualifier " mono-clonal " shows that the antibody proterties is available from the antibody population of homogeneity basically, and can not be interpreted as needing by any ad hoc approach Dispersal risk.For example, monoclonal antibody used according to the invention can be by first by Kohler etc., and prepared by the hybridoma method that 1975, Nature256:495 describes, maybe can for example, by recombinant DNA method (consulting U.S. Patent number 4,816,567), prepare." monoclonal antibody " can also be used such as being described in Clackson etc., 1991, Nature352:624-628 and Marks etc., and the technology in 1991, J.Mol.Biol.222:581-597 is separated from phage antibody library.

Monoclonal antibody specifically comprises the fragment (as long as they show the biologic activity of wanting) of such " chimeric " antibody (immunoglobulin (Ig)) and this antibody-like herein, heavy and/or the light chain of part or homology identical with sequence from corresponding in specific species or antibody that belong to specific antibodies class or subclass in described antibody, and the rest part of chain is identical with sequence from corresponding in another species or antibody that belong to another antibody class or subclass or homology (U.S. Patent number 4,816,567; With Morrison etc., 1984, Proc.Natl.Acad.Sci.USA81:6851-6855).

Term " multi-specificity antibody " is used with connotation the most widely and refers to have the specific antibody of multi-epitope.This type of multi-specificity antibody includes but not limited to comprise heavy chain variable domain (V _h) and light chain variable territory (V _l) (V wherein _hv _lunit has the multi-epitope specificity) antibody, there are two or more V _land V _hstructural domain and each V _hv _lunit in conjunction with the antibody of different epi-positions, there is two or more single variable domains and at least two single variable domains in conjunction with the antibody of different epi-positions, full length antibody, antibody fragment, binary, series connection antibody, linear antibody and trisome (triabody) as Fab, Fv, dsFv, scFv, the covalently bound or antibody fragment that mutually combines by noncovalent interaction.Used that other examples that maybe can make the antibody formation for producing multi-specificity antibody include but not limited to that the Fc of binary merges, series connection antibody and single-chain antibody (for example Db-Fc, taDb-Fc, taDb-CH3 and (scFV) 4-Fc), pestle shape structure (KnH) antibody, octopus antibody and DAF antibody.

" polyspecific molecule " refers to have the specific molecule of multi-epitope as used herein." multi-epitope specificity " refers to the ability of at least two different epi-positions on the same target molecule of specific combination or different target molecule." monospecific " only refers to the ability in conjunction with an epi-position.According to an embodiment, the polyspecific molecule is attached on each epi-position with the avidity of 5 μ M to 0.001pM, 3 μ M to 0.001pM, 1 μ M to 0.001pM, 0.5tM to 0.001pM or 0.1 μ M to 0.001pM.Term " dual specific " refers to the ability (for example, anti-il-i-beta/IL-18 bi-specific antibody) in conjunction with two kinds of epi-positions as used herein.Support maybe to transform to support that the example of the specific molecule of multi-epitope includes but not limited to antibody, affibody, immunoadhesin, peptide body and other Fc fusions.

The multivalent antibody of term " octopus " antibody or a plurality of antibody Zhi Han Fc district N-terminal two or more antigen binding sites in He Fc district (for example, WO01/77342, Wu etc. (2007) Nature Biotechnology and WO2007/024715) as used herein.In a preferred embodiment, the conformation of antibody polypeptides is VD1-(X1) n-VD2-(X2) n-Fc, and wherein VD1 is the first variable domain, VD2 is the second variable domain, a polypeptide chain in FcShi Fc district, X1 and X2 represented amino acid or polypeptide, and n is 0 or 1.In one embodiment, X1 or X2 are that CH1 structural domain, part CH1 structural domain, some other joint sequences for example, as GS joint or its some combinations (, the page 5 of WO2007/024715).

As used herein, when functional contact that be placed in another nucleotide sequence, nucleic acid is " effectively connecting ".For example, if expressed as participating in antibody secreted front albumen, the DNA of presequence or secretion leader sequence is effectively to be connected with the DNA of antibody; If promotor or enhanser have affected transcribing of sequence, with encoding sequence, be effectively to be connected; If perhaps ribosome bind site be positioned be beneficial to the translation with encoding sequence, be effectively to be connected.Generally speaking, " effectively connecting " refers to that the DNA sequence dna be connected is adjacency, and, in the situation that the secretion leader sequence, adjacency also is positioned at reading mutually.Yet enhanser can adjacency.By connecting on restriction enzyme site easily, realize connecting.If there is no this type of site, can be used synthetic oligonucleotide aptamers or joint according to routine operation.

" peptide body " refers to the fusion of peptide sequence and Fc structural domain.Consult the U.S. Patent number 6,660,843 (being incorporated herein by reference with its integral body) that Feige etc. is disclosed on December 9th, 2003.They comprise and are connected to N-terminal, C-terminal, amino acid side chain or more than one or more peptide on these sites.The peptide body technique makes it possible to the therapeutical agent of design integration peptide, the one or more parts of described peptide target or acceptor, tumor-homing peptide, membrane translocating peptide etc.Confirmed that the peptide body technique is effectively in a large amount of these quasi-molecules of design, described molecule comprises linear and two sulphur restriction peptides, " tandem polypeptide polymer " (on the strand of Fc structural domain more than a peptide).For example consult U.S. Patent number 6,660,843; Be published in the Application No. 2003/0195156 (corresponding to WO02/092620, being published on November 21st, 2002) on October 16th, 2003; Be published in the Application No. 2003/0176352 (corresponding to WO03/031589, being published on April 17th, 2003) on September 18th, 2003; Be filed in the United States serial 09/422,838 (corresponding to WO00/24770, being published on May 4th, 2000) on October 22nd, 1999; Be published in the Application No. 2003/0229023 on December 11st, 2003; Be published in the WO03/057134 on July 17th, 2003; Be published in the Application No. 2003/0236193 (corresponding to PCT/US04/010989, being filed on April 8th, 2004) on December 25th, 2003; Be filed in the United States serial 10/666,480 (corresponding to WO04/026329, being published on April 1st, 2004) on September 18th, 2003, wherein each is incorporated herein by reference with its integral body.

For this purpose, " pharmaceutical composition " is to adapt to and be suitable for the composition of using to Mammals especially people.Therefore, said composition can be used for the treatment of to disease or the pain disease in Mammals.In addition, the protein in said composition is carried out to a step or multistep purifying or separating step, in order to therefrom separate the pollutent that may disturb its therepic use.Generally speaking, pharmaceutical composition comprises therapeutic protein and pharmaceutically acceptable carrier or thinner.It is aseptic and can freeze-drying that said composition is generally.Pharmaceutical preparation has been described hereinafter in more detail.

" polynucleotide " or " nucleic acid " can Alternate as described here, refers to the polymer of the Nucleotide of any length, and comprises DNA and RNA.Nucleotide can be deoxyribonucleotide, ribonucleotide, modification Nucleotide or base and/or their analogue, maybe can or be integrated into any substrate in polymkeric substance by building-up reactions by DNA or RNA polymerase.Polynucleotide can comprise the Nucleotide of modification, as methylated nucleotide and their analogue.If present, can before or after the polymkeric substance assembling, give the modification to nucleotide structure.Nucleotide sequence can be cut off by the non-nucleotide component.Polynucleotide can also be modified after synthetic, as passed through to connect label.The modification of other types comprises, for example " cap ", substitute the Nucleotide of one or more natural generations with analogue, between Nucleotide, modify (internucleotide modification) as, for example, neutral (for example connects, methylphosphonate, phosphotriester, phosphoramidate, carbamate etc.) and electrically charged connection (as thiophosphoric acid (ester), phosphorodithioate etc.) those modifications, those modifications containing the suspension part, as, for example, protein (for example, nuclease, toxin, antibody, signal peptide, poly-L-Lysine etc.), there is intercalator (as acridine, psoralene etc.) those modifications, containing sequestrant (for example, metal, radioactive metal, boron, oxidized metal etc.) those modifications, those modifications containing alkide, containing the connection of modifying (for example, different nucleic acid of α etc.) those modifications, and the unmodified form of polynucleotide.In addition, usually being present in any hydroxyl in sugar can be replaced by for example phosphonate groups, phosphate group, by the protection of standard blocking group, or is activated with the outer Nucleotide of frontad and prepares extra connection, maybe can put together on solid or semisolid upholder.5 ' and 3 ' end OH can phosphorylation or partly alternative with organic cap group of amine or 1 to 20 carbon atom.Other hydroxyls can also be derived for the standard blocking group.Polynucleotide can also comprise the similar type of common ribose known in the art or ribodesose sugar, for example comprise 2 '-the O-methyl-, 2 '-O-allyl group, 2 '-fluoro-or 2 '-azido--ribose, carba sugars, α-different head sugar, epimerization are sugared as pectinose, wood sugar or lyxose, pyranose, furanose, sedoheptulose, non-ring analogues and alkali-free yl nucleosides acid-like substance, as methyl nucleoside.One or more phosphodiester bonds can be replaced by optional linking group.These optional linking groups include but not limited to phosphoric acid salt wherein by P (O) S (" thioate "), P (S) S (" dithioate "), " embodiment that (O) NR2 (" amidation "), P (O) R, P (O) OR ', CO or CH2 (" formacetal ") replace, wherein each R or R ' they are replacement or the non-substituted alkyl (1-20C.) of H or optional ether-containing key (O-), aryl, thiazolinyl, cycloalkyl, cycloalkenyl group or araldyl independently.It is all identical that all connections in polynucleotide do not need.Description above is applied to comprise all polynucleotide that relate to of RNA and DNA herein.

As used herein, " oligonucleotide " refer generally to short, that be generally strand, be generally synthetic polynucleotide, on described polynucleotide length generally but be not to be less than 200 Nucleotide.Term " oligonucleotide " and " polynucleotide " are not mutually exclusive.Above for the same oligonucleotide that also is applicable to fully of the description of polynucleotide.

Term " receptor binding domains ", for naming any native ligand of acceptor, comprises any zone or the derivative of cell adhesion molecule or this type of native ligand, and it retains at least one the qualitative receptor binding capacity to corresponding native ligand.This is defined in other definition and especially comprises the binding sequence of part to above-mentioned acceptor.

" secretory signal sequence " or " signal sequence " refer to encode nucleotide sequence of short signal peptide, described short signal peptide can be used for instructing new synthetic target protein matter through cytolemma, is generally procaryotic inner membrance or inner membrance and adventitia.Thus, target protein matter is secreted to the pericentral siphon of prokaryotic host cell or to substratum as light chain immunoglobulin or heavy chain polypeptide.Signal peptide by the secretory signal sequence coding can be endogenous for host cell, can be maybe external source, comprises that for polypeptide to be expressed be natural signal peptide.Secretory signal sequence generally is present in the aminoterminal of polypeptide to be expressed, and usually in biosynthesizing, in the process of secrete polypeptide from tenuigenin, by enzyme, is removed.Therefore, signal peptide generally is not present in the mature protein product.

The statement " single domain antibody " (sdAb) or " single variable domain (SVD) antibody " refer generally to single variable domain (V _hor V _l) can give the antibody of antigen combination.In other words, single variable domain need to be in order not interact with another variable domain in conjunction with target antigen.The example of single domain antibody includes but not limited to from natural for example, those antibody as camelids (yamma and camel) and selachian (, ginglymostoma cirratum) and carrys out those antibody (Nature (1989) 341:544-546 since the recombination method of people and mouse antibodies; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechno] (2003): 21:484-490; WO2005/035572; WO03/035694; Febs Lett (1994) 339:285-290; WO00/29004; WO02/051870).

As used herein, " therapeutic antibodies " be suffer from or the Mammals of easy infection disease or illness in treatment disease or the effective antibody of illness.Exemplary therapeutic antibodies comprises anti-il-i-beta antibody of the present invention and anti-IL-18 antibody, comprises anti-il-i-beta of the present invention and anti-IL-18 bi-specific antibody, and such antibody, and it comprises rhuMAb4D5

(Carter etc., 1992, Proc.Natl.Acad.Sci.USA, 89:4285-4289, U.S. Patent number 5,725,856); Anti-CD20 antibodies is as U.S. Patent number 5,736,137 in inosculating antibody CD20 " C2B8 ", as U.S. Patent number 5,721,108, chimeric or humanization variant or the Tositumomab of the 2H7 antibody in B1 anti-IL-8 (StJohn etc., 1993, Chest, 103:932 and international publication number WO95/23865); Comprise that the VEGF antibody of humanized and/or affinity maturation is as humanized VEGF antibody huA4.6.1AVASTIN ^tMinterior VEGF antibody (Kim etc., 1992, Growth Factors, 7:53-64, international publication number WO96/30046 and be disclosed in the WO98/45331 on October 15th, 1998); Anti-psca antibody (WO01/40309); Anti-cd40 antibody, comprise S2C6 and humanization variant (WO00/75348) thereof; Anti-CD11a antibody (U.S. Patent number 5,622,700, WO98/23761, Steppe etc., 1991, Transplant Intl.4:3-7 and Hourmant etc., 1994, Transplantation58:377-380); Anti-IgE (Presta etc., 1993, J.Immunol.151:2623-2632 and international publication number WO95/19181); Anti-CD18 (be disclosed in the U.S. Patent number 5,622,700 on April 22nd, 1997 or as be published in the WO97/26912 on July 31st, 1997); Anti-IgE (is disclosed in the U.S. Patent number 5 on February 3rd, 1998,714,338 or be disclosed in the U.S. Patent number 5 on February 25th, 1992,091,313, be published in the WO93/04173 on March 4th, 1993 or be filed in international application no PCT/US98/13410, the U.S. Patent number 5 on June 30th, 1998,714,338); Anti-Apo-2 receptor antibody (being published in the WO98/51793 on November 19th, 1998); Comprise cA2 cDP571 and MAK-195 (consult the U.S. Patent number 5 that is disclosed on September 30th, 1997,672,347, Lorenz etc. 1996, J.Immunol.156 (4): 1646-1653 and Dhainaut etc. 1995, Crit.CareMed.23 (9): 1461-1469) at interior anti-TNF-α antibody; Anti-tissue factor (TF) (the european patent number 0420937B1 that on November 9th, 1994 authorizes); Anti-human γ ₄-β ₇integrin (being published in the WO98/06248 on February 19th, 1998); Anti-EGFR (as being published in chimeric or humanized 225 antibody in the WO96/40210 on December 19th, 1996); Anti-cd 3 antibodies is as OKT3 (being disclosed in the U.S. Patent number 4,515,893 on May 7th, 1985); Anti-CD25 or anti-tac antibody are as CHI-621

with

(consulting the U.S. Patent number 5,693,762 that is disclosed on December 2nd, 1997); (Choy etc. 1996, Arthritis Rheum39 (1): 52-56) as cM-7412 antibody for anti-CD 4 antibodies; (Riechmann etc. 1988, Nature332:323-337 as CAMPATH-1H for anti-CD 52 antibody; Anti-Fe receptor antibody is as Graziano etc. 1995, J.Immunol.155 (10): the M22 antibody for Fc γ RI in 4996-5002; (Sharkey etc. 1995, Cancer Res.55 (23Suppl): 5935s-5945s as hMN-14 for anticancer embryonal antigen (CEA) antibody; The antibody for mammary epithelial cell (Ceriani etc. 1995, the Cancer Res.55 (23): 5852s-5856s that comprise huBrE-3, hu-Mc3 and CHL6; With Richman etc. 1995, Cancer Res.55 (23Supp): 5916s-5920s); In conjunction with the antibody of colon cancer cell, as C242, (Litton etc. 1996, Eur J.Immuno1.26 (1): 1-9); (Ellis etc. 1995, J.1mmunol.155 (2): 925-937) as AT13/5 for anti-CD38 antibody; (Jurcic etc. 1995, Cancer Res55 (23Suppl): 5908s-5910s and CMA-676 or CDP771 as Hu M195 for anti-CD 33 antibody; (Juweid etc. 1995, Cancer Res55 (23Suppl): 5899s-5907s as LL2 or LymphoCide for anti-CD22 antibody; Anti-EpCAM antibody is as 17-1A

anti-GpIIb/IIIa antibody is as ReoPro or c7E3Fab

anti-rsv antibodies is as MEDI-493

anti-CMV antibody as

anti-HIV antibody is as PRO542; Anti-hepatitis antibody is as anti-Hep B antibody

anti-CA 125 antibody OvaRex; Anti-idiotype GD3 epitope antibodies BEC2; Anti-α v β 3 antibody

anti-human renal cell carcinoma antibody is as ch-G250; ING-1; Anti-human 17-IA antibody (3622W94); Anti-human colorectum tumour antibody (A33); Anti-human melanoma antibody R24 for the GD3 Sphingolipids,sialo; Anti-human squamous cell carcinoma (SF-25); And anti-human human leucocyte antigen (HLA) antibody is as Smart ID10 and anti-HLA DR antibody Oncolym (Lym-1).

