CN1193353A - Allergen-XCD32 fusion proteins - Google Patents
Allergen-XCD32 fusion proteins Download PDFInfo
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- CN1193353A CN1193353A CN96196311A CN96196311A CN1193353A CN 1193353 A CN1193353 A CN 1193353A CN 96196311 A CN96196311 A CN 96196311A CN 96196311 A CN96196311 A CN 96196311A CN 1193353 A CN1193353 A CN 1193353A
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Abstract
Fusion proteins comprising one or more antigens and one or more moieties interacting with human Fc gamma receptor II (Fc gamma RII) (CD32).
Description
The present invention relates to the mixture of IgG and antigen/allergen (or antigen/allergenic molectron).It is about the fusion rotein between anti--CD32 molecule and the antigen/allergen (or antigen/allergenic molectron).Allergen this paper be defined as the atopy patient with anaphylaxis to its antigen of reacting.The employed antigen of this paper can be various sources, for example environment allergen (as room dirt mite, birch pollen, showy flowers of herbaceous plants powder, feline antigens, cockroach antigen) or food allergens (as milk, peanut, shrimp, soybean) or both molectrons, perhaps irrelevant antigen (as bovine serum albumin (BSA)).
Transformation reactions is a kind of disease, the wherein antibody-mediated effector cell's of IgE (mastocyte, basophilic leukocyte, eosinocyte) activation, and present by low and high-affinity receptor (B cell, monocyte, the dendritic cell) enhancement antigen of IgE.These effects of IgE can be offset by specific IgG antibodies, and described IgG antibody interacts with CD32 (Fc γ RII) on effect and inducing cell.Therefore, transformation reactions should be considered to have a kind of disease of unbalanced Th1 to the Th2 cell.Offset the IgE success or not in the IgG antibody body and depend on that specific IgE surpasses the relative concentration of specific IgG, also depends on the homotype of IgG.Everyone has low affinity to CD32 at the class homotype, and this can overcome by forming with allergenic mixture, but many allergens do not form enough big feasible can stably be attached on the CD32 and the mixture IgG molecule.
Two kinds of forms utilizing allergenic active inoculation have been used so far.The most so-called " immunotherapy ", this depends on the frequently immunity of allergen with relative high density.This technology only has certain effect in some case of minority allergic disease (as the sting transformation reactions) and rhinitis and conjunctivitis, not long ago some reports had shown the validity in asthma and atopic dermatitis.Classical subcutaneous route is used to use allergen, but recently this approach is used even topical application compares with oral, and the result generally speaking is positive still inconsistent.A kind of up-to-date immunotherapy, promptly so-called Saint-Remy technology (referring to for example EP 0 178 085 and 0 287 361), it uses external with relevant allergen compound self IgG antibody.This method makes to have littler side effect by the allergen that can use much lower amount.
Two kinds of therapies machine-processed unclear.In classical way, though the obvious increase of not every specific IgG is all relevant with the immunotherapy of success, therapy causes that specific IgG antibodies seems to have useful effect when increasing.A possible reason of this phenomenon is the lower affinity of IgG antibody to the CD32 on B cell, monocyte and the mastocyte.The Saint-Remy method is from the patient selection specific IgG antibodies, external subsequently it mixed with relevant allergen.By this way, they guarantee that allergen can not freely react with cell or at other antibody morphism on the cell (as the IgE on mastocyte).In addition, anti-individual group type antibody may produce at the specific IgG molecule, will stop transformation reactions subsequently, though this point is not also confirmed by experimental data.
The IgG molecule is instructing immune response at monocyte but not be important in the B cell.When general allergen-specific IgG replaced donor-specific allergen-specific IgG to use, therefore the Saint-Remy method should work.Yet, the problem of Saint-Remy method is the low affinity of IgG antibody to its Fc γ acceptor on the B cell, this is opposite with IgE, and the latter is easy in conjunction with nearly all antigen-be delivery cell (comprising the B cell), causes scattering transformation reactions and induces the Th2 cell.The purposes of mixture should be walked around low affinity problem, because the avidity that improves than mixture with single IgG molecular ratio; Yet the molectron of the allergen/IgG in mixture also is undesirable because in conjunction with the natural number of the allergenic epi-position of IgG molecule less than 5, very little so that can not strengthen the avidity of mixture to acceptor.
Adopt the present invention, the risk factors of classical immunotherapy are lowered, and the problem that is run in separating the specific IgG molecule is resolved with the low affinity problem of these IgG antibody to CD32.
IgE is bonded to and causes histamine release (when by antigen cross-linking) on mastocyte and the basophilic leukocyte.In addition, IgE can improve the antigen presentation (Fig. 1) of human B cell.In addition, when not having IgE, the B cell is antigen-the be delivery cell of non-constant, and except the B extracellular of small portion very, it is the specific antigens acceptor.Can be by pinocytosis generation antigen uptake, but this needs high antigen concentration and/or long incubation time.
Fig. 2 shows that the allergen pinocytosis that surpasses 5 days can imitate the antigen presentation that IgE-mediates, but the antigen presentation of IgG-mediation does not cause the hormesis of T cell.In fact, data show that compound allergen and IgG stop allergen to be presented.
In another serial experiment, these IgG-allergen mixtures have been studied to the antigen presentation of IgE-mediation or the effect of allergenic pinocytosis picked-up.Fig. 3 shows that the IgG mixture suppresses the antigen presentation that IgE-mediates with dose-dependent manner, and in addition, the antigen presentation after pinocytosis is suppressed.This effect is not antigen-specific (the IgG/BSA mixture suppresses the effect of Der P1-differential stimulus), and monomer (non-compound) IgG does not suppress antigen presentation.
The toxicity of having got rid of IgG/ allergen mixture by the experiment shown in Fig. 4.IgG3-Der P1 mixture with human monocyte's (in advance) incubation can cause a kind of normal t cell responses.The allergen that this means IgG/ allergen mixture inhibition B cell is presented, and stimulates monocyte and dendritic cell simultaneously.Similarly, aCD32 antibody or be coated on the hole at the bottom of the accumulative IgG of (hormesis is carried out therein) also suppress the antigen presentation (Fig. 5) of B cell.The efficient of CD32 oriented molecule inhibition antigen presentation depends on the crosslinked amount of CD32 molecule probably.Yet, to the guiding antigen of the CD32 on the B cell (as in natural IgG/ antigenic compound (Fig. 2) or in according to aCD32/ antigen coalescence protein of the present invention (Figure 13 a and 13b)) do not cause antigenic antigen presentation in the mixture.On the other hand, directed monocytic same compound thing causes antigen-specific T-cells reaction (Fig. 4,13a and 13b) really.This phenomenon can make an explanation by the existence of CD32B on the B cell, and monocyte and dendritic cell are expressed CD32A.Therefore, IgG/ antigenic compound and aCD32/ antigen coalescence protein instruct the immune response to the cell of monocyte and dendritic cell pedigree, this causes the t cell responses of main Th1 type B cell, main other APC that expresses CD32B does not present the antigen in the mixture, so these mixtures stop (further) to induce the Th2 cell response of B cell.Interaction between mixture and the CD32 does not rely on strong crosslinked (surpassing 2 CD32 molecules), can carry out stable interaction as long as the bonding strength of mixture is enough to make.In fact, the combination that has been presented at the very large mixture that causes CD32 multiple cross-linked (by coating accumulative IgG imitation) on the monocyte causes the downward modulation (table 1) for the important accessory receptor of the hormesis of antigen-specific T-cells (for example CD80).
