WO1996020215A2

WO1996020215A2 - Use of mhc-ii binding and/or mhc-ii mimicking molecules for the prevention and/or treatment of inflammatory diseases

Info

Publication number: WO1996020215A2
Application number: PCT/EP1995/005164
Authority: WO
Inventors: Roger Pascal Lauener
Original assignee: Laboratoires Om S.A.; Deutsche Om Arzneimittel Gmbh
Priority date: 1994-12-23
Filing date: 1995-12-27
Publication date: 1996-07-04
Also published as: CZ186897A3; HUT77468A; CA2208233A1; EP0800534A2; JPH10511652A; AU4347696A; SK80697A3; WO1996020215A3; BR9510554A; CN1171117A; 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

USE OF MHC-II BINDING AND/OR MHC-II MIMICKING

MOLECULES FOR THE PREVENTION AND/OR TREATMENT

OF INFLAMMATORY DISEASES

The present invention relates to the use of speci¬ fic molecules for interfering in the interaction between toxins like lipopolysaccharide (LPS) alone or as a complex with other molecules, such as CD14 and LBP, and its transdu- cer molecule. The invention further relates to the use of these molecules in the prevention and treatment of inflamma¬ tory diseases, like septic shock.

Lipopolysaccharide (LPS) is a constituent of the cell wall of Gram-negative bacteria. Infection with Gram- negative bacteria can result in a life-threatening disease, which is caused by specific binding of LPS to phagocytes, like monocytes, macrophages and granulocytes, which are thereby activated and secrete various cytokines, including tumor necrosis factor- (TNF-α) , interleukin 1 (IL-1) , IL-6, IL-8, and other mediators of inflammation. These substances, either by direct action or by activation of secondary media¬ tors, initiate a cascade of events resulting in disorders of the coagulation system, vasodilatation, multi-organ failure and, ultimately, septic shock (4, 5). In general the marked activation of phagocytic cells, resulting in secretion of a multitude of inflammatory mediators, is a central event in the pathogenesis of a pathologic condition called Systemic Inflammatory Reaction Syndrome (SIRS) . Besides activation by LPS, SIRS can develop as a result of various other clinical conditions, such as infection with bacteria or viruses, trauma, burns, pancrea¬ titis, graft-versus-host and host-versus-graft disease, hemophagocytosis and many more. Toxic shock, caused by exotoxins, like staphylococcal toxin A, from Gram-positive bacteria, is one example of an inflammatory disease. Other exotoxins, also known as superantigens, are staphylococcal toxin B and streptococcal toxins.

It has been demonstrated that LPS binds to the glycosylphosphatidylinositol (GPI)-anchored monocytic anti- gen CD14. CD14 is present on the surface of monocytes, macrophages and granulocytes, but is also found in a soluble form without the GPI anchor in the serum of healthy indivi¬ duals. Furthermore it has been demonstrated that activation of monocytes by LPS can be inhibited by anti-CD14 monoclonal antibodies. It was therefore suggested that CD14 would serve as a receptor for LPS (1) and mediates the effects of LPS to the cytoplasm. However, CD14-negative cells can also respond to LPS (2, 7, 8) . Furthermore, it became known that CD14 is a glyco- sylphosphatidylinositol (GPI)-anchored molecule, lacking a transmembrane and cytoplasmic domain (9) . Thus CD14 can not transduce a signal to the cytoplasm. It is a widely accepted hypothesis that GPI-linked proteins require associated transmembrane molecules for signal transduction. Thus, the real transducer molecule for LPS and possibly other SIRS stimuli, has not yet been identified.

In the case of cell activation by LPS, molecules other than CD14 have to be invoked to explain cellular activation by LPS (10) . It was proposed that LPS forms a complex with either membrane-bound or soluble CD14 and the LPS binding protein (LBP) . Other serum-derived molecules may also participate in this complex. The complex interacts with an as yet unidentified molecule on the cell surface, leading to the activation of the cells.

CD14 has been described as playing a key role in initiating cell activation by bacterial envelope products from Gram-positive as well as Gram-negative organisms (13) . Again, other membrane-bound or serum-derived molecules may be involved in the interaction with the cell-surface molecu¬ le which leads to cellular activation.