" target molecule " refers to can be in conjunction with the molecule of target recognition site.Target molecule: the interactional example of target table recognition site comprises antigen: antibody variable domains interacts, acceptor: ligand interaction, part: acceptor interaction, adhesin: adhesin interacts, vitamin H: strepto-biotin protein interaction etc.In one embodiment, target molecule is biological molecule.

Term " treatment significant quantity " refers to the amount for the disease in " alleviating " or " treatment " experimenter or Mammals or the effective present composition of illness.In one embodiment, " treatment significant quantity " be intended to comprise antibody described herein separately or with the amount of other active ingredients combinations, described amount for suppress or reduce IL-1 β and IL-18 and their acceptors in conjunction with being effectively or for treatment or to prevent to have the inflammatory conditions in the experimenter of this demand be effective.

" treatment " refers to that trial changes the clinical intervention of the experimenter's who treats natural process, and can be in order to prevent or to carry out in the clinical pathology process.The desired effects for the treatment of includes but not limited to prophylactic generation or sends out, relaxes any direct or indirect pathology consequence of symptom, minimizing disease, prevention transfer, reduction disease progression degree, improvement or relax disease condition and alleviate or improve prognosis.In some embodiments, antibody of the present invention is used for the progress that delays disease progression or slow down disease.Usually, the treatment of disease or illness relates to alleviating of one or more symptoms relevant to disease or illness or medical problem.In some embodiments, antibody of the present invention or composition can be used for preventing disease in experimenter or Mammals or morbidity or the recurrence of illness.For example, in suffering from the patient of autoimmune disease, antibody of the present invention can be used for prevention or treatment burst.Treat continuously or use the treatment referred on basis every day at least, there is no the interruption of a day or several days in treatment.Intermittent treatment or use or with intermittence form treatment or use to refer to not to be continuous but periodically treatment under state of nature.Treatment plan can be continuous or intermittence herein.

Term " variable " refers to the fact of some section of variable domains extensive different on sequence between antibody.V structural domain mediation antigen combination also limits the specificity of specific antibodies to its specific antigen.Yet mutability is not uniformly distributed on the amino acid span of variable domain.On the contrary, by having, high mutability is called as the shorter zone of " hypervariable region " and its separated geostationary fragment that is called as framework region (FR) forms in the V district.According to Antibody types, hypervariable region in a variable domain can act synergistically to facilitate the formation of antigen binding site on antibody (to consult Kabat etc. with the hypervariable region from another chain, Sequences of Proteins of Immunological Interest, the 5th edition .Public Health Service, National Institutes of Health, Bethesda, MD (1991)).Constant domain is not directly involved in the combination of antibody and antigen usually, but shows different effector functions, as antibody participates in antibody dependent cellular cytotoxicity (ADCC).

As used herein, " variant " or " change " heavy chain refers generally to have the heavy chain of the disulfide bonding ability of reduction, for example, has wherein made at least one cysteine residues can't form disulfide linkage.Preferably, described at least one halfcystine is positioned at the hinge area of heavy chain.

As used herein, term " carrier " is intended to the nucleic acid molecule that finger can be transported another nucleic acid of its connection.One class carrier is " plasmid ", the circular double stranded DNA ring that extra DNA section can be connected into.Another kind of carrier is phage vector.Another kind of carrier is virus vector, wherein extra DNA section can be connected into to viral genome.Self-replicating (bacteria carrier and episome (episomal) the Mammals carrier that for example, there is the bacterium replication orgin) in the host cell that some carrier can import at them.Other carriers (for example non-add body Mammals carrier) can be integrated in the tired group of base of host cell after importing host cell, and with host genome, are copied thus.In addition, some carrier can instruct the expression tired with its base effectively be connected.Examples of such carriers is called to " recombinant expression vector " (or summary be, " recombinant vectors ") herein.Generally speaking, the expression vector used in recombinant DNA technology is generally the form of plasmid.In this manual, because plasmid is the form that carrier is the most commonly used, so " plasmid " and " carrier " is used interchangeably.

Antibody with avidity " selective binding " target molecule significantly higher than its other molecules in conjunction with non-target molecule.Relative combination and/or avidity can be described by art-recognized several different methods, and described method includes but not limited to: enzyme-linked immunosorbent assay (ELISA) and fluorescent activation cell sorting art (FACS).In some embodiments, as measured through ELISA, antibody of the present invention with higher than its in conjunction with non-target molecule the concentration-response binding target molecule at least about 1 logarithm level.

Exemplary antibodies

The soluble human IL-1 β that optional and other molecules are puted together or human il-18 or its fragment can be used as producing the immunogen of antibody.The cell of perhaps or in addition, expressing people IL-I β or human il-18 can be used as immunogen.This type of cell can be maybe by recombinant technology, to have transformed to express the cell of people IL-1 β or human il-18 from natural origin.Other forms of people IL-1 β or human il-18 for the preparation of antibody are apparent for those skilled in the art.

A. polyclonal antibody

Preferably by repeatedly subcutaneous (sc) or intraperitoneal (ip) injection related antigen and adjuvant produce polyclonal antibody in animal.Use difunctional or derivatization reagent (for example maleimidobenzoyl sulfosuccinimide ester (puting together through cysteine residues), N-hydroxy-succinamide (through lysine residue), glutaraldehyde, succinyl oxide, SOCl ₂or R ¹n=C=NR, wherein R and R ¹different alkyl) by related antigen, with having in treating immune species that immunogenic protein puts together, may be useful, described protein is keyhole maple hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor for example.

By combination for example the protein of 100 μ g or 5 μ g or conjugate (respectively for rabbit or mouse) and 3 times of volumes Freund's complete adjuvant and at multidigit point intradermal injection solution, animal is produced to antigen, immunogen conjugates or derivative immune.Approximately, after one month, by the peptide in the Freund's complete adjuvant at multidigit point subcutaneous injection 1/5 to 1/10 original bulk or conjugate, animal is strengthened.After seven to 14 days, to the animal bloodletting and measure the antibody titers of serum.Animal is strengthened until titre reaches plateau.Preferably, by same antigen but be conjugated to different proteins and/or by the conjugate of different cross-linking reagents, animal strengthened.Conjugate can also be with the incompatible generation of protein blend in reconstitution cell.In addition, agglutinant is suitable for strengthening immunne response as alum.

B. monoclonal antibody

Monoclonal antibody can be used first by Kohler etc., 1975, Nature, and the hybridoma method that 256:495 describes produces, or for example, produces by recombinant DNA method (consulting U.S. Patent number 4,816,567).

In hybridoma method, mouse or other appropriate host animals carry out immunity with induction of lymphocyte as described above as hamster or macaque, described lymphocyte produce maybe can produce can specific combination for the antibody of immune protein.Perhaps, can be external to lymphocyte immunity.Use subsequently suitable fusogen, as polyoxyethylene glycol, lymphocyte and cancer cell of bone marrow are merged to produce hybridoma (Goding, 1986, Monoclonal Antibodies:Principles and Practice, 59-103 page (Academic Press)).

The hybridoma of preparation is thus inoculated and cultivated in the suitable culture medium of preferred one or more materials of containing the parental generation cancer cell of bone marrow that suppresses not merge, growing or surviving.For example; if the parental generation cancer cell of bone marrow lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT); substratum for hybridoma can comprise xanthoglobulin, aminopterin and thymidine (HAT substratum) usually so, and these substrates suppress the growth that HGPRT lacks cell.

Preferred hybridoma is effective integration, support the stable high level of selected antibody produced cell to produce antibody and responsive those hybridomas for substratum (as the HAT substratum).Wherein, preferred myeloma cell line is rat bone marrow tumour system, as from can be from Salk Institute Cell Distribution Center, San Diego, those rat bone marrow tumours of the MOPC-21 that Calif.USA obtains and MPC-11 mouse tumor are and can be from U.S. typical case culture center (American Type Culture Collection, Rockville, Md.USA) SP-2 obtained or those rat bone marrow tumour systems of X63-Ag8-653 cell.The different myeloma cell line of human myeloma and mouse-people has also been described for generation of human monoclonal antibodies (Kozbor, 1984, J.Immunol., 133:3001; Brodeur etc., 1987, Monoclonal Antibody Production Techniques and Applications, 51-63 page (Marcel Dekker, Inc., New York)).

Measure its generation for the monoclonal antibody of antigen of substratum of the hybridoma of wherein growing.Preferably, by immunoprecipitation or by external in conjunction with measuring, as the binding specificity of the monoclonal antibody produced by hybridoma is measured in radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Identified produce there is the specificity wanted, after the hybridoma of avidity and/or active antibody, can by the clone by the limiting dilution step carry out subclone and by standard method cultivated (Goding, above).Suitable culture medium for this purpose for example comprises D-MEM or RPMI-1640 substratum.In addition, hybridoma can be grown as ascites tumour in animal body.

By routine immunization sphaeroprotein purification step, for example a-protein-agarose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography from substratum, ascites fluid or serum appropriate separation by the monoclonal antibody of subclone secretion.

Use ordinary method (for example, by use can specific binding coding monoclonal antibody heavy chain and the oligonucleotide probe of the gene of light chain) DNA of monoclonal antibody of encoding can easily separate and check order.Hybridoma can be used as the preferred source of this type of DNA.Once after separating, DNA can be placed in to expression vector, the transfection subsequently of described expression vector to host cell as Bacillus coli cells, ape COS cell, Chinese hamster ovary (CHO) cell or otherwise can not produce in the myeloma cell of immunoglobulin (Ig) protein, to realize synthetic monoclonal antibody in recombinant host cell.The restructuring of antibody produces and will make a more detailed description hereinafter.

In another embodiment, antibody or antibody fragment can be described in McCafferty etc. from use, and 1990, Nature separates in the antibody phage library that the technology in 348:552-554 produces.Clackson etc., 1991, Nature, 352:624-628 and Marks etc., 1991, J.Mol.Bio1., 222:581-597 has described respectively the use phage library and has separated mouse and people's antibody.Follow-up publication has been described by chain and has been reorganized (Marks etc., 1992, Bio/Technology, 10:779-783), and combination is infected and the interior restructuring of body is used as the strategy (Waterhouse etc. that build very large phage library, 1993, Nuc.Acids.Res., 21:2265-2266) produce people's antibody of high-affinity (nM level).Therefore, these technology are the viable options for separating of traditional monoclonal antibody hybridoma technology of monoclonal antibody.

For example, also can substitute by the encoding sequence of employment heavy chain and light chain constant domain muroid sequence (U.S. Patent number 4,816,567 of homology; Morrison, etc., 1984, Proc.Natl.Acad.Sci.USA, 81:6851) or for example, by all or part of encoding sequence of immunoglobulin coding sequence covalent attachment NIg material (, protein domain) is carried out to modifying DNA.

Usually this type of NIg material is substituted to the constant domain of antibody, or an antigen binding site of the variable domain of alternative antibody, comprise the antigen binding site with a kind of antigen-specific and the chimeric bivalent antibody with antigen binding site of another different antigen-specifiies with generation.

C. humanization and people's antibody

Humanized antibody has one or more amino-acid residues from inhuman source.Inhuman amino-acid residue is commonly referred to " input " residue and usually takes from " input " variable domain.Generally can be according to Winter and colleague's thereof method (Jones etc., 1986, Nature, 321:522-525; Riechmann etc., 1988, Nature, 332:323-327; Verboeyen etc., 1988, Science, 239:1534-1536), by the corresponding sequence by rodent CDR or CDR sequence replacing people antibody, carry out humanization.Therefore, this type of " humanization " antibody is chimeric antibody (U.S. Patent number 4,816,567), wherein basically is less than whole person's variable domain and is substituted by the corresponding sequence from inhuman species.In fact, humanized antibody is people's antibody normally, and some of them CDR residue and possible some FR residues are by from inhuman, and for example in rodent animal antibody, the residue in similar site substitutes.

Being ready to use in people's light chain of generation humanized antibody and the selection of heavy chain variable domain is very important for reducing antigenicity.According to so-called " the suitableeest " method, for the whole library screening of known people's variable domain sequence the sequence of rodent animal antibody variable domain.Subsequently will be with the immediate human sequence of rodentine sequence as the people's framework (FR) (Sims etc., 1987, J.Immunol., the 151:2296 that are humanized antibody; Chothia etc., 1987, J.Mol.Biol., 196:901).Other method is used the specific framework from the consensus sequence of the specific subgroup of the light chain of everyone antibody or heavy chain.Same architecture can be used for several different humanized antibodies (Carter etc., 1992, Proc.Natl.Acad.Sci.USA, 89:4285; Presta etc., 1993, J.Immunol., 151:2623).

It is also important that humanized antibody retains the high-affinity of antigen and other useful biological properties.For this purpose, according to preferred method, by the method for analyzing parental array and multiple notional humanization product with the three-dimensional model parent and humanized sequence, prepare humanized antibody.Three-dimensional immunoglobulin (Ig) model is normally obtainable and know to those skilled in the art.Can obtain such computer program, its explanation and the possible three-dimensional conformation structure of showing selected candidate's immunoglobulin sequences.The check of these displayings allows to analyze residue may act in the function of candidate's immunoglobulin sequences, and analyzing influence candidate immunoglobulin (Ig) is in conjunction with the residue of the ability of its antigen.Like this, thereby can select and combine the antibody feature that the FR residue is realized expectation from acceptor and list entries, as the avidity that target antigen is raise.Generally speaking, the CDR residue directly and the most basically participates in affecting the antigen combination.