Except that antigen presentation, also suppress normal human B cell according to IgG/ antigenic compound of the present invention or aCD32/ antigen coalescence protein and produce Ig.The IgG that Figure 14 is presented at aCD32 antibody or accumulative coating exists the tonsilla B cell (interacting effectively with CD32) that stimulates with aCD40 and IL-4 down no longer can produce antibody.With aCD32F (ab) fragment handle with complete antibody the same effective (Fig. 5), show aCD40 and IL-4 stimulated after the restraining effect of Ig generation, do not need the crosslinked of acceptor.The B cell that is subjected to antigenic stimulation by its B-cell receptor (BCR) needs the co-crosslinking of BCR and CD32.The downward modulation that Ig produces is reversible and time-dependent manner.Stimulate when aCD32 antibody being joined the B cell in back 4 days when add IL-4 at aCD40, do not observe the restraining effect that Ig produces.Figure 16 shows that antigen-specificity that antibody produces induces the aCD32 that also is subjected to the B cell to handle inhibition.
In essence, the IgG molecule is by controlling the B cell response with the interaction of CD32.Especially, the effect of IgE antibody (this generally has positive acting to (the CD23 positive) B cell) can be offset by the IgG molecule.This stops organism to antigenic hyperergy immune response.In a large amount of transformation reactions that exist of IgE antibody, clearly natural IgG antibody can not be controlled immune response.What is interesting is that IgE is regulated by identical interleukin (being IL-4) with IgG4 in the mankind.Yet IgG4 has the minimum affinity to the IgG subclass of all CD32.
The present invention relates to fusion rotein, it comprises a) one or more antigens and b) (CD32) interactional part of one or more and human Fc gamma receptor II (FcPII), for example derive from the part of antibody molecule, hereinafter, this fusion rotein is abbreviated as " according to fusion rotein of the present invention ".
Overcome by the problem of IgG molecule according to fusion rotein of the present invention the low affinity generation of CD32.By having K
d＜10
-6ACD32 antibody and antigen combination, obtained negative sense (B cell) and two kinds of effects of forward of natural IgG molecule, described effect is included in and lacks antibody and cause Th1/Th0 to remember the immune selective stimulating effect of inductive under producing.This effect is harmless, and instruct immune response at the antigen of expressing CD32-be delivery cell, thereby, mainly express the inducing and activatory antigen presentation (as the result of the IL-12 that produces by CD32A-antigen expressed-be delivery cell) of the cell-mediated Th1 of causing cell of CD32A.
Preferably do not have strong crosslinked according to fusion rotein of the present invention.The number of the binding site of CD32 is preferably limited, with avoid pungency auxiliary-downward modulation of acceptor (as CD40, CD80 or CD86).
They have also destroyed the effector functions of mastocyte, and these cells carry the IgE-specificity part of fusion rotein.They have the feature of following uniqueness: 1) make the cell-mediated antigen presentation silence of B (the Th2 cell is induced in prevention); 2a) stop the IgE switch to be induced; 2b) stop the Ig in the B cell to produce; 3) stimulate T cellular compartment (hormesis of Th1 cell) by interaction with monocyte and/or dentritic cell; With 4) make carrying mastocyte silence the specific IgE of part of fusion rotein.
They comprise a) one or more antigens and b) one or more and the interactional part of human Fc gamma receptor II, for example derive from the part of antibody.
With the part of the interactional fusion rotein of Fc γ receptor II can be for example 1) fully or not exclusively (modification) mankind or humanization IgG antibody moiety, as long as still possible, this means that all or part of Fc fragment should exist with the interaction of these acceptors; Or 2) mankind or humanization aCD32 antibody moiety or its part, Fab fragment for example, its still specifically identification and in conjunction with Fc γ RII (CD32) antigen, as on B cell, mastocyte, monocyte and dentritic cell, expressing, for example Cao Zuo the mankind or humanization aCD32 or IgG antibody moiety or its part, it is with than natural aCD32 or the higher affinity identification Fc γ RII (CD32) of IgG antibody.
Described antigen can derive from complete protein or it still has the t cell epitope part of (being present on the sequence in the fusion rotein).The allergy patient that can use the hypersensitivity reaction with IgE-mediation is to its any antigen of reacting, as the allergen of atopic dermatitis, atopic asthma, rhinallergosis and allergic conjunctivitis.Prevailing environment allergen is: house dust mite, birch pollen, showy flowers of herbaceous plants powder, cat, cockroach.(for example: for house dust mite, main allergen is Der P1 to each such allergic effect original one or more " main allergens "; For birch pollen, main allergen is Bet V1).Yet complete antigen is optional, because fusion rotein only causes t cell responses usually and to the t cell responses of little (8-12 amino acid long) peptide.Therefore, the t cell epitope of selection can be included in every kind of allergenic fusion rotein, reduces the size and the molecular weight of mixture like this.Like this, fusion rotein can be to produce from one or more DNA skeletons that comprise t cell epitope, rather than produce from the gene of complete antigen.Overlapping cross reactivity epi-position between the allergen is preferred.Fusion rotein should be specifically in conjunction with CD32, and comprises one or more (for example two or more) t cell epitopes (its be one or more antigens/allergenic).In order to carry out correct antigen processing, the DNA skeleton longer slightly than actual t cell epitope preferably should be included in the preparation of fusion rotein.
To the gene Fusion of coding aCD32 antibody, preferably use the dna sequence dna of the weak point that derives from main allergenic clone gene, as main allergen I of house dust mite (Der P1) or birch pollen allergen (Bet V1).These short dna sequence dnas comprise the genetic code of one or more t cell epitopes, and described epi-position appears at antigen-present cell surface after processing, and causes the immune response of replying allergen-specific T-cells thus.For Der P1, most of t cell epitopes can be found on the 101-143 position with the sequence (SEQ.ID.No.1) of amino acid single-letter coded representation: QSCRRPNAQRFGISNYCQIYPPNANKIREALAQPQRYCRHYWT101 110 120 130 140
Especially use the 101-131 position of the sequence (SEQ.ID.No.2) of amino acid single-letter coded representation: QSCRRPNAQRFGISNYCQIYPPNANKIREAL101 110 120 130 comprises at least three t cell epitopes, and it is in conjunction with some HLAII quasi-molecules.
Fusion between for example aCD32 and antigen can be gone up at protein level (chemistry merge) or at gene level (recombination fusion protein) and be realized, the present invention also comprises and is used to produce the method for fusion rotein as defined above.It carries out in a conventional manner, preferably includes to adopt recombinant DNA technology or chemically crosslinked.
Recombination fusion protein is preferred.
Can utilize the preparation of recombinant DNA technology through currently known methods (as following method): from for example constant gene segment C of IV.3 cDNA PCR-amplification and clone, said constant gene segment C contains antigen-binding site and part Fc district, and the latter is in the CH2 exon downstream of aCD32 monoclonal antibody (for example cloning IV.3).Suitable RNA purifying is used as the source of PCR-amplification, for example, is used for the amplification of Der P1 gene from the house dust mite purified RNA.The allergen gene is connected into suitable mammalian expression vector with isolating heavy chain section, as p350.In addition, the fully corresponding light chain gene and the clone that separate IV.3 for example advance suitable mammalian expression vector, and as p345, it has the feature that is similar to p350.Two kinds of recombinant plasmids are used for being total to-transfection at for example COS cell, make and the recombination fusion protein that forms can be discharged in the developing medium.Preferably carry out the purifying of said product through immunoaffinity purification (based on the anti-light chain antibody columns that obtain in a large number easily).
