WO1996032418A1 - Anticorps anti-cd14 utilises dans l'induction de la secretion il-10 - Google Patents

Anticorps anti-cd14 utilises dans l'induction de la secretion il-10 Download PDF

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WO1996032418A1
WO1996032418A1 PCT/EP1996/001588 EP9601588W WO9632418A1 WO 1996032418 A1 WO1996032418 A1 WO 1996032418A1 EP 9601588 W EP9601588 W EP 9601588W WO 9632418 A1 WO9632418 A1 WO 9632418A1
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antibodies
cells
secretion
treatment
cell
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PCT/EP1996/001588
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Roger Pascal Lauener
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Laboratoires Om S.A.
Deutsche Om Arzneimittel Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to anti-CD14 antibodies for use in the induction and/or increase of interleukin-10 secretion.
  • the invention further relates to anti-CD14 antibodies for use in the treatment of inflammatory conditions based on increase of the IL-10 secretion, and in immunosuppression based on either increase of interleukin-10 secretion and/or the induction of T cell tolerance and/or anergy.
  • Interleukin-10 is one of the cytokines and is produced in the body by a number of different cell types, mainly by monocytes/macrophages and by some T cells. IL-10 may also be produced by non-immune cells, like keratinocytes and certain tumour cells. IL-10 has an effect on various target cells. It inhibits the production of pro-inflammatory cytokines in monocytes and macrophages, exerting an i munosuppressive and anti-inflammatory effect. Recently it has been found that I -10 can act directly on T-lymphocytes (1-5).
  • IL-10 for treating various medical indications like septic and toxic shock, tissue rejection, graft-versus-host disease, acute or chronic inflammation etc. has been described previously. In these applications IL-10 is always administered systemically (2-23) .
  • the increase and/or induction of IL-10 may contribute directly or indirectly to immunosuppression.
  • Inflammation is a uniform response of the human body to a variety of stimuli.
  • Phagocytes play a central role in the generation of inflammatory responses by secreting a variety of mediators of inflammation, such as IL-1, IL-6, TNF- ⁇ etc.. These mediators act directly on target cells, or indirectly by, for example, attracting other inflammatory cells, and thus contribute to the normal physiological inflammatory response helping the body's immune system to fight invading particles and microbes. However, the mediators may also participate in reactions detrimental to the human body (24-26) . Phagocytes can also secrete molecules which exhibit anti-inflammatory effects such as interleukin-10.
  • the specific immune response acts through various cells.
  • phagocytes Important phagocytes in the human body are monocytes/ macrophages (Mo) . While circulating in the blood stream these cells are called monocytes, after migrating from the blood stream to the various tissues they are called macrophages. Specialized forms of these cells are the Kupffer cells residing in the liver, lung macrophages, and many more.
  • the foreign molecule (antigen) is digested into small peptides, a process called antigen processing. These small peptides are then embedded in specialized molecules (Major Histocompatibility Complex, MHC) and transported to the cell surface, where the peptides, still embedded in MHC molecules, are presented to other cells (27-29) .
  • MHC Major Histocompatibility Complex
  • T cells have receptors which recognize a complex of a given peptide presented by an MHC molecule. Upon this interaction, the T cells are activated, proliferate and provide help to phagocytes, stimulate the secretion of antibodies, or kill virus-infected or tumor cells. Thus an immune reaction directed specifically against the foreign antigen has been started.
  • T cell receptor on the side of the T cell and an MHC-molecule with its antigen on the side of the antigen-presenting cell (APC) is not sufficient to activate a T cell.
  • APC-derived signals are required for initiating T cell activation. The requirement for such a "second signal” has been recognized more then 20 years ago (Bretscher and Cohn) .
  • APC-derived interleukins such as IL-1 and TNF- ⁇ have been found to contribute to the activation of T cells (29-33) .
  • T cell is activated by engagement of its T cell receptor by MHC+peptide in the absence of APC-derived factors, this T cell not only fails to proliferate, but it remains anergic for subsequent activation, too, even if for this subsequent activation APC contributing appropriate "second signals" are now present (30, 31, 33-40).
  • APC contributing appropriate "second signals" are now present (30, 31, 33-40).
  • induction of lack of immune response may be due to death of the responding T cells, and other circumstances where the T cells may remain alive but are no longer responsive.
  • This in vi £o-phenomenon may translate ji vivo to tolerance and/or anergy to a given antigen. Induction of tolerance and/or anergy is of great clinical relevance in the context of organ, bone marrow, blood and cell transplantation and in autoimmune diseases.
  • PBMC peripheral blood mononuclear cells
  • anti-CD14 antibodies are not restricted to cellular stimuli known to act through CD14.
  • the pro-inflammatory effects of T cell mitogens, such as superantigens and lectins (for example concanavalin A) which have not yet been described to act through CD14, are also downregulated by anti-CD14 antibodies.
