CN1171117A

CN1171117A - Use of MHC-II binding and/or MHC-II minicking molecules for prevention and/or treatment of inflammatory diseases

Info

Publication number: CN1171117A
Application number: CN95196985A
Authority: CN
Inventors: R·P·拉恩尔
Original assignee: Aum Drug Ltd By Share Ltd; Laboratories Om SA
Current assignee: Aum Drug Ltd By Share Ltd; OM Pharma SA
Priority date: 1994-12-23
Filing date: 1995-12-27
Publication date: 1998-01-21
Also published as: SK80697A3; JPH10511652A; WO1996020215A3; CA2208233A1; HUT77468A; WO1996020215A2; AU4347696A; BR9510554A; CZ186897A3; EP0800534A2; PL320922A1

Abstract

The invention relates to MHC-II binding and/or MHC-II mimicking molecules for use in interfering in the interaction between an activation stimulus for MHC-II bearing cells, such as phagocytes and cell-bound MHC-II molecules, in the interaction between lipopolysaccharide (LPS) or LPS in a complex with other molecules, such as CD14 and LBP, and cell-bound MHC-II molecules, or in the interaction between products from Gram-positive bacteria or complexes of products from Gram-positive bacteria with molecules such as CD14, and cell-bound MHC-II molecules. The MHC-II binding molecule may be any anti-MHC-II antibody or fragment thereof, or any molecule derived from such an antibody such as humanized, bispecific or other engineered molecules and the like. The MHC-II binding molecule may be selected from the group consisting of CD14, fragments thereof, modified versions thereof, or peptides having MHC-II binding properties.

Description

MHC-II combination and/or MHC-II model molecule are used to prevent and/or treat the purposes of inflammatory diseases

The present invention relates to utilize special molecular to disturb toxin such as lipopolysaccharides (LPS) itself or it and such as other the molecular complex body of CD14 and LBP and the interaction between its transducer cell.The invention still further relates to described molecule be used for the prevention and the treatment inflammatory diseases, as the purposes of septic shock.

LPS is the cell-wall component of gram negative bacterium.The infection of gram negative bacterium can cause life-threatening disease, this disease is by due to LPS and the cytophagous specific combination such as monocyte, scavenger cell and granulocyte, this bound energy activates described cell and makes it secrete various cytokines, comprises tumor necrosis factor-alpha, interleukin-11 (IL-1), IL-6, IL-8 and other inflammatory mediator.These materials or cause series reaction by direct effect or by intensifying secondary media, thus aggegation systemic disease, vasorelaxation disease caused, many organ defect are sick and finally cause septic shock disease (4,5).

Generally, the cytophagous remarkable activation that can cause secreting multiple inflammatory mediator is an important reaction in the pathogenic process of a kind of morbid state that is called as systemic inflammatory responses syndrome (SIRS).Except being activated by LPS, multiple other clinical disease also can cause SIRS, as infection, wound, burn, pancreatitis, graft versus host disease and host versus graft disease, the hemocytophagia etc. that caused by bacterium or virus.One of example of inflammatory diseases is the toxic shock that is caused by the extracellular toxin from staphylotoxin A of gram positive bacterium and so on.Known its has staphylotoxin B and streptococcus toxin for other extracellular toxin of superantigen.

Confirmed already that LPS can combine with the monocyte antigens c D14 of glycosyl-phosphatidyl inositol (GPI)-grappling.CD14 is present in monocyte, scavenger cell and granulocytic surface, but it also can be present in the serum of healthy individual with the soluble form of no GPI grappling.Confirm that in addition anti-CD14 monoclonal anti physical efficiency suppresses LPS to monocytic activation.Show that thus CD14 can play the effect (1) of LPS acceptor, and can mediate the effect of LPS pair cell matter.But, CD14-negative cells also can react (2,7,8) to LPS.

In addition, recognize that also CD14 is glycosyl-phosphatidyl inositol (GPI)-anchoring molecule (9) that a kind of shortage is striden film and cytoplasmic structure territory.Therefore, CD14 can not be to the tenuigenin transduction signal.A kind of received hypothesis is that the transmembrane molecule relevant with the protein requirement of GPI connection is so that carry out signal transduction.Therefore, the transduction molecule of LPS and other possible SIRS stimulation does not obtain identifying as yet.

Under situation,, press for other molecule (10) outside the CD14 for the cell-stimulating effect of explaining that LPS causes by the LPS active cells.Someone proposes, and LPS can form complex body with membrane-bound or soluble CD14 and LPS conjugated protein (LBP).Other molecule that comes from serum also can add this complex body.This compound physical efficiency combines with a kind of molecule that does not obtain as yet identifying on the cell surface, this cell is activated.

Confirmed that CD14 is causing play an important role (13) aspect the cell-stimulating effect that produces from the bacterium coating product of Gram-positive and gram negative bacterium.Similarly, other film in conjunction with or the molecule that comes from serum may participate in interaction with described cell surface molecule, thereby cause the activation of cell.