Perhaps, can produce now such transgenic animal (for example, mouse), after described transgenic animal immunity in the situation that do not produce the repertoire that endogenous immunoglobulin (Ig) can produce people's antibody.For example, the homozygous disappearance of having described heavy chain of antibody joining region (JH) gene in chimeric and germ line mutation body mouse has caused the inhibition fully that endogenous antibody is produced.Shifting ethnic group in this type of germ line mutation body mouse is that the immunoglobulin gene array can cause producing people's antibody after antigen is attacked.Such as consulting Jakobovits etc., 1993, Proc.Natl.Acad.Sci.USA, 90:2551; Jakobovits etc., 1993, Nature, 362:255-258; Bruggermann etc., 1993, Year in Immuno., 7:33; With Duchosal etc., 1992, Nature355:258.People's antibody also can come autophagy mattress body display library (Hoogenboom etc., 1991, J.Mol.Biol., 227:381; Marks etc., J.Mol.Biol., 1991,222:581-597; Vaughan etc., 1996, Nature Biotech14:309).

I. chimeric and humanized antibody

In certain embodiments, antibody provided herein is chimeric antibody.Some chimeric antibody for example is described in U.S. Patent number 4,816,567; With Morrison etc., Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)) in.In an example, chimeric antibody comprises inhuman variable region (for example, the variable region as monkey from mouse, rat, hamster, rabbit or non-human primate) and human constant region.In another example, chimeric antibody is " classification conversion " antibody, and wherein classification or subclass change from classification or the subclass of its parental antibody.Chimeric antibody comprises its Fab.

In certain embodiments, chimeric antibody is humanized antibody.Usually, the humanization non-human antibody, to reduce the immunogenicity to the people, and retain parent non-human antibody's specificity and avidity.Generally speaking, humanized antibody comprises one or more variable domains, HVR wherein, and for example, CDR (or its part) comes from the non-human antibody, and FR (or its part) is from human antibody sequence.Humanized antibody optionally also will comprise at least part of human constant region.In some embodiments, use for example, corresponding residue from non-human antibody's (, the antibody in HVR residue source) to substitute some the FR residues in humanized antibody, for example, to recover or to improve antibodies specific or avidity.

The method of humanized antibody and generation humanized antibody for example is summarized in Almagro and Fransson, in Front.Biosci.13:1619-1633 (2008), and such as also being described in Riechmann etc., Nature332:323-329 (1988); Queen etc., Proc.Nat ' l Acad.Sci.USA86:10029-10033 (1989); U.S. Patent number 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri etc., Methods36:25-34 (2005) (describing SDR (a-CDR) transplants); Padlan, Mol.Immunol.28:489-498 (1991) (describing " resurfacing "); Dall ' Acqua etc., Methods36:43-60 (2005) (describing " FR reorganization "); With Osbourn etc., Methods36:61-68 (2005) and Klimka etc., Br.J.Cancer, in 83:252-260 (2000) (describing " pathfinder selection " method to FR reorganization).

Can be used for humanized people's framework region includes but not limited to: the framework region (for example consulting the J.Immunol.151:2296 such as Sims (1993)) that uses " the suitableeest " method to select; Framework region from the consensus sequence of the specific subgroup from human antibody light chain or variable region of heavy chain (is for example consulted the Proc.Natl.Acad.Sci.USA such as Carter, 89:4285 (1992); With the J.Immunol. such as Presta, 151:2623 (1993)); People's ripe (body sudden change) framework region or people's germline framework region (for example consulting Almagro and Fransson, Front.Biosci.13:1619-1633 (2008)); For example, with the framework region that comes self-sizing FR library (consulting Baca etc., J.Biol.Chem.272:10678-10684 (1997) and Rosok etc., J.Biol.Chem.271:22611-22618 (1996)).

Ii. people's antibody

In certain embodiments, antibody provided herein is people's antibody.People's antibody can be used multiple technologies known in the art to produce.People's antibody general description is in van Dijk and van de Winkel, and Curr.Opin.Pharmacol.5:368-74 (2001) and Lonberg, in Curr.Opin.Immunol.20:450-459 (2008).

Can be by turning the tired animal of base, using the original preparation of immunity people antibody, describedly turn the tired animal of base and modified to respond antigen and attack and produce complete people's antibody or there is the complete antibody of people variable region.This type of animal is usually containing all or human immunoglobulin gene's seat part, and it has replaced endogenous immunoglobulin loci or its and has been present in the outer or random integration of karyomit(e) in the karyomit(e) of animal.In this type of transgenic mice, endogenous immunoglobulin loci is usually by inactivation.Summary obtains the method for people's antibody from transgenic animal, consults Lonberg, Nat.Biotech.23:1117-1125 (2005).Also can consult, for example describe XENOMOUSE ^tMthe U.S. Patent number 6,075,181 and 6,150,584 of technology; Describe the U.S. Patent number 5,770,429 of technology; K-M is described

the U.S. Patent number 7,041,870 of technology and description

the U.S. Patent Application Publication No. US 2007/0061900 of technology).Can also to the people variable region of the complete antibody by this class animal generation, further modify by for example combining different human constant regions.

Can also produce people's antibody by the method based on hybridoma.Human myeloma and the different myeloma cell line of mouse-people for generation of human monoclonal antibodies have been described.(for example consult Kozbor J.Immunol., 133:3001 (1984); Brodeur etc., Monoclonal Antibody Production Techniques and Applications, 51-63 page (Marcel Dekker, Inc., New York, 1987); With Boerner etc., J.Immunol., 147:86 (1991)).The people's antibody produced through the human B-lymphocyte hybridoma technology also is described in Li etc., and Proc.Natl.Acad.Sci.USA, in 103:3557-3562 (2006).Extra method comprises and for example is described in U.S. Patent number 7,189,826 (described and produce the mono-clonal human IgM antibody from hybridoma cell line) and Ni, Xiandai Mianyixue, 26 (4): those methods in 265-268 (2006) (having described people-people's hybridoma).People's hybridoma technology (trioma technology) also is described in Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): in 185-91 (2005).

The Fv clone variable domain sequence that can also be selected from the phage display library in people source by separation produces people's antibody.This type of variable domain sequence can combine with people's constant domain of wanting subsequently.From antibody library, select the technology of people's antibody that description is arranged hereinafter.

D. multi-specificity antibody

Multi-specificity antibody has at least two kinds of binding specificities of synantigen not.Although this quasi-molecule usually can be only for example, in conjunction with two kinds of antigens (, bi-specific antibody BsAb), there is extra specific antibody as also included by this statement used herein as three-specific antibody.The example of BsAb comprises that the antigen binding site had for IL-1 β reaches those BsAb for another antigen binding site of IL-18.In some embodiments, BsAb comprises for the first binding specificity of IL-1 β or IL-18 with for the second binding specificity of the activated receptor with tenuigenin ITAM motif.ITAM motif structure has two tyrosine that separated by 9-11 amino acid interval.General consensus sequence is YxxL/I (x) _6-8yxxL (Isakov, N., 1997, J.Leukoc.Biol., 61:6-16).The exemplary activated acceptor comprises Fc ε RI, Fc γ RIII, Fc γ RI, Fc γ RIIA and Fc γ RIIC.Other activated receptors comprise that for example CD3, CD2, CD10, CD161, DAP-12, KAR, KARAP, Fc ε RII, Trem-1, Trem-2, CD28, p44, p46, B-cell receptor, LMP2A, STAM, STAM-2, GPVI and CD40 (for example consult, Azzoni, Deng, 1998, J.Immunol.161:3493; Kita, etc., 1999, J.Immunol.162:6901; Merchant, etc., 2000, J.Biol.Chem.74:9115; Pandey, etc., 2000, J.Biol.Chem.275:38633; Zheng, etc., 2001, J.Biol.Chem.276:12999; Propst, etc., 2000, J.Immunol.165:2214).

In one embodiment, BsAb comprises for the first binding specificity of IL-1 β with for the second binding specificity of IL-18.Bi-specific antibody can be used as full length antibody or antibody fragment (as F (ab ') ₂bi-specific antibody) prepare.Can also prepare as pestle mortar structure or hinge-less antibody by bi-specific antibody.Bi-specific antibody, at Segal etc., is summarized in 2001, J.Immunol.Methods248:1-6.

Method for the preparation of bi-specific antibody is known in this area.The bi-specific antibody that tradition produces total length is based on two coexpressions that heavy chain immunoglobulin-light chain is right, and two chains have different specificity (Millstein etc., 1983, Nature, 305:537-539).Due to the random assignment of heavy chain immunoglobulin and light chain, these hybridomas (quadroma) produce the possible mixture of 10 kinds of different antibodies molecules, and a kind of correct dual specific structure that has is wherein only arranged.The suitable trouble of the purifying of the general correct molecule completed by the affinity chromatography step and productive rate are low.Similarly method is disclosed in WO93/08829 and Traunecker etc., and 1991, EMBO J., in 10:3655-3659.

According to different methods, the antibody variable domains (antibody-antigen binding site) with binding specificity of wanting merges with immunoglobulin (Ig) constant domain sequence.Fusion can be merged with the heavy chain immunoglobulin constant domain, comprises at least part of hinge, CH2 ,He CH3 district.Preferably have containing the first CH (CH1) in conjunction with essential site for light chain, it is present at least one fusion.By the coding heavy chain immunoglobulin syzygy DNA and, if required, the DNA of light chain immunoglobulin is inserted in independent expression vector, and cotransfection is to the appropriate host biology.When three kinds of antibody chains that use different ratios in structure provide optimum productive rate, this adjusts in embodiments in the mutual ratio of three kinds of antibody fragments great handiness is provided.Yet, when at least two antibody chains produce high yield with the expression of same ratio or when ratio does not have special significance, in an expression vector, the encoding sequence of two of insertions or all three antibody chains is feasible.

In another embodiment of present method, bi-specific antibody is comprised of (the second binding specificity is provided) the heterozygosis heavy chain immunoglobulin that has the first binding specificity on one arm and the heterozygosis heavy chain immunoglobulin-light chain on another arm.In the bispecific molecule of discovery due to half only, exist light chain immunoglobulin that easy separation method is provided, tired this this unsymmetrical structure contributes to dual specific compound and the undesired immunoglobulin chain composition wanted to separate.The method is disclosed in WO94/04690.Produce other details of bi-specific antibody method such as consulting Suresh etc., 1986, Methods in Enzymology.121:210.

According to the other method of describing in WO96/27011, can design interface between a pair of antibody molecule so that the per-cent of the heterodimer reclaimed maximizes from the recombinant cell culture thing.At least part of CH3 structural domain that preferred interface comprises the antibody constant domain.In this method, for example, with larger side chain (tyrosine or tryptophane), replace from one or more p1 amino acid side chain on first antibody molecule interface.By for example, replacing with less amino acid (L-Ala or Threonine), large amino acid side chain produces on second antibody molecule interface and the complementation " chamber " of the same or similar size of bulky side chain.This provides for increasing heterodimer and has been better than the mechanism of other undesired end products as the dimeric productive rate of homotype.

Bi-specific antibody comprises crosslinked or " different conjugate " antibody.For example, one of antibody in different conjugate can with avidin coupling, another and vitamin H coupling.For example, this antibody-like has been proposed by the undesired cell of immune system cell target (U.S. Patent number 4,676,980), and the treatment (WO91/00360, WO92/200373 and EP03089) of infecting for HIV.Can use any cross-linking method easily to prepare different conjugate antibody.Suitable linking agent is known in this area, and in for example being disclosed in U.S. Patent number 4,676,980 together with a large amount of crosslinking technological.

Also considered to have over two kinds of antibody of tiring.For example, according to Tutt etc., 1991, J.Immunol.147:60 can prepare three-specific antibody.

The engineered antibody with three or more functional antigen binding site that comprises " octopus antibody " can also be included in this (for example consulting US2006/0025576A1).

Antibody herein or fragment also comprise " double-acting FAb " or " DAF " (for example the consulting US2008/0069820) contained in conjunction with the antigen binding site of IL-1 β and IL-18.

E. the antibody that there is variable hinge area

Antibody of the present invention also can comprise the variant heavy chain, for example is filed in described in the patent application serial numbers 10/697,995 on October 30th, 2003.Antibody containing the variant heavy chain comprises the change that at least one forms the cysteine residues of disulfide linkage, makes cysteine residues can't form disulfide linkage.On the one hand, described halfcystine (therefore, this type of hinge area refers to " variant hinge area " herein and can refer to " hingless " again) in the hinge area of heavy chain.

In some respects, this immunoglobulin like protein lack usually can be in intermolecular (between two heavy chains) or molecule (between two cysteine residues in Single polypeptide chain) form the repertoire of the heavy chain cysteine residues of disulfide linkage.Generally also preferably, by the cysteine residues after changing (, causing and can't form disulfide linkage) disulfide linkage that forms is such disulfide linkage, when not existing in antibody, described disulfide linkage can not cause the normal physical chemistry of immunoglobulin (Ig) and/or the essence of biological property to be lost.Preferably, but not necessarily, causing the cysteine residues that can't form disulfide linkage is the halfcystine of the hinge area of heavy chain.

Antibody with variant heavy chain or variant hinge area is generally by express antibody in host cell, and described antibody is retrieved to produce from host cell, in described antibody, removed at least one, at least two, at least three, at least four or between between the heavy chain between two to 11 disulfide linkage.Can express described antibody from the polynucleotide of encoding antibody, the variant heavy chain that described antibody comprises the disulfide linkage ability with minimizing reclaims described antibody subsequently from the host cell that comprises polynucleotide.Preferably, the variant hinge area that described heavy chain comprises heavy chain immunoglobulin, wherein make at least one halfcystine of described variant hinge area can't form disulfide linkage.

Also expection can make any halfcystine in heavy chain immunoglobulin all can't form disulfide linkage, similar with hinge cysteine described herein, as long as this type of changes, basically can not reduce the biological function of immunoglobulin (Ig).For example, IgM and IgE lack hinge area, but each contains extra heavy chain structural domain; As long as the biological function of the antibody that basically can not reduce heavy chain and/or comprise heavy chain, can make in the method for the invention at least one (all halfcystines in some embodiments) in the halfcystine of heavy chain can't form disulfide linkage.

The heavy chain hinge cysteine is known in this area, Kabat for example, and 1991, " Sequences of proteins of immunological interest, " above describes.As known in the art, the number of hinge cysteine changes according to classification or the subclass of immunoglobulin (Ig).For example consult Janeway, 1999, Immunobiology, the 4th edition, (Garland Publishing, NY).For example, in human IgG I, two hinge cysteine are separated by two proline(Pro), and these match with its counterpart usually on the intermolecular disulfide bond of adjacent heavy chain.Other examples comprise IgG2, the IgG3 that contains 11 hinge cysteine that contains 4 hinge cysteine and the IgG4 that contains 2 hinge cysteine.

Therefore, method of the present invention is included in the heavy chain immunoglobulin that in host cell, expression comprises the variant hinge area, wherein make at least one halfcystine of variant hinge area can't form disulfide linkage, allow heavy chain and light chain to form mixture to form biologically activated antibody, and reclaim antibody from host cell.

Optional embodiment comprises these situations: wherein make at least 2,3 or 4 halfcystines can't form disulfide linkage; Wherein make from approximately two can't form disulfide linkage to about 11 halfcystines; And wherein make all halfcystines of variant hinge area can't form disulfide linkage.

The antibody be comprised of light chain and heavy chain of the present invention that the method according to this invention produces can be by single polynucleotide or coded by independent polynucleotide.