In addition, (for example derive from suitable phage display library, De Kruif etc., Proc. Natl. Acad. Sci.USA, 92 (1995) 3938-3942 are described) CD32 (scFv) specific single-chain antibody can be used for making recombination fusion protein (Fig. 6) by clone's allergen gene.From extracting DNA, separate and contain dna fragmentation and the clone who is integrated into half of a VL chain-synthetic VHDJH district through flexible joint (scFv fragment) in conjunction with phage.Adopt the synthetic oligonucleotide of the qualification of coding T-cell epitope to finish and for example Der P1 gene Fusion from Der P1 and other main allergen (height (T cell) the cross reaction begetting power by them is selected, so that cover patient as much as possible with a kind of fusion rotein).With these oligonucleotide with the scFv antibody fragment altogether-connect to advance in the suitable mammalian expression vector.Said recombinant vectors (for example plasmid) transfection is stably advanced in the suitable cell (as COS and/or Chinese hamster ovary celI), through above-described immune affinity chromatographic from the formed fusion rotein of cell conditioned medium liquid purifying.
Formed gene construct can be for example expressing in the Chinese hamster ovary celI, especially for scale operation; Yet, also be suitable through other generation system of recombinant DNA technology, especially when only using t cell epitope when (promptly not needing glycosylation).Can obtain aCD32 antibody from human Ig phage display library (it only contains F (ab) fragment of natural antibody).Yet the source of encoding antibody or antigen/allergenic genetic material is not crucial.
Adopt [journal of biological chemistry 173 (1978) 723-737] such as Calsson, [immunological method magazine 120 (1989) 133-143] described methods such as Cumber etc. [Enzymology method 112 (1985) 207-224] and Peeters are through being used for the preparation that the currently known methods of the fusion rotein between the Mc.a-CD32 and Der P1 for example carries out chemically crosslinked.
In brief, Der P1 and Mc.a-CD32 use SPDP (mol ratio is respectively 1/20 and 1/5) derivatize to introduce 2-pyridyl disulfide residue respectively individually.After gentleness reduction and gel chromatography desalination, with the Mc.a-CD32 (its disulphide group is reduced into mercapto groups) of SPDP-derivatize with the Der P1 of SPDP-derivatize with 1/1.6 mol ratio incubation.Then, the DerP1-Mc.a-CD32 product of formation is by gel-filtration (on the Superose-12 for example) and anion-exchange chromatography (on FPLC-MonoQ for example) purifying.
Initial substance is a known substances, perhaps can be in accordance with known methods or the similar approach preparation of the similar approach of currently known methods or (for example among the embodiment) described herein method.
To preventing and/or treating transformation reactions, particularly food allergy is useful according to fusion rotein of the present invention.It is common to suffering from specific allergenic allergic patient and the patient that suffers from one or more other allergenic a kind of like this reaction.Make by method of the present invention that to relate to two or more allergenic patients desensitizations simultaneously be possible, it is to carry out through using to comprise at various so allergenic antigenic fusion roteins.Preferably will be used for preventing and/or treating and have food allergy danger the transformation reactions of the ewborn infant of the transformation reactions of milk (for example to), perhaps prevent and/or treat the existing transformation reactions that has at the patient who is included in the allergenic transformation reactions in the employed specific fusion rotein according to fusion rotein of the present invention.
For these diseases, proper dosage certainly along with, for example, the disease of employed specific fusion rotein, host, method of application and expection treatment and changing.Yet in general, one to three inoculation in the 1-2 shows can obtain gratifying result, but if necessary, can carry out the additional inoculation of multiple.Shown that for these treatments fusion rotein of the present invention can be used with similar conventional dosage and the application program that uses.
Therefore the present invention also relates to the above fusion rotein that limits in the preventing and/or treating of transformation reactions (comprising food allergy) purposes and treat allergic method (this method comprises that the curee to this treatment of needs uses prevention or treatment and goes up the above fusion rotein that limits of significant quantity and the pharmaceutically acceptable carrier or the thinner of at least a routine) and with the above fusion rotein that limits as medicine (especially anti--allergy agent).
According to fusion rotein of the present invention can with pharmaceutically acceptable thinner and carrier and dispensable other mixed with excipients of routine, in parenteral, intravenously or intestines, use (for example through intramuscular or subcutaneous administration).Certainly, the concentration of fusion rotein is along with the character of employed compound, required treatment and medicament forms and change.
Like this, the present invention also comprises pharmaceutical composition, and it comprises the above fusion rotein that limits and at least a pharmaceutically acceptable carrier or thinner.
The invention still further relates to preparation and prevent and/or treat purposes on the transformation reactions medicine of (comprising food allergy) in manufacturing at the method (this method comprises the above fusion rotein that limits and pharmaceutically acceptable carrier or mixing diluents) of allergic medicine and the above fusion rotein that limits.
：aCD32 CD32 ( CD32 ) Ag APC -BCR BBetV1 BSA CNBR DerP1 ( Dermatophagoides pteronvssinus ) DPT DTT EBV Epstein-BarrELISA FACS FcγRII FcγII ( ＝CD32 ) Fig. FPLC GaM HAc HLA HPHT IC Ig IL-12 -12LST min MR PBS PCR pNPP sd SPDP 3- ( 2- ) N-
In following explanation the present invention but do not limit in the embodiments of the invention, all temperature all are degree centigrade.