The present invention has been found that, major histocompatibility complex II (MHC-II) molecule is that the LPS activating cells is necessary, and this molecule plays a part by the acceptor of LPS and the molecular molecular complex such as CD14 and LBP and/or the signal transduction factor.In human body, MHC is called as human leucocyte antigen (HLA).Have found that the LPS-reactivity depends on the expression of MHC II type molecule at cell surface.A kind of clone that is called as " THP-1MHC+ " in this article is that a kind of MHC II type is expressed monocytic series, and Tsuchiya etc. are referred to as THP-1 (3).The clone that another kind is called as " THP-1.6MHC-" in this article is a kind of MHC II type-negative monocytic series that is produced by spontaneous mutation by THP-1MHC+.THP-1MHC+ cell energy effect is in the LPS secrete cytokines, and the THP-1.6MHC-cell can not.The CD14-positive, the negative human peripheral blood mononuclear cells of MHC II-(PBMC) can not be reacted to LPS.Express and the LPS reactivity by can recover MHC II type with CIITA transfection THP-1.6MHC-cell, CIITA is a kind of can the coding to the cDNA of MHC-II molecule at the very crucial nf of the expression of cell surface.

Other SIRS stimulates the cytotropic transduction also can be numerator mediated by MHC-II.Verified in the past, extracellular toxin also can be incorporated on the MHC-II molecule of cell surface.The activity of other Gram-positive product to small part by CD14 mediation, therefore, the complex body of described product and CD14 probably also can with the MHC-II interaction of molecules, with active cells.

Therefore, can prevent and/or treat systemic inflammatory responses syndrome by disturbing by LPS and the interaction that combines with cell between the MHC-II such as the complex body (to call " CD14/LPS/LBP complex body " in the following text) that CD14 and LBP form.According to the present invention, can implement above-mentioned interference by two kinds of different modes.

At first, can use the MHC-II binding molecule such as anti-MHC-II antibody, CD14 or its peptide derivant to stop the CD14/LPS/LBP complex body to combine the combination of MHC-II with cell.This molecular energy and the competition of CD14/LPS/LBP complex body, thus this complex body combination stoped, but itself can not activate MHC-II.The transduction function that has stoped MHC-II thus.

Secondly, the compound physical efficiency of round-robin LPS or CD14/LPS/LBP is caught by the MHC-II model molecule.No longer can combine with soluble MHC-II or class MHC-II molecule bonded complex body with cell bonded MHC-II.Thereby prevent activation to this cell.

The MHC-II binding molecule comprises can stop LPS or CD14/LPS complex body and any molecule of MHC-II bonded.In practice, described molecule comprises the anti-MHC II antibody at the CD14/LPS of cell or LPS binding site, comprises monoclonal antibody and polyclonal antibody.Antibody fragment also is suitable for the binding molecule as MHC-II.

The MHC-II model molecule comprises that simultaneously soluble MHC-II molecule itself and any other can seal the molecule of the MHC-II binding site on LPS or the CD14/LPS complex body.This quasi-molecule can comprise complete MHC-II molecule or its fragment or subunit.In addition, can also adopt the peptide that can combine and don't can activate MHC-II with LPS or LPS/CD14/LBP complex body.Described peptide can with MHC-II portion homologous at least, and contain suitable D-amino acid, make this peptide have antagonistic properties.Described molecule can be connected for soluble form or with a kind of surface of carrier.

According to the information that the application provides, those skilled in the art can identify suitable combination and/or model molecule.

This quasi-molecule can be from any suitable source, as derives from people or other biology, and the preparation that may in all sorts of ways, and for example, extracts from the cell culture supernatant liquid of MHC-II positive cell or extracts from cell pyrolysis liquid.Optimum extracting method is an immune-affinity chromatography.Can also prepare suitable molecule with gene engineering, protein chemistry method or any other appropriate method.

According to the present invention, two types molecule can be used to prevent and/or treat inflammatory diseases, the inflammatory reaction that causes as the graft versus host disease after septic shock, the organ transplantation (for example bone marrow transplantation), graft-rejection, because of burn, unexpected injury, pancreatic infection etc.

The present invention also is suitable for preventing and/or treating other inflammatory reaction, for example postoperative inflammatory reaction, as capillary leaks syndrome, the allergic inflammation disease, autoimmune disease, as lupus erythematosus (LE) and hypotype thereof, scleroderma and hypotype thereof, eosinophil fascitis (eosinophilicfasciitis), Sjogren syndrome, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener ' s granulomatosis, temporal arteritis, polymyalgia rheumatica etc., similar rheumatism is as rheumatoid arthritis, juvenile chronic arthritis, Felty syndrome, Caplan syndrome, rheumatoid spondylitis (Marie-Strumpell-Bechterew disease), psoriasis, Reiter syndrome, Behcet syndrome.