  • anti-CD14 antibodies suppress the secretion of cytokines and other mediators of inflammation by monocytes/macrophages and other cells upon triggering with T cell mitogens, such as superantigens and lectins, like concanavalin A.
  • T cell mitogens such as superantigens and lectins, like concanavalin A.
  • anti-CD14 antibodies show an effect in the case of triggers that are not mediated by CD14.
  • PBMC peripheral blood mononuclear cells
  • the invention thus relates to the use of anti-CD14 antibodies in general for immunosuppression by the induction or increase of IL-10 secretion and/or the induction of T- cell tolerance and/or anergy.
  • CD14 (Mo2, My4, Leu M3) is a myeloid differentiation antigen detected on mature monocytes, macrophages, and on cells from myelomonocytic (M4) and monocytic (M5) leukemias (FAB classification) .
  • M4 and M5 leukemias FAB classification
  • CD14 is not detectable on immature leukemic cells, nor on the human cell lines U937 and HL60.
  • both cell lines express CD14 upon stimulation with 1,25-dihydroxyvitamin D 3 or dimethylformamide.
  • Membrane-bound CD14 has a molecular weight of approximately 52 kD.
  • a soluble form of CD14 (molecular weight 48-52 kD) has been detected in supernatants of CD14 expressing cells, as well as in plasma.
  • the CD14 protein has no transmembrane region, but is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • surface expression of CD14 is deficient in patients with paroxysmal nocturnal hemoglobinuria, an acquired disorder characterized by a selective lack of expression of GPI- anchored proteins.
  • the CD14 gene maps to a region on the long arm of chromosome 5 (5q23-q31) that encodes several myeloid-specific growth factors and growth-factor receptors, including IL-3, IL-4, granulocyte macrophage-colony stimulating factor, CSF-1, CSF-1 receptor and the receptor for platelet-derived growth factor (41-50) .
  • the IL-10 secretion is site-specific since it will only occur in the proximity of cells having CD14 on their cell surface, or by cells that have been stimulated, for example by LPS.
  • the various effects of the invention are a general phenomenon observed with various anti-CD14 antibodies, such as 3C10 (ATCC deposit accession number TIB 228) , My4 (Coulter) , T ⁇ k4 (Dako) etc..
  • various anti-CD14 antibodies may have various effects. Some may be more useful for specific applications than others. The skilled person will be capable of determining which anti-CD14 antibody is most suited for a particular purpose.
  • the induction or increase of IL-10 secretion and/or induction of T cell tolerance and/or anergy may be beneficial for a variety of clinical conditions, such as inflammatory conditions caused by lipopolysaccharide (LPS) , including, but not limited to sepsis and septic shock, inflammatory conditions caused by microbial exotoxins such as, e.g., the staphylococcal exotoxins, streptococcal exotoxin, and other toxins, allergic diseases, such as. e.g.
  • LPS lipopolysaccharide
  • asthma allergic rhinitis, dermatitis, autoimmune diseases, rheumatic diseases, tumors, graft-versus-host-disease following bone-marrow-transplantation, graft rejection following organ transplantation, systemic inflammatory response syndrome (SIRS) , pancreatitis, (severe) aplastic anemia, inflammations encountered after burns, trauma, surgery, including prevention and treatment of the capillary leak syndrome, neurological diseases in which immunological mechanisms play a role, including Alzheimer disease, arteriosclerosis, multiple sclerosis, any inflammatory or neoplastic condition not already mentioned above (1-25, 51, 52) .
  • cytokines such as IL-1, IL-6 and TNF- ⁇ for their growth.
  • IL-10 may inhibit their proliferation.
  • Anti-CD14 antibodies may therefore also be used for treatment of certain forms of malignant diseases.
  • novel anti-CD14 antibodies are provided. These antibodies were prepared by isolating soluble CD14 by immunoaffinity chromatography using the antibody 63D3 (available from the American Type Culture Collection under accession number HB 44) . The antigen was isolated under gentle conditions. In the material isolated CD14-molecules were identified. Using the material isolated by immunoaffinity chromatography containing CD14 molecules monoclonal antibodies were produced. It was found that the antibody was directed towards CD14-bearing monocytes. These novel anti-CD14 antibodies are another candidate for use in the present invention.
  • the invention further relates to pharmaceutical compositions for treating diseases wherein the induction and/or increase of IL-10 secretion may be beneficial.
  • compositions comprising one or more anti-CD14 antibodies as the active ingredient for inducing or increasing IL-10 secretion have the form of powders, suspensions, solutions, sprays, emulsions, infusions, aerosols, unguents or creams and can be used for local application, intranasal, rectal, vaginal and also for oral or parenteral (intravenous, intradermal, intramuscular, intrathecal etc.) administration, administration by means of inhalation etc..
