CN101506659A - Anti-drug antibody assay - Google Patents
Anti-drug antibody assay Download PDFInfo
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- CN101506659A CN101506659A CN200780030744.6A CN200780030744A CN101506659A CN 101506659 A CN101506659 A CN 101506659A CN 200780030744 A CN200780030744 A CN 200780030744A CN 101506659 A CN101506659 A CN 101506659A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
Abstract
The invention provides an antibody binding specifically to Cynomolgus IgG characterized by not binding to Human IgG, and a method for the immunologic al determination of an immune complex (DA/ADA complex) of a drug antibody (D A) and an antibody against said drug antibody (anti-drug antibody, ADA) in a sample of a monkey species using a double antigen bridging immunoassay.
Description
Invention field
The present invention comprises the method for measuring anti-drug antibodies and is used for the kit of such determination method.
Background of invention
Use the standard solid-phase immunoassay of monoclonal antibody relate to be adsorbed on antibody (capture antibody) on the solid phase, antigen and at another epi-position of antigen, puted together between the antibody (tracer antibody) of enzyme or detectable label and formed compound.Form sandwich body thus: solid phase-capture antibody-antigen-tracer antibody.In sandwich body, the enzymatic activity that antibody is puted together (or amount of detectable label) is proportional with the antigen concentration of hatching in the medium.A kind of sandwich assay is two antigen bridging immunoassays (double antigen bridging immunoassay), wherein catches and the different epi-position combinations of tracer antibody with antigen.Hoesel, people such as W. have reported the two antigen bridging determination methods of anti-EPO in J.Immunol.Methods 294 (2004) 101-110, used with amino and with the potpourri of the immobilization rhEPO of glycosyl coupling.Immunoassay such as two antigen bridging ELISA be the research patient for the immunogenic response of antibody drug in the common determination method type of application of institute.Mire-Sluis, people such as A.R. in J.Immunol.Methods 289 (2004) 1-16, have summed up design and have optimized the suggestion of detection at the immunoassay of host's antibody of biological technology products.According to people such as Mire-Sluis, well-known anti-drug antibody assay form demonstrates many shortcomings.Anti-drug antibody assay has mentioned in for example WO 2005/045058 and WO 90/006515.The anti-idiotype determination method has mentioned in for example US 5,219,730, WO 87/002778, EP 0 139 389 and EP 0 170302.Wadhwa, people such as M. have reported the method for the unnecessary antibody that detection, measurement and sign are induced by the therapeutic biological products in J.Immunol.Methods 278 (2003) 1-17.Described among the PCT/EP2007/001935 with two antigen bridging immunoassays carry out the immunoassay of immunoassays in the sample at the antibody of drug antibody.The immunoassay of measuring the people's antibody in the monkey has been described among the WO 2006/066912.The mensuration system that (for example US 2003/0068664) can the detection of active therapeutic antibodies in this area is known equally.Such system needs antigen to combine with solid phase, and therapeutic antibodies is the antigen combination of combination therewith, and detects the therapeutic antibodies that combines with solid phase by antigen.
The invention summary
The invention provides the antibody that combines, do not combine with machin (Cynomolgus) IgG specificity with human IgG.Preferred described antibody is monoclonal antibody.
In preferred embodiments, antibody according to the present invention is produced with clone 3.25.12 (DSMACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSM ACC2802) or 7.72.32 (DSM ACC2803).
The invention provides and use sandwich assay, (anti-drug antibodies, immune complex ADA) carries out the method for immunoassays to promptly two antigen bridging immunoassays to the sample Chinese traditional medicine antibody (DA) of monkey kind with at the antibody of described drug antibody.
Described immune complex further is abbreviated as the DA/ADA compound.
The invention provides the method for DA/ADA compound in the immunoassays sample, its use comprises sandwich type or two antigen bridging immunoassay of capture antibody and tracer antibody, be characterised in that one of described antibody, be tracer antibody or capture antibody, be the antibody that combines with machin IgG specificity, another antibody is the antibody that combines with the human immunoglobulin(HIg) specificity.
In a preferred embodiment of the invention, capture antibody is the anti-people Ig antibody that combines with the human immunoglobulin(HIg) specificity, and tracer antibody is the anti-machin IgG antibody that combines with machin IgG specificity.In a preferred embodiment of the invention, capture antibody is the anti-machin IgG antibody that combines with machin IgG specificity, and tracer antibody is the anti-people Ig antibody that combines with people Ig specificity.
Preferred anti-people Ig antibody combines with the human IgG specificity.Preferred anti-machin IgG antibody and/or anti-people Ig antibody are monoclonal antibody.Preferred described specificity does not combine with machin IgG in conjunction with the antibody of human immunoglobulin(HIg).Preferred described antibody in conjunction with human immunoglobulin(HIg) is produced by clone DSMACC2708.Preferred described antibody in conjunction with machin IgG does not combine with human IgG.Preferred described antibody in conjunction with machin IgG is produced by clone 3.25.12 (DSM ACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSM ACC2802) or 7.72.32 (DSM ACC2803).
In described mensuration process, between anti-machin IgG antibody, DA/ADA compound and anti-people Ig antibody, form compound, the amount of the compound of formation is associated with the concentration of DA/ADA compound, DA and/or ADA.
According to the present invention, can carry out direct sample analysis for the detection of the DA/ADA compound that forms.In such determination method, have only and contain drug antibody in the sample and anti-drug antibodies just can be seen positive signal.
According to the present invention, alternatively, sample analysis (can) with the drug antibody preincubate of scheduled volume after carry out.In such determination method, if contain anti-drug antibodies in the sample, just can see positive signal, exist/there is not drug antibody in the sample and rely on.
Preferred capture antibody is puted together by passive absorption and solid phase, therefore puts together in two different antibody sites and solid phase at least.Butler for example, J.E., Solid Phases in Immunoassay, in: Immunoassay, Diamandis, E.P. and Christopoulos, T.K. (volume) academic press, Santiago (1996) are described to some extent to passive absorption in the 205-225 page or leaf.
In a preferred embodiment of the invention, capture antibody by specificity in conjunction with to immobilization.Such combination to (first component/second component) is, for example streptavidin or avidin/biotin, antibody/antigen (are seen for example Hermanson, G.T. wait the people, BioconjugateTechniques, the academic press, 1996), agglutinin/polysaccharide, steroids/steroid binding protein, hormone/hormone receptor, enzyme/substrate, IgG/ albumin A and/or G etc.Preferred capture antibody and biotin-conjugated are fixed by immobilization avidin or streptavidin.
Preferred antibody is puted together puting together of gametophyte with it be to realize by chemical bond, carries out combination via N end and/or epsilon-amino (lysine), the epsilon-amino of different lysines, carboxyl, sulfydryl, hydroxyl and/or the phenol functional group of antibody amino acid backbone and/or the sugar alcohol base of antibody sugar structure.
In a preferred embodiment of the invention, tracer antibody and detectable mark are puted together, preferably by specificity in conjunction with to puting together.Such combination to (first component/second component) is, for example streptavidin or avidin/biotin, antibody/antigen, agglutinin/polysaccharide, steroids/steroid binding protein, hormone/hormone receptor, enzyme/substrate, IgG/ albumin A and/or G etc.Preferred tracer antibody is puted together by foxalin with at the antibody and the detectable mark of foxalin.Alternatively, tracer antibody and electrochemiluminescence mark are puted together, as the ruthenium bipyridyl complexes.