Can with the inventive method treatment and also have because of other disease due to the autoimmune mechanism at least in part: diabetes, Crohn disease, ulcerative colitis, digestive tract ulcer, kidney infect, as glomerulonephritis and ephritis, arteriosclerosis disease, multiple sclerosis, senile dementia, hyperthyroidism, thyroid function are low.

The present invention also can be used for the inflammatory reaction of one or more organs relevant with neoplastic disease, as leukemia, hemocyte knurl, cancer, fibroma, sarcoma and various types of histiocytosis.

The present invention can also be used to prevent and/or treat virus disease, as AIDS.Known LPS stimulates can strengthen duplicating of cell interior human immunodeficiency virus (HIV).Hormesis by MHC-II binding molecule of the present invention and/or model molecule prevention LPS can overcome this stimulation of duplicating.Therefore, MHC-II binding molecule of the present invention and/or model molecule can be used for stoping in the mode that stimulates by cell-stimulating the stimulation of virus replication are produced provide protection to the cell that HIV infects.

Based on the data that provides in an embodiment, it is contemplated that out following model with the LPS activating cells.On the one hand, LPS can be incorporated into film in conjunction with on the CD14 (mCD14), and this is a kind of interaction of being quickened by LPS conjugated protein (LBP) (14).The complex body that LPS and GPI grappling mCD14 and possible LBP form subsequently with stride the interaction of molecules of film MHC II type, cause signal transduction.This situation as shown in Figure 3A.On the other hand, LPS can be combined on the soluble cd 14 (sCD14) that is present in the serum with LBP, this complex body is incorporated on the lip-deep MHC II of the CD14-negative cells type molecule then, causes the CD14-feminine gender but MHC II type-positive cells is activated.This situation is shown in Fig. 3 B.Although the contact that illustrates betides between CD14 and the MHC-II, LBP and LPS also can participate in this interaction, and not obtain the molecule identified as yet the same with other for this.Gram positive bacterium can cause that also above-mentioned activation stimulates, but the effect of LBP is not determined as yet in this case.Here and be not precluded within not and form under the situation of complex body some with CD14 or LBP and activate and stimulate the possibility that directly acts on the MHC-II molecule.Confirmed that now this hormesis is numerator mediated by MHC-II.The transduction that described sealing or model molecule will stop this activation to stimulate.

The invention still further relates to MHC-II binding molecule, MHC-II model molecule and comprise one or both pharmaceutical composition in two types of molecules.Pharmaceutical composition contains one or more MHC-II binding molecules and/or MHC-II model molecule as activeconstituents, be used to disturb be used for such as the cytophagous cell that can express the MHC-II molecule such as the activation stimulator of LPS and the interaction between the cell bonded MHC-II molecule, the form of said composition is a powdery, suspension, solution, aerosol, emulsion, perfusion liquid, composition for inhalation, ointment or emulsion, and can be used for topical application, use in the nose, internal rectum uses, intravaginal uses, and also can be used for oral, parenteral (vein, subcutaneous, intramuscular, in the sheath etc.) or use through epidermis, suck use etc.Pharmaceutical composition of the present invention can prepare like this: with described active compound with have neutral feature can be medicinal vehicle (as water or anhydrous solvent, stablizer, emulsifying agent, stain remover, additive) merge (promptly by mix, dissolving etc.), if necessary and further with tinting material and seasonings merging.The concentration of activeconstituents can change between 0.001%-100% according to character and the administrated method handled in the pharmaceutical composition.The dosage of the activeconstituents of being taken also depends on concrete purposes and route of administration, but for instance, can change between 0.01 μ g-1mg/kg body weight, is preferably between 0.1 μ g-100 μ g/ kg body weight to change.

The present invention will be described with the following Examples for the general, and these embodiment are not used to limit the scope of the invention.1 explanation of embodiment example

Be the such hypothesis of excretory cytokine checking by MHC II type-positive (HLA-DR) and MHC II type-negative cells when relatively stimulating with LPS: when activating phagocytic cell by LPS, MHC-II is transducer and/or the acceptor that comprises the complex body of LPS, CD14, LBP and presumable other molecule.The secretion of cytokine can indicator cells activation.

Material and method

THP-1 ^MHC+Be a kind of people's of deriving from CD14-feminine gender, MHC II type-positive monocytic series (3).THP-1.6 ^MHC-Be a kind of THP-1 of spontaneous generation ^MHC+MHC II type-negative mutant, it is by repeating to limit the dilution clone.

By using CIITA (II type trans-activator; A kind of is the necessary nucleoprotein of MHC II type protein expression (12)) transfection THP-1.6 ^MHC-Cell is rebuild HLA-DR and is expressed, to obtain THP-1.6 ^MHC-CIITA.

Estimate HLA-DR and CD18 at THP-1 by fluidic cell metering method ^MHC+Cell (wild-type), THP-1.6 ^MHC-Cell (mutational cell line) and THP-1.6 ^MHC-CIITA is (with the THP-1.6 of CIITA transfection ^MHC-Cell) Biao Mian expression.

