WO1995032734A1 - Nouveaux procedes et composes destines a la modulation selective de la reaction des lymphocytes t specifiques d'un l'antigene - Google Patents

Nouveaux procedes et composes destines a la modulation selective de la reaction des lymphocytes t specifiques d'un l'antigene Download PDF

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Publication number
WO1995032734A1
WO1995032734A1 PCT/EP1995/002012 EP9502012W WO9532734A1 WO 1995032734 A1 WO1995032734 A1 WO 1995032734A1 EP 9502012 W EP9502012 W EP 9502012W WO 9532734 A1 WO9532734 A1 WO 9532734A1
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fcγrii
bridging
cells
antigen
composition according
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PCT/EP1995/002012
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English (en)
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Mark De Boer
Serge Barcy
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Innogenetics N.V.
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Priority to EP95921760A priority Critical patent/EP0759782A1/fr
Priority to AU26709/95A priority patent/AU2670995A/en
Publication of WO1995032734A1 publication Critical patent/WO1995032734A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • 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
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/283—Immunoglobulins [IGs], 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 Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • 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 the new finding that the bridging of Fc type II receptors on professional antigen presenting cells (APCs) impaires the expression of the essential co-stimulatory molecules B7-1/2 and causes the down modulation of the adhesion molecule ICAM-3 expression, resulting in the modulation of antigen-specific T-cell unresponsiveness.
  • the B7 molecules on APCs provide the essential co-stimulatory signal that determines whether TcR/CD3 signaling after activation of T cells with the specific antigen leads to full T-cell activation or T cell anergy.
  • the ICAM-3 molecule mediates cellular interactions of T cells and other lymphocytes at sites of inflammation and specific immune responses.
  • the present invention also relates to prophylactic and therapeutic methods to prevent or to treat allergic diseases.
  • the present invention further relates to the treatment of T-cell mediated autoimmune diseases.
  • the present invention also relates to prophylactic and therapeutic methods to prevent or to treat the rejection of solid organs or cells after allogeneic or xenogeneic transplantation.
  • Fc -receptors play important roles in several immunological processes such as phagocytosis of opsonized paniculate antigens, clearance of immune complexes, antibody- mediated cellular cytotoxicity, production of inflammatory mediators, and regulation of immunoglobulin synthesis (Mellman et al., J. Cell. Biol. 96:887 (1983); Kurlander et al., J. Immunol. 133:855 (1984); Shen et al., J. Immunol. 137:945 (1986); Graziano and Fanger, J. Immunol. 138:945 (1987); Zheng et al. , Eur. J. Immunol. 23:2826 (1993)).
  • Fc ⁇ -receptors on human leukocytes can be divided in three major classes, based on their molecular mass, specificity and affinity for IgG, cellular distribution, and reactivity with monoclonal antibodies (Van de Winkel et al., J. Leuk. Biol. 49: 11 (1991); Ravetch and Kinet, Annu. Rev. Immunol. 9:457 (1991)).
  • Fc ⁇ RI is a high affinity receptor for monomeric
  • IgG consists of three Ig-like extracellular domains and is expressed on monocytes and IFN- ⁇ activated neutrophils (Ierino et al. , J. Immunol. 150: 1794 (1993).
  • Fc ⁇ RII consists of two Ig- like extracellular domains. This receptor binds monomeric IgG only with low affinity, but has a high affinity for complexed IgG (Ierino et al. , J. Immunol. 150: 1794 (1993)).
  • Fc ⁇ RII is the most widely expressed FcR, it is present on monocytes, dendritic cells, granulocytes, platelets and B cells (Anderson and Looney, Immunology today 7:264 ( 1986); Nestle et al., J. Immunol. 151 :6535 (1993)).
  • Fc ⁇ RIII also contains two Ig-like extracellular domains and also binds monomeric IgG only with low affinity, but has a high affinity for complexed IgG (Ierino et al., J. Immunol. 150: 1794 (1993)).
  • F ⁇ RIII is expressed on neutrophils, eosinophils, a group of cells referred to as L cells which include natural killer cells and large granular lymphocytes, and on macrophages but not on monocytes (Anderson and Looney, Immunology today 7:264 (1986)).
  • TcR/CD3 complex has two functions in antigen-induced activation: a recognition function in which a specific antigen is recognized in the context of the appropriate MHC molecule, and a signalling function in which the recognition event is transmitted across the plasma membrane
  • T cells need a second signal in addition to the one mediated by the TcR/CD3 complex.