According to the present invention it has now been found that Major Histocompatibility Complex II (MHC-II) molecules are required for activation of cells by LPS, either by serving as receptor and/or as a signal transducing element for molecular complexes of LPS and molecules like CD14 and LBP. In humans MHC is known as Human Leucocyte Antigen (HLA) . It has been shown that LPS-responsiveness depends on expression of MHC class II-molecules on the cell surface. A cell line, referred to herein as ^»τHP-l^MHC+", is an MHC class II expressing monocytic cell line, described as THP-1 by Tsuchiya et al. (3) . A second cell line, referred to herein as "THP-1.6^MHC*", is an MHC class Il-negative mono¬ cytic cell line derived from THP-1^MHC"" by spontaneous mutati¬ on. THP-1^MHC+ cells secrete cytokines in response to LPS, whereas THP-1.6^MHC" cells do not. CD14-positive, MHC Il- negative human peripheral blood mononuclear cells (PBMC) are irresponsive to LPS, too. MHC class II-expression and LPS- responsiveness can be restored by transfecting THP-1.6^HHC' cells with CIITA, a cDNA encoding a nuclear factor essential for the expression of MHC-II molecules on the cell surface. The transduction of other SIRS stimuli to the cell may also be mediated by MHC-II molecules. It has already been demonstrated previously that exotoxins also bind to MHC-II molecules on the cell surface. The activity of other Gram positive products is mediated at least in part by CD14. and it is thus likely that the complex of these products with CD14 also interacts with MHC-II molecules to activate cells.

The prevention and/or treatment of systemic in¬ flammatory reaction syndrome may thus be performed by inter¬ fering in the interaction between the complex of LPS and other molecules, like CD14 and LBP (indicated hereinbelow as "CD14/LPS/LBP complex"), and cell-bound MHC-II. According to the invention this interference may be effected in two different ways.

First the binding of the CD14/LPS/LBP complex to cell-bound MHC-II may be blocked by MHC-II binding molecu¬ les, such as anti-MHC-II antibodies, CD14 or peptides deri¬ ved thereof. This type of molecule competes with the CD14/- LPS/LBP complex and thus prevents the complex from binding but does not itself activate the MHC-II. The transducer function of MHC-II is then blocked.

Second the circulating LPS or CD14/LPS/LBP complex may be captured by MHC-II mimicking molecules. Complexes binding to soluble MHC-II or MHC-II-like molecules are no longer able to bind to the cell-bound MHC-II. Thus activati¬ on of the cell is prevented.

MHC-II binding molecules comprise any molecule that is capable of blocking binding of LPS or the CD14/LPS- complex to MHC-II. In practice this will comprise anti-MHC- II antibodies, both monoclonal and polyclonal antibodies, directed to the CD14/LPS or LPS binding site of a cell. Antibody fragments are also suitable as MHC-II binding molecules. MHC-II mimicking molecules are meant to comprise both soluble MHC-II molecules themselves as well as any other molecule that is capable of blocking the MHC-II bin¬ ding site on LPS or the CD14/LPS complex. Molecules of this type may comprise complete MHC-II molecules or fragments or subunits thereof. Furthermore peptides capable of binding to LPS or the LPS/CD14/LBP complex without activating the MHC- II may be useful. Such peptides may be at least homologous to MHC-II and comprise suitable D-amino acids providing the peptide with antagonistic properties. The molecules may be in a soluble form or coupled to the surface of a carrier.

Based on the information given in this applicati¬ on, the skilled person will be able to identify suitable binding and/or mimicking molecules.

This type of molecule may originate from any suit- able source, either human or other, and be prepared by various means, such as isolation from the supernatant of a cell culture of MHC-II positive cells or from a cell lysate. An especially preferred isolation method is immunoaffinity chromatography. Suitable molecules may also be prepared by gene technology, by protein chemical methods or any other suitable method.

According to the invention these two types of molecules may be used in the prevention and/or treatment of inflammatory diseases, like septic shock, graft-versus-host disease after organ transplantations, like bone marrow transplantations, graft rejection reactions, inflammatory reactions resulting from burns, accidents, infections of the pancreas etc.. The invention is also suitable for the prevention and/or therapy of other inflammatory reactions occurring e.g. after surgery, like capillary leak syndrome, allergic diseases, autoimmune diseases, like Lupus Erythematodes (LE) and sub-forms thereof, sclerodermia and its sub-forms, eosinophilic fasciitis, Sjδgren Syndrome, polymyositis, dermatomyositis, periarteritis nodosa, Wegener's granuloma- tosis, arteritis temporalis, polymyalgia rheumatica etc., rheumatoid disorders, like rheumatoid arthritis, juvenile chronic arthritis, Felty syndrome, Caplan syndrome, ankylosating spondylitis (Marie-Strϋmpell-Bechterew disease), psoriasis, Reiter syndrome, Behcet syndrome.