Can can't form disulfide linkage by several different methods known in the art or by the halfcystine that apparent those methods make common participation disulfide linkage form after considering standard described herein to those skilled in the art.For example, can replace hinge cysteine as the Serine that can't form disulfide linkage with another amino acid.Can realize amino acid whose substituting as the directed mutagenesis of the nucleotide sequence of the hinge area to be finished of encoding by standard molecular biological technique.Suitable technology comprises and is described in Sambrook etc., 1989, Molecular Cloning:A Laboratory Manual, those technology in second edition.The oligonucleotide that comprises the composite coding hinge area for generation of the other technologies of the immunoglobulin (Ig) with variant hinge area, wherein use and substitute the codon that amino acid whose codon replaces treating alternative halfcystine.In the time of suitably, this oligonucleotide can be connected to subsequently and comprise the carrier main chain of other suitable antibodies sequences as variable region and Fc sequence.

In another embodiment, can lack hinge cysteine.Realize aminoacid deletion by standard molecular biological technique as the directed mutagenesis of the nucleotide sequence of the hinge area to be finished of encoding.Suitable technology comprises and is described in Sambrook etc., those technology above.Other technologies for generation of the immunoglobulin (Ig) with variant hinge area comprise the synthetic oligonucleotide that comprises coding hinge area sequence, have lacked the codon of halfcystine to be finished in described hinge area.In the time of suitably, this oligonucleotide can be connected to subsequently and comprise the carrier main chain of other suitable antibodies sequences as variable region and Fc sequence.

F. the bi-specific antibody that uses " projection in chamber " strategy to form

In some embodiments, use " projection in chamber " strategy to form bi-specific antibody of the present invention, described strategy is also referred to as for transforming interface between the first and second polypeptide " the pestle mortar structure " with different oligomerization.At least part of CH3 structural domain that preferred interface comprises the antibody constant domain.Reported that in the CH3 of Fc sequence structural domain " pestle mortar structure " sudden change has greatly reduced the formation (such as consulting Merchant etc., 1998, Nature Biotechnology, 16:677-681) of homodimer.Build " projection " by the p1 amino acid side chain of for example, replacing on the first polypeptide interface with larger side chain (tyrosine or tryptophane).Optionally by for example, replacing with less amino acid (L-Ala or Threonine), large amino acid side chain produces on the second polypeptide interface and the complementation " chamber " of the same or similar size of projection.While having the projection of correct position and size or chamber on the interface of the first or second polypeptide, only need on adjacent interfaces, transform respectively corresponding chamber or projection gets final product.Can be by synthetic method as the nucleic acid that changes coded polypeptide or synthesize to produce projection or chamber by peptide.For other descriptions of pestle mortar structure, consult U.S. Patent number 5,731,168; 5,807,706; 5,821,333.

In some embodiments, use " pestle mortar structure " technology to promote different dimerization for example, to produce the anti-Fc γ of total length dual specific RIIB and anti-" activated receptor " (IgER) antibody.In one embodiment, prepare anti-Fc γ IIB component (for example p5A6.11. pestle shape structure) by introduce " pestle shape structure " sudden change (T366W) to the Fc district, and the construct for preparing anti-IgER component (for example p22E7.11. mortar shape structure) by introducing " mortar shape structure " sudden change (T366S, L368A, Y407V).In another embodiment, by method as disclosed herein or by Merchant etc., (1998), above, or U.S. Patent number 5,731,168; 5,807,706; 5,821,333 disclosed method Xiang Qi Fc districts introduce " mortar shape structure " sudden change and prepare anti-Fc γ IIB component (as p5A6.11. mortar shape structure), and introduce the construct that " pestle shape structure " sudden change prepares anti-IgER component (as p22E7.11. pestle shape structure) in Qi Fc district.

Use general method of the different polymer of " projection in chamber " strategy preparation to be included in the polynucleotide of expressing coding the first polypeptide in or independent host cell, described the first polynucleotide are changed from original polynucleotide the projection of encoding, and second polynucleotide of expressing coding the second polypeptide, described the second polynucleotide are changed from original polynucleotide the chamber of encoding.Polypeptide is to express like this, expresses in common host cell and reclaim different polymer from the host cell culture, or expressing in independent host cell, and recovery and purifying, form subsequently different polymer.In some embodiments, the different polymer of formation is poly antibody, for example bi-specific antibody.Also consult the U.S. Patent Application Serial Number 13/092,708 of submitting on April 22nd, 2011.

In some embodiments, antibody combination pestle mortar structure strategy of the present invention and variant hinge area construct produce hingless bi-specific antibody.

G. immunoconjugates

The immunoconjugates that the present invention also provides the anti-il-i-beta antibody that comprises herein and/or anti-IL-18 antibody and one or more cytotoxic agents to put together, described cytotoxic agent for example, as chemotherapeutic or medicine, growth inhibitor, toxin (enzyme activity toxin of archon, bacterium, fungi, plant or animal-origin or its fragment) or radio isotope.

In one embodiment, immunoconjugates is antibody-drug conjugates (ADC), wherein antibody and one or more medicines are puted together, described medicine includes but not limited to that maytansinoid (consults U.S. Patent number 5,208,020,5,416,064 and European Patent EP0425235B1); Auristatin (MMAE and MMAF) (consulting U.S. Patent number 5,635,483 and 5,780,588 and 7,498,298) as monomethylauristatin drug moiety DE and DF; Dolastatin; The calicheamicin or derivatives thereof (is consulted U.S. Patent number 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman etc., Cancer Res.53:3336-3342 (1993); With Lode etc., Cancer Res.58:2925-2928 (1998)); Anthracycline antibiotics (is consulted Kratz etc., Current Med.Chem.13:477-523 (2006) as daunomycin or Dx; Jeffrey etc., Bioorganic & Med.Chem.Letters16:358-362 (2006); Torgov etc., Bioconj.Chem.16:717-721 (2005); Nagy etc., Proc.Natl.Acad.Sci.USA97:829-834 (2000); Dubowchik etc., Bioorg.& Med.Chem.Letters12:1529-1532 (2002); King etc., J.Med.Chem.45:4336-4343 (2002); With U.S. Patent number 6,630,579); Methotrexate; Vindesine; Taxan is as docetaxel, taxol, La Luotasai (larotaxel), for Si Tasai (tesetaxel) and Ao Tasai (ortataxel); Trichothecene; And CC1065.

In another embodiment, immunoconjugates comprises the antibody of puting together with enzyme activity toxin or its fragment as described herein, described enzyme activity toxin or its fragment include but not limited to diphtheria A chain, the non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa (Pseudomonas aeruginosa)), ricin A chain, abrin A chain, modeccin A chain, the α sarcina, aleurites fordii protein, oleanolic acid protein, dyers' grapes (Phytolaca americana) protein (PAPI, PAPII and PAP-S), balsam pear (momordica charantia) inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, spend more white tree toxalbumin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene (tricothecenes).

In another embodiment, immunoconjugates comprises the antibody of puting together to form the radioactivity conjugate as described herein and radioactive atom.Can obtain multiple radio isotope for generation of the radioactivity conjugate.Example comprises At ²¹¹, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and Lu radio isotope.When using the radioactivity conjugate to detect, it can comprise the radioactive atom for scintillation method research, for example tc99m or I123 or for nucleus magnetic resonance (NMR) imaging (also referred to as MRI (magnetic resonance imaging), mri) spin labeling, also as iodine 123, iodine 131, indium 111, fluorine 19, carbon 13, nitrogen 15, oxygen 17, gadolinium, manganese or iron.

Can use the conjugate of multiple bifunctional protein coupling agent Dispersal risk and cytotoxic agent, described bifunctional protein coupling agent is as N-succinimido-3-(2-sulfo-pyridine) propionic ester (SPDP), amber imines acyl-4-(N-maleimide ylmethyl (maleimidomethyl)) hexanaphthene-1-carboxylate salt (SMCC), 2-imino-thiophene (iminothiolane, IT), the dual-function derivative of imidoester is (as dimethyl adipimide hydrochloride (dimethyl adipimidate HCl), active ester (as two succinyl-suberates), aldehyde (as glutaraldehyde), the diazido compound is (as two (p-azido benzoyl) hexanediamine (bis (p-azido benzoyi) hexanediamine), two diazonium radical derivatives (as bis-(p-diazoniumbenzoyl)-ethylenediamine)), vulcabond is (as toluene 2, the 6-vulcabond) and two active fluorine cpd (as 1, 5-bis-fluoro-2, the 4-dinitrobenzene).For example, can be as Vitetta etc., describe and prepare the ricin immunotoxin in Science238:1098 (1987).The methyl diethylene triaminepentaacetic acid(DTPA) of carbon 14 marks (1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid, MX-DTPA) is the exemplary sequestrant that radioactive nuleus thuja acid and antibody are puted together.Consult WO94/11026.Joint can be " can cut joint " that is conducive to release cells toxin medicine in cell.For example, can use the responsive joint of acid-sensitive sense joint, peptase, photaesthesia joint, dimethyl joint or containing joint (Chari etc., the Cancer Res.52:127-131 (1992) of disulphide; U.S. Patent number 5,208,020).

Immunoconjugates or ADC are clearly contained but this type of conjugate of being not limited to prepare with linking agent herein, described linking agent includes but not limited to commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo--EMCS, sulfo--GMBS, sulfo--KMUS, sulfo--MBS, sulfo--SIAB, sulfo--SMCC, and sulfo--SMPB, and SVSB (succinimide-(4-vinyl sulfonic acid) benzoic ether) (succinimidyl-(4-vinylsulfone) benzoate), it is for example from Pierce Biotechnology, Inc., Rockford, IL., U.S.A can buy.

II. carrier, host cell and recombination method

The present invention also provides the polynucleotide of the separation of antibody as disclosed herein of encoding, the carrier that comprises polynucleotide and host cell and for generation of the recombinant technology of antibody.

Restructuring for antibody produces, and separates the polynucleotide of encoding antibody and is inserted into for further cloning (DNA amplification) or the replicable vector for expressing.Use ordinary method, for example by use can the specific binding encoding antibody the tired oligonucleotide probe of base can easily separate and the DNA of the encoding antibody that checks order.Many carriers are obtainable.Carrier component generally includes but is not limited to following one or more: signal sequence, replication orgin, one or more marker gene, enhancer element, promotor and transcription termination sequence.

(i) signal sequence element

The antibody of the present invention generation of not only can directly recombinating, can also be produced as the fusion antibody with heterologous antibody.In one embodiment, heterologous antibody is signal sequence or other antibody that have the specificity cleavage site at the N-terminal of mature protein or antibody.The signal sequence of (by the signal peptidase cutting) is preferably identified and processed to selected allos signal sequence by host cell.For None-identified and process for the prokaryotic host cell of natural antibody signal sequence, by the prokaryotic signal sequence substitution signal sequence that for example is selected from alkaline phosphatase, penicillinase, 1pp or heat-stable toxin II lead.For yeast secretary, can be by the signal replacing natural signals sequence of describing in for example yeast invertase lead, alpha factor lead (comprising yeast (saccharomyces) and kluyveromyces (Kluyveromyces) alpha factor lead) or acid p'tase lead, C.albicans glucoamylase lead or WO90/13646.In mammalian cell expression, mammalian signal sequence and viral secretory lead, for example herpes simplex gD signal is obtainable.The DNA of encoding antibody will be connected in the DNA frame of this type of prosoma.

In another embodiment, antibody produces and can occur in the tenuigenin of host cell, and does not therefore need to exist secretory signal sequence in each cistron.In this case, light chain immunoglobulin and heavy chain express, fold and assemble to form the functional immunity sphaeroprotein in tenuigenin.Some host strain (for example intestinal bacteria trxB bacterial strain) provides and is beneficial to the tenuigenin condition that disulfide linkage forms, and allows thus the correct folding and assembling (Proba and Plukthun, 1995, Gene, 159:203) of expressed protein subunit.

(ii) replication orgin component

The cloning and expression carrier contains the nucleotide sequence that can make carrier copy in one or more selected host cells.Usually, in cloning vector, this sequence is to make carrier be independent of the sequence that host chromosome DNA copies, and comprises replication orgin or autonomously replicating sequence.This type of sequence for various bacteria, yeast and virus is known.Replication orgin from plasmid pBR322 is suitable for most of gram negative bacterium, 2 μ plasmid starting points are suitable for yeast, and multiple viral starting point (SV40, polyoma, adenovirus, VSV or BPV) is useful for the cloning vector in mammalian cell.Usually, the replication orgin component is unwanted (only because it contains early promoter, so usually use the SV40 starting point) for mammalian cell.

(iii) Select gene component

The cloning and expression carrier can contain Select gene, also referred to as selected marker.Typical Select gene coding (a) is given microbiotic or other toxin, the protein of penbritin, Liu Suanyan NEOMYCIN SULPHATE, methotrexate or tetracyclin resistance for example, (b) protein that the extra-nutrition defective lacks, or the protein of the key nutrition that can't obtain from complex medium (c) is provided, as the gene of the D-alanine racemase of coding genus bacillus (Bacilli).

An example of selection scheme utilizes medicine to stop the growth of host cell.Those cells that utilize heterologous gene successfully to transform produce the protein of giving drug resistance and therefore survive in selection scheme.The example of this type of dominant selection is used medicine Liu Suanyan NEOMYCIN SULPHATE, mycophenolic acid and Totomycin.

For another example of the suitable selected marker of mammalian cell, be to make it possible to identify those of absorbing antigen nucleic acid of having the ability, as DHFR, thymidine kinase, metallothionein(MT)-I and metallothionein(MT)-II, preferably primates metallothionein gene, adenosine deaminase, guanylic acid decarboxylase etc.

For example, conversion has the cell of DHFR Select gene at first by all transformants are cultivated and identified in the substratum of the competitive antagonist methotrexate (Mtx) containing DHFR.When using wild-type DHFR, the appropriate host cell is Chinese hamster ovary (CHO) clone that lacks the DHFR activity.

Perhaps, can by containing to selected marker as for example, Growth of Cells in the substratum of the selective agent of aminoglycoside antibiotic (kantlex, Liu Suanyan NEOMYCIN SULPHATE or G418) select with encoding antibody, wild-type dhfr protein matter and another selected marker as glucosaminide 3 '-DNA sequence dna of phosphotransferase (APH) transforms or the host cell of cotransformation (especially containing endogenous DHFR wild-type host), referring to for example United States Patent (USP) 4,965,199.

Suitable Select gene for yeast is the trp1 gene (Stinchcomb etc., 1979, Nature, 282:39) that is present in yeast plasmid YRp7.The trp1 gene provides selected marker for the yeast mutants bacterial strain that lacks energy for growth in tryptophane (as ATCC No.44076 or PEP4-1.Jones, 1977, Genetics, 85:12).The existence of trp1 damage in the yeast host cell genome provides effective environment subsequently, and it grows to detect conversion for the situation by lacking tryptophane.Similarly, for example, with the yeast strain (bacterial strain that, there are ATCC accession number 20,622 or 38,626) of carrying the complementary Leu2 shortage of the tired known plasmid of Leu2 base.

In addition, the carrier from 1.6 μ m cyclic plasmid pKD1 can be used for transforming kluyveromyces.Perhaps, reported the expression system of K.lactis as extensive generation recombinant bovine rennin.Consult Van den Berg, 1990, Bio/Technology, 8:135.The suitable multiple copied expression vector of secreting ripe recombination human serum albumin for the kluyveromyces industrial strain is also disclosed.Consult Fleer etc., 1991, Bio/Technology, 9:968-975.