  • This co-stimulatory signal is normally provided by the cell surface of APCs (Springer et al. Annu. Rev. Immunol. 5:223 (1987)).
  • TcR/MHC-peptide interaction in the absence of the co-stimulatory signal results in T-cell inactivation in the form of clonal anergy (Mueller et al. Annu. Rev. Immunol. 7:445 (1989)).
  • CD2 with its ligand CD58 LFA-3
  • CDl la/CD18 LFA-1
  • IAM-1 CD54
  • VCAM-1 VCAM-1
  • the best candidate co-stimulatory signal that determines whether TcR- stimulation leads to full T-cell activation, or to T-cell anergy is generated by interaction of CD28 on the T cells with B7-1/2 on APCs. It has been demonstrated in vitro that cross- linking of the CD28 molecule can rescue mouse T-cell clones from becoming anergic (Harding et al. Nature 356:607 (1992)). In addition, it has been shown that B7-1 but not
  • ICAM-1 mediated co-stimulation of T cells can prevent the induction of alloantigen-specific tolerance (Vassiliki et al. J. Exp. Med. 178: 1753 (1993)).
  • the B7-CD28 interaction can result in a strong proliferative (Linsley et al. J. Exp. Med. 173:721 (1991); Gimmi et al. Proc. Natl. Acad. Sci. USA 88:6575 (1991); De Boer et al. Eur. J. Immunol. 22:3071 (1992)) as well as a cytolytic T-cell response (Van Gool et al. J. Immunol. 150:3254 (1993)). It has recently been demonstrated that there are at least two B7 molecules that can functionally interact with CD28 (Hathcock et al. Science 262:905 (1993); Freeman et al.
  • B7-1 originally named B7/BB 1 is a monomeric transmembrane glycoprotein with an apparent molecular mass of 45-65 KDa and is, like CD28, a member of the immunoglobulin superfamily (Freeman et al. J. Immunol. 143: 2714 (1989)).
  • the second B7 molecule named B70 or B7-2, is a transmembrane glycoprotein with an apparent molecular mass of approximately 70 KDa and is also a member of the immunoglobulin superfamily (Freeman et al.
  • B7-1 expression has also been found on cultured peripheral blood dendritic cells (Young et al. J. Clin. Invest . 90:229 (1992)) and on in vitro activated T cells (Azuma et al. J. Exp. Med. 177:845 (1993)).
  • the B7-1 molecule is constitutively expressed on dendritic cells, on monocytes / macrophages in inflammatory lesions, on a subset of B cells in and around germinal centers and, on T cells in chronical inflammatory lesions
  • B7-2 molecule seems to have a very similar distribution pattern as B7-1 , with the exception that induction of cell-surface expression seems to be faster (Freeman et al. Science 262:909 (1993)) and that it seems to be present on freshly isolated monocytes (Azuma et al. Nature 366:76 (1993)).
  • T-cell tolerance or anergy Incomplete activation of T cells in the absence of the co-stimulatory signals from the APC results in T-cell tolerance or anergy (Mueller et al. , Annu. Rev. Immunol. 7:445 (1989)).
  • T-cell anergy is characterized by the fact that anergic T cells do not anymore respond to normal activation signals, even in the presence of all the co-stimulatory signals.
  • Allergy Atopic allergic diseases i.e. asthma, allergic rhinitis, atopic dermatitis, and food and drug allergy, affect at least 20% of the population in the industialized world and are an important cause of morbidity, and for asthma mortality.
  • the initial factors involved in the allergic reaction are allergen-specific antibodies of the IgE type and mast cells, which express high affinity receptors for IgE and which are widely distributed in tissues that form our protective barrier against the outside such as mucosae.
  • an antigen the allergen
  • binding to and bridging of receptor-bound IgE on the mast cells results in activation of these cells.
  • This activation will result in the release of histamine that triggers an immediate response (within 20 minutes), as well as the release of soluble factors that initiate a local inflammatory response. This results in the influx of inflammatory cells that give subsequently rise to the late phase (6-24 hours) of the allergic response and to the chronic inflammation seen at places of repeated exposure to allergen. This late phase reaction is characterized by the occurence of eosinophils.
  • allergy is characterized by immediate and late phase hypersensitivity reactions to allergens. These immune reactions are associated with elevated serum levels of allergen- specific IgE and to eosinophilia, respectively. Both IgE production and eosinophil production are controlled by T helper (Th) lymphocytes and evidence is accumulating that the aberrant immunological characteristics of atopic allergy can be explained by the hyperactivation of a particular subset of Th cells (Th2).