Provide in other embodiment of the present invention with in sandwich type or the two antigen bridging immunoassay immunoassays monkey kind sample at antibody (anti-drug antibodies, method ADA) of drug antibody.
The invention provides the method for ADA in the immunoassays sample, use comprises sandwich type or two antigen bridging immunoassay of capture antibody and tracer antibody, be characterised in that one of described antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, another antibody is drug antibody.
In the preferred embodiment of immunoassays ADA, capture antibody is a drug antibody, and tracer antibody is the anti-machin IgG antibody that combines, do not combine with human IgG with machin IgG specificity.In other preferred embodiment of immunoassays ADA, capture antibody is the anti-machin IgG antibody that combines, do not combine with human IgG with machin IgG specificity, and tracer antibody is a drug antibody.In described mensuration process, between drug antibody, ADA and anti-machin IgG antibody, form compound, the amount of the described compound of formation is associated with the concentration of ADA.In the preferred embodiment of immunoassays ADA, described anti-machin IgG antibody is monoclonal antibody (anti-machin mAb).
Another embodiment of the present invention is hybridoma cell line 3.25.12 (DSM ACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSMACC2802), 7.72.32 (DSM ACC2803).
Another aspect of the present invention is the antibody compositions that is used for the method according to this invention, comprises the potpourri of the antibody of being produced by clone DSM ACC2799, clone DSM ACC2800, clone DSM ACC2801, clone DSM ACC2802 and/or clone DSM ACC2803.
Detailed Description Of The Invention
The invention provides the antibody that combines, do not combine with machin IgG specificity with human IgG.
According to the present invention, term " drug antibody " expression can be used to treat the antibody of disease individuality.In a kind of determination method of carrying out according to the present invention, drug antibody and capture antibody, or drug antibody and tracer antibody comprise " identical " antibody molecule respectively, for example use identical expression vector recombinant production, and comprise identical amino acid sequence.Drug antibody (therapeutic monoclonal antibodies) is widely used in treating multiple disease such as tumor disease (for example blood and solid malignant comprise non-Hodgkin lymphoma, breast cancer and colorectal cancer), immunity disease, nervous centralis disease, vascular diseases or infectious disease.For example at Levene, people such as A.P., Journal of the Royal Society ofMedicine 98 (2005) 145-152 describe to some extent to such antibody.Such antibody is for example at the antibody of CD20, CD22, HLA-DR, CD33, CD52, EGFR, G250, GD3, HER2, PSMA, CD56, VEGF, VEGF2, CEA, Levis Y antigen, IL-6 acceptor or IGF-1 acceptor.Therapeutic antibodies is at Groner, people such as B., Cu rr.Mol.Meth.4 (2004) 539-547; Harris, M. also describes among Lancet Oncol. (2004) 292-302 to some extent.
The example of therapeutic/drug antibody (preferred monoclonal antibody) is the antibody (mAb IL-6R) at the IL-6 acceptor.Such antibody is at for example Mihara, people such as M., Clin.Immunol.98 (2001) 319-326; Nishimoto, people such as N., Blood 106 (2005) 2627-2632; Clinical trial NCT00046774; Describe to some extent among the WO 2004/096274.
The example of therapeutic/drug antibody (preferred monoclonal) is the antibody (mAbIGF-1R) at the IGF-1 acceptor.Such antibody is described among the WO 2005/005635 to some extent at for example WO 2004/087756.
" anti-drug antibodies " is antibody, and it can be at any zone of drug antibody, for example the variable domains of drug antibody, constant domain or sugared structure.Such anti-drug antibodies may the immunogenic response as the patient (see Pan, people such as Y., FASEB be (1995) 43-49 J.9) occur in the Antybody therapy process.Most " anti-drug antibodies " combines with one or more complementary determining regions of drug antibody.Affinity between the antigen of anti-drug antibodies and its drug antibody is usually less than the affinity of drug antibody and its target antigen.
" anti-machin IgG antibody " is the antibody that combines with machin IgG (machin immunoglobulin G) specificity, is preferably monoclonal antibody (be monoclonal anti machin antibody, mAb＜CynoIgG 〉).The dissociation constant that such antibody combines with machin IgG (=KDiss.) be at least 10
-9Mol/L, more preferably KDiss. is few 10
-10Mol/L.Simultaneously, by 10
-8Mol/L or worse KDiss. for example 10
-5Mol/L guarantees the character that it does not combine with human IgG.Equally preferably, with the antibody that machin IgG combines, do not combine with human IgG, the difference to KDiss. between the reactivity of machin IgG and human IgG is at least 100 times respectively.
Preferred anti-machin IgG antibody also combines with marmoset IgG, rhesus macaque IgG and baboon IgG, dissociation constant (=KDiss.) be at least 10
-8Mol/l is preferably 10
-9Mol/L, more preferably KDiss. is at least 10
-10Mol/L.
In one embodiment, be monoclonal antibody according to antibody of the present invention.The antibody population by single kind cell or its filial generation production represented in the term of Shi Yonging " monoclonal " in this application, and combine with the single antigenic determinant of its target antigen.The term of Shi Yonging " does not combine with human IgG " or it is equal to the grammer term and represents the antibody that do not combine with the human IgG specificity in this application, and promptly its KDiss. is 10
-7Mol/L or poorer, for example 10
-5Mol/L.This does not comprise the polyclonal antibody group that machin is such, and it intersects absorption to remove the machin IgG that combines with human IgG with human immunoglobulin(HIg).Because the binding equilibrium that forms in this process, the absorption that intersects does not provide the polyclonal antibody that does not combine with human IgG group, let alone monoclonal antibody.Because many antibody that combine with human IgG do not intersect absorption under this balance, and stay in the solution, so stay in the antibody preparation that obtains.Therefore, immunoglobulin (Ig) can not got rid of the combination of any anti-human IgG fully, and still show the human IgG combination in case intersect absorption with human IgG.
Others of the present invention are clone DSM ACC2799, clone DSM ACC2800, clone DSM ACC2801, clone DSM ACC2802, clone DSM ACC2803, and the monoclonal antibody of being produced by clone DSM ACC2799 or clone DSM ACC2800 or clone DSMACC2801 or clone DSM ACC2802 or clone DSM ACC2803.Another aspect of the invention is antibody compositions, comprise the potpourri of the antibody of producing by clone DSMACC2799, clone DSM ACC2800, clone DSM ACC2801, clone DSMACC2802 and/or clone DSM ACC2803.The present invention also comprises composition, and it comprises the antibody by clone DSM ACC2799 or clone DSM ACC2800 or clone DSM ACC2801 or clone DSM ACC2802 or clone DSMACC2803 production.
Use is according to antibody of the present invention, can minimize or even gets rid of the change of background of individual human serum.
According to the present invention, term " monkey " refers to machin, marmoset, rhesus macaque and baboon." monkey " preferably represents machin, rhesus macaque and baboon.