Other diseases that may be treated according to the invention and at least partially result from autoimmune mechanisms are inter alia diabetes mellitus, morbus Crohn, colitis ulcerosa, digestive tract ulcers, renal infections, like glomerulonephritis and nephritis, arteriosclerotic disorders, multiple sclerosis, Alzheimer's disease, hyperthyreosis, hypothyreosis. The invention may also be used in inflammatory reactions in one or more human organs associated with onco¬ logical disorders, such as leukemia, blood cell tumors, carcinoma, fibroma, sarcoma, and various types of histiocy- tosis. The invention may also be used for the prevention and/or treatment of viral diseases such as AIDS. LPS-stimu- lation is known to increase intracellular Human Immunodefi¬ ciency Virus (HIV) replication. Blocking the stimulation by LPS by MHC-II binding and/or mimicking molecules of the invention will thus remove this replication stimulus. MHC-II binding and/or mimicking molecules of the invention may therefore be used to impart a protective effect on HIV- infected cells by preventing the stimulation of viral repli¬ cation by cell-activating stimuli. Based on the data presented in the examples the following model for activation of cells by LPS could be imagined. On the one hand, LPS may bind to membrane-bound CD14 (mCD14) , an interaction accelerated by Lipopolysaccha- ride Binding Protein LBP (14) . The complex of LPS and GPI- anchored mCD14 and possibly LBP would then interact with the transmembrane MHC class II-molecules, resulting in signal transduction. This situation is shown in figure 3A. On the other hand, LPS together with LBP may bind to soluble CD14 (sCD14) present in the serum, and this complex may then bind to MHC class II-molecules on the surface of CD14-negative cells, resulting in activation of CD14-negative, but MHC class II-positive cells. This situation is illustrated in the figure 3B. Although contact is shown between CD14 and MHC-II, LBP and LPS may also participate in this interaction as may other as yet unidentified molecules. The activation stimulus may also be components of Gram-positive bacteria, although in this case the role of LBP has not been determi- ned. The possibility that some activation stimuli act di¬ rectly on the MHC-II molecules without forming a complex with CD14 or LBP is herewith not excluded. It is now esta¬ blished that stimulation is mediated by MHC-II molecules. Blocking or mimicking these molecules thus will prevent the transduction of the activation stimulus.

The invention further relates to the MHC-II bin¬ ding molecules, to the MHC-II mimicking molecules and to pharmaceutical compositions comprising either or both types of molecules. Pharmaceutical compositions, comprising one or more MHC-II binding molecules and/or MHC-II mimicking mole¬ cules as the active ingredient for interfering in the inter¬ action between an activation stimulus, such as LPS, for cells expressing MHC-II molecules, such as phagocytes, and cell-bound MHC-II molecules have the form of powders, sus- pensions, solutions, sprays, emulsions, infusions, inha¬ lation compositions, unguents or creams and can be used for local application, intranasal, rectal, vaginal and also for oral, parenteral (intravenous, intradermal, intramuscular, intrathecal etc.) or transdermal administration, administra- tion by means of inhalation etc.. Pharmaceutical compositi¬ ons of the invention can be prepared by combining (i.e. by mixing, dissolving etc.) the active compound(s) with pharma¬ ceutically acceptable excipients with neutral character (such as aqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents, additives) , and further if necessa¬ ry coloring agents and flavoring agents. The concentration of the active ingredient in a pharmaceutical composition can vary between 0.001% and 100%, depending on the nature of the treatment and the method of administration. The dose of the active ingredient that is administered also depends on the specific application and route of administration, but may for example vary between 0.01 μg and 1 g per kg body-weig- ht, preferably between O.lμg and 100 μg per kg body-weight. The invention will be illustrated with reference to the following examples, which are not intended to limit the scope of the invention.

EXAMPLES

EXAMPLE 1 Introduction

The hypothesis that MHC-II is the transducer and/or receptor for a complex including LPS, CD14, LBP and possibly other molecules in the activation of phagocytic cells by LPS was tested by comparing the secretion of cyto- kines by MHC class II-positive (HLA-DR) and MHC class Il- negative cell lines upon stimulation with LPS. The secretion of cytokines is indicative of activation of the cells.

Materials and methods

THP-1^MHC* is a CD14-negative, MHC class II-positive onocytic cell line of human origin (3). THP-1.6^HHC' is a spontaneously derived, MHC class Il-negative mutant of THP- 1^MHC+, cloned by repeated limiting dilutions.

HLA-DR expression was reconstituted by transfection of THP-1.6^HHC' cells with CIITA (class II transactivator; a nuclear protein essential for the expression of MHC class II-proteins (12)) to yield THP- 1.6^HC"CIITA.

Expression of HLA-DR and CD18 on the surface of THP-l^MHt cells (wild-type), THP-1.6^MHC" cells (mutant cell line) and THP-1.6^HHC"CIITA (THP-1.6^MHC" cells transfected with CIITA) was assessed by flow cytometry.

The various cell lines were cultured at 10⁶ cells/ ml in medium supplemented by 10% FCS and stimulated with LPS in doses of 0, 1, 10, 100 ng/ml and 1 μg/ml. After 24 hours, the supernatants were harvested and assessed by ELISA for their content of TNF-α and IL-8.