Each clone is with 10 ⁶The density of cell/ml is cultivated in the substratum that has replenished 10%FCS, and with LPS respectively with 0,1,10, the dosage of 100ng/ml and 1 μ g/ml stimulates.Collect supernatant liquor after 24 hours and measure the content of its TNF-α and IL-8 by ELISA.

Result and discussion

LPS handles the vitality that can not damage cell.Confirm that by trypan blue dyeing the cell more than 95% is viable.

Although three expression in the clone that are expressed in of CD18 are similarly, by THP-1.6 ^MHC-The HLA-DR amount of cell expressing is starkly lower than THP-1 ^MHC+Cell expressing.

Stimulating THP-1 with LPS ^MHC+During cell, the reaction of this cell is TNF secretion-α and IL-8 (Fig. 1).Forming correlated is to use LPS, even the LPS of high dosage stimulation HLA-DR-negative cells is THP-1.6 ^MHC-, can not induce its TNF secretion-α or IL-8.Yet, can recover HLA-DR-with the CIITA transfection and express and the LPS-reactivity.Conclusion is that the expression of HLA-DR molecule is that LPS activated mononuclear cell is necessary.Example 2 explanations

In order to confirm the discovery of example 1, use available from suffering from MHC-II type defective-a kind of rare inherited disease, its show as the serious compressibility immune deficiency of child's early stage appearance-patient's cell confirm the result that obtained with the clone system.The expression of CD-14 is unaffected in this patient's body.There is under the condition of heavy dose of LPS PBMC and the individual PBMC of contrast that cultivates MHC II type defective type patient, and measuring the content of TNF-α and IL-1 in the supernatant liquor of described culture.

Material and method

In the contrast body of MHC-II defective type patient and health, obtain peripheral blood lymphocytes (PBMC).

Measure HLA-DR in the expression (histogram of hatching is in the left side of Fig. 2) on described patient's PBMC surface with in the expression (grey histogram) on the PBMC surface of the contrast of health by direct IF staining and fluidic cell metering method.

With 10 ⁶The concentration of cell/ml is cultivated in the substratum that has replenished 10% normal people AB-positive serum available from patient's PBMC (thick line) with available from the PBMC (fine rule) of contrast, and respectively with 0,1,10 and the LPS of 100ng/ml stimulate.Gather in the crops supernatant liquor after 24 hours, and survey its TNF-alpha content by ELISA.

Result and discussion

Just as desired, because the LPS stimulation, from the PBMC TNF secretion-α and the IL-1 of healthy individual.Yet the PBMC of MHC II type-defective of patient can not stimulate the cytokine (Fig. 2) of secreting significant quantity because of LPS.Confirmed the requirement of expressing MHC II type molecule is not limited to the THP-1 of clone thus ^MHC+Family.

Data show, film is not necessary (THP-1 during by the LPS activating cells in conjunction with CD14 ^MHC+Cell is not expressed CD14), and amount also not enough (expressing the CD14 of normal level from MHC II type defective type patient's PBMC).

Yet above-mentioned experiment does not relate to the effect of soluble cd 14.In all experiments, adopt the substratum that has replenished the serum that contains soluble cd 14.Therefore, these experiments are multiple, cultivate THP-1 in the substratum of serum-free ^MHC+, THP-1.6 ^MHC-And THP-1.6 ^MHC-The CIITA cell.Handle the TNF-α that can not cause secreting significant quantity with heavy dose of LPS.

Above result shows that MHC II type molecule has importantly participated in the LPS-reactivity.The HLA-DR negative cells can not stimulate secrete cytokines because of LPS.Perhaps, people can say that the reaction that lacks LPS is relevant with a factor of combining closely with MHC II type, and are to be regulated by CIITA, rather than the expression of shortage MHC II type itself causes suppressing the LPS-reactivity.Yet, the shortage of LPS reaction is not limited to the secretion of independent a kind of cytokine, because the secretion of TNF-α and IL-1 and IL-8 also is suppressed.In addition, although further investigate, do not find the molecule outside the MHC II type so far as yet by the CIITA adjusting.At last, disease of patient is not to rise because of a kind of defective relevant with CIITA.The expression that transfection can not recover MHC II type molecule from this patient's the B cell that is transformed by EBV.Example 3 explanations

Interact with the physics between 3 kinds of different methods checking MHC II type molecules and the CD14.

At first, with lysate and MHC II specific antibody or the control antibodies and the radioactivity CD14 (CD14 of MHC II positive cell line and MHC II negative cells system ^*) or the radiocontrast molecule cultivate together.Have only by the interaction between albumin A agarose and MHC II specific antibody and just can make MHC II and CD14 ^*The complex body precipitation.When adopting contrast molecule or control antibodies, in this precipitation, radioactivity should not arranged.If there is not MHC II, in described precipitation, do not have radioactivity yet.

Secondly, MHC II is cultivated with radioactivity CD14.Suppose to form MHCII/CD14 ^*Complex body.Theoretically, this species complex can be with to any has specific antibody and the albumin A agarose precipitates in two kinds of components of this complex body.