(iv) promotor component

The cloning and expression carrier generally contains promotor host living beings identification and that effectively be connected with antibody nucleic acid.The promotor that prokaryotic hosts is applicable to using comprises that phoA promotor, β-lactamase and Lac operon system, alkaline phosphatase, tryptophane (trp) promoter systems, hybrid promoter are as the tac promotor.Yet other known bacterium promotors are also suitable.The promotor of using in bacterial system also comprises Shine-Dalgarno (S.D.) sequence effectively be connected with the DNA of encoding antibody.

For Eukaryotic promoter sequence, be known.In fact all eukaryotic genes have be positioned at its transcription initiation site upstream approximately 25 to 30 base places be rich in the AT district.Another sequence that is found in 70 to 80 base places, the initial upstream of many genetic transcriptions is the CNCAAT district, and wherein N can be any Nucleotide.3 ' the end at most of eukaryotic gene is the AATAAA sequence, and described sequence is for add the signal of poly A tail to 3 ' end of encoding sequence.All these sequences all are suitable for inserting in carrier for expression of eukaryon.

The example that is used for the suitable promoter sequence of yeast host comprises 3-phosphoglycerate kinases or other glycolytic ferments, as the promotor of Hydratase, phosphoenolpyruvate, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, glucose phosphate isomerase and glucokinase.

Other Yeast promoters (it is to have the inducible promoter of being controlled the additional advantage transcribe by growth conditions) are the promoter regions of the enzyme of alcohol dehydrogenase 2, different cell pigment (isocytochrome) C, acid p'tase, degrading enzyme, metallothionein(MT), glyceraldehyde-3-phosphate dehydrogenase and responsible maltose and the galactose utilization relevant to nitrogen metabolism.Suitable carrier and promotor for yeast expression also are described in EP73,657.The yeast enhanser is also together advantageously used with Yeast promoter.

Transcribe and be subject to following promoter regulation from the antibody of carrier in mammalian cell, for example, available from as polyomavirus, bird pox virus, adenovirus (as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably be the promotor of the tired group of viral base of simian virus 40 (SV40), available from the allos mammalian promoter, the promotor of actin promoter or immunoglobulin promoter for example, available from the promotor of heat-inducible promoter, as long as this type of promotor and host cell systems are compatible.

The early promoter of SV40 virus and late promoter can be used as the SV40 restriction fragment and obtain easily, and described SV40 restriction fragment also contains SV40 virus replication starting point.The immediate early promoter of human cytomegalic inclusion disease virus can be used as HindIII E restriction fragment and obtains easily.In mammalian hosts, use bovine papilloma virus to be disclosed in U.S. Patent number 4,419 as the system of vector expression DNA, in 446.The modification of this system is described in U.S. Patent number 4,601, in 978.Also consult Reyes etc., 1982, Nature297:598-601, it is about humanβ-interferon cDNA expression under the thymidine kinase promoter from hsv is controlled in mouse cell.Perhaps, can use Rous sarcoma virus long terminal repetition as promotor.

(v) enhancer element component

Usually increase higher eucaryote transcribing the DNA of code book invention antibody by insert enhancer sequence in carrier.Many enhancer sequence are at present known to mammalian genes (sphaeroprotein, elastoser, albumin, α-fetoprotein and Regular Insulin).Yet, usually can use the enhanser from eukaryotic cell virus.Example is included in the sub-enhanser of SV40 enhanser, cytomegalovirus early promoter of replication orgin rear side (bp100-270), at polyoma enhanser and the adenovirus enhanser of replication orgin rear side.Also can consult Yaniv, 1982, Nature297:17-18 is about for activating the enhancer element of eukaryotic promoter.Enhanser can be spliced into carrier in 5 ' or 3 ' position of antibody coding sequence, but be preferably placed on the position of promotor 5 '.

(vi) Transcription Termination component

Also will comprise and stop transcribing the sequence essential with stable mRNA for the expression vector of eukaryotic host cell (yeast, fungi, insect, plant, animal, people or from the karyocytes of other multicellular organisms).This type of sequence can, available from the 5 ' non-translational region of eucaryon or viral DNA or cDNA, be 3 ' non-translational region usually once in a while.The Nucleotide section that the untranslated part transcription at the mRNA of encoding antibody is the polyadenylation fragment is contained in these zones.A kind of useful Transcription Termination component is Trobest polyadenylation district.Consult WO94/11026 and expression vector disclosed herein.

(vii) adjusting of translation intensity

Can also express immunoglobulin (Ig) of the present invention from such expression system, can regulate the light chain of expression and the quantitative proportion of heavy chain in described expression system, so that the maximum production of the full length antibody of secretion correct assembling.This type of adjusting realizes by the translation intensity of regulating light chain and heavy chain simultaneously.

Be disclosed in Simmons etc. for a kind of technology of regulating translation intensity, U.S. Patent number 5,840,523 and Simmons etc., 2002, J.Immunol.Methods, in 263:133-147.It utilizes the variant in intracistronic translation initiation district (TIR).For given TIR, can produce a series of amino acid or the nucleotide sequence variant of the translation intensity with certain limit, such facilitated method is provided thus, regulate the expression level wanted of this factor for special chain by described method.Although the silence preferably in nucleotide sequence changes, the conventional induced-mutation technique that can change by the codon that causes changing aminoacid sequence produces the TIR variant.Variation in TIR comprises, for example changes the number of Shine-Dalgamo sequence or the change of spacing, and the change in signal sequence.A kind of preferred method that produces the mutant signal sequence is the section start generation " codon bank " at encoding sequence, and it does not change the aminoacid sequence (it is reticent changing) of signal sequence.This can realize by the 3rd nucleotide position that changes each codon; In addition, some amino acid, as leucine, Serine and arginine have a plurality of first and second nucleotide position, this can increase the complexity that produces described bank.This mutafacient system is described in detail in Yansura etc., and 1992, METHODS:A Companion to Methods in Enzymol., in 4:151-158.

Preferably, produce the one group of carrier that there is the TIR intensity of certain limit for each cistron wherein.This restriction group provides the expression level of each chain under multiple TIR intensity combination and the comparison of full length product output.As Simmons etc., U.S. Patent number 5,840,523 and Simmons etc., 2002, J.Immunol.Methods, describe in detail in 263:133-147, can quantitatively measure TIR intensity by the expression level to reporter gene.For the purposes of the present invention, in carrier for the combination of the translation intensity of specific a pair of TIR by (N-is light, and M-is heavy) representative, wherein N is the relative TIR intensity of light chain and M is the relative TIR intensity of heavy chain.For example, (3-is light, and 7-is heavy) means that carrier provides the relative TIR intensity that is about 3 for light chain expression, and heavy chain expression is about to 7 relative TIR intensity.Comparison based on translation intensity, select individual TIR and the expression vector establishment body of the present invention wanted to be combined.

(viii) selection of host cell and conversion

The suitable host cell that is used for the DNA of clone or expression carrier herein is above-described prokaryotic organism, yeast or higher eukaryotic cell.Suitable prokaryotic organism comprise archeobacteria (Archaebacteria) and eubacterium (Eubacteria) for this purpose, as Gram-negative or Gram-positive biology, for example, enterobacteriaceae (Enterobacteriaceae), as escherichia (Escherichia), intestinal bacteria for example, intestinal bacteria belongs to (Enterobacter), Erwinia (Erwinia), Kleb (Klebsiella), sex change bacterium (Proteus), Salmonellas (Salmonella), Salmonella typhimurium (Salmonella typhimurium) for example, Bacterium prodigiosum (Serratia), Serratia marcescans for example, and bacillus dysenteriae (Shigella), and genus bacillus (Bacilli) (for example is published in the DD266 on April 12nd, 1989 as subtilis (B.subtilis) and Bacillus licheniformis (B.licheniformis), disclosed Bacillus licheniformis 41P in 710), pseudomonas (Pseudomonas) is as Pseudomonas aeruginosa (P.aeruginosa) and streptomycete (Streptomyces).A kind of preferred escherichia coli cloning host is intestinal bacteria 294 (ATCC31,446), although other bacterial strains are also suitable as intestinal bacteria B, intestinal bacteria X1776 (ATCC31,537) and large intestine bar mattress W3110 (ATCC27,325).These examples are exemplary rather than restriction.Also preferably secrete the proteolytic ferment of minimum for host cell, and expect that extra proteinase inhibitor is incorporated in cell culture.Prokaryotic host cell also can be included in the sudden change in Trx and/or gsh approach.

Except prokaryotic organism, eukaryotic microorganisms, as filamentous fungus or yeast are suitable clone or expressive hosts for the antibody coding carrier.In the low eucaryon host microorganism of waiting, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or common bread yeast are the most frequently used.Yet, usually can obtain and use herein many other genus and species and bacterial strain, as schizosaccharomyces pombe bacterium (Schizosaccharomyces pombe); Genus kluyveromyces (Kluyveromyce) host, for example Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis) (ATCC12,424), K.bulgaricus (ATCC16,045), K.wickera mii (ATCC24,178), K.waltii (ATCC56,500), K.drosophilarum (ATCC36,906), K.thermotolerans and Kluyveromyces marxianus (K.marxianus); Ascus mattress yeast belong (yarrowia) (EP402,226); Pichia pastoris phaff (Pichia pastoris) (EP183,070); Candidiasis (Candida); Trichodermareesei (Trichoderma reesia) (EP244,234); Neurospora crassa (Neurospora crassa); Permitted prosperous yeast belong (Schwanniomyces) as Schwanniomyces occidentalis; And filamentous fungus, neurospora (Neurospora) for example, Penicillium (Penicillium), Tolypocladium and Aspergillus (Aspergillus) host is as Aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger).

Come from multicellular organism for the suitable host cell of expressing glycosylated antibodies.The example of invertebral zooblast comprises plant and insect cell.Identify many baculovirus bacterial strains and variant and coveted the insect host cell of the corresponding license of noctuid (Spodoptera frugiperda) (caterpillar), Aedes aegypti (Aedes aegypti) (mosquito), Aedes albopictus (Aedes albopictus) (mosquito), drosophila melanogaster (Drosophila melanogaster) (fruit bat) and silkworm (Bombyxmori) from the host as meadow.Can openly obtain the Various Diseases toxic bacterial strain for transfection, for example the L-1 variant of autographa california (Autographa californica) NPV and the Bm-5 bacterial strain of BmSNPV, and this viroid can be used as virus herein according to the present invention, be particularly useful for the transfection of greedy noctuid (Spodoptera frugiperda) cell in meadow.Also can utilize the plant cell cultures of cotton, corn, Ma Lingzhu, soybean, morning glory, tomato and tobacco as the host.

Be widely used the vertebrate host cell, and the breeding (tissue culture) that vertebrate cells is cultivated has become ordinary method.The example of useful mammalian host cell line is the monkey kidney CV1 system transformed by SV40 (COS-7, ATCC CRL1651); Human embryo kidney (HEK) system (293 or 293 cells of subclone for cultivating in suspension culture, Graham etc., J.Gen Virol.36:59 (1977)); Baby hamster kidney cell (BHK, ATCC CCL10); Chinese hamster ovary cell/-DHFR (CHO, Urlaub etc., 1980, Proc.Natl.Acad.Sci.USA77:4216); The mouse sertoli's cell (TM4, Mather, 1980, Biol.Reprod.23:243-251); Monkey-kidney cells (CV1ATCC CCL70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); Human cervical carcinoma cell (HELA, ATCC CCL2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL34); Buffalo mouse liver cell (BRL3A, ATCC CRL1442); Human pneumonocyte (W138, ATCC CCL75); Human liver cell (Hep G2, HB8065); MMT (MMT060562, ATCC CCL51); TRI cell (Mather etc., 1982, Annals N.Y.Acad.Sci.383:44-68); The MRC5 cell; The FS4 cell; Murine myeloma cell, as NSO (as RCB0213,1992, Bio/Technology10:169) and SP2/0 cell (for example, SP2/0-Ag14 cell, ATCC CRL1581); Rat myeloma cell, for example, as YB2/0 cell (, YB2/3HL.P2.G11.16Ag.20 cell, ATCC CRL1662); With people's liver cancer system (Hep G2).Chinese hamster ovary celI is to put into practice preferred cell of the present invention to be, wherein CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics12:555 (1986)), and Lec13 as exemplary host cell is.In the situation that CHO-K1, DUK-B11, DG44 or CHO-DP12 host cell can be changed so that the ability of the protein that their shortage fructose (fucosylate) is wherein expressed these.

The present invention also is applicable to hybridoma.Term " hybridoma " refers to the hybrid cell line produced by the immortal cell line of fusion immunoglobulin (Ig) origin and antibody produced cell.Term comprises the offspring that different hybridization myelomatosis merges, and it is with people's cell and is the result merged with the rat bone marrow tumour cell of plasma cell fusion subsequently, is commonly referred to trioma.In addition, term means and comprises any infinite multiplication hybrid cell line that produces antibody, such as quadroma (such as consulting Milstein etc., 1983, Nature, 537:3053).Hybrid cell line can be any species that comprise people and mouse.

In the preferred embodiment of major part, mammalian cell is non-hybridoma mammalian cell, and the nucleic acid that the external source of its purpose antibody that has been encoded is separated transforms." exogenous nucleic acid " or " heterologous nucleic acids " means such nucleotide sequence, and described nucleotide sequence is external for cell, or with the cell homology but be arranged in host cell nucleic acid and usually can not find on the position of this nucleic acid.

(ix) cultivate host cell

Cultivated the gene of the sequence that in the time of suitably, the described conventional nutritional medium of improvement is wanted for evoked promoter, selection transformant or amplification coding for generation of antibody and in conventional nutritional medium with above-mentioned expression or cloning vector transformed host cell.

Can in multiple substratum, cultivate the host cell for generation of antibody of the present invention.Commercially available substratum, as Ham ' s F10 (Sigma), Minimal Essential Medium ((MEM) (Sigma), RPMI-1640 (Sigma)) and Dulbecco ' s Modified Eagle ' sMedium, ((DMEM) Sigma) is suitable for cultivating host cell.In addition, be described in Ham etc., 1979, Meth.Enz.58:44, Barnes etc., 1980, Anal.Biochem.102:255, U.S. Patent number 4,767,704; 4,657,866; 4,927,762; 4,560,655; Or 5,122,469; WO90/03430; WO87/00195; Or United States Patent (USP) Re.30, any substratum in 985 all can be used as the substratum of host cell.While needing, available hormone and/or other somatomedins (son as tired as Regular Insulin, Transferrins,iron complexes or epidermal growth), salt (as sodium-chlor, calcium, magnesium and phosphoric acid salt), damping fluid (as HEPES), Nucleotide (as adenosine and thymidine), microbiotic are (as GENTAMYCIN ^tMmedicine), trace elements (being defined as generally the mineral compound that the final concentration with micro-molar range exists), and glucose or equivalent energy source supplement these substratum arbitrarily.Also can comprise that any other must supplement by proper concn well known by persons skilled in the art.Culture condition as temperature, pH etc. is that host cell is selected previous those conditions of using for expression, and is apparent for those of ordinary skill.

All substratum provide at least one component from one or more following classifications usually:

1) energy derive is generally the form of carbohydrate, as glucose;

2) all indispensable amino acids are generally basic group that 20 seed amino acids add halfcystine;

3) VITAMIN and/or with other organic compound of lower concentration demand;

4) free fatty acids; With

5) trace elements, wherein trace elements is defined as usually with the mineral compound of low-down concentration demand or the element of natural generation, is generally micro-molar range.