  • Pharmaceutical compositions of the invention can be prepared by combining (i.e.
  • the active compound(s) by mixing, dissolving etc.) the active compound(s) with pharmaceutically acceptable excipients with neutral character (such as aqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents, additives) , and further if necessary 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 can further be varied between 0.01 ⁇ g and 1 g per kg body-weight, preferably between O.l ⁇ g and 1 mg per kg body-weight.
  • the whole antibody may be used, or any fragment of the antibody molecule or molecules derived from the original antibody (e.g. humanized, bispecific or other engineered antibodies and the like) as long as the specificity for CD14 and its IL-10 secretion stimulating or inducing effects and/or its immunosuppressive effects are maintained.
  • the invention is not limited to antibodies, although these are specifically preferred, but also relates to other CD14-binding molecules having the same physiological effect as the antibodies.
  • Hybridomas 3C10 and 63D3, both secreting anti-CD14 antibodies were obtained from the American Type Culture Collection (ATCC, MD, USA) . The cells were grown in culture and supernatants were collected and frozen. Anti-CD14 antibodies were purified from the supernatants from the hybridomas 3C10 and 63D3 using protein G coupled to Sepharose 4 Fast Flow (Pharmacia) following the manufacturer's instruction. A second purification step was performed using goat-anti-mouse immunoglobulin linked to a Carbolink affinity column (Pierce) , following the manufacturer's guidelines. With this affinity column, mouse anti-human CD14 antibodies were purified.
  • the purified 3C10- and 63D3-antibodies were each coupled to a HiTrap column (CNBr-activated sepharose, Pharmacia) , according to the manufacturer's guidelines.
  • CD14 protein was isolated from human plasma (fresh frozen plasma, Swiss Red Cross) . Approximately 15 ⁇ g protein per ml of fresh frozen human plasma were obtained. The proteins isolated by affinity chromatography were checked by SDS-PAGE.
  • CD14 antigen prepared as described above was incubated with a macrophage cell line originating from Balb/c mice, which is part of this kit, in a concentration of 2 ⁇ g antigen per ml cell suspension, for 48 hours.
  • the spleen was removed from a Balb/c mouse after sacrificing the animal, and a suspension of spleen cells was prepared.
  • the spleen cells were added to the macrophage cell line. After addition of another 10 ⁇ g of CD14 antigen, the cells were incubated at 37°C for 4 days. Then, the cells were fused with cells of the mouse B-myeloma cell line Ag8 (obtained from Dr. J. Guzman, Infectiology Laboratory, University Children's Hospital, Zurich, and Prof. Hengartner, Institute of Pathology, Zurich University Hospital) . After fusion, the cells were distributed in eight 96-well plates for further culture.
  • the cells were kept in HAT (hypoxanthine aminopterin thymidine)-medium, allowing only cells derived from the fusion of a myeloma cell and a B cell to survive, whereas unfused B cells or myeloma cells died.
  • HAT hyperxanthine aminopterin thymidine
  • 143 of the 480 wells seeded cell growth was observed.
  • the hybridoma cells from these 143 wells were transferred in 24-well plates cultured in HT-medium for two weeks. Subsequently, IMDM-medium was used.
  • the supernatants of the wells showing cell growth were assessed in an ELISA (Boehringer Mannheim) for the presence of antibodies (regardless of the specificity of these antibodies) .
  • ELISA Boehringer Mannheim
  • hybridomas was performed by limiting dilution.
  • the cells were diluted to a concentration of 1 cell per ml medium. Subsequently, this cell suspension was cultured in a 96-well plates at 100 ⁇ l per well. Thus statistically, every tenth well contains one hybridoma cell, and the probability of a well containing two cells is only 1:100.
  • hybridoma cell lines thus obtained were again subjected to analysis of binding to human PBMC, followed by further rounds of limiting dilution.
  • the antibodies secreted by hybridoma 3B9 bind to epithelial cells transfected with cDNA for CD14
  • the cDNA for CD14 was obtained by polymerase chain reaction amplification and inserted into the plasmid pCEP4 (InVitrogen) .
  • 293 cells embryonic kidney, human: European collection of animal cell cultures (ECACC) No. 85120602) transfected with the ⁇ -, ⁇ - and invariant chains of human MHC class II were a kind gift of Dr. J. Neefjes, Netherlands Cancer Institute.
  • These cells were additionally transfected with the CD14 cDNA incorporated into the pCEP4 vector by liposomal transfection using Lipofectin (Gibco Life Technologies) , Cells successfully transfected were selected by culture in the presence of hygromycin B (100 ⁇ g/ml) .