Preferred anti-people Ig antibody (Ig represents immunoglobulin (Ig)) is such antibody, and it combines with epitope specificity on not being present in the machin immunoglobulin (Ig), in WO 2006/066912 this is described to some extent.This epi-position is characterised in that itself and MAB＜H-Fc γ pan〉M-R10Z8E9, also claim MAB＜h-Fc gamma〉M-R10Z8E9, or be called for short MAB M-R10Z8E9 combination.In according to the preferred embodiments of the invention, anti-people Ig antibody is further characterized in that the epi-position of its combination is identical with MAB M-R10Z8E9.MAB M-R10Z8E9 is by such clone production, and this cell lies in and was preserved in German microbial preservation center (DSMZ) on Dec 22nd, 2004, and preserving number is DSM ACC2708.Preferred anti-people Ig antibody comprises variable heavy chain and the light chain domain of MAB M-R10Z8E9.More preferably anti-people Ig antibody comprises the CDR district and the inhuman framework of MAB M-R10Z8E9 variable heavy chain and light chain domain.Preferred anti-people Ig antibody is monoclonal antibody (anti-people Ig mAb).
Preferably use
Instrument assessment antibody in conjunction with character, KDiss. particularly.In the method, the variation evaluation by surface plasma body resonant vibration (SPR) is in conjunction with character.Can be easily with the antibodies studied on solid phase (being called chip), and assessment monoclonal antibody, polyclonal antibody or even comprise the combination that the serum of IgG wraps the chip of quilt therewith.
According to the present invention, the solid support that is used for immunoassay has extensive description (seeing for example Butler, J.E., Methods 22 (2000) 4-23) in state of the art.
Hage for example, D.S. has described different immunoassays ratio juris in Anal.Chem.71 (1999) 294R-304R.Lu, people such as B. have reported that in Analyst.121 (1996) 29R-32R the orientation of antibody fixes, to be used for immunoassay.Wilchek for example, M. and Bayer, E.A., Methods Enzymol.184 (1990) 467-469 have reported the immunoassay of avidin-biotin mediation.
The term that uses among the present invention " two antigen bridging immunoassay " expression sandwich type immunoassay, wherein antigen combines with two kinds of different antibodies, its separately with antigen in the different epi-position combinations of non-overlapping or interference.In this determination method, form the sandwich body comprise capture antibody, antigen and tracer antibody, antigen thus with it two kinds of antibody bridgings of combination get up.
As protein, monoclonal antibody and its constant domain comprise many reactive side chains and binding partners coupling, and binding partners is as surface, protein, polymkeric substance (for example PEG), cellulose or polystyrene, enzyme or in conjunction with right member.The chemical reaction group of antibody is for for example, amino (epsilon-amino of lysine, alpha-amido), mercapto (cystine, halfcystine, methionine), carboxylic acid group's (aspartic acid, glutamic acid) and sugar alcohol base.Aslam for example, M. and Dent, A., Bioconjuation, wheat Courlene publishing company (MacMillan Ref.Ltd.) (1999), the 50-100 page or leaf is described to some extent to such method.
Term " sample " comprises the material from any amount of monkey.Such material includes but not limited to from the whole blood of such individuality, serum or blood plasma, and these are the most widely used sample sources in the routine before clinical.
Term " solid phase " refers to comprise non-fluid substance: particle (comprising particulate and pearl), its by as make such as materials such as polymkeric substance, metal (paramagnetic, ferromagnetic particle), glass and potteries; Gelatinous mass such as silica gel, aluminium oxide and polymer gel; Kapillary, it can be made by polymkeric substance, metal, glass and/or pottery; Zeolite and other porous mass; Electrode; Microwell plate; Solid detector bar (strip); Cuvette, pipe or other spectrometer sampling receptacle.The difference on the inert solid surface that may touch in solid phase element in the determination method and the determination method is that " solid phase " element surface comprises at least one expection and the interactional part of capture antibody.Solid phase can be a retaining element, as pipe, bar, cuvette or microwell plate, maybe can be unfixed element, as pearl and particulate.Particulate can also be used for even phase determination method form as solid phase.Can use and allow protein and other material is non-covalent or the multiple particulate of covalent attachment.Such particle comprises: polymer beads such as polystyrene and polymethylmethacrylate; Gold grain such as gold nano grain and aurosol; With ceramic particle such as silica gel, glass and metal oxide particle.See for example Martin, people such as C.R., Analytical Chemistry-News ﹠amp; Features70 (1998) 322A-327A, it incorporates this paper by reference into.
The example of detectable mark is chromogen (fluorescence or luminophore and dyestuff), enzyme, NMR-reactive group or metallic particles, haptens, for example foxalin.Detectable mark can also be the photoactivated cross-linking group, for example azido or aziridinyl (azirine).Metallo-chelate that can the electricity consumption chemiluminescence detection also is preferred signal emission group, preferred especially ruthenium chelate, for example ruthenium (dipyridine)
3 2+Chelate.The ruthenium labelling groups that is fit to has for example been described among EP 0 580 979, WO 90/05301, WO 90/11511 and the WO92/14138.
Immunoassay is known by the technician.Summed up the method for implementing such determination method in the relevant textbook, and practical use and operation.The example of relevant textbook has Tijssen, P., Preparation of enzyme-antibody or other enzyme-macromoleculeconj ugates, in: Practice and theory of enzyme immunoassay, Burdon, R.H. and v.Knippenberg, P.H. (volume), like to think only your company (Elsevier), Amsterdam (1990) 221-278 page or leaf; Colowick, S.P. and Caplan, N.O. (volume), " Methodsin Enzymology ", the academic press, in relate to the multireel of immunologic detection method, particularly roll up 70,73,74,84,92 and 121.
Can be produced by hybridoma cell line 3.25.12 (DSM ACC2799), 3.29.15 (DSM ACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSMACC2802), 7.72.32 (DSM ACC2803) according to antibody of the present invention, these hybridoma cell lines itself also are an aspect of of the present present invention.According to other antibody of the present invention, i.e. the antibody that combines, do not combine with machin IgG specificity with human IgG, can use-case such as embodiment 3 in the method for general introduction obtain.
Alternatively, for example can use such method, wherein the epi-position of two kinds of antibody combining with same target antigen with the competition experiments system measurement earlier of the first step is overlapping.For this purpose, for example by enzyme immunoassay, the antibody studied of test combines the degree of immobilization target antigen with the known antibodies competition, for example according to the competition combination degree of the antibody of clone production of the present invention.For this purpose, with suitably the known antibodies and the excessive antibody of studying of immobilized target antigen and mark pattern are hatched.Under the antibody existence of studying by being determined at and non-existent situation, the amount of the antibodies of mark pattern can be assessed the degree that the antibody of being studied can be replaced the combination of known antibodies.If replace more than 20%, preferably more than 30% in same concentration; Perhaps the antibody of being studied is in higher concentration, and preferred 10
3-10
5When doubly being in excess in known antibodies, if displacement is then overlapping for epi-position takes place preferably more than 80% more than 70%, two kinds of antibody all with the identical of same epi-position or lap combination.In second step of described method, use the antibody of identifying like this.Measure combining of the antibody identified in the first step and human IgG this moment.Can carry out such mensuration with ELISA for example, immunoassay or with surface plasma body resonant vibration.Do not combine with human IgG if measure it, then defining such antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity.Alternatively, can be according to antibody of the present invention by measuring it respectively for the KDiss. of machin IgG and human IgG and contrast these numerical value and obtain identifying.