Medium optimization is containing serum, for example, lower than approximately 5%, and preferably lower than 1%, the protein of more preferably 0 to 0.1% serum, and other animal-origins.Yet, as needs, can use the protein of animal-origin.In a preferred embodiment of the invention, cell culture medium comprises the acid of excess of ammonia base.The excessive amino acid provided for example can be selected from Asn, Asp, Gly, Ile, Leu, Lys, Met, Ser, Thr, Trp, Tyr and Val.Preferably, excessive Asn, Asp, Lys, Met, Ser and the Trp of providing.For example, can use one times or amino acid, VITAMIN, trace elements and other nutrient media componentses of twice of the range of definition in European patent EP 307,247 or U.S. Patent number 6,180,401.By reference these two pieces of Piece file mergences are entered to this paper.

The protein of wanting for expression also can add the cultivation of the mammalian cell of the carbohydrate of wanting at specific position, can use multiple culture condition, as long as pay special attention to cultivated host cell.Suitable culture condition for mammalian cell is (W.Louis Cleveland etc. known in the art, 1983, J.Immunol.Methods56:221-234) or can easily determine and (for example consult by the technician, Animal Cell Culture:A Practical Approach second edition, Rickwood, D. and Hames, B.D., editor Oxford University Press, and change according to selected particular host cell New York (1992)).

(x) antibody purification

When using recombinant technology, can be in cell, at periplasmic space, produce antibody, or described antibody direct secretion is in substratum.If produce antibody in cell, the first step is for example removed particulate fragment, the fragment that described particulate fragment is host cell or cracking by centrifugal or ultrafiltration.Carter etc., 1992, Bio/Technology10:163-167 has described the method for separation antibody, and described antibody-secreting is in colibacillary periplasmic space.In brief, melt cell mass approximately 30 minutes existing under sodium-acetate (pH3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF).Can be by centrifugal removal cell debris.At first antibody-secreting in substratum the time, is generally used commercially available protein compression strainer, for example Amicon or Millipore Pellicon ultra-filtration equipment concentrated supernatant thing from this type of expression system.Can in above-mentioned any step, add proteinase inhibitor, as PMSF is hydrolyzed with arrestin matter, and can comprise microbiotic, to prevent the growth of external contaminant.

Can use the antibody compositions that for example prepared from cell by hydroxyapatite chromatography, gel electrophoresis, dialysis and affinitive layer purification, affinity chromatography is preferred purification technique.A albumen depends on kind and the isotype of any immunoglobulin fc region existed in antibody as the suitability of affinity ligand.A albumen can be used for the antibody of purifying based on people γ 1, γ 2 or γ 4 heavy chains (Lindmark etc., 1983, J.Immunol.Meth.62:1-13).Recommend G albumen for all mouse isotype and people γ 3 (Guss etc., 1986, EMBO J.5:15671575).The matrix of affinity ligand combination is often agarose, but also available other matrix.Stable matrix physically, as controlled pore glass or poly-(vinylbenzene divinyl) benzene allow sooner than the attainable flow velocity of agarose, process period is shorter.Antibody comprises C _hduring 3 structural domain, Bakerbond ABX ^tMresin (J.T.Baker, Phillipsburg, N.J.) is for purifying.Also can obtain the other technologies for protein purification according to antibody to be recycled, as the chromatography on the fractional separation on ion exchange column, ethanol precipitation, reversed-phase HPLC, silicon-dioxide, heparin SEPHAROSE ^tMon chromatography, positively charged ion or anionite-exchange resin (as the poly aspartic acid post) on chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

In one embodiment, can use and be adsorbed to the upper purifying glycoprotein of lectin substrate (for example, the lectin affinity column), to remove the glycoprotein containing trehalose from preparation, enrichment is without the glycoprotein of trehalose thus.

(xi) antibody activity is measured

Can characterize its physical/chemical of immunoglobulin (Ig) of the present invention and biological function by many measure method known in the art.In one aspect of the invention, the selectivity in conjunction with target is important than other to compare antibodies immunogen of the present invention.

In certain embodiments of the invention, analyze its biologic activity of immunoglobulin (Ig) produced herein.In some embodiments, its antigen-binding activity of test immunoglobulin (Ig) of the present invention.Known in the art and herein available antigen binding assay include but not limited to use as following technology any directly or the competitive binding assay method, as western trace, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " interlayer " immunoassay, immune precipitation determination, fluorescence immunoassay and A protein immunization are measured.Illustrative antigen binding assay is provided in the embodiment part hereinafter.

Can, by a series of assay methods, include but not limited to that N-terminal order-checking, amino acid analysis, non-sex change size exclusion high pressure liquid chromatography (HPLC) (HPLC), mass spectrum, ion exchange chromatography and papain digestion further characterize the immunoglobulin (Ig) of purifying.Method for quantification of protein is known in the art.For example, its quantitative intensity of protein example that can relatively express on the SDS-PAGE of Coomassie blue stain.Perhaps, for example by the analysis of western blot gel and/or AME5-RP, measure testing goal particular bands (for example total length band).

Pharmaceutical preparation

Can there is the treatment preparation that acceptable carrier, vehicle or stablizer on the antibody of wanting degree purity and optional physiology carry out Dispersal risk/a plurality of antibody by mixing, its form that is freeze-dried preparation or the aqueous solution (Remington ' s Pharmaceutical Sciences the 16th edition, Osol, A. edits (1980)).Acceptable carrier, vehicle or stablizer are nontoxic to the experimenter on dosage used and concentration, and comprise damping fluid, as phosphoric acid salt, Citrate trianion and other organic acids; Antioxidant, comprise xitix and methionine(Met); Sanitas is (as stearyl dimethyl benzyl ammonium chloride; Meton; Benzalkonium chloride, benzethonium chloride; Phenol, butyl alcohol or benzylalcohol; Alkyl to this manthanoate of hydroxyl as methyl or propyl group to this manthanoate of hydroxyl; Catechol; Resorcinol; Hexalin; The 3-amylalcohol; And m-cresol); Lower molecular weight (lower than about 10 residues) antibody; Protein, as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, as polyvinylpyrrolidone; Amino acid, as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate, comprise glucose, seminose or dextrin; Sequestrant, as EDTA; Sugar, as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; Form the counter ion of salt, as sodium; Metal complex (for example, zinc-protein complex); And/or nonionogenic tenside, as TWEEN ^tM, PLURONICS ^tMor polyoxyethylene glycol (PEG).Exemplary pharmaceutically acceptable carrier also comprises insterstitial medicine dispersion agent herein, Unidasa glycoprotein as active as soluble neutrality (sHASEGP), the soluble PH-20 Unidasa of people glycoprotein for example, as rHuPH20 (

baxter International, Inc.).Describe some exemplary sHASEGPs and using method in United States Patent (USP) publication number 2005/0260186 and 2006/0104968, comprised rHuPH20.On the one hand, sHASEGP and one or more extra glycosaminoglycanases combine as chondroitinase.

At U.S. Patent number 6,267, the antibody preparation of exemplary freeze-drying has been described in 958.The water antibody preparation is included in U.S. Patent number 6,171,586 and WO2006/044908 in describe those, rear a kind of preparation comprises the Histidine acetate buffer solution.

Preparation herein also can contain more than a kind of essential active compound of specific adaptations disease for the treatment of, those active compounds that preferably have not negative impact complementary activity each other.For example, described preparation also can comprise another kind of antibody or chemotherapeutic.This quasi-molecule suitably exists so that the expection purpose is effectively measured to combination.

For example also can for example, by condensation technique or by interfacial polymerization at the colloid drug delivery system (, liposome, albumin microsphere, microemulsion, nanoparticles and nanocapsule) or comprise respectively activeconstituents in the minigel of large emulsion preparation, Walocel MT 20.000PV or gelatin-minigel and poly-(methylmethacylate) minigel for example.Remington ' s Pharmaceutical Sciences the 16th edition, Osol, A. edits in (1980) this type of technology that discloses.

Being ready to use in the preparation of using in body must be aseptic.This can filter easily and complete by aseptic filter membrane.

Can prepare sustained release preparation.The suitable example of sustained release preparation comprises the semipermeability matrix of the solid hydrophobic polymkeric substance that contains antibody, and described matrix is the form of formed article, for example film or minigel.The example of sustained-release matrix (for example comprises polyester, hydrogel, poly-(2-hydroxyethyl-methacrylic acid ester), or poly-(vinyl alcohol)), polylactide (U.S. Patent number 3,773,919), Pidolidone and the multipolymer of γ ethyl Pidolidone salt, nondegradable ethylene-vinyl acetate, degradable lactic acid-hydroxyacetic acid (lactic acid-glycolic acid) multipolymer, as LUPRON DEPOT ^tM(the Injectable microspheres body formed by lactic acid-hydroxyacetic acid copolymer and leuprorelin acetate), and poly--D-(-)-3-hydroxybutyric acid.Work as polymkeric substance, when as ethylene-vinyl acetate and lactic acid-hydroxyacetic acid, making it possible to discharge molecule and surpass 100 days, the time that some hydrogel release protein is shorter.When encapsulated antibody retains in vivo for a long time, they are trapped in 37 ℃ and expose in moisture and sex change or cohesion cause the variation that bioactive forfeiture and immunogenicity are possible.Strategy that can be reasonable in design according to related mechanism is for stabilization.For example, if find that aggegation mechanism is the intermolecular S-S key formed by the sulfo-disulfide exchange, so can be by modifying sulfhydryl residue, freeze-drying from acidic solution, control water-content, by suitable additive and develop specific polymer matrix composition and realize stabilization.

The non-therapeutic purposes of antibody

Antibody of the present invention can be used as the affinity purification agent.In the method, use method well known in the art that antibody is fixed on to solid phase, on SephadexTM resin or filter paper.Fixing antibody is contacted with the sample that contains antigen to be purified, then use suitable solvent (it can remove all substances beyond antigen to be purified in sample basically) washing upholder, described antigen to be purified is in conjunction with fixing antibody.Finally, with another kind of suitable solvent, as glycine buffer, pH5.0 washs upholder, this will be from antibody released antigen.

Antibody also can be used in diagnostic assay, for example, for detection of the expression of purpose antigen in specific cells, tissue or serum.For diagnostic use, usually use detectable part traget antibody.Can obtain many markers, it generally can be divided into following classification:

(a) radio isotope, as ³⁵s, ¹⁴c, ¹²⁵i, ³h and ¹³¹i.For example can use Current Protocols in Immunology, the 1st volume and the 2nd volume, Coligen etc., editor Wiley-Interscience, New York, N.Y., the technology labelled with radioisotope antibody of describing in Pubs. (1991), and use scintillation counting to measure radioactivity.

(b) can obtain fluorescent marker, as Rare Earth Chelate (europium inner complex) or fluorescein and derivative, rhodamine and derivative thereof, dansyl, Liz amine, phycoerythrin and texas Red.For example can use Current Protocols in Immunology, above disclosed technology is conjugated to fluorescent marker on antibody.Can use the photofluorometer quantitative fluorescence.

(c) can obtain multiple enzyme-substrate marker, and U.S. Patent number 4,275,149 provides the summary of some these markers.The general catalysis of described enzyme can be used the chemical conversion of the chromogenic substrate of multiple technologies mensuration.For example, but the colour-change of this enzyme catalytic substrate, and its available spectrophotometer is measured.Perhaps, enzyme can change fluorescence or the chemoluminescence of substrate.The technology changed for quantitative fluorescence has above been described.Chemical luminous substrate is by chemical reaction by electron excitation, the light that then emission can be determined (for example, using the chemoluminescence meter) or provide energy to fluorescent receptor.The example of enzyme labelling thing comprises luciferase (for example, Photinus pyralis LUC and bacterial luciferase; U.S. Patent number 4,737,456), luciferin, 2,3-bis--hydrogen phthalazine diketone, malate dehydrogenase (malic acid dehydrogenase), urease, peroxidase for example, as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, carbohydrate oxydase (, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)), heterocycle oxydase (as uriKoxidase and XOD), lactoperoxidase, microperoxisome etc.At O ' Sullivan etc., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, Methods in Enzym. (editor J.Langone and H.Van Vunakis), Academic press, New York, described enzyme be conjugated to the technology on antibody in 73:147-166 (1981).

The example of enzyme-substrate combination comprises, for example:

1) horseradish peroxidase (HRPO) utilize the hydrogen peroxide oxidation dyestuff former (for example, orthophenylenediamine (OPD) or 3,3 ', 5,5 '-tetramethyl benzidine hydrochloride (TMB));

2) alkaline phosphatase (AP), utilize p-nitrophenyl phosphate as chromogenic substrate; With

3) beta-D-galactosidase (β-D-Gal), have chromogenic substrate (for example, p-nitrophenyl-beta-D-galactosidase) or fluorogenic substrate 4-methyl umbelliferone (methylumbelliferyl)-beta-D-galactosidase.

Those skilled in the art can obtain many other enzyme-substrate combinations.For these general summaries, consult U.S. Patent number 4,275,149 and 4,318,980.

Sometimes, marker indirectly and antibody put together.The technician knows the multiple technologies for realizing that this is puted together.For example, antibody can with biotin-conjugated, and the marker of any three kinds of wide variety mentioned above can put together with avidin, vice versa.Vitamin H is optionally in conjunction with avidin, so this marker is puted together antibody with its indirect mode.Perhaps, in order to realize indirectly puting together of marker and antibody, antibody and little haptens (for example, digoxin) are puted together, and one of dissimilar marker mentioned above for example, is puted together with antihapten antibody (, anti digoxin antibody).Be stranded this, can realize indirectly puting together of marker and antibody.

In another embodiment of the present invention, antibody does not need mark, and can use the traget antibody of binding antibody to detect its existence.

Can be at any known measuring method, as used antibody of the present invention in competitive binding assay, direct and indirect sandwich assay and immunosorbent assay.Zola, Monoclonal Antibodies:A Manual of Techniques, 47-158 page (CRC Press, Inc.1987).

Antibody also can be used in in-vivo diagnostic mensuration.Generally speaking, with radionuclide (as ¹¹¹in, ⁹⁹tc, ¹⁴c, ¹³¹i, ¹²⁵i, ³h, ³²p or ³⁵s) traget antibody, in order to can be used immanoscintiography location antigen or express the cell of described antigen.

Purposes in the body of antibody

In another embodiment, anti-il-i-beta of the present invention and/or anti-IL-18 antibody/a plurality of antibody and therapeutical agent are used jointly, to strengthen the function of therapeutical agent.For example, to the anti-Fc γ of administration RIIB, with blocking-up IgG, in conjunction with Fc γ RIIB, prevent that thus the Immunosuppression of Fc γ RIIB mediation from replying.This result causes the cytotoxicity of IgG therapeutic antibodies to strengthen.For example, when therapeutic antibodies is special to tumour antigen, jointly use the cytotoxicity that anti-Fc γ RIIB of the present invention and antitumor-antigen antibody have strengthened antitumor-antigen antibody.

Developed therapeutic antibodies (above having described many described therapeutic antibodies) and permitted being used for the treatment of various diseases, having comprised cancer.For example,

(Rituximab) (IDEC Pharm/Genentech, Inc.) is used for the treatment of B cell lymphoma, AVASTIN ^tM(bevacizumab) (Genentech, Inc.) is used for the treatment of metastatic colorectal cancer, and

(Trastumab) (Genentech, Inc.) is the Humanized anti-HER 2 monoclonal antibody that is used for the treatment of metastatic breast cancer.Although thoroughly do not understand by treat the mechanism of all mab treatment cancers of developing for this reason, but at least in some cases, the part effect of Antybody therapy is attributable to (Houghton etc., 2000 of raising of immunological effect subfunction, Nature Medicine, 6:373-374; Clynes etc., 2000, Nature Medicine, 6:433-446).