  • Th2 Th cells regulate immune responses via cytokines that they produce upon recognition of specific antigen presented by antigen presenting cells (APC).
  • APC antigen presenting cells
  • Th cells In response to various antigens, Th cells produce many cytokines simultaneously (type 0 profile). However, in response to intracellular micro-organisms the production of cytokines of the Th cells is biased to high levels of interferon (IFN)- ⁇ and low levels of interleukin (IL)-4 and IL-5 (type 1 cytokine profile, Thl). Such a Thl response is protective because IFN- ⁇ stimulates the intracellular killing of microbes by phagocytic cells. In contrast, in response to certain helminth types the Th cell response is biased to low levels of IFN- ⁇ and high levels of IL-4 and IL-5 (type 2 cytokine profile, Th2).
  • IFN interferon
  • IL-4 and IL-5 type 1 cytokine profile, Thl
  • IL-4 induces B cells to secrete IgE, provided the level of secreted IFN- ⁇ is low, whereas IL-5 promotes the production of eosinophils in the bone marrow.
  • the Th2 response is protective, because both IgE and eosinophils are considered to contribute to the expelling of the helminths.
  • APC-derived factors include IL-12 and prostaglandin (PGE)-2, that skew T cell cytokine production towards Thl and Th2 profiles, respectively.
  • IL-12/PGE-2 production ratio in APC will results in IL-4 dominated T cell responses, whereas a high IL-12/PGE-2 production ratio will result in IFN- ⁇ -dominated T cell responses.
  • Immunotherapy which presently consists of the subcutaneous administration of increasing doses of intact or chemically modified allergens, has shown to result in a down- regulation of allergen-induced T-cell proliferation and release of cytokines and histamine- releasing factors by the allergen-specific T cells (Varney et al., J. Clin. Invest. 92:644 (1993); Secrist et al., J. Exp. Med. 178:2123 (1993)).
  • CD28 molecule of the T cells (Harding et al. Nature 356:607 (1992); Vassiliki et al. J. Exp. Med. 178: 1753 (1993)).
  • TcR/CD3 complex Incompatibility for the histocompatibility antigens, both major (MHC) and minor antigens, is the cause for graft rejection. Both CD4+ helper T cells (Th) and CD8 + cytotoxic T cells (CTL) are involved in the rejection process. Activation of T cells after transplantation is the result of ligand-receptor interactions, when the TcR/CD3 complex recognizes its specific alloantigen in the context of the appropriate MHC molecule. To induce proliferation and maturation into effector cells, T cells need a second signal in addition to the one mediated by the TcR/CD3 complex.
  • Th CD4+ helper T cells
  • CTL cytotoxic T cells
  • Fc-receptors Activation of a certain type of Fc-receptors is reported to occur only when several Fc regions within an antigen-antibody complex simultaneously bind to several Fc-receptors, causing them to be cross-linked (US Patent No. 4,753,927). Such Fc-receptor cross-linking by several Fc regions appears to be the critical signal required to activate certain types of Fc- receptors.
  • the activation of lymphocytes via Fc-receptors is generally viewed as a strong pro-inflammatory event.
  • Fc ⁇ RII CD32
  • Fc ⁇ RII CD32
  • Fc ⁇ RII CD32
  • Fc ⁇ RII bridging compositions which are characterized by their specific capacity to impaire the activation of antigen-presenting cells (APCs) to stimulate the activation of antigen-specific T-cells, resulting in modulation of T-cell responsiveness.
  • APCs antigen-presenting cells
  • Fc ⁇ RII bridging compositions which are characterized in that they either (i) prevent the up-regulation (or prevent the expression or down-modulate the expression) of the co-stimulatory B7 molecules on professional APCs, and/or, (ii) they down-modulate (or impaire the expression of or prevent the up-regulation of) the adhesion molecule ICAM-3 on professional APCs.
  • the present invention is based in essence on the new finding that bridging several Fc ⁇ RII (CD32) molecules on profesional antigen presenting cells (APCs) prevents the up- regulation of the essential co-stimulatory molecules B7-1/2 and causes the down modulation of the adhesion molecule ICAM-3, resulting in the modulation of antigen-specific T cell unresponsiveness.