The present invention has reported the method for immunity of measuring the compound of drug antibody and anti-drug antibodies, and described compound is expressed as the DA/ADA compound hereinafter.In more detail, the present invention comprises with the sandwich type immunoassay to monkey kind sample Chinese traditional medicine antibody (DA) with at the antibody (anti-drug antibodies of described drug antibody, ADA) method that immune complex (DA/ADA compound) carries out immunoassays, this method comprises capture antibody and tracer antibody, one of wherein said antibody is the antibody that combines with machin IgG specificity, preferably do not combine with human IgG, and another described antibody is the antibody that combines with the human IgG specificity, preferably do not combine with machin IgG.In one embodiment, capture antibody is the antibody that combines with the human IgG specificity, do not combine with machin IgG, and tracer antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity.In different embodiments, capture antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and tracer antibody is the antibody that combines with the human IgG specificity, do not combine with machin IgG.In another embodiment of the present invention, antibody that combines with machin IgG specificity and/or the antibody that combines with the human IgG specificity are monoclonal antibody.
In the preferred embodiment of this method, the drug antibody preincubate of sample and scheduled volume.This makes no matter whether to have drug antibody in the sample, all can detect anti-drug antibodies, is DA/ADA compound (seeing for example Fig. 5 and 6) because this method detects.
According to the height homogeneity between people or humanization drug antibody and the machin IgG, the major antigen determinant of drug antibody is its complementary determining region.Anti-drug antibodies is preferably discerned these antigenic determinants.So, thereby complementary determining region, the also combination with it of most anti-drug antibodies identification relative medicine antibody.The formation of anti-drug antibodies-drug antibody compound shielding epitope, thus in the antagonism sandwich assay to the mensuration of described compound.
For example as described at WO 2006/066912, anti-human IgG antibody is the antibody that combines with such epitope specificity, and described epi-position is not present on the immunoglobulin (Ig) that obtains from machin.In preferred embodiments, anti-human IgG antibody is further characterized in that, its epi-position with the antibodies of being produced by clone DSM ACC2708 is identical, just combine with such epi-position, described epi-position is present in all subclass of G class human immunoglobulin(HIg), and except on the IgG that is present in chimpanzee, on the immunoglobulin (Ig) of most experiments animal, do not exist.In other preferred embodiment, anti-human IgG antibody is the antibody of being produced by clone DSM ACC2708.
Find that surprisingly use the method according to this invention has prevented the incomplete detection for anti-drug antibodies.For example, if capture molecules and anti-drug antibodies be in conjunction with identical antigenic determinant, i.e. the CDR of drug antibody, then combining of drug antibody and capture molecules can these epi-positions of sealing, be about to them and shield, thereby stoped the combination of anti-drug antibodies and be detected with respect to anti-drug antibodies.This causes the incomplete detection to the anti-drug antibodies in the sample.Anti-drug antibodies has low binding affinity for drug antibody usually, so bound drug antibody needs the cooperation of two antigen binding domains of anti-drug antibodies.So, can not combine the low detection that also may cause resisting drug antibody with capture molecules.Therefore, the CDR by anti-drug antibodies catches and is not suitable for measuring.
Therefore, an aspect of of the present present invention is to monkey kind sample Chinese traditional medicine antibody (DA) with at the antibody (anti-drug antibodies of described drug antibody with the sandwich immunoassay method that comprises capture antibody and tracer antibody, ADA) method that immune complex (DA/ADA compound) carries out immunoassays, one of wherein said antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and another antibody is the antibody that combines with the human IgG specificity, do not combine with machin IgG.In the embodiment in this regard, the antibody that combines, do not combine with human IgG with machin IgG specificity is produced by clone DSM ACC2799 or DSM ACC2800 or DSM ACC2801 or DSM ACC2802 or DSM ACC2803.The antibody that in another embodiment in this regard, combine with the human IgG specificity, does not combine with machin IgG is produced by clone DSM ACC2708.
In an embodiment of this method, the amount of the compound of formation is associated with the concentration of DA/ADA compound, DA and/or ADA.
In another embodiment, capture antibody combines with anti-drug antibodies or DA/ADA compound, not with the CDR of anti-drug antibodies or combining with CDR next-door neighbour's framework region on the sequence or on the geometry.
Another aspect of the present invention be with the sandwich immunoassay method that comprises capture antibody and tracer antibody in the monkey kind sample at the antibody (anti-drug antibodies of drug antibody, ADA) carry out the method for immunoassays, wherein capture antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and tracer antibody is a drug antibody.Also have be on the one hand with the sandwich immunoassay method that comprises capture antibody and tracer antibody in the monkey kind sample at the antibody (anti-drug antibodies of drug antibody, ADA) carry out the method for immunoassays, wherein tracer antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, and capture antibody is a drug antibody.
According to preferred hybridoma cell line 3.25.12 of the present invention, 3.29.15,4.38.30,7.57.41 and 7.72.32 according to Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure be preserved in German microbial preservation center (Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, DSMZ):
| The clone | Preserving number | Preservation day |
| 3.25.12 | DSM ACC2799 | 2006.08.24 |
| 3.29.15 | DSM ACC2800 | 2006.08.24 |
| 4.38.30 | DSM ACC2801 | 2006.08.24 |
| 7.57.41 | DSM ACC2802 | 2006.08.24 |
| 7.72.32 | DSM ACC2803 | 2006.08.24 |
| MAB M-R10Z8E9 | DSM ACC2708 | 22.12.2004 |
The antibody that can be got by described clone is embodiment of the present invention.
Provide hereinafter embodiment and accompanying drawing to help the understanding of the present invention, true scope of the present invention is illustrated by the claims of enclosing.Should understand in the case of without departing from the spirit of the present invention and can make modification the method for listing.
Description of drawings
Fig. 1: the determination method that detects the DA/ADA compound---do not add DA.
Biotinylated anti-people Ig antibody is fixed on the microwell plate (SA-MTP) of streptavidin bag quilt.Drug antibody/anti-drug antibodies (DA/ADA) compound is immobilized anti-people Ig antibody (Bi; Biotinylated) institute catch.(Dig with the foxalin mark; The digoxigenin glycosidation) anti-machin IgG antibody and anti-foxalin antibody horseradish peroxidase (POD) conjugate (anti-DIG antibody of polyclone and POD put together pAb＜Dig〉POD) detect the DA/ADA compound of combination.With with the chemically conjugated human IgG of machin IgG as standard.
Fig. 2: the determination method that detects the DA/ADA compound---add DA.
Before sample analysis, use damping fluid dilution monkey serum sample, the admixture drug antibody based on PBS.After 15 minutes preincubates, with above-mentioned elisa assay sample (seeing the explanation of Fig. 1).
Fig. 3: with the determination method of anti-machin antibody test ADA.
Biotinylated drug antibody is combined (Bi with the microwell plate (SA-MTP) of streptavidin bag quilt; Biotinylated).Anti-drug antibodies (ADA) combines with the immobilization drug antibody.(Dig with the foxalin mark; The digoxigenin glycosidation) anti-machin IgG antibody and anti-foxalin antibody horseradish peroxidase conjugate (pAb＜Dig〉POD) detect the ADA of combination.With with the chemically conjugated anti-human IgG antibody of machin IgG as standard.
Fig. 4: the typical curve of DA/ADA compound determination method.
Damping fluid dilution and the chemically conjugated human IgG of machin IgG based on PBS with containing 1% (v/v) machin serum have provided its optical density (OD) under a plurality of concentration among the figure.