Embodiment: preparation and purifying Dre P1--monoclonal anti CD32 fusion rotein A) method:
By using SPDP (a kind of difunctional coupling reagent) that Der P1 is covalently bound to Mc.a-CD32.In brief, use SPDP (mol ratio is respectively 1/20 and 1/5) derivatize Der P1 and Mc.a-CD32 respectively, to introduce 2-pyridyl disulfide residue.After gentleness reduction and gel chromatography desalination, with the Mc.a-CD32 (whole disulphide groups are reduced into mercapto groups) of SPDP-derivatize with the Der P1 of SPDP-derivatize with 1/1.6 mol ratio incubation.Then, the DerP1-Mc.a-CD32 conjugate of formation passes through at gel-filtration on the Superose-12 and the anion-exchange chromatography purifying on FPLC-MonoQ.B) material: a) SPDP: molecular weight 312.4; 25.61mM dimethyl formamide stock solution (8mg/ml) just prepared before coupling.B) Der P1: by the immune affinity chromatographic purifying on covalently bound Mc.a-Der P1 (4C1/BS/3F8) to CNBR-Sepharose 4B.Molecular weight 28kD; Iso-electric point 5.0 (PHAST-IEF); Sterile filtration is diluted with PBS.C) monoclonal anti-CD32 (IV.3): homotype: mouse IgG2b-κ chain; Molecular weight 150kD; Iso-electric point 6.2 (PHAST-IEF); On Protein-A and HPHT,, and dialyse with PBS from the medium supernatant purifying.Reaction mixture A:
In 1.2ml Der P1 solution (0.84mg), add 23.4 μ l SPDP stock solution (20 molar excess), under room temperature (22 ℃), in 2.5ml taper reaction flask (Pierce), stirred the mixture 45 minutes.Reaction mixture is added into 2.5ml with PBS and unconjugated SPDP.Upward remove the N-hydroxy-succinamide of release at the disposable desalting column of 9.1mlPharmacia PD-10 (Sephadex G-25) (crossing) through desalination with 50ml PBS balance.After this component that contains 2-pyridyl disulfide-activatory Der P1 with 3.5ml PBS wash-out and collection.Concentration is about 0.17mg/ml, is equivalent to 0.6mg.The estimation of the degree that replaces by the 2-pyridyl disulfide:
100 μ l samples are added into 150 μ l with PBS, add 50 μ l 150mMDTT with the PBS dilution.By the absorbancy (molar extinction coefficient: 8080M that is determined at 343nm
-1Cm
-1) measure to add the concentration of the pyridine-2-thioketones that discharges behind the DTT, its 2-that is equivalent to introduce pyridyl-disulphide residue.The OD 343nm that measures is increased by 0.074 (proofreading and correct dilution), and the degree that calculates replacement is 1.5Mol 2-pyridyl disulfide/Mol Der P1.Reaction mixture B:
In 0.5ml Mc.a-CD32 solution (4.2mg), add 5.5 μ l SPDP stock solution (5 molar excess), at room temperature stirred the mixture 45 minutes.Reaction mixture is added to the disposable desalting column of 2.5ml Pierce GF-5 (with 10ml 0.1M sodium acetate/HAc damping fluid, 0.1M NaCl, pH 4.5 balances are crossed) on, after this, with 1.5ml acetate buffer (pH 4.5) wash-out and collect the component of desalination, filter with 0.22 μ m MILLEX-GV (low protein binding) filter.The introducing of SH-group:
By protein-bonded 2-pyridyl disulfide group being transformed into protein-bonded mercapto groups with the DTT reduction.In room temperature under agitation with 1.5ml desalination sample with 1ml 62.5mMDTT (diluting) incubation 30 minutes (final DTT concentration 25mM) with acetate buffer.Because under acid pH, the 2-pyridyl disulfide increases its Electron Affinities (electrophilicity), reduce under low pH and still can carry out, and the natural protein disulfide linkage is unaffected.
The OD 343nm that measures is increased by 0.447, and the degree that calculates replacement is 7Mol mercapto groups/Mol Mc.a-CD32.
Reaction mixture is added on the Pharmacia PD-10 post (crossing with 50ml PBS balance), after this, with 3.5ml PBS wash-out and collect the component of desalination.With CENTRIPLUS-10 (YM10 film) thickener sample concentration is arrived 0.6ml.Concentration is about 3.1mg/ml, is equivalent to 1.86mg.C) coupling method:
3.3ml (0.56mg) Der P1 (pyridine-disulphide activatory) and 0.6ml (1.86mg) Mc.a-CD32 (SH-activatory) are mixed, in 5ml Pierce reaction flask, stirred 2 hours down, stirred 15 hours at+4 ℃ in room temperature (22 ℃).The molecular ratio of Der P1/Mc.a-CD32 is 1.6/1 in reaction mixture.By measuring because linked reaction is monitored in the increase of the OD 343nm that the increase of the pyridine-2-thioketones that discharges causes:
Time OD (343nm)
Beginning 0.1112
15 minutes 0.1257
30 minutes 0.1342
45 minutes 0.1412
60 minutes 0.1470
75 minutes 0.1519
120 minutes 0.1590D) purifying of conjugate: Superose-12:
With one 0.22 μ m MILLEX-GV filter filter reaction mixture (3.9ml), and be concentrated into 0.5ml with the Centriplus-10 thickener.The ultimate density of conjugate is 3.7mg/ml, is equivalent to 1.9mg.
Sample is added on Superose-12 (HR10/30) post of crossing with the PBS balance (Pharmacia).Column volume 24ml; Flow velocity 0.2ml/ minute; Volume/component 0.4ml; Inking chart speed 1.5mm/ minute; Pressure 0.3MPa; Scanning wavelength 280nm.The high molecular weight component of wash-out is divided into 3 groups according to elution profile:
1.2ml A group: component #21-23
1.2ml B group: component #24-26
1.2ml C group: component #29-31
Each is organized sterile filtration (0.22 μ m MILLEX-GV), and under aseptic condition, store in+4 ℃.Total protein concentration is about: A group: 100 μ g/ml; B group: 200 μ g/ml; C group: 390 μ g/ml.FPLC-Mono?O：
1ml C group (390 μ g) 20mM thanomin/HCl, 0.01%NaN
3PH of buffer 9.0 dialysis are through the ion-exchange chromatogram purification on FPLC-Mono Q (HR5/5) anionite.Initial buffer A: 20mM thanomin/HCl, 0.01%NaN
3PH 9.0; Restriction buffer B: A+0.5M NaCl pH 9.0; Flow velocity 1ml/ minute, volume/component 1ml; Inking chart speed 5mm/ minute; Pressure 1.2Mpa; Scanning wavelength 280nm.After this, a beginning linear gradient program 30 minutes (conductivity 50-1260 μ S/cm).Relevant peak wash-out (under 340 μ S/cm) in component #8.Protein concn is about 45 μ g/ml (OD 280nm, E
1%14.0).With PBS dialysis sample and sterile filtration.The protein concn of the material of final purifying is 20 μ g/ml (OD 280nm).E) assay determination: a) total protein concentration:
According to the method for Bradford, use BIO-RAD protein determination test kit I to estimate total protein concentration; Standard substance: ox IgG.B) measure Der P1, Mc.a-CD32 and Der P1-Mc.a-CD32 conjugate through ELISA
On PVC droplet plate, carry out solid phase ELISA.To be coated with damping fluid 0.1MNaHCO
3/ Na
2CO
3, 0.01%NaN
3PH 9.6, and the PBS with 0.05%Tween-20 is as washing soln, and 2% foetal calf serum in lavation buffer solution is as the thinner of sample, vitamin H and enzyme conjugate.Substrate is the 1mg/mlp-NPP with 1M diethanolamine/HCl damping fluid (pH 9.8) dilution.Stop bath is 2M NaOH.In humanized chamber, carry out all incubations.Sample processing and be Beckman Biomek-1000 laboratory work station at the equipment of the OD of 405nm reading.Quantitative evaluation: Beckman Immunofit; Fitting of a curve: 4-parameter reasoning.B) 1) measure Der P1 (Fig. 7):