  • CD32 Fc ⁇ RII
  • APCs profesional antigen presenting cells
  • the present invention thus relates more particularly to an Fc ⁇ RII (CD32) bridging composition characterized in that it impaires (for impairing) the capacity of antigen presenting cells (APCs) to stimulate the activation of antigen-specific T-cells, resulting in modulation of T-cell responsiveness.
  • Fc ⁇ RII CD32
  • APCs antigen presenting cells
  • Fc ⁇ RII (CD32) bridging within the context of the present invention refers to the fact that incubation of professional APCs carrying the Fc ⁇ RII (CD32) with a suitable bridging agent results in the prevention of the up-regulation of the essential co ⁇ stimulatory molecules B7-1/2 by these professional APCs and/or results in the down modulation of the adhesion molecule ICAM-3 by these professional APCs.
  • the suitable bridging agents according to the present invention are listed below. Most preferably several Fc ⁇ RII (CD32) molecules are bridged by several Fc regions.
  • the terms “bridging” or “bridged” used in the present invention have the same meaning as “cross-linking” or “cross-linked”. The term cross-linking is preferably not used further in the specification to avoid confusion with the strict meaning of the term “cross- linking" to couple 2 or more protein molecules together, for instance to obtain a state of agreggation of the bridging agent as is explained further.
  • APCs refers to the impaired expression of B7-1/2 molecules as detailed in the Examples section upon applying certain Fc ⁇ RII bridging agents.
  • the expression of B7-1/2 molecules may be measured by analyses such as shown in the Examples section (e.g. FACS analysis) or by means of any other technique known in the art or as indicated below.
  • ICAM-3 molecules upon applying certain Fc ⁇ RII bridging agents or methods such as described in the Examples section or as indicated below.
  • the expression of ICAM-3 may be measured by analysis methods such as shown in the Examples section, as indicated below or by any other technique known in the art.
  • the Fc ⁇ RII bridging agents/compositions of the present invention aim upon binding to the Fc ⁇ RII on professional APCs and activating this receptor in such a way that the up- regulation of B7-1/2 co-stimulatory molecules is prevented and/or that of the adhesion molecule ICAM-3 is down-modulated resulting in an impaired capacity of the APCs to induce T-cell activation and thus resulting in modulation of T-cell responsiveness.
  • Such Fc ⁇ RII bridging compositions according to the present invention should comprise at least one of the bridging agents as an active principle, with said agents being chosen from the group consisting of:
  • a recombinant fusion protein of 2 or more human IgG Fc parts - a liposomal vesicle containing any of the foregoing agents as detailed below.
  • a selected agent may be confirmed as being a proper Fc ⁇ RII bridging agent according to the present invention by means of the following test system:
  • the amount of B7-1 , B7-2 and/or ICAM-3 expressed is measured by any of the techniques known in the art (such as ELISA or immunofluorescence (FACS) analysis in combination with suitable monoclonal antibodies or ligand antigens as described in the Examples section) on the cell surface of profesional antigen presenting cells (or APCs, e.g. monocytes cultured in the presence of IFN- ⁇ or GM-CSF) in the presence of the Fc ⁇ RII bridging agent to be tested in comparison to appropriate control conditions and/or agents.
  • FACS immunofluorescence
  • compositions within the meaning of the present invention differ in that a composition should comprise at least one FcR-bridging agent as an active principle in addition to other component(s).
  • FcR-bridging agent as an active principle in addition to other component(s).
  • the preferred composition or formulation of said compositions of the invention are detailed extensively below.
  • aggregated may mean aggregated by means of any chemical cross-linking agent known in the art or aggregated by any other means such by immobilizing the IgG's or fragments thereof to a solid phase as is explained below and in the examples section.
  • “Monoclonal antibodies to the Fc ⁇ RII” refer to monoclonal antibodies which are specifically directed to the Fc ⁇ RII. These monoclonal antibodies can be prepared as described in international application WO 88/00052 (describing die production of monoclonal antibodies specifically directed to the Fc ⁇ RI) or Anderson et al. ((1986) J. Biol. Chem. 261: 12856).
  • the Fc ⁇ RII monoclonal antibodies according to the present invention should bind to a site on the Fc ⁇ type II receptor (CD32) such that it preventis the up-regulation of B7-1/2 and/or causes the down-modulation of ICAM-3 by the antigen presenting cells incubated with said Fc ⁇ RII monoclonal antibodies.