Fig. 5: in machin, carry out HuMab＜IGF-1R〉single dose of drug dynamics research (3mg/kg; Intravenous injection), detect DA/ADA compound in this study sample.
8 time points between 0h after the administration and 1176h are collected blood serum sample, with elisa assay (as shown in Figure 1), do not add drug antibody.The amount (the OD signal of 405nm) of DA/ADA compound was mapped with respect to the time after the administration.
Fig. 6: in machin, carry out HuMab＜IGF-1R〉single dose of drug dynamics research (3mg/kg; Intravenous injection), detect DA/ADA compound in this study sample.
8 time points between 0h after the administration and 1176h are collected blood serum sample, with elisa assay (as shown in Figure 2), add drug antibody.The amount (the OD signal of 405nm) of DA/ADA compound was mapped with respect to the time after the administration.
Fig. 7: the contrast that monoclonal anti machin IgG and polyclone machin IgG detect for machin IgG.
Damping fluid dilution and the chemically conjugated human IgG of machin IgG based on PBS with containing 1% (v/v) machin serum have provided its optical density (signal (median)) under a plurality of concentration among the figure.
Embodiment
Preparation machin IgG and machin Fc segment
A) preparation machin IgG
Machin serum warp
380 degreasings are with ammonium sulfate (ad 2.0M) precipitation.To be deposited in phosphate buffer and homogenize, with phosphate buffer, pH 7.0 dialysis.With the DEAE ion-exchange chromatography at pH 7.0 separating mixtures, by the IgG in the concentrated and purified effluent of gel filtration.
B) preparation machin Fc
In the presence of the 15mM halfcystine, at 37 ℃, pH 7.0 usefulness papains (the every mg IgG of 4mU papain) are with the IgG segmentization of purifying in a).After 80 minutes, potpourri and iodoacetamide (ad 30mM) are hatched at 25 ℃, subsequently to contain 30mM NaCl, the 10mM HEPES damping fluid dialysis of pH 7.5.With Q-agarose ion-exchange chromatography separating mixture.The Fab fraction reaches the salt gradient wash-out Fc fraction of 1M sodium chloride with concentration in effluent.At last, the Fc fraction is dialysed with phosphate buffer, by the gel filtration purifying.
Produce monoclonal anti machin IgG antibody
A) immune mouse
With 100 μ g machin IgG (machin immunoglobulin G) or the machin Fc that mixes with CFA (Freund's complete adjuvant), the female Balb/c or the NMRI mouse in 8-12 age in week carried out initial immunity in the peritonaeum respectively.After 4,7 and 10 weeks, the machin IgG that every mouse mixes with IFA (incomplete Freund) with 100 μ g carries out immune step in three peritonaeums again.Merging first three day then, each is in PBS (phosphate buffered saline(PBS) with 100 μ g; Add antihistamine and adrenaline) in machin IgG carry out the intravenous booster immunization.
B) fusion and clone
According to Galfr é, G. and Milstein, C., Methods Enzymol.73 (1981) 3-46 will be according to a) splenocyte and the myeloma cell of mice immunized are merged.With about 1 * 10
8Splenocyte and about 2 * 10
7Myeloma cell (P3x63-Ag8.653, ATCC CRL1580) mixes, centrifugal (10 minutes, 300 * g, 4 ℃).With the RPMI 1640 nutrient culture media washed cells of no FCS (hyclone) once, centrifugal once more with 400 * g in the sharp bottom tube of 50mL afterwards.After this, (polyglycol, molecular weight 4 000g/mol), mix by imbibition to add 1mL PEG.After 1 minute, dropwise add the RPMI1640 that 5mL does not have FCS 37 ℃ of water-baths, mix suspension, fill it up with to 50mL with the RPMI 1640 that contains 10% (v/v) FCS, centrifugal then.The cell of precipitation is resuspended with the RPMI1640 that contains 10%FCS, containing growth factor interleukin 6 (IL-6, hypoxanthine 100U/mL)-azaserine is selected bed board in the nutrient culture media (100mmol/L hypoxanthine, 1 μ g/mL azaserine is among the RPMI1640 that contains 10%FCS).After about 10 days, measure the specific antibodies synthetic (referring to embodiment 3) of primary culture.Combine with machin IgG showing and not with the primary culture usefulness flow cytometer (FACSAria of the normal IgG cross reaction of people, BD Biological Science Co., Ltd (BD Biosciences)) carries out individuation by unicellular being deposited in the 96 porocyte culture plates, contain growth factor interleukin 6 (100U/mL) in the nutrient culture media.Produce clone's (table 1) of preservation according to this experimental program.Be used for clone of the present invention and be deposited in German microbial preservation center (DSMZ) (table 1).
Table 1:
Anti-machin mAb clone
| The clone | IgG class and subclass | Immunogene | Preserving number | Preservation day |
| 3.25.12 | IgG1，κ | IgG | DSM ACC2799 | 2006.08.24 |
| 3.29.15 | IgG1，κ | IgG | DSM ACC2800 | 2006.08.24 |
| 4.38.30 | IgG1，κ | IgG | DSM ACC2801 | 2006.08.24 |
| 7.57.41 | IgG2a，κ | Fc | DSM ACC2802 | 2006.08.24 |
| 7.72.32 | IgG1，κ | Fc | DSM ACC2803 | 2006.08.24 |
C) produce immunoglobulin (Ig) by cell culture supernatant
The hybridoma cell line that produces is with 1.0 * 10
5To 2.2 * 10
5The initial cell density (living cells) of the every mL of individual cell (depending on individual cell lines) is being added the RPMI1640 inoculation of medium of 10%FCS, with the time of stirring technique amplification 9 to 16 days (depending on individual cell lines).In the culture supernatant of results, the monoclonal anti bulk concentration reaches every mL 36 to 61 μ g.The purifying antibody that the culture supernatant obtains according to the standard protein chemical method for example according to Bruck, people such as C., Methods Enzymol.121 (1986) 587-596 carries out.
Embodiment 3
Detect the screening method of anti-machin IgG antibody
A) preliminary screening and the preferential antibody that combines of machin IgG
For measuring the specificity of antibody in the Hybridoma Cell Culture thing supernatant, streptavidin (MicroCoat will recombinate, Bernried company, batch MC 1098) wraps the MTP (microwell plate) of quilt in advance respectively with the biotinylated machin IgG that is among the PBS that adds 1.0% (w/v) BSA II, 250ng/mL or biotinylated human IgG, 250ng/mL wrap by (the every hole of 100 μ L, incubated at room 60 minutes, follow vibration), use 0.9% (w/v) NaCl/0.05% afterwards
20 washings three times.Next step, every hole adds 100 μ L antibody-solutions to be measured (culture supernatant), and incubated at room 60 minutes is followed vibration.Pass through with 0.9% (w/v) NaCl/0.05%
After three times the step of 20 washings, every hole adds the F (ab ') of the polyclone sheep anti mouse Fc gamma antibodies of 100 μ L horseradish peroxidase-labeled
2Segment, to detect the sample antibody of combination, incubated at room 60 minutes is followed vibration.Subsequently, by above washing.At last, every hole adds 100 μ L
(German Luo Shi diagnostic products company limited (Roche Diagnostics GmbH), catalog number (Cat.No.) 1684302).After the incubated at room 30 minutes, commercial microwell plate ELISA read in the plate device to measure 405 and 492nm[405/492] delustring (OD) located.The fine antibody that combines and human IgG is shown nothing/low cross reactivity with machin IgG is selected in this screening.The antibody of selecting is proceeded the mensuration of step b).