  • the antibody 3B9 binds to 293 cells transfected with CD14 cDNA, but not to CD14-negative 293 wild-type cells nor to 293 cells transfected with the cDNAs for the MHC class II-molecules as shown in Fig. 6.
  • PBMC peripheral blood mononuclear cells
  • Hybridoma supernatants 3C10, 3B9, 2D11 were added at 1:1 (vol:vol) . After 24 hours, the cell culture supernatants were harvested and assessed for their content of TNF- ⁇ and IL-10 using commercially available ELISAs (R&D Systems) according to the manufacturer's guidelines. For experiment 5, purified 3C10 monoclonal antibody was used. Purification of the antibody from the hybridoma supernatant was performed using a protein G-column (Pharmacia) according to the manufacturer's guidelines. Purified 3C10 antibody was used at 5 ⁇ g/ml. Hybridoma 3C10 and 63D3 were obtained from ATCC and cultured in tissue culture flasks under the conditions suggested by ATCC.
  • Anti-CD14 antibodies inhibit LPS-induced secretion of TNF- ⁇
  • Anti-CD14 antibodies augment and/or induce the secretion of IL-10
  • Anti-CD14 antibodies decrease the ratio TNF- ⁇ /IL-10 It was thus found that treatment with anti-CD14 antibodies has an immunoregulatory effect, in that secretion of the proinflammatory cytokine TNF- ⁇ is downregulated and secretion of the "anti-inflammatory" and "immunosuppressive" cytokine IL-10 is enhanced. To better demonstrate this effect of the anti-CD14 antibodies the ratio of secreted TNF- ⁇ /IL-10 was calculated. It is submitted that this ratio is a useful indicator for the anti-inflammatory effect of these antibodies. The results for the experiments 2, 3 and 4 are depicted in the figures 2c, 3c and 4c, respectively.
  • Anti-CD14 antibodies inhibit secretion of cytokines upon stimulation bv triggers other than LPS:
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • My4 (Coulter) and control IgG2 (R&D) were used at 25 ⁇ g/ml, Tiik4 (Dako) at 3.6 ⁇ g/ml.
  • Interleukin-6 was determined by ELISA using a kit from Boehringer-Mannheim following the manufacturer's guidelines. Table 2 shows the results.
  • CD14 antibody inhibits cellular activation not only when triggered by LPS, but also when triggered by other stimuli, such as the superantigen TSST-1.
  • Treatment with an anti-CD14 antibody furthermore results in prevention of upregulation and/or downregulation of secretion of interleukin-6, a molecule involved in stimulation of immunoglobulin-secretion by B cells.
  • Interleukin-6 is an important trigger for secretion of immunoglobulins by B cells; prevention of upregulation and/or downregulation of secretion of interleukin-6 thus further supports the observation that treatment with anti-CD14 antibodies results in suppressing the immune response, not only by inhibiting T cell proliferation, but also by indirectly affecting secretion of immunoglobulins.
  • Treatment with anti-CD14-antibodies not only results in a modified cytokine secretion but also in various other effects, such as an upregulation or downregulation of the expression of the surface molecules.
  • PBMC peripheral blood mononuclear cells
  • monocytes were prepared as described elsewhere (53) . Briefly, peripheral blood mononuclear cells were isolated from venous blood of healthy human donors by Ficoll-Hypaque density gradient centrifugation. The cells were then resuspended in RPMI 1640 supplemented with 10% AB+ serum. For isolation of monocytes, the cells were adhered for 1 h at 37°C in a 5% C0 2 atmosphere in 100 x 15 mm plastic Petri dishes (Falcon) , each containing 10-15xl0 7 mononuclear cells.
  • the dishes were extensively washed with warm medium, then incubated with cold PBS on ice for 15 min.
  • Adherent monocytes were subsequently recovered by vigorous pipetting, washed and resuspended in complete medium at 1x10° cells/ml. Cell viability, as determined by trypan blue exclusion, was always > 95%.
  • PBMC Cell culture Cells
  • lxlO 6 cells/ml Toxic shock syndrome toxin-1
  • SEB staphylococcal toxin B
  • Ttik4 Anti-CD14, Dako
  • Flow cytometry analysis Cells in staining buffer (RPMI 1640- 2.5% FCS, containing 0.01% sodium azide) were incubated with appropriate dilutions of FITC- or phycoerythrin-conjugated antibodies for 40 min at 4°C.
  • Antibodies used were: anti- membrane TNF- ⁇ mAb (R&D) ; anti-CD80 (Ancell) .
  • PE- and FITC- conjugated murine IgG mAbs of unrelated specificity (Becton Dickinson & Co.) were used as controls. The cells were then extensively washed and fixed in paraformaldehyde.