  • the selection of such monoclonal antibodies can be performed by measuring the amount of B7-1 , B7-2 and/or
  • ICAM-3 produced by profesional antigen presenting cells (e.g. monocytes cultured in the presence of IFN- ⁇ or GM-CSF) in the presence of the monoclonal antibodies to be tested in comparison to control monoclonal antibodies by any of the techniques known in the art (e.g. by ELISA or immunofluorescence analysis as described in the Examples section or as described above).
  • profesional antigen presenting cells e.g. monocytes cultured in the presence of IFN- ⁇ or GM-CSF
  • Bivalent or multivalent monoclonal antibodies to the Fc ⁇ RII in particular of the IgA or IgM isotype, can be prepared as described in international application WO 88/00052 where the supernatants of the hybridoma clones are directly screened for the production of specific antibodies of the IgM or IgA isotype using specific reagents.
  • IgA secreting hybridoma cells can be obtained from an IgG secreting hybridoma cell line by limiting dilution cloning and selection of Ig-isotype switch variants by methods known to those skilled in the art.
  • Fc ⁇ Rll-specific antibodies of the IgA or IgM isotype can be prepared using recombinant DNA technology by expressing the antigen-binding variable region of the said antibodies in a vector containing the cDNA encoding for the IgA or IgM constant region by methods known to those skilled in the art.
  • These constant regions can be of mouse origin, but more preferably are of primate or human origin.
  • Bivalent or multivalent monoclonal antibodies to Fc ⁇ RII or functionally active fragments (such as F(ab') 2 fragments) of such antibodies can also be prepared by conjugating antibodies with known coupling or cross-linking agents such as protein A, carbodiimide, N-succinimidyl-2(2- pyridythio) propionate (SPDP) Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu et al (1985) Proc. Natl. Acad. Sci. U.S.A. 82:8648).
  • such fragments can be generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases.
  • the expression "functionally active fragment of said bivalent or multivalent antbody to the Fc ⁇ RII” may refer to an F(ab') 2 fragment of said antibody or conjugated Fab fragments of said multivalent or bivalent monoclonal antibody (ies), provided that the resulting antibody fragments have the effect of preventing the up-regulation of B7-1/2 and/or cause the down-modulation of ICAM-3 expression by the APCs incubated with said antibodies as described above. Conjugated antibody fragments may be obtained as detailed below.
  • a liposome vesicle comprising any of the foregoing agents refers more particularly to liposome vesicles having Fc regions of any of the foregoing bridging agents sticking out of the liposome.
  • Liposome vesicles according to this aspect of the invention may be prepared by any method for preparing liposomes known to the man skilled in the art such as described by A. Gabizon et al., Cancer Research 42:4734 (1982); D.S. Cafiso, Biochem Biophys Acta 649:129 (1981) and F. Szoka, Ann Rev Biophys Eng 9:467 (1980).
  • Other drug delivery systems known in the art may also be applicable and are described in e.g. M.J.
  • the present invention further contemplates an Fc ⁇ RII bridging composition as defined above, further characterized in that said Fc ⁇ RII bridging agent prevents the up-regulation (or impaires the expression) of B7-1/2 molecules by these APCs.
  • the present invention contemplates also any of the Fc ⁇ RII bridging composition as defined above, further characterized in that said Fc ⁇ RII bridging agent causes the down modulation (or impaires the expression) of ICAM-3 molecules by these APCs.
  • the present invention relates to any composition as defined above, further characterized in that said composition comprises a specific antigen or antigen-complex combined with an agent capable of bridging Fc ⁇ RII molecules as defined above.
  • the term “combined” refers to any type of combination known in the art, more particularly implies covalently attaching or cross-linking said antigen or antigen-complex to said bridging agent preferentially via chemical cross-linking. Said antigen or antigen-complex may thus be attached or aggregated to said bridging agent by means of any technique known in the art using any type of attaching agent or cross-linking agent known in the art.
  • Liposome vesicles as bridging agents will preferably contain said antigen or antigen-complex inside of the liposome vesicle.
  • Such vesicles may be prepared by any of the techniques known in the art for liposome vesicle preparation.
  • the present invention relates to an Fc ⁇ RII bridging composition as defined above, further characterized in that said specific antigen or antigen-complex causes allergic diseases (often referred to as allergen).
  • allergen examples of such antigens that can act as allergens in humans are the major allergen of cat hair and dander, house dust mite antigen, pollen antigens, and bee venom.
  • the present invention also relates to an Fc ⁇ RII bridging composition as defined above, further characterized in that said specific antigen is an alloantigen.
  • alloantigen refers to foreign MHC antigens, recognized by specific T cells and responsible for the onset of transplant rejection.