Results and discussion Viability of the cells was not compromised by the

LPS treatment. More than 95% of the cells were viable as shown by trypan blue staining.

While expression of CD18 was similar in the three cell lines, THP-1.6^MHC" cells expressed significantly lower levels of HLA-DR than THP-1^HHC+ cells.

Upon stimulation of THP-1^HHC* cells with LPS the cells responded by secretion of TNF-α and IL-8 (fig. 1) . By contrast, the HLA-DR-negative cell line THP-1.6^HC" could not be induced to secrete TNF-α nor IL-8 by stimulation with LPS, even using high doses of LPS. However, HLA-DR- expression and LPS-responsiveness were both restored by transfection with CIITA. It was concluded that expression of HLA-DR molecules was required for activation of monocytic cells by LPS.

EXAMPLE 2 Introduction

To confirm the finding of example l the results obtained with the cell line system were verified using cells from a patient with MHC class II-deficiency, a rare inherited disease manifesting as severe combined immuno¬ deficiency in early childhood. CD14-expression is not affected in these patients. PBMC of a patient with MHC class II-deficiency and of a control individual were cultured in the presence of increasing doses of LPS and the levels of TNF-α and of IL-1 were measured in the supernatants of these cultures. Materials and methods

Peripheral Blood Mononuclear Cells (PBMC) were taken from a patient with MHC-II deficiency and from a healthy control. The expression of HLA-DR on the surface of both

PBMC of the patient (hatched histogram, left panel of Fig. 2) and of the healthy control (grey histogram) was assessed by direct immunofluorescence staining and flow cytometry. PBMC from the patient (thick line) and from the control (thin line) were cultured at 10⁶ cells/ml in medium supplemented by 10% normal human AB-positive serum and stimulated with 0, l, 10 and 100 ng/ml of LPS. After 24 hours the supernatants were harvested and assessed by ELISA for their content of TNF-α.

Results and discussion

PBMC from healthy individuals secreted TNF-α and IL-1 in response to LPS, as expected. However, the patient's MHC class II-deficient PBMC did not secrete significant levels of cytokines in response to LPS (fig. 2) . Thus it was shown that the requirement for expression of MHC class II- molecules was not limited to the THP-1^MHC+ family of cell lines. The data show that membrane-bound CD14 is neither required for activation of cells by LPS (THP-1^MHC+ cells do not express CD14) nor sufficient (PBMC from MHC class II- deficient patients express normal levels of CD14) .

However, the experiments outlined above do not address the role of soluble CD14. In all these experiments media supplemented with serum containing soluble CD14 have been used. The experiments were therefore repeated, culturing THP-l^MHt , THP-1.6^MHC" and THP-1.6^HHC'CIITA cells in serum-free medium. Treatment with increasing doses of LPS did not result in secretion of significant levels of TNF-α. The results indicate that MHC class II-molecules are crucially involved in LPS-responsiveness. HLA-DR negative cells could not secrete cytokines upon stimulation with LPS. One could argue that lack of response to LPS is related to a factor closely associated with MHC class II and regulated by CIITA, too, rather than lack of MHC class II- expression per se causing depressed LPS-responsiveness. However, the defect in the response to LPS is not limited to the secretion of one cytokine alone since secretion of TNF-α as well as IL-1 and IL-8 was depressed. Furthermore no molecule regulated by CIITA other than MHC class II has been found to date, despite extensive investigations. Finally, the patient's disease is not due to a defect related to

CIITA. Transfection of Epstein Barr Virus (EBV)-transformed B-cells from this patient did not restore expression of MHC class-II molecules.

EXAMPLE 3 Introduction

The physical interaction between MHC class II- molecules and CD14 was demonstrated in three different ways. First, lysates of MHC II positive and MHC II negative cell lines were incubated with MHC II specific or control antibodies and radioactive CD14 (CD14^*) or a radioactive control molecule. Complexes between MHC II and CD14^* can only be precipitated by interaction of protein A- agarose and MHC II specific antibodies. No radioactivity should be found in the precipitate when the control molecule or the control antibody is used. If no MHC II is present, no radioactivity may be found in the precipitate.

Second, MHC II was incubated with radioactive CD14. Presumably MHC II/CD14^* complexes will be formed. In theory, these complexes may be precipitated by means of antibodies that are specific for either of the two partners in the complex, and protein A-agarose.

Third, it was tested whether anti-CD14 antibodies could block the interaction of MHC II and CD14^*. The principle of these experiments is further illustrated in figure 4. Methods

1. Production of radioactive CD14

Recombinant CD14 has been produced by in vitro transcription/translation from a full length CD14 cDNA, that was prepared by means of PCR techniques and the nucleotide sequence of which was verified, using the TNT T7 coupled reticulocyte lysate system following the manufacturer's protocol (Promega, Switzerland) in the presence of ³⁵S- methionine. As control, radioactively labeled luciferase has been produced by in vitro transcription/translation using the same system.