(Omalizumab) (Genentech, Inc.) is used for the treatment of allergic anti-IgE antibodies.

The treatment potential of this bifunctional antibody comprises participation inflammation and/or allergic signal attenuation.For example, when by IgE and allergen (FcFR) activation, mastocyte and basophil secretion inflammatory mediator and cytokine, it acts on vascular and the myocyte goes up and raise inflammatory cell.Inflammatory cell is secreted conversely inflammatory mediator and is raised inflammatory cell in causing the time-continuing process of long-term inflammation.Result is that the method for the mastocyte activation that control IgE induces provides methods for the treatment of, with the initial allergic disease for the treatment of by the blocking-up inflammatory response.As mentioned above, bifunctional antibody comprises antibody or its fragment, or its selective binding IL-1 β, and comprises antibody or its fragment, its selective binding IL-18.

The antibody that extra bifunctional antibody example (for example, bi-specific antibody) comprises selective binding IL-1 β or the antibody of its fragment and second selective binding IL-18 or the combination of its fragment.In some embodiments, antibody of the present invention activates inhibition Fc γ RIIB acceptor for the Mammals utilizing Antybody therapy, with the B cell activation that suppresses proinflammatory disease signal and/or mediate by activated receptor.Therefore, antibody is used for the treatment of inflammatory condition and/or autoimmune disease, as those diseases of above identifying.

In order to prevent or treat disease, the suitable dosage of antibody will depend on that the type of disease to be treated, seriousness and the course of disease, the administration of antibodies of disease are to be preventative purpose or therapeutic purpose, previous tretament, patient's clinical history and the reaction of antagonist, and attending doctor's judgement.Antibody is suitable for once or in a series of treatments using to the patient.

According to type and the seriousness of disease, approximately (for example, 0.1-20mg/kg) antibody is the initial candidate dosage for using to the patient to 1 μ g/kg to 15mg/kg, for example no matter by one or many, uses separately, or passes through continuous infusion.According to factor mentioned above, typical every per daily dose is approximately changing in 1 μ g/kg to 100mg/kg or higher scope.For several days or interior repetitive administration of longer time, according to circumstances, continued treatment was until occur the expection of disease symptoms is suppressed.Yet other dosages are useful.Can and measure the process of easily monitoring this treatment by routine techniques.

Dosage and administration of antibodies composition are prepared, applied to mode that should be consistent with the medical practice with good.The factor of considering in this context comprise clinical setting, illness cause, the delivery of agents of treated particular condition, the specific Mammals for the treatment of, individual patient site, application process, use and arrange and other known factors of clinicist." the treatment significant quantity " of antibody to be administered determined by these Considerations, and is prevention, improvement or treatment disease or the required minimum of illness.Antibody does not need, but optionally by one or more reagent preparations of the illness of discussing for prevention or treatment at present.The significant quantity of these other reagent of class depends on type and other factors discussed above of amount, illness or the treatment of the antibody existed in preparation.Generally with the same dose with use above and route of administration or above using dosage approximately 1 to 99% use these reagent.

For example, (for example relate to inflammatory cell in order to treat, the autoimmune disease of white corpuscle) adhesion, migration and activation, as rheumatoid arthritis and lupus, antibody herein can with for example anti-LFA-1 antibody (as anti-CD11a or anti-CD18) or anti-ICAM antibody, as ICAM-1 ,-2 or-3 uses jointly.Additional agents for the antibody combined treatment rheumatoid arthritis with herein comprises Enbrel ^tM, DMARDS, for example methotrexate and NSAID (non-nonsteroidal antiinflammatory medicine).Also can use except antibody herein more than a kind of these type of other promoting agents.In addition, Regular Insulin can be used for treating diabetes, and anti-IgE is used for the treatment of asthma, and anti-CD11a is used for the treatment of psoriatic, and anti-α 4 β 7 and tethelin (GH) are used for the treatment of inflammatory bowel.

In addition, preparation is suitable for using together with the hypoglycemic agents with significant quantity.For this purpose, term " hypoglycemic agents " refers to for regulating the compound of glucose metabolism, preferred oral reagent.What more preferably for the mankind, use is Regular Insulin and sulfonylurea oral hypoglycemic herein, and it causes pancreatic secretion Regular Insulin.Example comprises Glyburide, Glipizide and gliclazide.In addition, the reagent that strengthens the Regular Insulin susceptibility is insulin sensitizing agent, as biguanides (comprising N1,N1-Dimethylbiguanide and phenformin) and thiazolidinedione (thiazolidenediones) as REZULIN ^tMthe insulin sensitizing agent reagent of (troglitazone) trade mark, and in conjunction with other compounds of PPAR-γ nuclear receptor in its definition, and be also preferred.

By any suitable technology, comprise in parenteral, nose, oral cavity or by any other suitable pathways to the administration hypoglycemic agents.Most preferably, by oral cavity route, use.For example, the MICRONASE of 1.25,2.5 and 5mg sheet agent concentration of the strong marketization of A Pu ^tMtablet (Glyburide) is suitable for oral administration.For this treatment, for the common maintenance dose of type ii diabetes generally from or the scope of about every day 1.25 to 20mg in, its can be used as that single dose provides or suitably the time in one day gradation provide.Physician′sDesk?Reference，2563-2565(1995)。Other examples that can be used for the tablet based on Glyburide of prescription comprise GLYNASE ^tMtrade mark medicine (A Puqiang) and DIABETA ^tMtrade mark medicine (Hoechst-Roussel).GLUCOTROL ^tM(Pratt) be Glipizide (1-cyclohexyl-3-(p-(2-(5-methylpyrazine imidazole carboxamide) ethyl) phenyl) sulphonyl) urine) trade mark of tablet, can obtain with 5-and 10-mg intensity, and also the type ii diabetes patient who needs hypoglycemic treatment after dietary control or the patient who has stopped other sulfonylureas are responded be prescribed.Physician′s?Desk?Reference，1902-1903(1995)。Except sulfonylurea, also can use other hypoglycemic agentss, for example, for example, as biguanides (, N1,N1-Dimethylbiguanide and phenformin) or thiazolinedione (, troglitozone), or affect the other drug of insulin function.If thiazolidinedione is used together with peptide, identical or level use lower level a little with the level with at present used, the described thiazolidinedione of capable of regulating is for obtaining independent use peptide or use the effect that can observe together with diketone.Troglitazone (REZULIN ^tM) self exemplary dosage used is about 100-1000mg every day, more preferably every day 200-800mg, and this scope is applicable herein.For example consult Ghazzi etc., Diabetes, 46:433-439 (1997).Can use other thiazolinedione except the thiazoline diketone with low dosage more, it is stronger insulin sensitizer.

Finished product

In another aspect of this invention, provide and contained the finished product that are used for the treatment of, prevent and/or diagnose the material of above-mentioned illness.Described finished product comprise on container and this container or label or the package insert relevant to this container.Suitable container for example comprises, bottle, bottle, syringe, IV solution bag etc.Can be from multiple material, as glass or standby this container of plastics.Described container preserve separately or with the composition of the another kind of combination of compositions of effective treatment, prevention and/or diagnosis situation, and can there is sterile access port (for example, this container can be intravenous solution bag or the bottle with the transparent stopper of hypodermic needle).At least one promoting agent in composition is antibody of the present invention.Label or package insert mean that said composition is used for the treatment of selected illness.In addition, finished product can comprise the first container that (a) wherein contains composition, and wherein said composition comprises antibody of the present invention; (b) wherein contain second container of composition, wherein said composition comprises other cytotoxic agents or other treatment agent.Finished product in the embodiment of the present invention can further comprise package insert, and it means that described composition can be used for treating particular condition.Perhaps, or in addition, finished product can further comprise second (or the 3rd) container, and it comprises pharmaceutically acceptable damping fluid, as bacteriostatic water (BWFI), phosphate-buffered salt, Ringer's solution and the glucose solution for injection.It also comprises other materials of wanting from business and user's position, comprises other buffering salts, thinner, strainer, pin and syringe.

The therapeutic antibodies composition generally is placed in the container with sterile access port, for example intravenous solution bag or have the bottle of the transparent stopper of hypodermic needle.

The present invention also provides finished product and containing to be used for the treatment of the test kit of the material of cancer for example or disease.Described finished product comprise the container with label.Suitable container comprises for example bottle, bottle and test tube.Container as described in can be as standby as glass or plastics from multiple material.This container is preserved the composition that comprises antibody described herein.Promoting agent in composition is specific antibody.Label on container means that described composition is used for the treatment of or prevents specified disease or illness, and also indication is used for the specification sheets in body, as described above those.

Second container that test kit of the present invention comprises said vesse and comprises damping fluid.It also comprises the other materials of wanting from business and user perspective, comprises the package insert that other damping fluids, thinner, strainer, pin, syringe and use are introduced.

Should be understood that any above-mentioned finished product can comprise the immunoconjugates of alternative IL-1 β of the present invention and/or IL-18 antibody, or comprise immunoconjugates of the present invention except IL-1 β and/or IL-18 antibody.

Embodiment

Be hereinafter the example of the inventive method and composition, and provide herein them for the illustrative purpose, be not intended to limit the scope of the invention.If provide in this article general explanation basis upper, should understand and can put into practice multiple other embodiments.The disclosure of all patents cited herein and scientific literature clearly is incorporated herein by reference with its integral body.

Use the commercial reagent of reference in embodiment according to the specification sheets of manufacturers, except as otherwise noted.Those cell deriveds of identifying by the ATCC accession number in embodiment and specification sheets full text hereinafter are U.S. typical case culture centers (American Type Culture Collection, Manassas, VA) of Virginia, USA Manassas.

Method

The colitis that dextran sulfate sodium (DSS) is induced

Body weight keeps not processed or optionally in its tap water, accepts 3.5%DSS5 days at wild-type and the knock-out mice of the age between the 19-25 gram and gender matched.Assessed losing weight of mouse since the 4th day every day, and at the 8th day by its execution.Then collect colon, the scoring and for organ culture or histopathology.Complete as follows the scoring to the colonic inflammation degree: after removing ight soil by cleaning down, the degree scoring colon based on wall thickening.Scoring is changed to 4 from 0, and the Normal Colon scoring is 0, and is 4 in the scoring that whole colon has a wall thickened.

Colon for the cytokine spectrum is cultivated

Cleaning, from the colon of mouse, is vertically opened, and is placed in the RPMI substratum that contains 1% penicillin/streptomycin solution.After 37 ℃ of incubations that spend the night, collect substratum and clarified, then use BioRad Bio-Plex technology (Luminex) analysis of cells single or that the 23-plex assay method is passed through based on xMAP tired sub.

The AAV2/5 subretinal injection

By peritoneal injection ketamine/xylazine (80mg/kg: 15mg/kg) anesthetized animal.Under dissecting microscope, by the 30g insulin needle for generation of through the puncture of sclera, allow use 33-gauge Hamilton pin and micro-automatic injector (World Precision Instruments, Sarasota, the FL) 1x10 through subretinal injection 1 μ l ¹²aAV2/5 genome particle/ml (Genedetect, Bradenton, FL).The formation of retina bubble shows to inject successfully.

The Western trace

1x10 with 1 μ l ⁹aAV2/5-IL1 β or AAV2/5 blank virus (Genedetect) are through the subretinal injection Balbc mouse in 10 week age.After the injection of 4 months, some mouse are stood strong illumination (ILE, 8000lux) 3.5 hours, then are placed in dark 48 hours.Cut eyes and containing proteinase inhibitor (Protease Inhibitor Cocktail set I, Calbiochem, Gibbstown, NJ) cell lysis buffer solution (Cell Signaling Technologies, Danvers, MA) in, eyeshade (eye cups) (eyes are removed cornea and lens) is shredded to 1 hour, and freezing in-80 ℃.BCA assay method (Tbermo/Pierce, Rockford, IL) is carried out quantitatively the protein in sample.To be loaded at the 10ug protein containing boiling 3 minutes in Lammeli ' the s damping fluid of b-mercaptoethanol in each swimming lane of 10-20%tris-glycine gels (Invitrogen, Carlsbad, CA) and electrophoresis 1.5 hours under 125mW.Protein transduction is moved to upper 1 hour of 0.2um hole nitrocellulose filter in transfering buffering liquid (Invitrogen) under 25mW.With in the PBS/0.1%Tween-20 (PBST) containing 5% breast, sealing trace 1 hour, and by the 0.2ug/ml goat anti-il-i-beta (R&amp in 1% breast/PBST; D cat#AF-401-NA) or the large mouse-anti Caspase 1 of 0.5ug/ml (Genentech, clone4B4.2) add 1 hour.In PBST, the washing trace is 4 times, each 5 minutes.Add anti-goat HRP (1: 5000R& D) or the mouse HRP of the Chinese People's Anti-Japanese Military and Political College (Thermo/Pierce) 45 minutes and in PBST the washing 5 times, each 5 minutes.Utilize ECL Plus and hyperfilm (GE Healthcare, Buckinghamshire, UK) development trace.

Immunohistochemistry

Utilize the 1x10 of 1 μ l ⁹aAV2/5-IL1 β or AAV2/5 blank virus (Genedetect) are through the subretinal injection Balbc mouse in 10 week age.After injecting 7 weeks, take out whole eyes and at room temperature fixedly spend the night in 10% neutral buffered formalin.Process and cut into slices and be embedded in paraffin, then with anti-CD45, dye and utilize the DAB colour developing.

Fluorescence angiography (FA)

By peritoneal injection ketamine/xylazine (80mg/kg: 15mg/kg) anesthetized animal utilize 1% mydriacyl (Bausch and Lomb, Rochester, NY) expansion eyes.Utilize the artificial tears that eyes are kept to moistening.Utilize the injectable fluorescein of 100 μ l10%AK-Fluor (Akorn, Buffalo Grove, IL) through the peritoneal injection mouse.Utilize Heidelberg Spectralis HRA/OCT photographic camera (Heidelberg Engineering, Vista, CA) to obtain image.

Light Harmony body section radiography (OCT)

By peritoneal injection ketamine/xylazine (80mg/kg: 15mg/kg) anesthetized animal utilize 1% mydriacyl (Bausch and Lomb, Rochester, NY) expansion eyes.Utilize the artificial tears that eyes are kept to moistening.Utilize Heidelberg Spectralis HRA/OCT photographic camera (Heidelberg Engineering, Vista, CA) to obtain image.Measuring result is 15.2mm ²the mean thickness of 19 sections on the area retina.Thickness comprises retina and choroid.