  • FITC-labeled goat-anti-mouse antibody Immunotech
  • Percentages of positive cells and mean fluorescence intensity (MFI) were analyzed by a FACScan (Becton Dickinson & Co.); for analysis of expression of molecules on monocytes a gate on the monocyte population was applied, as defined by forward and side light scatter. Five thousand cells were counted.
  • CD80 (also denominated B7) is a molecule expressed on antigen presenting cells. By interacting with the CD28- antigen expressed on T cells it delivers a costimulatory signal critical for activation of T cells. Inhibiting this interaction by supplying a soluble ligand for CD80 (such as CTLA4Ig) competing with CD28 for binding to CD80 results in inhibition of delivery of a second signal necessary for T cell activation and hence anergy and/or tolerance and/or immunosuppression (54) .
  • a soluble ligand for CD80 such as CTLA4Ig
  • PBMC normal human PBMC were isolated from venous blood by Ficoll-Hypaque density gradient centrifugation. PBMC were cultured at lxlO 6 cells/ml medium (RPMI 1640, supplemented with 10% human AB+ serum) alone, or in the presence of stimuli (TSST-1; Sigma; 100 ng/ml; SEA; 1 ⁇ g/ml) . Where indicated anti-CD14 antibodies (My4, Coulter; 25 ⁇ g/ml) have been added. After 24 h the cells were harvested, and stained for CD14 (anti-CD14-FITC antibody from Becton, Dickinson & Co. ; isotype control) .
  • MFI mean fluorescence intensity
  • This example shows that treatment with anti-CD14- antibodies inhibits cellular activation (in the form of CD80 expression) not only when triggered by LPS, but also when triggered by other stimuli, such as the superantigens TSST-1 or SEA, and results in a prevention of upregulation and/or a downregulation of CD80. Since CD80 is a signal required for activation of T-cells a decrease in its expression may result in an immunosuppressive effect.
  • Treatment with anti-CD14 antibodies prevents superantjgen-triggered upregulation of membrane-TNF- ⁇ Stimulation of normal human monocytes with the superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcal exotoxin B (SEB) results in upregulation of membrane-bound TNF- ⁇ , as indicated by an increase in fluorescence intensity upon flow cytometry analysis as shown in Fig. 7.
  • Treatment with anti-CD14 antibodies inhibits TSST-1- and SEB-triggered upregulation of membrane-TNF- ⁇ .
  • treatment with anti- CD ⁇ -antibodies inhibits cellular activation not only when triggered by LPS, but also when triggered by other stimuli, such as the superantigens TSST-1 and SEB. Furthermore it follows that treatment with anti-CD14-antibodies results in prevention of upregulation and/or a downregulation of membrane-TNF- ⁇ , a signal involved in activation of T-cells.
  • TNF- ⁇ Activated T cells express receptors for TNF- ⁇ , and TNF- ⁇ enhances the T cell response (56, 57) ; thus, by influencing TNF- ⁇ -production, treatment with anti-CD14 antibodies will also affect the antigen-specific immune response. 4. Effect of treating PBMC from a patient with septic shock in vitro with anti-CD14 antibodies
  • Venous blood was taken from a patient with septic shock. Blood cultures were positive for meningococci, and the patient suffered from multiorgan failure. 5 days after admission to the hospital, venous blood was taken and peripheral blood mononuclear cells were isolated by Ficoll- Hypaque density gradient centrifugation. PBMC were cultured at lxlO 6 cells per ml medium (RPMI 1640, supplemented with 10% AB+ human serum) . No exogenous stimuli were added to the cell cultures.
  • the cells were cultured in medium alone, or with the addition of antibodies (control IgGl and IgG2, 25 ⁇ g/ml; R&D), I0M2 (anti-CD14, 25 ⁇ g/ml; Immunotech) ; My4 (anti-CD14, 25 ⁇ g/ml; Coulter), 2D11 (anti-CD14, produced in our laboratory, supernatant, 1:1 vol/vol) .
  • the supernatants were harvested, and levels of TNF- ⁇ were determined by ELISA (R&D) . The results are shown in Figure 8.
  • PBMC purified from this blood sample secreted upon in vitro culture significant levels of TNF- ⁇ , despite the fact that at the time of blood sampling the patient had received antibiotic treatment for 5 days and LPS was not anymore detectable in the patient's serum at this time point.
  • In vitro-treatment with anti-CD14 antibodies resulted in downregulation of this TNF- ⁇ -secretion.
  • this effect of anti-CD14 antibodies cannot easily be explained by blocking CD14 (CD14 being the receptor for LPS) ; rather, treatment with anti-CD14 antibodies results in deactivation of monocytes/macrophages.
  • treatment with anti-CD14 antibodies downregulates and/or prevents upregulation of various second signals (e.g. membrane-bound molecules such as CD80, membrane-bound molecules such as TNF- ⁇ , IL-1, etc.) normally provided by resting and/or activated antigen-presenting cells. This results in immunosuppression and/or anergy and/or tolerance, as discussed above.