2. Production of cell lvsates

So-called 293 cells are derived from human embryo- nal kidney transformed with human adenovirus type 5 DNA (ATCC designation CRL 1573) ; wild-type 293 cells do not express MHC class II-molecules as detected by FACS-analysis. These were taken as MHC Il-negative cells. 293 cells trans- fected with cDNA from the α-, β and invariant (i) chains of human MHC class II were obtained form Dr. Jacques Neefjes, Netherlands Cancer Institute, Amsterdam. These do express MHC II and are therefore used as MHC II positive cells. Lysates from MHC class II-positive and MHC class Il-negative cell lines were produced as follows. 5 x 10⁶ cells were lysed in a Petri dish in 1 ml of lysis buffer (Boehringer Mannheim, cellular labeling and immunoprecipitation kit) . After 30 minutes lysed cells were collected and subjected to sonification (3x15 seconds) , followed by 30 minutes of incubation on ice and subsequent centrifugation. The supernatants were subjected to immunoprecipitation as described below.

3. Immunoprecipitation in Experiment 1

Immunoprecipitation was performed using the rea- gents and following the protocol from the "Cellular labeling and immunoprecipitation kit" form Boehringer Mannheim (Swit¬ zerland) . In brief, the samples were precleared using 50μl protein A-agarose suspension. After removal of this protein A-agarose by centrifugation the supernatants were incubated with the following antibodies: L243 (anti-HLA-DR, ATCC designation HB 55); 1B5 (anti-MHC class II, obtained from Dr. Jacques Neefjes, Netherlands Cancer Institute, Amster- dam) ; and anti-CD3 (Pharmingen, San Diego, USA/AMS Biotech¬ nology, Switzerland) . After 1 hour, 50 μl protein A-agarose was added, and the samples were incubated for 3 hours followed by 6 washes.

Then the pellets were resuspended in 30 μl of buffer (50 mM Tris, pH 7.5, 20 mM NaCl, 1 mM EDTA, 2 mM PMSF) , and 5 μl from the solution obtained in the in vitro transcription/translation reaction containing radioactively labeled CD14 were added, and this mixture was incubated for 30 minutes at room temperature. After centrifugation, the pellets were washed 2x with wash buffer #2 followed by 2 washes with wash buffer #3 (buffers #2 and #3 originate from the cellular labeling and immunoprecipitation kit of Boeh¬ ringer Mannheim, Switzerland) . The pellets were then dissol¬ ved and boiled in standard SDS-PAGE loading buffer and subjected to gel electrophoresis on a 10% SDS polyacrylamide-gel, followed by autoradiography.

4. Purification of MHC class II-molecules

MHC class II-molecules were extracted and purified from THP-1 cells as described by Gorga et al. (15) with minor modifications. L243 (anti-HLA-DR, ATCC designation HB 55) was immobilized on a protein A-Sepharose 4B column by crosslinking with dimethylpimelimidate. THP-1 cells were lysed on ice in Tris-HCl pH 8.0 containing 0.1 mM PMSF. All subsequent steps were carried out at 4°C. The lysate was centrifuged at 3000xg and the pellet washed until the supernatant was clear. The pooled supernatants were centrifuged at 160000xg for 40 min and the pellet redissolved by the addition of Nonidet P40 to 4% final concentration. After centrifugation at 160000xg for 2 hours, the supernatant was applied to series of columns in the following order: Sepharose 4B (10ml) , normal rabbit serum- Affigel 10 (10ml), Protein A-Sepharose 4B (2ml) L243- Sepharose 4B (12ml) . The columns were washed with 10 mM Tris-HCl/0.1% Nonidet P40 pH 8.0 (5 vols.); 10 mM MOPS/140 mM NaCl/0.1% deoxycholate pH 8.0 (2 vols.); lOmM Tris- HC1/0.1% deoxycholate pH 8.0 (4 vols.). The L243 column was then disconnected from the other columns and eluted rapidly with 50 mM glycine/0.1% deoxycholate pH 11.5. 10 ml fractions were collected and adjusted to pH 7.0 to 8.0 as soon as they were eluted with 2M glycine pH 2.0. The eluted HLA-DR molecules were concentrated by ultrafiltration with 30 kDalton cut-off membranes. After washing three times in Tris-HCl/0.1% deoxycholate pH 8.0, the protein was rediluted in the same buffer.