  • the present invention relates to an Fc ⁇ RII bridging composition as defined above, further characterized in that said specific antigen is an antigen causing the autoimmune attack on the body's own tissue (often referred to as an autoantigen).
  • the present invention relates to any of the compositions as defined above, further characterized in that said Fc ⁇ RII bridging agent consists of aggregated human IgG molecules or aggregated Fc fragments of human IgG molecules. According to yet another preferred embodiment, the present invention relates to any of the compositions as defined above, further characterized in that said Fc ⁇ RII bridging agent consists of a bivalent or multivalent Fc ⁇ RII specific monoclonal antibody, or functionally active fragments of said Fc ⁇ RII specific monoclonal antibody.
  • the present invention relates to any composition as defined above, further characterized in that said Fc ⁇ RII bridging agent consists of a recombinant fusion protein of two or more human IgG Fc parts.
  • the present invention also relates to a recombinant vector, particularly for cloning and/or expression, with said recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral promoter sequence followed by the nucleotide sequences comprising the nucleic acid sequence encoding such a recombinant fusion protein comprising at least two human IgG parts, and with said recombinant vector allowing the expression of said recombinant protein as defined above in a prokaryotic, or eukaryotic host or in living mammals when injected as naked DNA.
  • vector may comprise a plasmid, a cosmid, a phage, or a virus.
  • bacteria such as E. coli or in eukaryotic cells such as in S. cerevisiae, or in cultured vertebrate or invertebrate hosts such as insect cells, Chinese
  • Hamster Ovary (CHO), COS, BHK, and MDCK cells, the following steps are carried out: transformation of an appropriate cellular host with a recombinant vector, in which a nucleotide sequence coding for a fusion protein of two or more human IgG Fc parts has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence.
  • a recombinant vector in which a nucleotide sequence coding for a fusion protein of two or more human IgG Fc parts has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucle
  • the present invention relates to any of the compositions as defined above, further characterized in that said Fc ⁇ RII bridging agent consists of a liposome vesicle comprising at least one Fc ⁇ RII bridging agents with said agents being chosen from the group consisting of:
  • a recombinant fusion protein of 2 or more human IgG Fc parts - a liposome vesicle comprising any of the foregoing agents as detailed above.
  • the present invention also relates to methods of preparing liposomes containing an Fc ⁇ RII bridging agent as detailed above.
  • the present invention also contemplates any method for preparing any of the Fc ⁇ RII bridging compositions as defined above. Examples of the preparation of some of the Fc ⁇ RII bridging agents and compositions according to the present invention are disclosed above, in the Examples section or are within the knowledge of the man skilled in the art.
  • Target cells for treatment with the Fc ⁇ RII bridging compositions according to the present invention may include professional antigen presenting cells (APCs) such as human leukocytes, preferably macrophages, monocytes, dendritic cells, Langerhans cells or B cells. These target cells may possibly be activated before or during treatment with e.g. IFN- ⁇ , or GM-CSF. If desired, target cells to be treated may be derived from the donor of a graft in transplantation.
  • APCs professional antigen presenting cells
  • compositions of this invention will be administrated at a certain concentration that is therapeutically effective for the envisaged treatment.
  • the composition may be formulated using a variety of acceptable excipients known in the art.
  • the compositions are administered by injection, either intravenously, intradermally, intramuscular or subcutaneously. Methods to accomplish this administration are known to those of ordinary skill in the art. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes.
  • formulants may be added to the composition or bridging agents of the invention.
  • a liquid formulation is preferred.
  • these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents.
  • carbohydrates include sugar or sugar alcohols such as mono, di, or polysaccharides, or water soluble glucans.
  • the saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof.
  • Sucrose is most preferred.
  • “Sugar alcohol” is defined as a C 4 to C s hydrocarbon having an -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is most preferred.
  • These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation.
  • the sugar or sugar alcohol concentration is between 1.0 w/v% and 7.0 w/v% , more preferably between 2.0 and 6.0 w/v% .
  • amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added.
  • Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000.
  • PVP polyvinylpyrrolidone
  • PEG polyethylene glycol
  • a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Most any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer.
  • the concentration is from 0.01 to 0.3 molar.
  • Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268, 110.
  • composition comprises antibodies as an active principle these can be chemically modified by covalent conjugation to a polymer to increase its circulating half-life, for example.
  • Preferred polymers, and methods to attach them to peptides are shown in US Patent Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546 which are all hereby incorporated by reference in their entireties.
  • Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG).
  • PEG is soluble in water at room temperature and has the general formula : R(O-CH 2 -CH 2 ) n O-R where R can be hydrogen, or a protective group such as an alkyl or alkanol group.
  • the pretective group has between 1 and 8 carbons, more preferably it is methyl.
  • n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500.
  • the PEG has a preferred average molecular weight between 1000 and 40,000, more preferably between 2000 and 20,000, most preferably between 3,000 and 12,000.
  • PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with a free amino group on the inhibitor.
  • the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention.
  • Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body.
  • the POG has a preferred molecular weight in the same range as PEG.
  • the structure for POG is shown in Knauf et al. , 1988, J. Bio. Chem. 263 : 15064-15070, and a discussion of POG/IL-2 conjugates is found in US Patent No. 4,766, 106, both of which are hereby incorporated by reference in their entireties.
  • the liquid pharmaceutical composition is preferably lyophilized to prevent degradation and to preserve sterility.
  • Methods for lyophilizing liquid compositions are known to those or ordinary skill in the art.
  • the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients.
  • a sterile diluent Finger's solution, distilled water, or sterile saline, for example
  • the composition is preferably administered to subjects using those methods that are known to those skilled in the art.
  • compositions are administered so that aggregated IgG antibodies are given at a dose between l ⁇ g/kg and 20mg/kg, more preferably between 20 ⁇ g/kg and lOmg/kg, most preferably between 1 and 7mg/kg.
  • Therapy with the Fc ⁇ RII bridging compositions or the Fc ⁇ RII bridged cells according to the present invention may be performed in conjunction with any other known techniques or compounds for treatment of inflammatory, allergic or autoimmune diseases or for treatment of transplantation rejection, such as for instance immunosuppressive and anti- inflammatory drugs (e.g.
  • CsA cyclosporin A
  • methotrexate prednisolone
  • dexamethasone FK506, rapamycin
  • cyclooxygenase inhibitors such as indomethacin and corticosteriods
  • immunomodulatory cytokines such as IFN- ⁇ , IL-10 or IL-12.
  • the new Fc ⁇ RII bridging compositions according to the present invention may further also be used for any other human or animal treatment or diagnostic purpose for which they are possibly suited.
  • the present invention relates to the use of any of the Fc ⁇ RII bridging compositions as defined above as a medicament, more particularly for treating T-cell mediated diseases, even more particularly for the preparation of a medicament for treating T-cell mediated diseases.
  • An Fc ⁇ RII bridging composition to be used according to this aspect of the invention comprises particularly new Fc ⁇ RII bridging agents or compositions according to the above specified aspects of the present invention.
  • the present invention relates to the use of any of the Fc ⁇ RII bridging compositions as defined above, for the modulation of antigen-specific T-cell responsivenes, more particularly for the preparation of a medicament for the modulation of antigen-specific T-cell responsivenes.
  • the present invention relates to the use of any of the Fc ⁇ RII bridging compositions as defined above, for inducing T-cell tolerance or anergy, more particularly for the preparation of a medicament for inducing T-cell tolerance or anergy.
  • T-cell anergy and "T-cell tolerance” are reviewed above.
  • the invention provides a method for the induction of antigen-specific T-cell tolerance or T-cell anergy by modulating the co-stimulatory function of professional APCs by providing to a subject in the need of such a treatment an effective amount of a specific antigen combined with an agent capable of bridging the Fc ⁇ RII with at least one of the above-described functional effects of preventing the essential co-stimulatory molecules B7-1 and -2 from being up-regulated and by down-modulating the adhesion molecule ICAM-3.
  • Said Fc ⁇ RII-bridging agent is selected from the group consisting of: aggregated human IgG molecules; aggregated Fc fragments of human IgG molecules; a bivalent monoclonal antibody specific to the Fc ⁇ RII; a functionally active fragment of the latter antibodies; a multivalent monoclonal antibody specific to the Fc ⁇ RII; a recombinant fusion protein of 2 human IgG Fc parts; liposome vesicles containing at least one of the foregoing brdiging agents; all as detailed above.
  • Fc ⁇ RII bridging agents or compositions to be used according to this particular aspect of the present invention may comprise known FcR bridging compositions, agents or principles or in a preferred way specific Fc ⁇ RII bridging compositions particularly aimed at in the present invention (resulting in the prevention of expression of B7 molecules and/or the down-modulation of ICAM-3 molecules expression) as detailed above.