  • various second signals e.g. membrane-bound molecules such as CD80, membrane-bound molecules such as TNF- ⁇ , IL-1, etc.
  • PBMC preactivated with LPS results in deactivation of the cells, even if at the time of antibody-treatment no exogenous stimulus persists
  • THP-1 cells were obtained from ATCC. THP-1 cells were cultured for 5 days in the presence of vitamin D3 (Calcijex, Roche: 10 ' °!-) . The cells were stimulated with LPS (1 ng/ml; Sigma) for 6h. After extensive washing the cells were resuspended in medium (RPMI 1640, supplemented by 10% FCS) with the addition of My4 (anti-CD14 antibody, Coulter; 25 ⁇ g/ml) , or with the addition of 3C10 (anti-CD14 antibody, ATCC; supernatant, 1:1 vol/vol) , or with the addition of control antibody (mix of IgGl and IgG2, each 20 ⁇ g/ml; R&D), and cultured for another 20 h.
  • vitamin D3 Calcijex, Roche: 10 ' °!-
  • TNF- ⁇ (pg/ml) treatment control ab My4 3C10
  • Anti-CD14 antibodies inhibit the physical interaction between CD14 and MHC class Il-molecules. and lack of
  • MHC class Il-molecules results in vivo in an increased IL-10-production in response to LPS
  • LPS-receptor CD14 has to interact with other molecules proposed to serve as signal transducers following engagement of CD14 by LPS and/or by LPS-binding protein.
  • treatment of PBMC with anti-CD14 antibodies results in an increased secretion of IL-10 in response to LPS;
  • MHC class Il-molecules participate in signal transduction upon engagement of CD14 by LPS and/or LPS- binding protein;
  • iii) - at least some - anti-CD14 antibodies interrupt the physical interaction between CD14 and MHC class Il-molecules.
  • mice MHC class II-positive C57BL/6 mice and MHC class II-knock out mice (B6AAO) in vivo with LPS and compared the serum levels of IL-10 2h after LPS-injection.
  • mice and B6AA0 mice were injected with 2.8 mg/kg of LPS (E.coli 0111:B4) diluted in sterile NaCl 0.9%, or with 0.9% NaCl only. 5 mice for each group were examined. After 2 h, the mice were sacrificed and bled. The blood was allowed to coagulate at room temperature and subsequently centrifuged to obtain the serum. Levels of IL-10 were determined in the serum by ELISA (Biosource) , following the manufacturer's guidelines. The mean +/- standard error of the levels of IL- 10 measured in the sera from the 5 mice of each group is indicated in table 5.
  • Peripheral blood mononuclear cells were isolated from blood of healthy volunteer donors as described for the other examples.
  • PBMC peripheral blood mononuclear cells were isolated from blood of healthy volunteer donors as described for the other examples.
  • PBMC peripheral blood mononuclear cells were cultured in RPMI 1640 supplemented with 10% normal human AB+ serum in a 96 well plate (0.2x10° cells/well) .
  • Cells were stimulated with staphylococcal exotoxin B (SEB, 1 ⁇ g/ml; Sigma) .
  • SEB staphylococcal exotoxin B
  • the anti-CD14 antibodies My4, Coulter; Ttik4, Dako
  • IgG2, R&D control antibody
  • Neutralizing anti-interleukin-10 antibody (R&D) was used at lOO ⁇ g/ l.
  • Anti-CD14 antibodies affect immune response in vivo
  • This example was performed to study immunomodulating effects of a monoclonal antibody against human CD14. As this monoclonal antibody cross-reacts with rabbit CD14 this example was performed in rabbits.
  • the aim of the example was to investigate whether intravenous anti-CDl4 administration affects the immune response induced after an (primary or secondary) ovalbumin immunization. Effects of anti-CD14 administration on in vitro ovalbumin or mitogen induced lymphocyte proliferation, in vivo produced (ovalbumin specific) IgG levels and in vivo Delayed Type Hypersensitivity response against ovalbumin were measured.
  • test Mab was a monoclonal Mouse Anti-Human Monocyte CD-14, Clone TtiK4 (Dako) , Ig fraction, without sodium azide.
  • control Mab was monoclonal Mouse IgG2a (R&D) , anti-Keyhole Limpet Hemocyanin (KLH) , code No. MAB003,
  • ovalbumin for i.v. immunization Inject® Ovalbumin, product number 77120, Pierce, Rockford III., U.S.A. was used.
  • SPPF Specific Pathogen Free
  • Housing conditions were conventional. Feed and water were provided ad libitum. The general condition and behaviour of all animals was checked and recorded daily. Body weight were recorded at days 0, 7, 15, 21 and 28 of the study.