5. Immunoprecipitation in Experiment 2 5 μl from the solution containing radioactively labeled CD14 obtained in the in vitro transcription/ translation reaction were mixed with 1 μl of the solution containing approximately lOng MHC class II-molecules and incubated for 30 minutes at room temperature. Then, the antibodies were added and the samples were incubated for 1 hour at 4°C. After addition of 50 μl protein A-agarose the samples were incubated for another 3 hours at 4°C. After centrifugation, the pellets were washed 2x with wash buffer #2 followed by 2 washes with wash buffer #3 (cellular labelling and immunoprecipitation kit, Boehringer Mannheim, Switzerland) . The pellets were then dissolved and boiled in standard SDS-PAGE loading buffer and subjected to gel electrophoresis on a 10% SDS polyacrylamide-gel, followed by autoradiography.

6. Immunoprecipitation in Experiment 3

Before adding the radioactively labeled CD14 to the immunoprecipitation samples as described for the other immunoprecipitation experiments, the solution containing CD14 was incubated with a cocktail of anti-CD14 antibodies for 10 minutes at room temperature. The cocktail consisted of 10 μg of each of the following anti-CD14 antibodies: RM052 (Immunotech, Switzerland) ; LeuM3 (Beckton & Dickinson, Switzerland); MY4 (Coulter, Swtizerland) ; Tϋk 4 (Dako, Switzerland) ; and 100 μl of the supernatant of the following anti-CD14 hybridomas: 3C10, 63D3 (both obtained from ATCC) . As a control, PBS in the same volume as the volume of the antibody cocktail, was added to 5μl of the solution containing radioactively labeled CD14.

The antibodies used for the immunoprecipitation were: MY4 (anti-CD14) and L243 (anti-MHC II).

Results

EXPERIMENT 1

Demonstration by coprecipitation of MHC class II-molecules and CD14 using lysates from MHC class II-positive and MHC class II-neσative cells The results of this experiment are shown in figure

5. On the left side of the panel, the results from the experiments performed using lysate from 293-cells transfec- ted with MHC class II (i.e. cotransfected with the α, β, and i-chain of MHC class II) are depicted, on the right side the results using lysate of the MHC class Il-negative 293 wild- type cells are visible.

In lanes 1 and 2 only faint bands presumably corresponding to radioactive CD14 (lane 1) respectively to luciferase (lane 2) were unspecifically precipitated by the control antibody anti-CD3. Bands corresponding to radioacti¬ vely labeled CD14 (CD14^*) are clearly visible in lanes 3 and 5; two different antibodies recognizing MHC class II- molecules (lane 3: L243; lane 5: 1B5) have (co-)precipitated CD14^*. The coprecipitating effect of these anti-MHC class II antibodies is specific for CD14, since no significant amount of a radioactively labeled control protein (luciferase) is coprecipitated by these antibodies (lanes 4 and 6) . The coprecipitation of CD14 by anti-MHC class II antibodies is dependent on the presence of MHC class II-molecules, since there is no precipitation of CD14 by anti-MHC class II antibodies in the absence of MHC class II-molecules (using lysate from MHC class Il-negative 293 cells, lanes 7-9) . EXPERIMENT 2

Demonstration bv coprecipitation of MHC class II-molecules and CD14 using MHC class II-molecules purified bv an iromuno- affinitv column The results are shown in figure 6. On the left side of the panel the results from the immunoprecipitation experiments performed in the presence of purified MHC class II-molecules are depicted, on the right side the results from the experiments performed in the absence of purified MHC class II-molecules, i.e. using the buffer as negative control, are visible.

In lanes 1 and 4 faint bands corresponding to CD14 unspecifically precipitated by the control antibody (anti- CD3) are visible. In lane 3 a band corresponding to CD14 appears upon precipitation by an anti-CDl4 antibody. In lane 2 the band corresponding to CD14 is of greater intensity than in lane 3, although in this experiment precipitation has been performed with an anti-MHC class II-molecules. In the absence of purified MHC class II-molecules, CD14 is strongly precipitated by anti-CD14 antibodies (lane 6), whereas coprecipitation of CD14 with an anti-MHC class antibody does not exceed background level (lane 5) .

EXPERIMENT 3 The physical interaction between CD14 and MHC class II- molecules can be inhibited bv anti-CD14 antibodies

The results are shown in figure 7. Lanes 1 and 5 show that the anti-CD14 antibody MY4 precipitates the radio- actively labeled CD14 produced by in vitro transcripti- on/translation, independently of the presence (lane 1) or absence (lane 5) of MHC class II-molecules (control) . CD14 is strongly precipitated by L243, an anti-MHC class II antibody, provided MHC class II molecules are present (lane 2) , but only in background amounts in the absence of MHC class II-molecules (lane 6) . If the CD14 is treated first with a cocktail of anti-CD14 antibodies, previously to being mixed with the cell lysate containing MHC II-molecules, CD14 cannot be precipitated by anti-MHC class II antibodies. Lane 4 shows that a buffer (control) has no effect on the copre¬ cipitation of CD14 by anti-MHC class II antibodies. Lanes 7 and 8 show the results of the same experiments as in lanes 3 and 4 but performed in the absence of MHC class II-molecu- les.