B) screening does not have the antibody of detectable cross reactivity to human IgG
In order identifying in the antibody of selecting a) from preliminary screening human IgG to be shown the antibody that does not have detectable cross reactivity, to carry out determination method described below.Streptavidin (MicroCoat will recombinate, Bernried company, batch MC1098) MTP that wraps quilt in advance is with the biotinylated machin IgG that is among the PBS (phosphate buffered saline(PBS)) that contains 1.0% BSA II, 250ng/mL wraps by (the every hole of 100 μ L, incubated at room 60 minutes, follow vibration), use 0.9% (w/v) NaCl/0.05% subsequently
20 washings three times.Next step adds every hole 100 μ L antibody-solutions (culture supernatant) to be measured and 50 μ L PBS (contrast signal) respectively, or 50 μ L human IgG solution (80mg/mL; Final concentration in the determination method: 27mg/mL; Measured signal), incubated at room 60 minutes is followed vibration.Pass through with 0.9% (w/v) NaCl/0.05%
After three times the step of 20 washings, every hole adds the F (ab ') of the polyclone sheep anti mouse Fc gamma antibodies of 100 μ L horseradish peroxidase-labeled
2Segment, to detect the sample antibody of combination, incubated at room 60 minutes is followed vibration.Subsequently, by above washing.At last, every hole adds 100 μ L
(German Luo Shi diagnostic products company limited, catalog number (Cat.No.) 1684302).After the incubated at room 30 minutes, the delustring of reading in the plate device to measure [405/492] nm place at commercial microwell plate ELISA.Selecting measured signal to show with relevant contrast signal does not have the antibody of significant difference to make further purposes.Quantizing angle, this equals cross reactivity with human IgG and is estimated as＜and 0.001%.
(the definition of " not having significant difference ": the 90-110% of measured signal=contrast signal.)
Embodiment 4
The conjugate of preparation machin IgG (Cyno-IgG) and human IgG (H-IgG)
A) preparation Cyno-IgG-SATP
Machin IgG by ion-exchange chromatography and gel filtration purifying from machin serum is with the 30mM kaliumphosphate buffer, pH 7.1 dialysis, and adjusting protein solution to the protein concentration that obtains is about 10mg/ml.N-succinimide-3-acetyl thio propionic ester (SATP) is dissolved in DMSO, in the mol ratio adding antibody-solutions with 1:5 (IgG:SATP).Potpourri is at 25 ℃, and pH 7.1 was hatched 60 minutes.By adding L-lysine to final concentration is that 10mM stops reaction, and pH is adjusted into pH 6.1, and to contain 200mM NaCl and 1mM EDTA, the 10mM kaliumphosphate buffer of pH 6.1 is dialysed, and removes excessive SATP.
B) preparation H-IgG-MH
The human IgG that passes through the ion-exchange chromatography purifying from human serum is with the 30mM kaliumphosphate buffer, and pH 7.1 dialyses, and adjusting protein solution to the protein concentration that obtains is about 20mg/ml.(Maleimidohexanoyl-N-hydroxysuccinimide ester MHS) is dissolved in DMSO, in the mol ratio adding antibody-solutions with 1:6 (IgG:MHS) with maleimide hexanoyl-N-hydroxy-succinamide ester.Potpourri is at 25 ℃, and pH 7.1 was hatched 60 minutes.By adding L-lysine to final concentration is that 10mM stops reaction, and pH is adjusted into pH 6.1, and to contain 200mMNaCl and 1mM EDTA, the 10mM kaliumphosphate buffer of pH 6.1 is dialysed, and removes excessive MHS.
C) put together Cyno-IgG-SATP and H-IgG-MH
Add 2.5% (v/v) 1M hydroxylamine solution, pH 7.5, hatched 60 minutes at 25 ℃, and be Cyno-IgG-SH with the Cyno-IgG-SATP deacetylation.The antibody of deacetylation mixes with H-IgG-MH that (mol ratio of Cyno-IgG-SH:H-IgG-MH=1:1) final concentration to total IgG is about 7mg/ml.PH is adjusted into 7.1,25 ℃ of mixtures incubated.Put together process with analytical solvent resistant column (for example TSK 3000) analysis.Generally speaking, after 40 minutes, be that 2mM stops to put together process by adding halfcystine to final concentration.After hatching 30 minutes, adding N-methyl maleimide (NMM) to final concentration is 5mM, and pH is adjusted into 7.5.25 ℃ hatch 60 minutes after, S-300 separates conjugate by gel filtration chromatography with the propylene sephadex, removes unconjugated antibody.
The monoclonal anti machin Fc antibody of preparation digoxigenin glycosidation
A) preparation monoclonal anti machin Fc antibody
The fermentation supernatant of monoclonal anti machin Fc antibody is concentrated about 10 times, move to and contain 20mMTRIS, 1M ammonium sulfate, the damping fluid of pH 9.0, last sample is to albumin A-agarose column.To use the 0.2M sodium citrate, the eluent of pH 3.0 wash-outs is with phosphate buffer, and pH 7.5 dialyses.By using immobilized antibody to carry out immunoadsorption separation ox IgG pollutant (from the FCS in the fermentation liquor) at ox IgG.
B) the digoxigenin glycosidation of monoclonal anti machin Fc antibody
The monoclonal anti machin Fc antibody-solutions that will be in the phosphate buffer is adjusted into pH 8.1, and concentration is about 2mg/ml.Foxalin-3-O-methyl carbonyl-EACA-N-hydroxy-succinamide ester is dissolved in DMSO, in the mol ratio adding antibody-solutions with 1:5.Stop reaction by adding L-lysine after 60 minutes, to contain 150mM NaCl, excessive labelled reagent is removed in the 50mM kaliumphosphate buffer dialysis of pH 7.5.
Embodiment 6
By
Combination/the specificity of system evaluation antibody
All measurements are all with using the CM5 chip
2000 instruments carry out.Realize the bag quilt of antibody by the standard amine coupling to chip.Except as otherwise noted, all incubation step are all carried out in HBS damping fluid (pH 7.4 for HEPES, NaCl) in 25 ℃.Different monoclonal anti machin IgG antibody, MAB M-R10Z8E9 and the anti-people Fc of polyclone gamma antibodies (Dianova company, Germany) with saturating capacity are separately fixed at by the amine coupling on the different slots (channel) of same CM5 chip.All animal blood serums all are diluted to final concentration in containing the HBS damping fluid of 1mg/ml Sensor Chip CM 5 be 1%.Analyzed combination in 60 seconds by injecting 1 to 100 serum that dilutes and hatching.Dissociate by measuring in 180 seconds with HBS damping fluid washing chip surface.With
BIAevaluation software, with blue Mil's model of fit (the Langmuir fitting model) computational solution of 1:1 from constant value (=KDiss.).For all animal blood serums, this calculating all is the hypothesis of 15mg/ml based on the IgG level.Selection begins to inject the amount (RU of table 2) that the signal value of test antibody after 80 seconds is used for contrasting the IgG of combination.