The 3rd, whether the check anti-CD 14 antibody can stop MHC II and CD14 ^*Interact.

In Fig. 4, these experiment principle are described further.Method 1. production radioactivity CD14

Produce recombinant C D14 by in-vitro transcription/translation by total length CD14 cDNA.Described cDNA prepares with round pcr, and with the reticulocyte lysate system that TNT T7 engages, measures its nucleotide sequence by the scheme (Promega, Switzerland) that the manufacturer recommends under the condition that the 35S-methionine(Met) is arranged.In contrast, produce radiolabeled luciferase with identical system by in-vitro transcription/translation.2. production cell lysate

So-called 293 cells are the usefulness human adenovirus 5 type DNA cell transformed (ATCC called after CRL 1573) that derive from people embryo's kidney; Find that by facs analysis wild-type 293 cells are not expressed MHC II type molecule.This cell is used as MHC II negative cells.With the α that comes from human MHC II type-, 293 cells of the cDNA transfection of β and constant (i) chain are so kind as to give by Jacques doctor Neefjes (Netherland Cancer Institute, Amsterdam).This cell can be expressed MHC II, therefore is used as MHC II positive cell.The lysate for preparing positive MHC II type negative cells system in by the following method from MHC II type.

In culture dish, in 1ml lysis buffer (Boehringer Mannheim, cell marking and immunoprecipitation test kit) lining cracking 5 * 10 ⁶Individual cell.Collect the cracked cell after 30 minutes and carry out sonic treatment (3 * 15 seconds), then cultivated 30 minutes on ice, centrifugal then.As follows supernatant liquor is carried out immunoprecipitation.

3. in experiment 1, carry out immunoprecipitation

Use " cell marking and immunoprecipitation test kit " available from Boehringer Mannheim (Switzerland), carry out immunoprecipitation by the scheme that described reagent is recommended.In brief, with 50 μ l albumin A-agarose suspension sample is carried out preclearing.Centrifugal this albumin A-agarose of removing, then with supernatant liquor with following antibody incubation: L243 (anti--HLA-DR, ATCC names HB55); IB5 (anti--MHC II type, be so kind as to give Netherland Cancer Institute, Amsterdam by Jacque doctor Neefjes); With anti--CD3 (Pharminger, San Diego, USA/AMS Biotechnology, Switzerland).Add 50 μ l albumin A-agaroses after 1 hour, and with this sample incubation 3 hours, with after scouring 6 times.

To precipitate resuspending then in 30 μ l damping fluid (50mM Tris, pH7.5,20mMNaCl, 1mM EDTA, 2mM PMSF) in, and add the solution that contains radio-labeling CD14 that 5 μ l obtain in in-vitro transcription/translation reaction, at room temperature this mixture of incubation is 30 minutes.Wash 2 times with the #2 lavation buffer solution after centrifugal, and then wash 2 times (#2 and #3 damping fluid are derived from cell marking and the immunoprecipitation test kit of Boehringer Mannheim, Switzerland) with the #3 lavation buffer solution.Be dissolved in precipitation in the standard SDS-PAGE sample loading buffer and boil, electrophoresis on 10% sds page carries out radioactive automatic developing then.4. purifying MHC II type molecule

Use it for from the THP-1 cell after the method (15) of Gorga etc. improved a little and extract and purifying MHC II type molecule.By crosslinked with L243 (anti--HL-DR, ATCC names HB55) immobilization on albumin A-Sepharose 4B post with dimethyl-g diacid imidization thing (dimethypimelimidate).The THP-1 cell is placed on ice cracking in the Tris-HCl that contains 0.1mM PMSF (pH8.0).All steps are subsequently all carried out under 4 ℃.With the centrifugal gained lysate of 3000xg, washing precipitation is clarified until supernatant liquor.With the supernatant liquor collected centrifugal 40 minutes with 160000xg, and the dissolution precipitation again of the mode by adding Nonidet P40 with 4% final concentration.After centrifugal 2 hours, supernatant liquor is added on a series of post in the following sequence: Sepharose 4B (10ml), normal rabbit serum Affigel 10 (10ml), albumin A-Sepharose 4B (2ml) L243-Sepharose4B (12ml) with 160000xg.With these posts of following solution washing: 10mM Tris-HCl/0.1% Nonidet P40pH8.0 (5 times of volumes); 10mM MOPS/140 mM NaCl/0.1% deoxycholate salt pH8.0 (2 times of bodies); 10mM Tris-HCl/0.1% deoxycholate salt pH8.0 (4 times of volumes).The L243 post is separated with other post, use 50mM glycine/0.1% deoxycholate salt pH11.5 wash-out rapidly.Collect the 10ml fraction, and after it is by 2M glycine pH2.0 wash-out, transfer to pH7.0-8.0.By block the HLA-DR molecule that membrane ultrafiltration concentrates wash-out with 30kDal.In Tris-HCl/0.1% deoxycholate salt pH8.0, wash 3 times, then albumen is diluted in the same damping fluid again.5. in experiment 2, carry out immunoprecipitation