Electroretinogram ERG (ERG) record

Mouse adapts to darkling 24 hours before ERG, with the balance retina, reacts.Once adapt to dark, all subsequent steps all carry out darkling, only with red light, throw light on.Utilize ketamine and xylazine (75-80mg/kg: 7.5-15mg/kg) through peritoneal injection, carry out anesthetized animal.The constant temperature heating plate that use is connected on its control device maintains 37 ℃ by mouse temperature.Utilize 1% coromegine expansion pupil and utilize 0.5% a Proparacaine HCl anesthesia anterior corneal surface.Use Espion E2 (Diagnosys LLC, Lowell, MA) visual electro-physiology system to record the ERG of two eyes simultaneously.By mouse, be placed on platform and with reference to electrode through subcutaneous insertion forehead and ground-electrode is inserted to the afterbody substrate.Gonak hypermellose solution is placed on cornea, and to set up electrically contacting between cornea and platinum electrode, and the protection eyes avoid drying in experimentation.Mouse is placed in to ColorDome desktop Ganzfeld stimulator in the whole field, and uses 1x10 ^-5-5cd/m ²white light in scope: 3 flash intensities are stimulated, and allow to pause 2 minutes between flash of light, to rebuild baseline response.At 0.15-1000Hz place band-pass filter signal and at the 2kHz place, sample.

The method of phage elutriation IL-1 β

For the fixing some phage display synthetic antibody libraries of people IL-1 β elutriation.By the recovery storehouse of measuring the special phage clone to IL-1 β and the enrichment of the ratio in those phage clones recovery storehouses special in conjunction with bovine serum albumin being determined to the antibody displaying phage library special to IL-1 β in third round with in the later several rounds.At elsewhere people such as (, 2004) Sidhu, described synthetic unprocessed

the structure of antibody phage display libraries.Several take turns elutriation after, identified and showed the antibody variable heavy chain structural domain special to IL-1 β and the phage clone of light chain structural domain.Determined the DNA sequence dna of variable heavy chain and it has been reconstructed into to human IgG1's expression vector, to allow the instantaneous antibody expression in mammalian cell.Use A albumen antibody purification from the cell cultures growth medium, for suppress the follow-up test of assay method and the assay method based on functioning cell at soluble protein binding affinity assay method, receptor-ligand.

Competition suppresses people IL-1 β in conjunction with people IL-1RI or IL-1RII

In phosphate buffered saline (PBS) (PBS) by NeutrAvidin (Pierce, Rockford, IL) be diluted to 2 μ g/mL and be coated on ELISA flat board (the high combination flat board in 384-hole, Nunc in 4 ℃ are spent the night the incubation process, Neptune, New Jersey).After utilizing lavation buffer solution (PBS/0.05%Tween-20) to wash three times, with dull and stereotyped 1 to 2 hour of PBS/0.5% bovine serum albumin (BSA) sealing.This step and all follow-up hatching are all at room temperature carried out on orbital shaker.Use the biotinylated people IL-1 of maleimide PEG-vitamin H (Pierce) β (R&amp according to the guidance of manufacturers; D Systems, Minneapolis, MN) be diluted to 400ng/ml in measuring damping fluid (PBS/0.5%BSA/0.05%Tween-20).The NeutrAvidin flat board of washing sealing, and in 1-2 hour hatches process, biotinylated people IL-1 β is caught to flat board.Use 3-amino-3-deoxidation digoxin half succinyl-acid amides succinimide ester (Invitrogen, Eugene, OR) digoxigenin labeled people IL-1RI and IL-1RII (R&amp according to the guidance of manufacturers; D Systems).Assess by dilution antibody in wide region and by them and the people IL-1RI of equivalent DIG mark or IL-1RII mixing (final concentration is respectively 1 μ g/ml or 60ng/ml) ability that antibody blocking IL-1RI and IL-1RII are combined with IL-1 β.Add mixture and allow to hatch 1-2 hour in the washing flat board.Then use monoclonal anti DIG antibody (Jackson ImmunoResearch, West Grove, PA) that horseradish peroxidase (HRP) is puted together to detect IL-1RI or the IL-1RII of dull and stereotyped combination.After hatching 1 hour and carrying out extra washing step, add tetramethyl benzidine (TMB, Kirkegaard; Perry Laboratories, Inc., Gaithersburg, MD), and allow about 10 minutes of colour developing.By adding 1M phosphoric acid termination reaction.Use microplate reader (450nm, 650nm reference) to read optical density(OD), and four parameter fittings of use curve (Kaleidagraph, Synergy Software, Reading, PA) are determined the antibody concentration that produces the inhibition of half maximum combined.Consult Figure 21.

Competition suppresses mouse IL-1 β in conjunction with mouse IL-1RI or IL-1RII

Use the ability of similar method assessment antibody blocking mouse IL-1 β in conjunction with mouse IL-1RI and IL-1RII.Use sulfo-NHS-LC-vitamin H (Pierce) biotinylation mouse IL-1 β (R&amp according to the guidance of manufacturers; D Systems) and with the concentration of 400ng/ml catch to the NeutrAvidin flat board.Dilute antibody in wide region, and by mouse IL-1RI-or the IL-1RII-human IgG1 Fc fused protein (R&amp of itself and equivalent; D Systems; Final concentration is respectively 1 μ g/ml or 60ng/ml) mix, and hatch 1-2 hour on prepared flat board.The acceptor of goat Anti-TNF-α human IgG Fc antibody (Jackson ImmunoResearch) the detection combination of using HRP to put together.Developed the color as mentioned above and data analysis.Consult Figure 21.

Competition suppresses human il-18 in conjunction with human il-18 Ra

Total measuring method basic with for assessment of inhibition people IL-1 β/IL-1R in conjunction with described identical.With the coated ELISA flat board of NeutrAvidin (Pierce), will use the biotinylated human il-18 (R&amp of sulfo-NHS-LC-vitamin H (Pierce); D Systems) be diluted to 400ng/ml and it is caught to flat board.Use 3-amino-3-deoxidation digoxin half succinic diamide succinimide ester (Invitrogen, Eugene, OR) to utilize digoxin (DIG) mark human il-18 Ra-human IgG1 Fc (R& D Systems).The antibody of dilution mixes with isopyknic DIG-IL-18Ra-Fc (final concentration is 1 μ g/ml).Use anti-DIG antibody (Jackson ImmunoResearch) to detect the acceptor of combination.Developed the color as mentioned above and data analysis.

Embodiment 1: the IL-1 β combined in inflammatory bowel and IL-18 blocking-up

In clinical study, the inventor has found in regional ileitis that the cell of expressing IL-1 β and IL-18 significantly increases, and finds that in regional ileitis Serum IL-18 Level significantly increases (seeing Fig. 4).In the clinical front mouse model of IBD, IL-1 β and the secretion increase (see Fig. 5) of IL-18 from colon in the Isolated colon cultivation have been found.For IL-1 β, positive cell is positioned at active inflammation place, seldom or the zone (Fig. 5, upper figure) that does not have positive cell to be positioned at not enliven the inflammation evidence.For IL-18, positive cell on morphology with marrow sample dendritic cell (arrow) consistent (Fig. 5, figure below) in the marginarium of follicular dendritic cell (arrow) and lymphoid follicle.The IL-18 positive cell is also referred to as colon epithelial cell.These results have represented 21 parts of regional ileitis clinical samples assessing.

The mouse model of studying comprises colitis (in WT B6 female mice), the adopting property transfer of T-cell and the piroxicam IL-10KO (seeing Fig. 6,7 and 8) that DSS-induces.The inventor has proved that blocking-up IL-1 β, IL-18 or both (in the situation that ASC KO research) reduce inflammatory reaction ((IL-1 β, IL-18, TNF α, IL-17, IL-6) and colon scoring (seeing Fig. 9-13) in the DSS of colitis model.

Embodiment 2: the IL-1 β combined in relevant macular degeneration of age and IL-18 blocking-up

Early-stage Study has reported that IL-1 β increases in the vitreous humor of suffering from diabetic retinopathy and uveitic patient.Yet IL-1 β and IL-18 in moist or dryness AMD do not reported in research.Originally studies show that IL-1 β level increases (seeing Figure 14) in the vitreum of AMD patient subgroups.In clinical front mice study, the inventor is presented in the mouse eyes and crosses the beta induced retina inflammation of expression IL-1, and IL-18 crosses expression and do not induce retina inflammation (seeing Figure 15-18).In addition, the inventor shows that IL-1 β and IL-18 all affect retinal function, as the ERG record is measured (seeing Figure 19).Based on these research, the inventor reaches a conclusion, and expects that the IL-1 β of single and combination and IL-18 blocking-up improve sight sensor function and CNV/ oedema (seeing Figure 20).

Embodiment 3: the IL-1 β combined in diabetes B and IL-18 blocking-up

Inventor's hypothesis, target is determined the function that IL-1 β can protect β cell in the patient who suffers from diabetes B.It is reported that IL-1 β reduces in vitro the insulin secretion of pancreatic beta cell and changes the function of multiple β cell.In addition, it is reported and utilize the IL-1Ra treatment can prevent or improve diabetes animal model, and it is reported that IL-1Ra has reduced the β cell from suffering from diabetes B patient acquisition.See Figure 28.

It is reported gene pleiomorphism relevant to central obesity and metabolic syndrome (Carter etc., 2008) in IL-1 β/IL-18 approach.In addition, it is reported that IL-1 β reduces the insulin secretion of pancreatic beta cell (Lewis and Dinarello in vitro, 2006), and it is reported that Kineret (IL-1Ra) improves the too high and β emiocytosis function (Larsen etc., 2007) of blood sugar in the patient.In addition; it is reported that the IL-1 that increases and IL-18 serum level have reduced the ratio of IL-1Ra and IL-18BP in T2DM patient; and IL-1Ra and IL-18BP protect and avoid STZ or higher fatty acid hyperglycemia of inducing (Sandberg etc., 1994) in preclinical models.

In addition, Larsen etc. use and within 13 weeks, carry out double blind (Larsen etc., 2007) by using a Kineret every day in suffering from the patient of diabetes B.This treatment has improved the too high and β cell Regular Insulin secretion capacity of blood sugar and has reduced the mark of systemic inflammation.Yet, still needing to determine that anti-IL-1 treatment is to recover the possible beneficial effect of β cell mass and function in suffering from the patient of diabetes B, described anti-IL-1 treatment has the longer transformation period and uses within the longer time.

Embodiment 4: anti-IL-1b and/or anti-IL18 in piroxicam IL-10KO IBD model

IL-10-/-idiopathic colitis occurred in mouse.Yet sickness rate and seriousness are inconsistent, cause it to be difficult to the therapeutics of as the model of IBD, testing us.By piroxicam feed IL-10-/-mouse, piroxicam may aggravate chronic intestinal inflammations and may make colitis outbreak synchronization in these mouse, as shown in (2002) such as Berg.Therefore, this acute DSS model with IBD is contrary, and this is the chronic inflammation model of IBD.

Female IL-10KO (Genentech) mouse in 6 week age is divided into following treatment group;

Use how much dilutions with the concentration of 200ppm, the piroxicam pulvis to be mixed with pulverous rodent diet.In brief, the mouse diet of equivalent is joined in piroxicam, then thoroughly mix.The mouse diet that adds continuously equivalent, mix after each dilution, until mix the mouse diet of whole amounts.After overnight fasting, with the diet containing piroxicam, feed mouse 11 days, returned normal diet at the 12nd day.With weekly 3 times above shown in amount in 400 μ l PBS continue 6 weeks through all processing of peritoneal injection.Weigh every day animal when research finishes, it being put to death for analyzing.Before starting the piroxicam treatment, by tail cant, in whole tail vein, collect 100 μ l blood for FACS and serum.Then, within the 5th week after starting experiment, collect 100 μ l blood.In these researchs, analyze the treatment effect to visible colon scoring, colon and blood-serum P K.See Figure 29.

Measure the level of cytokine profiles in the IL-10KO mouse of accepting and do not accept the piroxicam treatment.See Figure 13.As indication, in the piroxicam treatment group, IL-1 β and IL-18 raise (with respect to the WT animal), and TNF α, IL-12 and IL-17 are suitable between group.

For histopathological analysis, fixing organization in 10% formalin, be embedded in it in paraffin subsequently for section and phenodin and eosin dyeing.In near-end, centre and DC and rectum, evaluate histopathology is marked and is marked in 1 to 3 scope.Amount to the scoring of each colon section to obtain the total points of every animal.The histologic characteristics that same individual scoring is all and there is no the information of experimental group.

The result that has shown visible colon scoring and histological score in Figure 30.Assessed the serum level of IL-1 β and IL-18.Utilize the combination therapy of anti-il-i-beta and anti-IL-18 antibody to cause the damage of colon is significantly reduced statistically.Combination therapy is equally effective with the TNFRII-Fc treatment.The combination blocking-up of these results proof IL-1 β and IL-18 is the effective treatment for IBD.The combination blocking-up of IL-1 β and IL-18 also provides the treatment more safer than TNF-α-block.

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Claims (26)

1. the method for the treatment of disease in the patient, described method comprises to described patient uses significant quantity:

A.IL-1 β/IL-18 bi-specific antibody; Or

B. in conjunction with the antibody of IL-1 β and IL-18 activity; Or

C. in conjunction with the antibody of IL-1 β with in conjunction with the antibody of IL-18;

Wherein the described antibody of part a, b or c can neutralize or block IL-1 β and IL-18 activity in cell or tissue.

2. the process of claim 1 wherein that described antibody is humanized.

3. the process of claim 1 wherein that the described antibody of part (b) is bifunctional antibody.

4. the process of claim 1 wherein that at least one antibody of part (c) is monoclonal.

5. the process of claim 1 wherein that each antibody of part (c) is monoclonal.

6. the process of claim 1 wherein part (c) described antibody simultaneously or continuous administration.

7. the method for claim 6, wherein said antibody was used in 1 hour.

8. the process of claim 1 wherein that described disease is Immunological diseases or autoimmune disease or inflammation or self inflammatory disease.

9. the process of claim 1 wherein that described disease is the disease of inflammation corpusculum mediation.

10. the process of claim 1 wherein that described disease is the IL-1 ss related diseases.

11. the process of claim 1 wherein that described disease is the IL-18 relative disease.

12. the process of claim 1 wherein that described disease is IL-1 β/IL-18 relative disease.

13. the method for claim 8, wherein said disease is age-related macular degeneration (AMD).

14. the method for claim 8, wherein said disease is diabetes B (T2D).

15. the method for claim 8, wherein said disease is inflammatory bowel (IBD).

16. the method for claim 15, wherein said IBD is regional ileitis (CD).

17. the method for claim 15, wherein said IBD is ulcerative colitis (UC).

18. the process of claim 1 wherein that described patient resists the TNF treatment and do not produce and reply.

19. the method for the treatment of disease in the patient, described method comprises to described patient uses the monoclonal antibody in conjunction with IL-1 β of significant quantity and in conjunction with the monoclonal antibody of IL-18.

20. the method for neutralization or blocking-up IL-1 β and/or IL-18 activity in cell or tissue, described method comprises makes described cell or tissue contact with the monoclonal antibody in conjunction with IL-18 with the monoclonal antibody in conjunction with IL-1 β, and neutralizes thus or block described activity.

21. the method for claim 19, wherein in conjunction with the described monoclonal antibody of IL-1 β and in conjunction with the described monoclonal antibody of IL-18 simultaneously or continuous administration.

22. the method for claim 20, wherein said cell simultaneously or with the described monoclonal antibody in conjunction with IL-1 β, with the described monoclonal antibody in conjunction with IL-18, contact continuously.

23. the antibody of neutralization or blocking-up IL-1 β and IL-18 activity.

24. the antibody of claim 1, wherein said antibody is bi-specific antibody.

25. the antibody of claim 1, wherein said antibody is humanized.

26. the antibody of claim 1, wherein said antibodies IL-1 β and IL-18.

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