Table 2:
Animal blood serum and monoclonal anti machin IgG antibody and the sero-fast binding signal of the anti-people Fc of polyclone γ [RU]
Table 2 show monoclonal anti machin IgG antibody not with the human serum cross reaction.Only detected combining of its IgG that comprises with machin, rhesus macaque and baboon serum.Different with monoclonal anti machin IgG antibody, the high response of the serum of the anti-people Fc of polyclone antibody demonstration and people, dog and all test monkey kinds.
Embodiment 7
Measure the DA/ADA compound---do not add DA
In the first step, biotinylated MAB M-R10Z8E9 is bonded in the hole of microwell plate (SA-MTP) of streptavidin bag quilt.Remove excessive not binding antibody by washing.Afterwards, in the hole, hatch monkey serum sample and reference standard (the machin IgG in 1% machin serum is chemically conjugated for human IgG and admixture).After the unconjugated material of flush away, in conjunction with the DA/ADA compound with the anti-machin antibody test of digoxigenin glycosidation, then hatch with the anti-foxalin antibody of horseradish peroxidase-labeled.Antibody-enzyme conjugate catalysis
The chromogenic reaction of substrate.With ELISA read the plate device at wavelength 405nm place (with reference to wavelength: 490nm ([405/490] nm)) measuring-signal.Light absorption value with duplicate each blood serum sample of mensuration.Fig. 1 shows the scheme of this test macro of illustration.
Embodiment 8
Measure the DA/ADA compound---add DA
Before the sample analysis, the monkey serum sample is diluted the admixture drug antibody in the damping fluid based on PBS.After 15 minutes preincubates, by above-mentioned elisa assay sample.Fig. 2 shows the scheme of this test macro of illustration.
Embodiment 9
Detect the DA/ADA compound in the machin pharmacokinetic study sample
Use that (WO 2005/005635 at IGF-1R; 3mg/kg; People's antibody iv) carries out machin blood serum sample single dose of drug dynamics research (PK=pharmacokinetics), analyze with above-mentioned ELISA: i) detect the DA/ADA compound, do not add drug antibody (Fig. 5) and ii) detect the DA/ADA compound, add drug antibody (Fig. 6).8 time points between 0h after the administration and 1176h are collected blood serum sample and analysis.The amount (the OD signal at 405nm place) of DA/ADA compound was mapped with respect to the time after the administration.As shown in Figure 5, if form the AD/ADA compound in the body, and without external adding drug antibody, then in the blood serum sample only between 336h and 672h (peak shape) detect positive signal.Do not exist under the situation of anti-drug antibodies, do not have immune complex to form, detect less than positive signal (time point before the 336h).672h or afterwards after the administration owing to do not have medicine in the sample, can not detect compound.When in blood serum sample, adding drug antibody, all can detect/can detect the DA/ADA compound of interior formation of body and external formation as the preincubate step.As shown in Figure 6, positive signal only depends on the generation of ADA.Two width of cloth figure (Fig. 5 and 6) are all relevant well with medicine-time curve.
Carry out the determination method that ADA detects with anti-machin antibody
In the first step, biotinylated drug antibody (at people's antibody of IGF-1R) is attached in the hole of microwell plate (SA-MTP) of streptavidin bag quilt.Excessive unconjugated antibody is removed in washing.Afterwards, hatch monkey serum sample (dilution is 20 times in based on the damping fluid of PBS) and reference standard.After the unconjugated material of flush away, use the anti-drug antibodies (ADA) of the anti-machin antibody test combination of digoxigenin glycosidation, then use the anti-foxalin antibody incubation of horseradish peroxidase-labeled.Antibody-enzyme conjugate catalysis
The chromogenic reaction of substrate.Read the plate device with ELISA and measure the signal at wavelength 405nm place (with reference to wavelength: 490nm).Light absorption value with triplicate each blood serum sample of mensuration.Fig. 3 shows the scheme of this test macro of illustration.
Embodiment 11
Preparation is at the polyclonal antibody of machin IgG
A) purifying polyclonal antibody from rabbit anteserum
According to standard method, rabbit is carried out immunity with machin Fc.For from five original serum, remove fat composition wherein with Aerosil (1.5% (w/v)) degreasing, with ammonium sulfate (1.7M) precipitation immunoglobulin (Ig) with the rabbit of machin Fc immunity.Through acid treatment (30min., pH 5.5) and to add 50mM NaCl, after the 15mM kaliumphosphate buffer dialysis of pH 7.0, in pH 7.0 separating mixtures, the IgG fraction in the effluent (the anti-machin IgG of=rabbit polyclonal antibody) is concentrated into about 25mg/ml with the DEAE ion-exchange chromatography.
B) the anti-machin IgG of the multi-clone rabbit antibody (pAb＜Cyno-IgG 〉) of preparation affinity purification, it does not have cross reactivity to human IgG
The concentrated IgG fraction of step a) moved to add 150mM NaCl, the 50mM kaliumphosphate buffer system (PBS) of pH 7.5.With prior art machin IgG and NHS agarose are puted together, prepare immunosorbent, be filled in the post, with the 50mM kaliumphosphate buffer balance of adding 150mM NaCl pH 7.5 with immobilization machin IgG.
Sample on the 10mg IgG/ml immunosorbent is extremely used in the post of PBS balance.Successively with PBS, add 0.05% (w/v)
20 0.5M NaCl and 30mM NaCl wash post.IgG with 3mMHCl and 1M propionic acid wash-out combine with the affinity substrate specificity dialyses with PBS.
For removing the antibody that human IgG is had cross reactivity, to the affinity column with immobilization human IgG, this affinity column gets for prior art non-specific human IgG and NHS agarose being puted together preparation with sample on the antibody of affinity purification.Use the PBS balance columns.Sample is to post on will about 6mg IgG/ml immunosorbent.Specific polyclonal IgG fraction is in effluent.With adding 0.05% (w/v)
After 20 0.5M NaCl, 30mM NaCl, 1M propionic acid and PBS make column regeneration, repeat immunoadsorption twice, to remove the antibody that human IgG is had cross reactivity fully for the IgG of non-specific binding.
The anti-machin IgG of the purifying polyclone antibody that human IgG is not had a cross reactivity that obtains is concentrated into about 4mg/ml, is stored in-80 ℃.
Embodiment 12
With
Antibodies/the specificity of the anti-machin Fc of system evaluation multi-clone rabbit antibody (pAb＜Cyno-Fc 〉)
Whole measurements is all with using the CM5 chip
2000 instruments carry out.Realize the bag quilt of antibody by the standard amine coupling to this chip.Except as otherwise noted, all hatching all carried out at 25 ℃ in HBS damping fluid (pH 7.4 for HEPES, NaCl).The anti-machin IgG of the polyclone of saturating capacity antibody is fixed on the CM5 chip by the amine coupling.All animal blood serums all are diluted to final concentration in containing the HBS damping fluid of 1mg/ml Sensor Chip CM 5 be 1%.Analyzed combination in 60 seconds by injecting 1 to 100 serum that dilutes and hatching.Dissociate by measuring in 180 seconds with HBS damping fluid washing chip surface.With
BIAevaluation software, with the blue Mil's model of fit of 1:1 computational solution from constant value (=KDiss.).For all animal blood serums, this calculating all is the hypothesis of 15mg/ml based on the IgG level.Selection begins to inject the amount (RU of table 3) that the signal value of test antibody after 80 seconds is used for contrasting the IgG of combination
Table 3:
The binding signal [RU] of the anti-machin IgG of animal blood serum and polyclone antibody
Table 3 show the anti-machin IgG of polyclone antibody not with the human serum cross reaction.Only detected combining of its monkey IgG that comprises with marmoset, machin, rhesus macaque and baboon serum.Different with monoclonal anti machin IgG antibody, the anti-machin IgG of polyclone antibody shows the reactivity with the NMRI mice serum.And the anti-machin IgG of polyclone antibody do not combine with human IgG, and and monkey IgG and mouse IgG between reactive difference be at least 100 times.