The solution that contains radiolabeled CD14 that 5 μ l are obtained in in-vitro transcription/translation reaction mixes with the solution that 1 μ l contains about 10ng MHC II type molecule, and incubation 30 minutes at room temperature.Add antibody then, and with this sample 4 ℃ of following incubations 1 hour.Add behind 50 μ l albumin A-agaroses again in 4 ℃ of following incubation samples 3 hours.Centrifugal back is washed precipitation 2 times with the #2 lavation buffer solution, then washes (cell marking and immunoprecipitation test kit, Boehringer Mannheim, Switzerland) 2 times with the #3 lavation buffer solution again.Then with resolution of precipitate in standard SDS-PAGE sample loading buffer and boil, on 10% sds page, carry out gel electrophoresis, carry out radioactive automatic developing then.6. in experiment 3, carry out immunoprecipitation

To with the identical immunoprecipitation sample of other experiment in add radiolabeled CD14 before, the solution that will contain CD14 is with a kind of anti-CD14 mixtures of antibodies incubation 10 minutes at room temperature.Described mixture contains each 10 μ g:RMO 52 (Immunotech, Switzerland) of following anti-CD14 antibody; LeuM3 (Beckton ﹠amp; Dickinson, Switzerland); MY4 (Coulter, Switzerland); Tuk4 (Dako, Switzerland); Supernatant liquor with the following anti-CD14 hybridoma of 100 μ l: 3C10,63D3 (all available from ATCC).In contrast, PBS being added to 5 μ l with the volume identical with mixtures of antibodies contains in the solution of radiolabeled CD14.

The antibody that is used for immunoprecipitation is: MY4 (anti-CD14) and L243 (anti--MHCII).The result tests 1 usefulness positive from MHC II type in the lysate checking MHC II type molecule of MHC II type negative cells and the cosedimentation of CD14

This result of experiment as shown in Figure 5.Shown in the left side of figure is to be used for the result of experiment that the lysate of 293-cell of personal MHC II type transfection (promptly using α, β and the i chain cotransfection of MHC II type) carries out, and shown in the right side of figure is the result that the lysate with negative 293 wild-type cells of MHC II type obtains.

Have only faint band in the

swimming lane

1 and 2, may be by control antibodies anti--CD3 divides in addition and radioactivity CD14 (swimming lane 1) and luciferase (swimming lane 2) carry out due to the non-special

precipitation.In swimming lane

3 and 5 corresponding to radio-labeling CD14 (CD14 ^*) be with high-visible; Two kinds of different antibodies (swimming lane 3:L243 of identification MHC II type molecule; Swimming lane 5:IB5) has (altogether-) sedimentary CD14 ^*The co-precipitation of these anti--MHC II type antibody is specific to CD14, because the radiolabeled reference protein (luciferase) that does not have a significant quantity is by these antibody co-precipitation (

swimming lane

4 and 6).Anti--MHC II type antibody depends on the existence of MHC II type molecule to the co-precipitation of CD14, because the precipitation (being used to the lysate from negative 293 cells of MHC II type, swimming lane 7-9) that produces at the anti--MHC II type antibody that do not have MHC II type to divide the period of the day from 11 p.m. to 1 a.m to have no way of.Experiment 2 usefulness are by the MHC II type molecule checking MHC II type molecule of immune affinity chromatographic column purifying and the co-precipitation of CD14

The result as shown in Figure 6.Shown in the left side of figure be that the result who carries out immunoprecipitation under the situation of MHC II type molecule of purifying is being arranged, shown in the right side of figure is the experiment that divides the period of the day from 11 p.m. to 1 a.m to carry out in the MHC II type that does not have purifying, promptly with the result of damping fluid as negative control.

In

swimming lane

1 and 4, only as seen be equivalent to by the non-sedimentary specifically faint band of control antibodies (anti-CD3).In swimming lane 3, owing to a band that is equivalent to CD14 has appearred in the precipitating action of anti-CD14 antibody.In swimming lane 2, the band that is equivalent to CD14 is more obvious than the band in the swimming lane 3, although in this experiment be with anti--MHC II type molecule carries out sedimentary.Under the condition of the MHC of no purifying II type molecule, CD14 can effectively be precipitated (swimming lane 6) by anti-CD14 antibody, but does not surpass background level (swimming lane 5) by the co-precipitation that anti--MHC type antibody and CD14 carry out.

Experiment 3

Can interact with the physics that anti-CD14 antibody suppresses between CD14 and the MHC II type molecule

The result as shown in Figure 7.