  • the rabbits were allocated to four groups (A-D) , proportionately by weight class and each animal was uniquely identified by an animal identification number applied with a marker pen in the ear.
  • the different groups were treated according to the design presented in table 6 and as described hereunder.
  • Ovalbumin immunizations were performed with doses of 300 ⁇ g in 500 ⁇ l 0.15 M NaCl solution at days 0 and 15. Each immunization consisted of two subcutaneous injections, at both upper axilla, with a volume of 250 ⁇ l each. At day 0 and day 28 (all animals were intradermally injected with 50 ⁇ l of 0.15 M NaCl containing different doses of ovalbumin (vehicle control: 0 ⁇ g or 3 ⁇ g or 10 ⁇ g or 30 ⁇ g ovalbumin) on a shaven flank to assess the DTH response after 24 hours.
  • ovalbumin vehicle control: 0 ⁇ g or 3 ⁇ g or 10 ⁇ g or 30 ⁇ g ovalbumin
  • PBMC peripheral blood mononuclear cells
  • Ovalbumin-specific IgG antibodies were measured in the plasma samples obtained at days 0, 15 and 28 using a sandwich ELISA. Briefly, flatbottom microtiter plates (NUNC Immuno Plate, Roskilde, Denmark) were coated overnight (4 ⁇ C) with 100 ⁇ l/well ovalbumin diluted to 5 ⁇ g/ml in carbonate buffer pH 9.6.
  • Table 8 shows DTH responses measured 24 hours after intradermal ovalbumin challenges at day 28 of the study. Presented are the numbers of animals which showed an induration and/or oedema at the injection site. Table 8

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Abstract

L'invention se rapporte à des anticorps anti-CD14 utilisés dans l'induction et/ou l'augmentation de la sécrétion de l'interleukine-10 et/ou l'induction de l'immunosuppression, par exemple, dans la prévention et/ou le traitement de l'inflammation. En outre, l'invention se rapporte à des compositions pharmaceutiques qui induisent et/ou augmentent la sécrétion de l'interleukine-10, comportant des anticorps anti-CD14 et/ou des fragments et/ou leurs versions modifiées, étant combinées avec un excipient adéquat.
PCT/EP1996/001588 1995-04-13 1996-04-15 Anticorps anti-cd14 utilises dans l'induction de la secretion il-10 WO1996032418A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU56885/96A AU5688596A (en) 1995-04-13 1996-04-15 Anti-cd14 antibodies for use in the induction of il-10 secre tion

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP95200950 1995-04-13
EP95200950.4 1995-04-13

Publications (1)

Publication Number Publication Date
WO1996032418A1 true WO1996032418A1 (fr) 1996-10-17

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AU (1) AU5688596A (fr)
WO (1) WO1996032418A1 (fr)

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WO1998022580A2 (fr) * 1996-11-18 1998-05-28 The Wellesley Hospital Foundation Proteine immunotrope (cd14) associee a la lactation bovine, gene codant celle-ci et application de cette proteine dans une activation de lymphocytes b
WO2000020562A1 (fr) * 1998-10-04 2000-04-13 Tel-Aviv Medical Center Research & Development Fund Nouvelle population de lymphocytes exprimant mo2 intracellulaire
EP1213586A1 (fr) * 1999-09-17 2002-06-12 Mochida Pharmaceutical Co., Ltd. Procede de mesure fractionnel de proteine de cd14 soluble
EP1496066A1 (fr) * 2002-03-25 2005-01-12 Japan Science and Technology Agency Anticorps reconnaissant des celllules hepatiques humaines proliferatives, cellules hepatiques humaines proliferatives et cellules hepatiques humaines fonctionnelles
US7462642B2 (en) 2002-03-22 2008-12-09 Cancer Research Technology Limited Anti-cancer combinations
US7510830B2 (en) * 2000-07-28 2009-03-31 Cancer Research Technology Limited Cancer treatment by combination therapy
US7585893B2 (en) 2002-11-01 2009-09-08 Cancer Research Technology Limited Anti-cancer composition comprising DMXAA or related compound
US7592310B2 (en) 1998-05-27 2009-09-22 Gemma Biotechnology Ltd. Induction of antibiotic proteins and peptides by LAIT/sCD14-protein
US7863322B2 (en) 2001-09-03 2011-01-04 Cancer Research Technology Limited Anti-cancer combinations
CN103733068A (zh) * 2011-08-12 2014-04-16 三菱化学美迪恩斯株式会社 术后感染症的诊断方法