Coating: 100 μ l/ hole Mc.a-Der P1 (5H8), 10 μ g/ml, spend the night at+4 ℃.After the washing, add 100 μ l/ hole samples:
A) Der P1 (link coupled initial substance) is (250-0.49ng/ml) as standard substance
B) Superose-12A group (4000-7.81ng/ml)
C) Superose-12B group (4000-7.81ng/ml)
D) Superose-12C group (4000-7.81ng/ml)
In+37 ℃ of incubations 2 hours.After the washing, add Mc.a-DerP1 (the 4C1/B8/3F8)-biotin conjugate of 1/500 dilution ,+37 ℃ of incubations 2 hours with 100 μ l/ holes.After the washing, add the streptavidin-alkaline phosphatase conjugate of 1/1000 dilution ,+37 ℃ of incubations 1 hour with 50 μ l/ holes.After the washing, add substrate, in 37 ℃ of incubations 60 minutes and termination with 100 μ l/ holes.From the typical curve quantitative evaluation:
Superose-12A group: 16.1 μ g/ml
Superose-12B group: 54.9 μ g/ml
Superose-12C group: 135 μ g/mlb) 2) measure Mc.a-CD32 (mouse IgG2b) (Fig. 8):
Coating: 100 μ l/ hole goat Pc.a-mouse IgG2b (Southern), 5 μ g/ml, spend the night at+4 ℃.After the washing, add sample with 100 μ l/ holes:
A) Mc.a-CD32 (link coupled initial substance) is (1000-1.96ng/ml) as standard substance
B) Superose-12A group (8000-15.63ng/ml)
C) Superose-12B group (8000-15.63ng/ml)
D) Superose-12C group (8000-15.63ng/ml)
+ 37 ℃ of incubations 2 hours.After the washing, add the goat Pc.a-mouse IgG2b-alkaline phosphatase conjugate (Southern) of 1/1000 dilution ,+37 ℃ of incubations 2 hours with 50 μ l/ holes.After the washing, add substrate, at room temperature incubation 15 minutes and termination with 100 μ l/ holes.From the typical curve quantitative evaluation:
Superose-12A group: 17.7 μ g/ml
Superose-12B group: 58.8 μ g/ml
Superose-12C group: 190 μ g/ml
Estimate the mol ratio of Der P1/Mc.a-CD32 in conjugate according to ELISA:
Superose-12A group: 4.9/1
Superose-12B group: 5.0/1
Superose-12C group: 3.8/1b) 3) detection of Der P1--Mc.a-CD32 conjugate: b) 3) 1) coating: Mc.a-Der P1 (mouse IgG2a) is (Fig. 9):
Coating: 100 μ l/ hole Mc.a-Der P1 (5H8), 10 μ g/ml, spend the night at+4 ℃.After the washing, add sample with 100 μ l/ holes:
A) Der P1 (link coupled initial substance) (4000-7.81ng/ml)
B) Superose-12A group (4000-7.81ng/ml)
C) Superose-12B group (4000-7.81ng/ml)
D) Superose-12C group (4000-7.81ng/ml)
+ 37 ℃ of incubations 2 hours.After the washing, add the goat Pc.a-mouse IgG2b-alkaline phosphatase conjugate (Southern) of 1/1000 dilution ,+37 ℃ of incubations 2 hours with 50 μ l/ holes.After the washing, add substrate, at room temperature incubation 30 minutes and termination with 100 μ l/ holes.Detect Mc.a-Der P1-bonded Der P1--Mc.a-CD32 conjugate by the distribution of Mc.a-CD32 in each Superose-12 purified components group.B) 3) 2) coating: Pc.a-mouse 1gG2b (Figure 10):
Coating: 100 μ l/ hole goat Pc.a-mouse IgG2b (Southern), 5 μ g/ml, spend the night at+4 ℃.After the washing, add sample with 100 μ l/ holes:
A) Der P1 (link coupled initial substance) (1000-1.95ng/ml)
B) Superose-12A group (8000-15.63ng/ml)
C) Superose-12B group (8000-15.63ng/ml)
D) Superose-12C group (8000-15.63ng/ml)
+ 37 ℃ of incubations 2 hours.After the washing, add Mc.a-DerP1 (the 4C1/B8/3F8)-biotin conjugate (mouse IgGI) of 1/500 dilution ,+37 ℃ of incubations 2 hours with 100 μ l/ holes.After the washing, add the streptavidin-alkaline phosphatase conjugate of 1/1000 dilution ,+37 ℃ of incubations 1 hour with 50 μ l/ holes.After the washing, add substrate with 100 μ l/ holes.In 37 ℃ of incubations 60 minutes and termination.Detect Pc.a-mouse IgG2b-bonded Der P1--Mc.a-CD32 conjugate by the distribution of Der P1 in each Superose-12 purified components group.C) measure the molecular weight of Der P1--Mc.a-CD32 conjugate through natural polypropylene acrylamide gel gradient electrophoresis
With natural PHAST gel 4-15% gradient (Pharmacia) (separating ranges: 1000kD-150kD), (test kit Pharmacia) is relatively estimated the molecular weight of the Der P1--Mc.a-CD32 conjugate in Superose-12 purified components group with natural polymer amount standard protein.Detect: silver dye (silver-colored transfection reagent box, Pharmacia) (figure .11) estimates:
Superose-12A group: at the band of MW 700kD
The Superose-12B group: at MW 460kD, the band of 330kD
The Superose-12C group: at MW 330kD, the band of 170kD
The molecular weight of the Der P1--Mc.a-CD32 conjugate of final FPLC-Mono Q purifying is estimated as 330kD (Figure 12).F) test-results: a) antigen-differential stimulus effect of the T cell clone CFTS4:3.1 of employing aCD32 Der P1 fusion rotein:
Be used to stimulate Der P1-specific T-cells to clone CFTS4:3.1 the aCD32 (Medarex clone IV.3) of the Der P1 chemically crosslinked of above and purifying with the LST of standard with different concns.In first group of test, use chemistry to merge the A group and the B group of preparation, the A group contains the single band of 700kD (with 2 aCD32 molecules of 10 Der P1 molecules fusions), B group is made up of two bands of 460kD and 330kD (being respectively 1 aCD32 and 10 Der P1 and 1 aCD32 and 5 Der P1 molecules).
In second group of test, use the protein purification of C group, its single band by about 330kD (the Der P1 of 1 aCD32 molecule and 5 molecules merges) is formed.With monocyte and B cell at room temperature with the various component preincubation shown in Figure 13 a and the 13b 1 hour.Stimulate in contrast, between complete incubation period, 100 μ g/ml DPT are joined the T cell and add in the antigen presenting cell.The B cell can stimulate and has DPT but do not have A group, the CFTS4:3.1 of B group or C group, and monocyte can stimulate and has DPT and have the A group, the CFTS4:3.1 that B group and C organize.This has confirmed the former discovery of relevant above alleged natural IgG allergen mixture.In addition, this also shows, and is crosslinked optional to the antigenic stimulation that is caused by monocyte CD32, because all components fully stimulates the T cell with being equal to.B) the IgE synthetic suppresses:
Exist and do not exist commercially available aCD32 antibody (with shown in concentration (Figure 14) add) stimulate the human tonsilla B cell of purifying down with aCD40 and IL-4.After 9 days, culture supernatants is used to test IgE and IgG1 content.All aCD32 antibody suppress IgE in the dose-dependently mode and IgGI produces.Figure 15 shows, and is synthetic even the Fab fragment of Medarex clone IV.3 also can suppress IgE.This explanation be can't help its BCD activatory B cell through blocking its antibody generation with the monomer interaction of CD32.To the B cell that stimulates in antigen-specificity mode, the co-crosslinking between CD32 and BCR is essential.This means, absorb antigen (as Der F1) at for example B cell by IgE and CD23, then become the Der P1-specificity T h2 cell of stimulation, cause the B cell to produce in the transformation reactions of IgE, can be in the B cell surface and interaction CD32 so that these B cells stop to produce.In fact, even in relevant B cell T cell interaction, aCD32 antibody blocking antibody synthesizes (Figure 16).