EXAMPLE 4 Introduction

To demonstrate the role of MHC II in the activati- on of cells in vivo MHC class II knock-out mice were used. If MHC II is involved in the activation mechanism by LPS, mice lacking MHC II should not show the usual physiological effects of LPS stimulation. A similar experiment was perfor¬ med in vitro by using blood of the same mice.

Methods

For the in vivo experiment 100 μg of LPS .E.coli 0111:B4, diluted in sterile 0.9% NaCl) were injected intra¬ venously in wild-type C57BL/6 and B6-Aa°/Aa° MHC class II knock-out mice (Hoffmann-La Roche; ref. 16) . After 2 hours the mice were sacrificed and bled sterily. The blood was allowed to coagulate at room temperature and was centrifuged for 5 minutes at 13.000 rp . Then the serum was removed. The content of TNF-α, a marker for activation of phagocytes, was determined in the serum by ELISA (Biosource) . Figure 8 shows the results.

For the in vitro experiment heparinized whole blood from wild-type MHC II positive C57BL/6 mice (black circles in Figure 9) and B6-Aa°/Aa° mice (open squares) , in which MHC class II-expression is lacking after targeted disruption of the MHC class II gene Aa¹, was incubated in the presence of 0, 0.1, 1, 10 and 100 ng/ml LPS. After 4 hours incubation, the level of TNF-α was assessed in plasma by ELISA (Biosource) . The results are shown in figure 9. Clearly the TNF-α secretion is higher in mice or cells expressing MHC class II molecules. EXAMPLE 5 Introduction

The in vivo effect of MHC II-binding and MHC II- mimicking molecules was assessed as follows.

Method

Wild-type C57B1/6 mice were weighed individually and injected with an LD₅₀ determined in preliminary experiments. Before injection of LPS mice were pretreated with:

Group 1: soluble MHC class II molecules in a dose range from

1 μg to 1 mg/kg body weight;

Group 2: anti-MHC class II antibodies in the same dose range; and Group 3*: saline only (control group) ;

As a control three other groups got soluble MHC class II, anti-MHC II or saline, respectively without LPS challenge afterwards.

Survival was monitored for 7 days. Mice were observed on a regular basis during the first 48 hours to note symptoms.

One sub-group of mice was sacrificed and bled before LPS- induced death occurred and levels of TNF-α were determined in the serum from these animals by ELISA.

Results

Mortality as well as TNF-α serum levels were significantly decreased in the groups of mice treated with soluble MHC class II molecules or anti-MHC II antibodies (results not shown) as compared. The control group treated with MHC class II molecules or anti-MHC II antibodies but without LPS challenge did not show significant differences from the mice treated with saline without LPS challenge. References

1. Wright, S.D., Ramos, R.A. , Tobias, P.S., Ulevitch, R.J. & Mathison, J.C., Science 249, 1431-1433 (1990)

2. Couturier, C. , Jahns, G., Kazatchkine, M.D. & Haeffner Cavaillon, N. , Eur. J. Immunol. 22., 1461-1466 (1992).

3. Tsuchiya, M. , et al., Int. J. Cancer 2.6, 171-176 (1980)

4. Bone, R., Chest 101, 802-808 (1991)

5. Glauser, M. , Zanetti, G. , Baumgartner, J. & Cohen, J. , Lancet 338. 732-736 (1991)

6. Schumann, R.R., et al. Science 249. 1429-1431 (1990)

7. Frey, E.A. , et al. J. Exp. Med. 176. 1665-1671 (1992)

8. Haziot, A., Rong, G.W. , Silver, J. & Goyert, S.M., J. Immunol. 151. 1500-1507 (1993)

9. Haziot, a., et al., J. Immunol. 141. 547-552 (1988)

10. Tobias, P.S. & Ulevitch, R.J. , Immunobiology 187. 227- 232 (1993).

11. Marrack, P. & Kappler, J. , Science 248. 705-711 (1990)

12. Steimle, V., Otten, L.A. , Zufferey, M. & Mach, B. , Cell 75, 135-146 (1993)

13. Pugin, J., et al., Immunity 1 , 509 (1994)

14. Hailmann, E. , et al., J. Exp. Med. 179. 269-277 (1994)

15. Gorga, J.C. et al. , J. Biol. Chem. 262. 16087-16094 (1987)

16. Kδntgen et al.. International Immunology 5 , 957-964 (1993)

Claims

1. MHC-II binding and/or MHC-II mimicking molecu¬ les for use in interfering in the interaction between an activation stimulus for MHC-II bearing cells and cell-bound MHC-II molecules.