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WO1994028025A1 (fr) * 1993-05-28 1994-12-08 The Scripps Research Institute Procedes et compositions d'inhibition de l'activation des cellules induites par cd14

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Cited By (26)

* Cited by examiner, † Cited by third party
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EP1801216A3 (fr) * 1996-11-18 2007-11-07 The Arthritis & Autoimmunity Research Centre Foundation Protéine immunotrope (CD14) associée à la lactation bovine, gène codant celle-ci et application de cette protéine dans une activation des lymphocytes B
WO1998022580A3 (fr) * 1996-11-18 1998-10-01 Wellesley Hospital Found Proteine immunotrope (cd14) associee a la lactation bovine, gene codant celle-ci et application de cette proteine dans une activation de lymphocytes b
US6093693A (en) * 1996-11-18 2000-07-25 The Wellesley Hospital Foundation B cell activation
AU732476B2 (en) * 1996-11-18 2001-04-26 Wellesley Hospital Foundation, The Bovine lactation associated immunotropic protein (CD14), encoding gene and application in B cell activation
WO1998022580A2 (fr) * 1996-11-18 1998-05-28 The Wellesley Hospital Foundation Proteine immunotrope (cd14) associee a la lactation bovine, gene codant celle-ci et application de cette proteine dans une activation de lymphocytes b
US6676985B1 (en) 1996-11-18 2004-01-13 The Arthritis & Autoimmunity Research Centre Foundation Bovine lactation associated immunotropic protein (CD14), encoding gene and application in B cell activation
CZ300752B6 (cs) * 1996-11-18 2009-08-05 The Arthritis & Autoimmunity Research Centre Foundation Použití polypeptidu k aktivaci B bunek, zpusob prímé aktivace, indukce rustu a diferenciace B bunek in vitro a pro výrobu vakcíny, in vitro zpusob obohacení krevního séra pacienta o savcí B bunky, vakcína, prípravek pro deti k aktivaci B bunek a zpus
US7592310B2 (en) 1998-05-27 2009-09-22 Gemma Biotechnology Ltd. Induction of antibiotic proteins and peptides by LAIT/sCD14-protein
WO2000020562A1 (fr) * 1998-10-04 2000-04-13 Tel-Aviv Medical Center Research & Development Fund Nouvelle population de lymphocytes exprimant mo2 intracellulaire
EP1213586A4 (fr) * 1999-09-17 2003-02-05 Mochida Pharm Co Ltd Procede de mesure fractionnel de proteine de cd14 soluble
EP1213586A1 (fr) * 1999-09-17 2002-06-12 Mochida Pharmaceutical Co., Ltd. Procede de mesure fractionnel de proteine de cd14 soluble
US6916628B1 (en) 1999-09-17 2005-07-12 Mochida Pharmaceutical Co., Ltd. Method for the measurement of soluble CD14 proteins separately
US7510830B2 (en) * 2000-07-28 2009-03-31 Cancer Research Technology Limited Cancer treatment by combination therapy
US7863322B2 (en) 2001-09-03 2011-01-04 Cancer Research Technology Limited Anti-cancer combinations
US7863321B2 (en) 2001-09-03 2011-01-04 Cancer Research Technology Limited Anti-cancer combinations
US7863320B2 (en) 2001-09-03 2011-01-04 Cancer Research Technology Limited Anti-cancer combinations
US7868040B2 (en) 2001-09-03 2011-01-11 Cancer Research Technology Limited Anti-cancer combinations
US7868039B2 (en) 2001-09-03 2011-01-11 Cancer Research Technology Limited Anti-cancer combinations
US7462642B2 (en) 2002-03-22 2008-12-09 Cancer Research Technology Limited Anti-cancer combinations
EP1496066A1 (fr) * 2002-03-25 2005-01-12 Japan Science and Technology Agency Anticorps reconnaissant des celllules hepatiques humaines proliferatives, cellules hepatiques humaines proliferatives et cellules hepatiques humaines fonctionnelles
EP1496066A4 (fr) * 2002-03-25 2006-09-13 Japan Science & Tech Agency Anticorps reconnaissant des celllules hepatiques humaines proliferatives, cellules hepatiques humaines proliferatives et cellules hepatiques humaines fonctionnelles
JPWO2003080670A1 (ja) * 2002-03-25 2005-07-21 独立行政法人科学技術振興機構 増殖性ヒト肝細胞を認識する抗体と、増殖性ヒト肝細胞および機能性ヒト肝細胞
US7585893B2 (en) 2002-11-01 2009-09-08 Cancer Research Technology Limited Anti-cancer composition comprising DMXAA or related compound
CN103733068A (zh) * 2011-08-12 2014-04-16 三菱化学美迪恩斯株式会社 术后感染症的诊断方法
CN103733068B (zh) * 2011-08-12 2016-04-13 美迪恩斯生命科技株式会社 术后感染症的诊断方法
US9506923B2 (en) 2011-08-12 2016-11-29 Lsi Medience Corporation Method of diagnosing surgical site infections

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