Table 1
| Stimulate | ％CD80 | ％CD40 | ?％HLA- ??? |
| 0 day (newly isolating) | ???1 | ???26 | ???99 |
| 1 day IFN-γ | ???31 | ???82 | ???99 |
| 1 day IFN-γ+accumulative IgG | ???13 | ???61 | ???99 |
| 1 day IFN-γ+accumulative IgG+aCD32 | ???32 | ???82 | ???99 |
Exist or do not exist 5 μ g/ml accumulative IgG (to be applied to each hole, spending the night under 4 ℃) and/or 10 μ g/ml mono-clonal mouse anti-human aCD32 (Medarex IV.3) under, the monocyte (the 91%CD14 positive) that will derive from normal donor through elutriation and 100 U/ml IFN-γ incubations 24 hours, then dyeing is used for the facs analysis of cue mark.IFN-γ-inductive CD80 and CD40 rise effect can be by strong acceptor and the crosslinked inhibition of accumulative IgG on the monocyte.Preincubation with aCD32 is offset by the accumulative IgG, illustrates 1) inhibition that causes by the accumulative IgG mediate by CD32 and 2) only the combination of CD32 is not caused the downward modulation of accessory receptor.
HLA-DR expresses the influence that is not subject to processing.Description of drawings: Fig. 1: Der-P1-(3) NIP mixture is to the combination of the CD23 of relevant antigen presentation: A figure=fluorescence; B figure=propagation.
With preformed IC ' s pulse EBV B cell, described IC ' s by the IgE of constant density (▲ 7.5 μ g/ml, ● 5.0 μ g/ml, ■ 2,5 μ g/ml) and Der-P1-(3) NIP of variable concentrations form.
A figure has shown combining of preformed IC ' s and B cell.IC ' s with identical MR connects with a line, and actual MR (0.1,0.25,0.5,1,7) represents with the Arabic numerals near line.When Der P1 concentration was about 0.2 μ g/ml, IgE was in conjunction with reaching platform (plateau).IgE when not having DerP1-(3) NIP is in conjunction with the box indicating that has on the Y-axle of the right.
B figure has shown by the antigen presentation with the EBV-B cell of the irradiation of identical IC ' s pulse.Here, wire is represented the mixture made with identical IgE concentration not to be subjected to the MR of IC ' s to influence (relation conefficient=0.96) to emphasize antigen presentation.Monomer complex [the highest Der P1-(3) the NIP concentration of each line] also is effectively and is passed T cell.Do not exist IgE to divide the period of the day from 11 p.m. to 1 a.m, all do not having the T cytositimulation with DerP1-(3) the NIP concentration (black region) of any use.Data presentation was at the 5th day
3The H-thymidine mixes the (mean value of three repeated holes ± sd).Fig. 2: with the antigen presentation of the B cell of non-pulse relatively:
The dose-dependently of staying free Der P1-(2) the NIP inducing T cell in the developing medium between whole stimulation period stimulates.With autologous (irradiation) EBV-B cell as antigen-be delivery cell.The not obvious enhancement antigen of IgE that is present in the mixture is presented, and gives T cell (comparing with free Der P1-(2) NIP) but IgGI and IgG3 are present in the antigen presentation that stops Der P1-(2) NIP among IC ' s.The result was with the 5th day
3The H-thymidine mixes the expression (mean value of three repeated holes ± sd).Fig. 3: the antigen presentation restraining effect of IgG-Der P1-(3) NIP mixture:
When not having IgG3, use IgE-Der P1-(3) NIP mixture to find good T cytositimulation effect.Cause the dose-dependent inhibition effect of the antigen presentation of IgE-mediation with preformed IgG3-Der P1-(3) the NIP mixture titration of identical (1: 1) ratio.In order to obtain this effect, when the T cytositimulation, need the IgG3 mixture to be present in the substratum.Because IgG3 to the low affinity of the CD32 on the B cell, is unsuccessful with the pre-cultivation of IgG3 mixture B cell or " pulse ".When not having IgE and also using IgG3-BSA-(3) NIP mixture, observe similar antigen presentation restraining effect.Fig. 4: the antigen presentation of the monocytic IgG3-mediation of the fresh mankind:
When not having or existing uncorrelated gathering IgG, use preformed IgG3-Der P1-(3) NIP mixture pulse human monocyte (ratio 1: 1) 1 hour on ice.Then wash the monocyte of pulse, and mix with T cell clone that Der P1 specificity HLA-DP matches.After 5 days, with
3The H-thymidine mixes measures propagation.Induce allergen-specific T-cells reaction by the IgG3 mixture that the human monocyte presents, it can be blocked by accumulative IgG, and the interactional specificity of IgG3 and monocyte is described.Data are represented with the mean value ± sd of three repeated holes.Fig. 5: DPT-specific T-cells propagation; ACD32 or accumulative IgG are to the influence of antigen presentation:
With (solid bars) or one night of need not (hollow strips) 250 μ g/ml DPT pulses human EBV B cell.After the irradiation, the T cytomixis with B cell and Der P1-specific T-cells clone made cell proliferation 5 days.Exist different concns by the crosslinked aCD32 antibody of GaM the time, observe the dose-dependently inhibited proliferation.Assemble when aCD32 or non--specificity and to observe identical effect when IgG is applied to culture hole.Fig. 6: the example that derives from the recombination fusion protein between aCD32 scFv (Fab) fragment of phage display library
Can obtain the Fab fragment from phage display library (92 (1995) 3938-3942 are described for De Kruif etc. for example, Proc. Natl. Acad. Sci.USA).Yet, can substitute aCD32 Fab fragment with any molecule (or part of molecule) of human CD32 effective interaction.Allergen can be any allergic protein or protein group zoarium of causing.In addition, allergen can be substituted by the allergen fragment that contains t cell epitope.Recombinant protein can not relied on the expression system generation of glycosylation and other posttranslational modification by any available.This fused protein also can be used for (excessively) disease that to produce undesirable Ig molecule be feature (for example rheumatoid arthritis, the graft host repels (graft-versus-host) disease) or any other disease of playing a role of autoantibody wherein.In these cases, allergen can be substituted by simple infinite joint, to make up two kinds of aCD32 parts.Yet, when allergen is substituted by " autoantigen " that cause disease, can obtain best result.Fig. 7: the Der P1 Fig. 8 in the Der P1--Mc.a-CD32 conjugate component group of sandwich-ELISA mensuration Superose-12 purifying: the Mc.a-CD32 Fig. 9 in the Der P1--Mc.a-CD32 conjugate component group of sandwich-ELISA mensuration Superose-12 purifying: the Der P1--Mc.a-CD32 conjugate in the component group of sandwich-ELISA mensuration Superose-12 purifying
Coating: Mc.a-Der P1.Figure 10: the Der P1--Mc.a-CD32 conjugate in the component group of sandwich-ELISA mensuration Superose-12 purifying
Coating: Pc.a-mouse IgG2b.The silver of Figure 11: A., B and C group component dyes:
Swimming lane 1:350ng Der P1
Swimming lane 2:50ng Mc.a-CD32
Swimming lane 3:500ng high molecular mark
The initial substance of swimming lane 4:500ng Superose-12
Swimming lane 5:100ng Superose-12A group
Swimming lane 6:100ng Superose-12B group
Swimming lane 7:200ng Superose-12C group
Swimming lane 8:250ng high molecular mark
The high molecular mark:
669kD: thyroglobulin
440kD: ferritin
232kD: catalase
140kD: serum lactic dehydrogenase
67kD: the silver of the fusion rotein of bovine serum albumin Figure 12: C group purifying dyes:
Swimming lane 1:250ng high molecular mark
The material of swimming lane 2:45ng FPLC Mono-S purifying.Figure 13 a and 13b:Ag-specific T-cells propagation:
Figure 13 a:APC-Mo 250495 and CFB4:2
Figure 13 b: the influence of the CD32/Der P1 of monomer purifying
The LST[Van Reijsen of use standard, F.C. etc., (1992) transformation reactions clinical immunology magazine, 90 184], will be used to stimulate Der P1-specific T-cells clone CFTS4:3.1 with the aCD32 (Medarex clones IV.3) of the Der P1 chemically crosslinked of purifying with different concentration.First group of experiment (Figure 13 a) in, use chemistry to merge the A group and the B group of preparation: the A group contains the single band (being that 2 aCD32 molecules and 10 Der P1 molecules merge) of 700kD, the B group is by forming at two bands of 460kD and 330kD (being respectively 1 aCD32 and 10 Der P1 and 1 aCD32 and 5 Der P1 molecules merges).