Swimming lane

1 and 5 shows, anti-CD14 antibody MY4 can precipitate the radiolabeled CD14 by in-vitro transcription/translation generation, and with (swimming lane 1) arranged or do not have (swimming lane 5) MHC II type irrelevant molecule (contrast).Under the condition that MHC II type molecule is arranged, a kind of resisting-MHC II type antibody is effectively deposit C D14 (swimming lane 2) of L243, but only is equivalent to background level (swimming lane 6) under the condition of no MHC II type molecule.If with at first handle CD14 before the cell lysate that contains MHC II molecule mixes with the anti-CD14 mixtures of antibodies, CD14 just can not be precipitated by anti--MHC II type antibody.Swimming lane 4 shows that damping fluid (contrast) does not have influence for the co-precipitation of CD14 and anti-MHC II type

antibody.Swimming lane

7 and 8 expressions be and result in the identical experiment of

swimming lane

3 and 4 that but this experiment is to carry out under the condition of no MHCII type molecule.Example 4 explanations

In order to confirm the MHC II effect of activating cells in vivo, use MHC II type to knock out (knock-out) mouse.If MHC II participates in the activation mechanism that LPS causes, the mouse that lacks MHCII will should not show the physiologic effect that common LPS stimulates.Blood with same mouse carries out similar experiment in vitro.Method

When in carrying out body, testing, wild-type C57BL/6 and B6-Aa °/Aa ° MHC II type knock-out mice (Hoffmann-La Roche are arrived in 100 μ g LPS (intestinal bacteria 0111:B4 dilutes) intravenous injection in aseptic 0.9%NaCl; Document 16).After 2 hours, kill mouse and bloodletting by aseptic technique.At room temperature allow hemopexis, and with 13, centrifugal 5 minutes of 000rpm.Remove serum deprivation then.By the content of TNF α in ELISA (Biosource) the mensuration serum, it is a phagocytic cell activated sign.The result as shown in Figure 8.

In order to carry out experiment in vitro, have 0,0.1,1,10 and the condition of 100ng/ml LPS under the whole blood available from positive C57 BL/6 mouse (bullet among Fig. 9) of wild-type MHC II and B6-Aa °/Aa ° knock-out mice (hollow square frame) of incubation heparinization, in described knock-out mice, destroy MHC II type gene A a in orientation ¹The back lacks MHC II type and expresses.Incubation passed through the content that ELISA (Biosource) surveys TNF-α in the blood plasma after 4 hours.The result as shown in Figure 9.

Clearly, TNF-α secretion is more in the mouse that can express MHC II type molecule or cell.Example 5 explanations

Act in the body with following method mensuration MHC II binding molecule and MHCII model molecule.Method

Wild-type C57B1/6 mouse is carried out independent weighing, and the LD in preliminary experiment, to determine ₅₀Inject.Before injection LPS, mouse is cooked following pre-treatment: the 1st group: soluble MHC II type molecule, dosage range are 1 μ g-1mg/kg body weight; The 2nd group: anti--MHC II type antibody, dosage identical with the 1st group; And the 3rd group: salt solution (control group) is only arranged;

Three groups of in contrast other carry out pre-treatment with soluble MHC II type, anti-MHC II or salt solution respectively, but after this stimulate without LPS.

Monitor 7 days survival rate.Observed, note symptom in 48 hours under normal operation in beginning.Induce at LPS and the mouse of a subgroup to be killed and bloodletting before causing death, measure from the TNF-alpha content in the serum of above-mentioned mouse by ELISA.

The result

Comparatively speaking, mortality ratio and the content of TNF-α in serum of organizing with the mouse of soluble MHC II type molecule or anti-MHC II antibody treatment obviously reduces (result does not show).With MHC II type molecule or anti--MHC II antibody treatment but without LPS stimulated control group with brine treatment but do not have significant difference between the mouse without the LPS stimulation.Reference 1.Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J. ﹠amp; Mathison, J.C., Science 249,1431-1433 (1990) 2.Couturier, C., Jahns, G., Kazatchkine, M.D.﹠amp; Haeffner Cavaillon, N., Eur.J.Immunol.22,1461-1466 (1992).3.Tsuchiya, M., etc., Int.J.Cancer 26,171-176 (1980) 4.Bone, and R., Chest 101,802-808 (1991) 5.Glauser, M., Zanetti, G., Baumgartner, J.﹠amp; Cohen, J., Lancet 33R, 732-736 (1991) 6.Schumann, R.R., etc., Science 249,1429-1431 (1990) 7.Frey, and E.A., etc., J.Exp.Med.176,1665-1671 (1992) 8.Haziot, A., Rong, G.W., Silver, J.﹠amp; Goyert, S.M., J. Immunol.151,1500-1507 (1993) 9.Haziot, a., etc., J.Immunol.141,547-552 (1988) 10.Tobias, P.S.﹠amp; Ulevitch, R.J., Immunobiology 187,227-232 (1993) .11.Marrack, P.﹠amp; Kappler, J., Science 248,705-711 (1990) 12.Steimle, V., Otten, L.A., Zufferey, M.﹠amp; Mach, B., Cell 75,135-146 (1993) 13.Pugin, J., etc., Immunity 1,509 (1994) 14.Hailmann, E., Deng, J.Exp.Med.179,269-277 (1994) 15.Gorga, J.C. etc., J.Biol.Chem.262,16087-16094 (1987) 16.K ntgen etc., International Immunology 5,957-964 (1993)

Claims

1.MHC-II binding molecule and/or MHC-II model molecule, what be used to disturb the cell that is used to have MHC-II intensifies stimulator and the intermolecular interaction of cell bonded MHC-II.

