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  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2833—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
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• • A—HUMAN NECESSITIES
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  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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.
• LPS lipopolysaccharide
• the invention further relates to the use of these molecules in the prevention and treatment of inflamma ⁇ tory diseases, like septic shock.
• Lipopolysaccharide 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- ⁇ ) , interleukin 1 (IL-1) , IL-6, IL-8, and other mediators of inflammation.
• TNF- ⁇ tumor necrosis factor-
• IL-1 interleukin 1
• IL-6 interleukin 6
• IL-8 interleukin 8
• SIRS Systemic Inflammatory Reaction Syndrome
• 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.
• 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.
• CD14-negative cells can also respond to LPS (2, 7, 8) .
• CD14 is a glyco- sylphosphatidylinositol (GPI)-anchored molecule, lacking a transmembrane and cytoplasmic domain (9) .
• GPI glyco- sylphosphatidylinositol
• LPS LPS binding protein
• 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.
• MHC-II Major Histocompatibility Complex II
• LPS Human Leucocyte Antigen
• a cell line referred to herein as » ⁇ HP-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.
• MHC-II binding molecu ⁇ les such as anti-MHC-II antibodies, CD14 or peptides deri ⁇ ved thereof.
• 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.
• 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.
• 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.
• 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.
• 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..
• 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.
• autoimmune diseases like Lupus Erythematodes (LE) and sub-forms thereof, sclerodermia and its sub-forms, eosinophilic fasciitis, Sj ⁇ gren 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-Str ⁇ mpell-Bmürew disease), psoriasis, Reiter syndrome, Behcet syndrome.
• Sj ⁇ gren Syndrome polymyositis, dermatomyositis, periarteritis nodosa, Wegener's granuloma- tosis, arteritis temporalis,
• 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) .
• mCD14 membrane-bound CD14
• LBP Lipopolysaccha- ride Binding Protein LBP
• 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.
• 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 ⁇ g and 1 g per kg body-weig- ht, preferably between O.l ⁇ g and 100 ⁇ g 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.
• 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.
• 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.
• CIITA class II transactivator; a nuclear protein essential for the expression of MHC class II-proteins (12)
• THP-l MHt cells wild-type
• THP-1.6 MHC" cells mutant cell line
• THP-1.6 HHC" CIITA THP-1.6 MHC” cells transfected with CIITA
• the various cell lines were cultured at 10 6 cells/ ml in medium supplemented by 10% FCS and stimulated with LPS in doses of 0, 1, 10, 100 ng/ml and 1 ⁇ g/ml. After 24 hours, the supernatants were harvested and assessed by ELISA for their content of TNF- ⁇ and IL-8.
• LPS treatment More than 95% of the cells were viable as shown by trypan blue staining.
• THP-1.6 MHC cells expressed significantly lower levels of HLA-DR than THP-1 HHC+ cells.
• THP-1 HHC * cells Upon stimulation of THP-1 HHC * cells with LPS the cells responded by secretion of TNF- ⁇ and IL-8 (fig. 1) .
• the HLA-DR-negative cell line THP-1.6 HC" could not be induced to secrete TNF- ⁇ nor IL-8 by stimulation with LPS, even using high doses of LPS.
• 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.
• 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- ⁇ and of IL-1 were measured in the supernatants of these cultures.
• PBMC Peripheral Blood Mononuclear Cells
• PBMC of the patient hatchched 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 thin line and from the control (thin line) were cultured at 10 6 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- ⁇ .
• MHC class II- molecules 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.
• 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.
• 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 35 S- methionine. As control, radioactively labeled luciferase has been produced by in vitro transcription/translation using the same system.
• 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 ⁇ -, ⁇ 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.
• Immunoprecipitation was performed using the rea- gents and following the protocol from the "Cellular labeling and immunoprecipitation kit" form Boehringer Mannheim (Swit ⁇ zerland) .
• the samples were precleared using 50 ⁇ l 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 ⁇ l protein A-agarose was added, and the samples were incubated for 3 hours followed by 6 washes.
• pellets were resuspended in 30 ⁇ l of buffer (50 mM Tris, pH 7.5, 20 mM NaCl, 1 mM EDTA, 2 mM PMSF) , and 5 ⁇ l 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.
• buffer 50 mM Tris, pH 7.5, 20 mM NaCl, 1 mM EDTA, 2 mM PMSF
• 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
• 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.
• the solution containing CD14 was incubated with a cocktail of anti-CD14 antibodies for 10 minutes at room temperature.
• the cocktail consisted of 10 ⁇ g of each of the following anti-CD14 antibodies: RM052 (Immunotech, Switzerland) ; LeuM3 (Beckton & Dickinson, Switzerland); MY4 (Coulter, Swtizerland) ; T ⁇ k 4 (Dako, Switzerland) ; and 100 ⁇ l of the supernatant of the following anti-CD14 hybridomas: 3C10, 63D3 (both obtained from ATCC) .
• PBS in the same volume as the volume of the antibody cocktail, was added to 5 ⁇ l of the solution containing radioactively labeled CD14.
• the antibodies used for the immunoprecipitation were: MY4 (anti-CD14) and L243 (anti-MHC II).
• EXPERIMENT 3 The physical interaction between CD14 and MHC class II- molecules can be inhibited bv anti-CD14 antibodies
• 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.
• 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.
• Wild-type C57B1/6 mice were weighed individually and injected with an LD 50 determined in preliminary experiments. Before injection of LPS mice were pretreated with:
• Group 1 soluble MHC class II molecules in a dose range from
• Group 2 anti-MHC class II antibodies in the same dose range
• Group 3* saline only (control group) ;
• mice One sub-group of mice was sacrificed and bled before LPS- induced death occurred and levels of TNF- ⁇ were determined in the serum from these animals by ELISA.

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