In second group of experiment (Figure 13 b), use C group protein purification (CP230595), form by the single band of about 330kD (the Der P1 of 1 aCD32 molecule and 5 molecules merges).
With monocyte and B cell various components at room temperature to that indicated in the drawings preincubation together 1 hour.Stimulate in contrast, between complete incubation period, 100 μ g/ml DPT are joined the T cell and add antigen-be in the delivery cell.According to describing (Van Reijsen etc., the same), after 5 days with
3The H-thymidine mixes measures T cytositimulation (4 hole mean values ± sd).The B cell can stimulate CFTS4:3.1 (DPT is arranged but do not have A group, B group, or C group), and monocyte can stimulate CFTS4:3.1 (DPT is arranged and the A group is arranged, B group and C group).This is corresponding to the former discovery [Bheekha Escura, R. etc., immunology (1995) 86 343] with natural IgG allergen mixture.In addition, this explanation, to monocytic antigenic stimulation, crosslinked optional more than two CD32 molecules is because all components is equivalent stimulating on the T cell.Figure 14: IgE and IgG1 synthetic restraining effect separately:
Exist and do not exist commercially available aCD32 antibody (with shown in concentration add) under, as Armerding, D. etc., immunobiology, 188 (1993) 259-273 are described, stimulate the human tonsilla B cell of purifying with aCD40 and IL-4.After 9 days, culture supernatants is used to test IgE and IgG1 content according to describing (Armerding, D. etc., the same).The result produces expression (mean value ± sd) with the antibody of 3 groups of components of 9 multiple.All aCD32 antibody suppress IgE in the dose-dependently mode and IgG1 produces.The restraining effect of IgM and IgA is similar.Figure 15: the IgE synthetic suppresses:
Exist and do not exist commercially available aCD32 antibody (Medarex IV.3) or its Fab fragment (with shown in concentration add) under, as Armerding, D. etc., same as above, stimulate the human tonsilla B cell of purifying with aCD40 and IL-4.After 9 days, culture supernatants is used to test IgE and IgG1 content according to describing (Armerding, D. etc., the same).The result produces expression (mean value ± sd) with the antibody of 3 groups of components of 9 multiple.Complete Medarex clone IV.3 or its Fab fragment both can suppress IgE synthetic (IgG1, IgM and IgA).Figure 16: the DPT specific IgE is induced; The influence of aCD32:
With (solid bars) or one night of need not (hollow strips) 250 μ g/ml DPT pulses human tonsilla B cell.After irradiation,, make cell proliferation and produced antibody 9 days B cell and Der P1-specific T-cells clone's (CFTS4:3.1) T cytomixis.After 9 days, according to Armerding, D. etc., the same description is used to test IgE and IgG1 content with culture supernatants.The result produces expression (mean value ± sd) with the antibody of 3 groups of components of 9 multiple.When having the aCD32 antibody of different concns, observe dose-dependently IgE and produce restraining effect.Even the B cell T cell interaction that this explanation is relevant, aCD32 antibody also blocking antibody are synthetic.
Sequence table (1) general information:
(i) applicant:
(A) title: Sandoz company limited.
(B) street: Lichtstrasse 35
(C) city: Basle
(E) country: Switzerland
(F) zip code (ZIP): CH-4002
(G) phone: 61-324 5269
(H) fax: 61-322 7532
(A) name: Mudde, Geert C.
(B) street: Ruzickagasse 88-104/Haus 39
(C) city: vienna
(E) country: Austria
(F) zip code (ZIP): A-1230
(ii) denomination of invention: fusion rotein
(iii) sequence number: 2
(iv) computer-reader form:
(A) media type: floppy disk
(B) computer: IBM PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0
Version #1.25 (EPO)
(the v) data of current application:
Application number: WO PCT/EP96/......
(vi) in the data of first to file:
(A) application number: GB 9516760.7
(B) information of the applying date: 16-8-1995 (2) SEQ ID NO:1:
(i) sequence signature:
(A) length: 43 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: the unknown
(ii) molecule type: peptide
(iii) suppose: not
(iii) antisense: not
(v) clip types: inside
(vi) primary source:
(A) organism: Dermatophagoides pteronyssinus
(xi) sequence description: SEQ ID NO:1:Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr1 5 10 15Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala
20??????????????????25??????????????????30Gln?Pro?Gln?Arg?Tyr?Cys?Arg?His?Tyr?Trp?Thr
The information of 35 40 (2) SEQ ID NO:2:
(i) sequence signature:
(A) length: 31 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: the unknown
(ii) molecule type: peptide
(iii) suppose: not
(iii) antisense: not
(v) clip types: inside
(vi) primary source:
(A) organism: Dermatophagoides pteronyssinus
(xi) sequence description: SEQ ID NO:2:Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr1 5 10 15Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu
20??????????????????25??????????????????30
Claims (13)
1. fusion rotein, it comprises
A) one or more antigens and
B) (CD32) interactional part of one or more and human Fc gamma receptor II (Fc γ RII).
2. according to the fusion rotein of claim 1, wherein said antigen is the allergen in atopic dermatitis, atopic asthma, rhinallergosis or allergic conjunctivitis.
3. according to the fusion rotein of claim 1, it is to produce from one or more DNA skeletons that comprise t cell epitope, rather than produce from the gene of complete antigen.
4. according to the arbitrary desired fusion rotein of claim 1 to 3, the wherein said and interactional part of Fc γ RII is the mankind or humanization aCD32 antibody, or still discerns and be attached to the part of these antibody on Fc γ RII (CD32) antigen specifically.
5. according to the arbitrary desired fusion rotein of claim 1 to 3, the wherein said and interactional part of Fc γ RII is the mankind or humanization IgG antibody, or still discerns and be attached to the part of these antibody on Fc γ RII (CD32) antigen specifically.
6. according to the arbitrary desired fusion rotein of claim 1 to 3, the wherein said and interactional part of Fc γ RII is the mankind or humanization aCD32 or IgG antibody or its part of operating, and it is to discern Fc γ RII (CD32) than natural aCD32 or the higher affinity of IgG antibody.
7. pharmaceutical composition, it comprises according to the fusion rotein of claim 1 and at least a pharmaceutically acceptable carrier or thinner.
8. produce the method according to the fusion rotein of claim 1, this method comprises uses recombinant DNA technology or chemically crosslinked.
According to the fusion rotein of claim 1 in the purposes that prevents and/or treats on the transformation reactions (comprising food allergy).