2. MHC-II binding and/or MHC-II mimicking molecu¬ les for use in interfering in the interaction between an activation stimulus for phagocytes and cell-bound MHC-II mo¬ lecules.

3. MHC-II binding and/or MHC-II mimicking ole- cules for use in interfering 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.

4. MHC-II binding and/or MHC-II mimicking molecu- les for use in interfering in the interaction between pro¬ ducts from Gram-positive bacteria or complexes of products from Gram-positive bacteria with molecules such as CD14, and cell-bound MHC-II molecules.

5. MHC-II binding and/or MHC-II mimicking molecu- les as claimed in any one of the claims 1-4, wherein the

MHC-II binding molecule is an anti-MHC-II antibody or frag¬ ment thereof, or any molecule derived from such an antibody such as humanized, bispecific or other engineered molecules and the like.

6. MHC-II binding and/or MHC-II mimicking molecu¬ les as claimed in any one of the claims 1-4, wherein the MHC-II binding molecule is selected from the group consis¬ ting of CD14, fragments thereof, modified versions thereof, or peptides having MHC-II binding properties.

7. MHC-II binding and/or MHC-II mimicking molecu¬ les as claimed in claim 1, 2, 3 or 4, wherein the MHC-II mimicking molecule is selected from the group consisting of soluble complete MHC-II, one or more soluble subunits of MHC-II, fragments of complete MHC-II or subunits thereof, modified versions of complete MHC-II or subunits thereof, peptides having the LPS or LPS/CD14/LBP complex binding properties of MHC-II or subunits thereof.

8. MHC-II binding and/or MHC-II mimicking molecu¬ les as claimed in claim 1, 2, 3, 4 or 7 wherein the MHC-II mimicking molecule is coupled to a carrier.

9. MHC-II binding and/or MHC-II mimicking molecu¬ les as claimed in any one of the claims 1-8 for use in the prevention and/or treatment of inflammatory diseases; septic shock; graft-versus-host disease after organ transplantati- ons, like bone marrow transplantations; graft rejection reactions; inflammatory reactions in one or more human organs resulting from burns, accidents, infections of the pancreas, such as adult respiratory distress syndrome (ARDS) etc. ; inflammatory reactions occurring in one or more human organs after surgery, like capillary leak syndrome; allergic diseases in one or more human organs; inflammatory reactions in one or more human organs associated with autoimmune diseases, like Lupus Erythematodes (LE) and sub-forms there¬ of, sclerodermia and its sub-forms, eosinophilic fasciitis, Sjogren Syndrome, polymyositis, dermatomyositis, periarteri- tis nodosa, Wegener's granulomatosis, arteritis temporalis, polymyalgia rheumatica etc. ; inflammatory reactions in one or more human organs associated with rheumatoid disorders, like rheumatoid arthritis, juvenile chronic arthritis, Felty syndrome, Caplan syndrome, ankylosating spondylitis (Marie- Strumpell-Bechterew disease) , psoriasis, Reiter syndrome, Behcet syndrome; inflammatory reactions in one or more human organs associated with diseases which at least partially result from autoimmune mechanisms, such as diabetes elli- tus, morbus Crohn, colitis ulcerosa, digestive tract ulcers, renal inflammations, like glomerulonephritis and nephritis, arteriosclerotic disorders, multiple sclerosis, Alzheimer's disease, hyperthyreosis, hypothyreosis; inflammatory reacti¬ ons in one or more human organs associated with oncological disorders, such as leukemia, blood cell tumors, carcinoma, fibroma, sarcoma, and various types of histiocytosis; and viral diseases such as AIDS.

10. MHC-II binding molecules.

11. MHC-II mimicking molecules.

12. Pharmaceutical compositions comprising as the active ingredient MHC-II binding and/or MHC-II mimicking molecules together with a suitable excipient.

13. Use of MHC-II binding and/or MHC-II mimicking molecules for the preparation of a pharmaceutical compositi¬ on for interfering in the interaction between an activation stimulus for MHC-II bearing cells and cell-bound MHC-II molecules.

14. Use of MHC-II binding and/or MHC-II mimicking molecules for the preparation of a pharmaceutical compositi¬ on for interfering in the interaction between an activation stimulus for phagocytes and cell-bound MHC-II molecules.

15. Use of MHC-II binding and/or MHC-II mimicking molecules for the preparation of a pharmaceutical compositi¬ on for interfering in the interaction between lipopolysac¬ charide (LPS) or LPS in a complex with other molecules, such as CD14 and LBP, and cell-bound MHC-II molecules.

16. Use of MHC-II binding and/or MHC-II mimicking molecules for the preparation of a pharmaceutical compositi¬ on for interfering 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.