The PCT/RO/134 table
B. (attached sheet) are identified in preservation
Applicant: Flax Huffmun-Laroqie Co., Ltd
Applicant's reel number: 23897WO-ASK
International application no: PCT/EP2007/007803
Other preservation thing
The clone of mentioning below all is preserved in
Germany microbial preservation center (DSMZ)
The D-38124 Brunswick
Cot Er Oudelu 1b
Preservation day preserving number
24.08.2006 DSM ACC2799
24.08.2006 DSM ACC2800
24.08.2006 DSM ACC2801
24.08.2006 DSM ACC2802
24.08.2006 DSM ACC2803
C. separate statement (attached sheet)
Applicant: Flax Huffmun-Laroqie Co., Ltd
Applicant's reel number: 23897WO-ASK
International application no: PCT/EP2007/007803
Expert's scheme statement of the biomaterial of preservation described in the instructions
The statement that relates to the biomaterial of preservation all is included in the instructions.Following separate statement need not the part into instructions, should be considered as " other statements ".This only relates to expert's scheme.
Following separate statement relates to the biomaterial of preservation mentioned in instructions the 12nd page table:
MAB M-R10Z8E9 DSM ACC2708
3.25.12 DSM ACC2799
3.29.15 DSM ACC2800
4.38.30 DSM ACC2801
7.57.41 DSM ACC2802
7.72.32 DSM ACC2803
Separate statement is as follows:
Specify for CA (Canada):
Specify for Canada, regulation according to Canadian Patent method patent detailed rules and regulations the 107th and 108, be awarded Canadian Patent or under day out of court or situation up to the application through abandoning and can not recovering again or be withdrawn, only provide the mode (detailed rules and regulations 104 (4)) of sample, the biological material specimens of institute's preservation is provided by Independent Expert to council's appointment.Specify for EP (European patent):
Specify for EPO, regulation according to EPC detailed rules for the implementation 28 (3), up to the application obtain the European patent mandate open or if the application is out of court, recall or the situation of deemed withdrawal under, from submit 20 years, only provide the mode (EPC detailed rules and regulations 28 (4)) of sample, the biological material specimens of institute's preservation is provided by expert to claimant's appointment.
Specify for SG (Singapore):
The applicant states hereby, according to nineteen ninety-five the 3rd section of Patent Law detailed rules and regulations the 4th scheme, only provides above-mentioned culture samples to the expert.
Claims (15)
1. the antibody that combines with machin IgG specificity is characterised in that it does not combine with human IgG, and is monoclonal antibody.
2. monoclonal antibody, it is produced by clone DSM ACC2799 or clone DSMACC2800 or clone DSM ACC2801 or clone DSM ACC2802 or clone DSM ACC2803.
With the two antigen bridging immunoassays that comprise capture antibody and tracer antibody to monkey kind sample Chinese traditional medicine antibody (DA) with at the antibody (anti-drug antibodies of described drug antibody, ADA) method that immune complex (DA/ADA compound) carries out immunoassays, be characterised in that one of described antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, another antibody is the antibody that combines with the human immunoglobulin(HIg) specificity.
4。The described method of claim 3 is characterised in that capture antibody is the antibody that combines with the human IgG specificity, and tracer antibody is the antibody that combines, do not combine with human immunoglobulin(HIg) with machin IgG specificity.
5. the described method of claim 3 is characterised in that capture antibody is the antibody that combines, do not combine with human immunoglobulin(HIg) with machin IgG specificity, and tracer antibody is the antibody that combines with the human IgG specificity.
6. each described method of claim 3 to 5 is characterised in that described antibody that combines with machin IgG specificity and/or the described antibody that combines with the human immunoglobulin(HIg) specificity are monoclonal antibody.
7. each described method of claim 3 to 6 is characterised in that the amount of the compound of formation is associated with the concentration of DA/ADA compound, DA and/or ADA.
8. claim 3 is characterised in that the described drug antibody preincubate of sample and scheduled volume to each described method.
With the two antigen bridging immunoassays that comprise capture antibody and tracer antibody in the monkey kind sample at the antibody (anti-drug antibodies of drug antibody, ADA) carry out the method for immunoassays, be characterised in that capture antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, tracer antibody is a drug antibody.
With the two antigen bridging immunoassays that comprise capture antibody and tracer antibody in the monkey kind sample at the antibody (anti-drug antibodies of drug antibody, ADA) carry out the method for immunoassays, be characterised in that tracer antibody is the antibody that combines, do not combine with human IgG with machin IgG specificity, capture antibody is a drug antibody.
11. each described method of claim 3 to 10, be characterised in that capture antibody by specificity in conjunction with to immobilization.
12. the described method of claim 11 is characterised in that capture antibody and biotin-conjugated, fixes by immobilization avidin or streptavidin.
13. each described method of claim 3 to 12, be characterised in that tracer antibody by specificity in conjunction with to puting together with detectable mark.
14. the described method of claim 13 is characterised in that tracer antibody and foxalin put together, by realizing being connected with detectable mark at the antibody of foxalin.
15. hybridoma cell line 3.25.12 (DSM ACC2799), 3.29.15 (DSMACC2800), 4.38.30 (DSM ACC2801), 7.57.41 (DSM ACC2802), 7.72.32 (DSM ACC2803).
16. be used for the antibody compositions of the described method of claim 3 to 14, be characterised in that it comprises the potpourri of the antibody of being produced by clone DSM ACC2799, clone DSM ACC2800, clone DSM ACC2801, clone DSM ACC2802 and/or clone DSM ACC2803.
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| EP06019016.2 | 2006-09-12 | ||
| EP06019016 | 2006-09-12 | ||
| EP06020797 | 2006-10-04 | ||
| EP06020797.4 | 2006-10-04 | ||
| PCT/EP2007/007803 WO2008031532A1 (en) | 2006-09-12 | 2007-09-07 | Anti-drug antibody assay |
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| CN101506659B CN101506659B (en) | 2012-12-12 |
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| CN105051544A (en) * | 2013-03-20 | 2015-11-11 | 豪夫迈·罗氏有限公司 | Specific detection of rat antibodies in mouse serum |
| CN105624118A (en) * | 2009-10-19 | 2016-06-01 | 弗·哈夫曼-拉罗切有限公司 | Non-cross-reactive anti IgG antibodies |
| CN109358192A (en) * | 2018-02-08 | 2019-02-19 | 中国科学院上海药物研究所 | A kind of device and method, the preparation method and application of the device removing free drug in anti-medicine antibody test sample |
| CN110546506A (en) * | 2017-03-31 | 2019-12-06 | 埃博灵克斯股份有限公司 | Improved immunogenicity assays |
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| EP1658096A1 (en) * | 2003-08-25 | 2006-05-24 | PanGenetics B.V. | Method of inducing immune tolerance |
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