10. prevent and/or treat purposes on the transformation reactions medicine of (comprising food allergy) according to the fusion rotein of claim 1 in manufacturing.
11. fusion rotein according to claim 1 as medicine.
12. treat allergic method, this method comprise the curee to this treatment of needs use prevention or treatment go up significant quantity according to the fusion rotein of claim 1 and the pharmaceutically acceptable carrier or the thinner of at least a routine.
13. preparation is at the method for allergic medicine, this method comprises fusion rotein and pharmaceutically acceptable carrier or mixing diluents according to claim 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9516760.7 | 1995-08-16 | ||
| GBGB9516760.7A GB9516760D0 (en) | 1995-08-16 | 1995-08-16 | Organic compounds |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1193353A true CN1193353A (en) | 1998-09-16 |
Family
ID=10779307
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96196311A Pending CN1193353A (en) | 1995-08-16 | 1996-08-16 | Allergen-XCD32 fusion proteins |
Country Status (16)
| Country | Link |
|---|---|
| EP (1) | EP0846178A1 (en) |
| JP (1) | JPH11513025A (en) |
| KR (1) | KR19990036440A (en) |
| CN (1) | CN1193353A (en) |
| AU (1) | AU6873596A (en) |
| BR (1) | BR9610239A (en) |
| CA (1) | CA2227795A1 (en) |
| CZ (1) | CZ43798A3 (en) |
| GB (1) | GB9516760D0 (en) |
| HU (1) | HUP9802349A3 (en) |
| IL (1) | IL122898A0 (en) |
| NO (1) | NO980566L (en) |
| PL (1) | PL324897A1 (en) |
| SK (1) | SK19398A3 (en) |
| TR (1) | TR199800214T1 (en) |
| WO (1) | WO1997007218A1 (en) |
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| GB9724531D0 (en) | 1997-11-19 | 1998-01-21 | Smithkline Biolog | Novel compounds |
| EP1044263A2 (en) | 1997-12-02 | 2000-10-18 | Medarex, Inc. | CELLS EXPRESSING ANTI-Fc RECEPTOR BINDING COMPONENTS |
| EP1221317A1 (en) * | 2000-12-28 | 2002-07-10 | SHAN-Beteiligungsgesellschaft m.b.H. | Vaccines containing hybrid polypeptides consisting of at least two different allergenic proteins |
| US8025873B2 (en) | 2002-06-20 | 2011-09-27 | Paladin Labs, Inc. | Chimeric antigens for eliciting an immune response |
| US8029803B2 (en) | 2002-06-20 | 2011-10-04 | Paladin Labs, Inc. | Chimeric antigens for eliciting an immune response |
| US8007805B2 (en) | 2003-08-08 | 2011-08-30 | Paladin Labs, Inc. | Chimeric antigens for breaking host tolerance to foreign antigens |
| BG65715B1 (en) * | 2003-09-04 | 2009-08-31 | Чавдар ВАСИЛЕВ | Device for selective suppression of pathologic dna-specific b cells |
| BG65954B1 (en) * | 2005-01-05 | 2010-07-30 | Чавдар ВАСИЛЕВ | Means for selective suppression of the activity of pathologic autoreactive b-cells |
| US9636415B2 (en) | 2006-03-03 | 2017-05-02 | S-Target Therapeutics Gmbh | Bispecific molecule binding TLR9 and CD32 and comprising a T cell epitope for treatment of allergies |
| EP1829895A1 (en) * | 2006-03-03 | 2007-09-05 | f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. | Bispecific molecule binding TLR9 and CD32 and comprising a T cell epitope for treatment of allergies |
| AU2012382406B2 (en) | 2012-06-15 | 2017-12-07 | Immunomic Therapeutics, Inc. | Nucleic acids for treatment of allergies |
| CA2878771A1 (en) | 2012-07-13 | 2014-01-16 | S-Target Therapeutics Gmbh | Immunoregulatory vaccine |
| AU2013340899B2 (en) * | 2012-11-01 | 2017-10-05 | Veterinaerinstituttet | New fusion proteins for the treatment of allergic diseases |
| US9005630B2 (en) | 2012-11-01 | 2015-04-14 | Veterinaerinstituttet | Fusion proteins for the treatment of allergic diseases |
| ES2665780T3 (en) | 2013-05-21 | 2018-04-27 | Tyg Oncology Ltd. | Immunogenic composition of gastrin peptide |
| CN113186167B (en) * | 2021-04-28 | 2021-11-30 | 中国食品药品检定研究院 | Method for determining biological activity of anti-CD 20 monoclonal antibody drug ADCP |
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|---|---|---|---|---|
| WO1994024281A1 (en) * | 1993-04-14 | 1994-10-27 | Immulogic Pharmaceutical Corporation | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
| CA2188812A1 (en) * | 1994-05-26 | 1995-12-07 | Mark De Boer | New methods and compounds for the selective modulation of antigen-specific t-cell responsiveness |
-
1995
- 1995-08-16 GB GBGB9516760.7A patent/GB9516760D0/en active Pending
-
1996
- 1996-08-16 AU AU68735/96A patent/AU6873596A/en not_active Abandoned
- 1996-08-16 CN CN96196311A patent/CN1193353A/en active Pending
- 1996-08-16 SK SK193-98A patent/SK19398A3/en unknown
- 1996-08-16 KR KR1019980701110A patent/KR19990036440A/en not_active Application Discontinuation
- 1996-08-16 CZ CZ98437A patent/CZ43798A3/en unknown
- 1996-08-16 IL IL12289896A patent/IL122898A0/en unknown
- 1996-08-16 EP EP96929263A patent/EP0846178A1/en not_active Withdrawn
- 1996-08-16 JP JP9508953A patent/JPH11513025A/en active Pending
- 1996-08-16 PL PL96324897A patent/PL324897A1/en unknown
- 1996-08-16 BR BR9610239A patent/BR9610239A/en not_active Application Discontinuation
- 1996-08-16 WO PCT/EP1996/003616 patent/WO1997007218A1/en not_active Application Discontinuation
- 1996-08-16 CA CA002227795A patent/CA2227795A1/en not_active Abandoned
- 1996-08-16 HU HU9802349A patent/HUP9802349A3/en unknown
- 1996-08-16 TR TR1998/00214T patent/TR199800214T1/en unknown
-
1998
- 1998-02-10 NO NO980566A patent/NO980566L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| TR199800214T1 (en) | 1998-06-22 |
| BR9610239A (en) | 1999-06-15 |
| NO980566D0 (en) | 1998-02-10 |
| HUP9802349A3 (en) | 1999-04-28 |
| NO980566L (en) | 1998-02-10 |
| CZ43798A3 (en) | 1998-06-17 |
| SK19398A3 (en) | 1998-09-09 |
| IL122898A0 (en) | 1998-08-16 |
| MX9801253A (en) | 1998-05-31 |
| EP0846178A1 (en) | 1998-06-10 |
| HUP9802349A2 (en) | 1999-01-28 |
| GB9516760D0 (en) | 1995-10-18 |
| KR19990036440A (en) | 1999-05-25 |
| CA2227795A1 (en) | 1997-02-27 |
| WO1997007218A1 (en) | 1997-02-27 |
| AU6873596A (en) | 1997-03-12 |
| JPH11513025A (en) | 1999-11-09 |
| PL324897A1 (en) | 1998-06-22 |
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