2. with MHC-II binding molecule and/or MHC-II model molecule, be used to disturb the interaction that is used between cytophagous activation stimulator and the cell bonded MHC-II molecule.

3.MHC-II binding molecule and/or MHC-II model molecule are used to disturb lipopolysaccharides (LPS) or and the LPS of other molecular composition complex body such as CD14 and LBP and the interaction between the cell bonded MHC-II molecule.

4.MHC-II binding molecule and/or MHC-II model molecule are used to disturb from the product of gram positive bacterium or by from product of gram positive bacterium and interaction such as molecular complex body of CD14 and cell bonded MHC-II molecule.

5. as each MHC-II binding molecule and/or MHC-II model molecule among the claim 1-4, wherein said MHC-II binding molecule is a kind of resisting-MHC-II antibody or its fragment, or any by such antibody deutero-molecule, as humanized, dual specific or other engineering molecule etc.

6. as each MHC-II binding molecule and/or MHC-II model molecule among the claim 1-4, wherein said MHC-II binding molecule is selected from CD14, its fragment, its modified forms or has the peptide of MHC-II binding characteristic.

7. as claim 1,2,3 or 4 MHC-II binding molecule and/or model molecule, wherein said MHC-II model molecule are selected from the peptide of the modified forms of the fragment of one or several subunit of the complete MHC-II of solubility, MHC-II, complete MHC-II or its subunit, complete MHC-II or its subunit, the LPS with MHC-II or its subunit or LPS/CD14/LBP complex body binding characteristic.

8. as claim 1,2,3,4 or 7 MHC-II binding molecule and/or MHC-II model molecule, wherein said MHC-II model molecule is connected with a kind of carrier.

9. each MHC-II binding molecule and/or MHC-II model molecule among the claim 1-8 are used to prevent and/or treat inflammatory diseases; Septic shock; Organ transplantation is as the graft versus host disease after the bone marrow transplantation; Graft-rejection; Because of the inflammatory reaction of one or more organs on burn, unexpected injury, the human body that pancreatic infection caused, as adult respiratory distress syndrome (ARDS) etc.; After operation, come across the inflammatory reaction of one or more human organs, as capillary leaks syndrome; Betide the anaphylaxis of one or more organs of human body; The inflammatory reaction of one or more organs relevant with autoimmune disease is as lupus erythematosus (LE) and hypotype, scleroderma and hypotype thereof, eosinophil fascitis, Sjogren syndrome, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener ' s granulomatosis, temporal arteritis, polymyalgia rheumatica etc.; The inflammatory reaction of one or more organs relevant with similar rheumatism is as rheumatoid arthritis, juvenile chronic arthritis, Felty syndrome, Caplan syndrome, rheumatoid spondylitis (Marie-Strumpell-Bechterew disease), psoriasis, Reiter syndrome, Behcet syndrome; The inflammatory reaction of one or more organs relevant with to small part being the disease that causes because of autoimmune mechanism, as diabetes, Crohn disease, ulcerative colitis, digestive tract ulcer, ephritis, as glomerulonephritis and ephritis, arteriosclerosis disease, multiple sclerosis, senile dementia, hyperthyroidism, thyroid function hang down inferior; The inflammatory reaction of human body one or more organs relevant with neoplastic disease is as leukemia, hemocyte tumour, cancer, fibroma, sarcoma and various types of histiocytosis; And virus disease, as AIDS.

10.MHC-II binding molecule.

11.MHC-II model molecule.

12. contain pharmaceutical composition as the MHC-II binding molecule of activeconstituents and/or MHC-II model molecule and a kind of suitable vehicle.

13. MHC-II binding molecule and/or MHC-II model molecule are used to prepare a kind of purposes that is used for disturbing the activation stimulator and the interactional pharmaceutical composition between the cell bonded MHC-II molecule of the cell that has MHC-II.

14. MHC-II binding molecule and/or MHC-II model molecule are used to prepare a kind of purposes that is used for disturbing the interactional pharmaceutical composition between cytophagous activation stimulator and the cell bonded MHC-II molecule.

15. MHC-II binding molecule and/or MHC-II model molecule be used to prepare a kind ofly be used for disturbing lipopolysaccharides (LPS) or and form the LPS of complex body and the purposes of the interactional pharmaceutical composition between the cell bonded MHC-II molecule such as other molecule of CD14 and LBP.

16. with MHC-II in conjunction with and/or the MHC-II model molecule be used to prepare and a kind ofly be used for disturbing from the product of gram positive bacterium or by the complex body that forms from the product of gram positive bacterium and such as other molecule of CD14 and the purposes of the interactional pharmaceutical composition between the cell bonded MHC-II molecule.