  • the present invention relates to the use of any of the Fc ⁇ RII bridging compositions as defined above for treating allergic diseases, more particularly for the preparation of a medicament for treating allergic diseases in humans, such as asthma, allergic rhinitis, atopic dermatitis, food and drug allergy.
  • the invention also provides a method for treating allergic diseases in humans, wherein said method comprises providing to a subject in the need of such a treatment an effective amount of a specific antigen combined with an agent capable of bridging Fc ⁇ RII molecules with the above-described functional effects of preventing the essential co-stimulatory molecules B7-1 and -2 from being up-regulated and by down-modulating the adhesion molecule ICAM-3, wherein said Fc ⁇ RII-bridging agent is selected from the group consisting of: aggregated human IgG molecules; aggregated Fc fragments of human IgG molecules; a bivalent monoclonal antibody specific to the Fc ⁇ RII; a multivalent monoclonal antibody specific to the Fc ⁇ RII, or funtionally active fragments of the latter antibodies; a recombinant fusion protein of 2 or more human IgG Fc parts; liposomes containing any of the foregoing as detailed above, possibly in combination with an allergen and other immunomodulatory agents as detailed above.
  • Fc ⁇ RII bridging agents or compositions to be used according to this particular aspect of the present invention may comprise known FcR bridging agents or compositions or in a preferred way, specific Fc ⁇ RII bridging agents or compositions particularly aimed at in the present invention (resulting in the prevention of B7-1/2 molecules expression and/or the down-modulation of ICAM-3 molecules expression) as detailed above.
  • the present invention relates to the use of any of the Fc ⁇ RII bridging compositions as defined above, for preventing rejection of solid organs, tissues and cells after transplantations, more particurlarly for the preparation of a medicament for preventing rejection of solid organs, tissues or cells after transplantation in humans.
  • the invention also provides a method for preventing rejection of solid organs, tissues or cells after transplantation in humans, wherein said method comprises providing to a subject in the need of such a treatment an effective amount of a specific alloantigen combined with an agent capable of bridging Fc ⁇ RII molecules with the above-described functional effects of preventing the essential co-stimulatory molecules B7-1 and -2 expression and by down-modulating the adhesion molecule ICAM-3 expression, wherein said Fc ⁇ RII-bridging agent is selected from the group consisting of: aggregated human IgG molecules; aggregated Fc fragments of human IgG molecules; a bivalent monoclonal antibody to the Fc ⁇ RII; a multivalent monoclonal antibody to the Fc ⁇ RII, or functionally active fragments of the latter antibodies; a recombinant fusion protein of 2 or more human IgG Fc parts; liposomes containing any of foregoing as detailed above, possibly in combination with an alloantigen and an immunosuppressive or immunomodul
  • Fc ⁇ RII bridging agents or compositions to be used according to this particular aspect of the present invention may comprise known FcR bridging agents or principles or in a preferred way specific Fc ⁇ RII bridging agents or compositions particularly aimed at in the present invention (resulting in the prevention of B7-1/2 molecules expression and/or the down-modulation of ICAM-3 molecules expression) as detailed above.
  • Target cells for treating in vitro or in vivo with the Fc ⁇ RII bridging agents or compositions according to the present aspect of the invention are preferably professional APCs treated with any form of aggregated or immobilized human IgG's, or aggregated Fc fragments of human IgG; or soluble human IgG's in the presence of antibodies to human IgG; or in the presence of bivalent or multivalent Fc ⁇ RII monoclonal antibodies, or in the presence of functionally active fragments of the latter antibodies, or in die presence of a recombinant fusion protein of 2 or more human IgG Fc parts, or in the presence of liposomes containing any of the foregoing.
  • aggregated may mean aggregated by means of any known chemical cross- linking agent.
  • immobilized may refer to any known immobilization method on any known subtrate such as a microtiter plate, a membrane (e.g. nylon or nitrocellulose), a microsphere (bead) or inserted into a liposome vesicle. Prior to application to the membrane or fixation it may be convenient to modify the IgG in order to facilitate fixation or improve its binding efficiency to Fc-receptors.
  • the present invention relates to the use of any of the Fc ⁇ RII bridging compositions as defined above for treating autoimmune diseases, more particularly for the preparation of a medicament for the treatment of autoimmune diseases, such as thyroiditis, rheumatoid arthritis, systemic lupus erythematosus
  • the invention also provides a method for the treatment of autoimmune diseases, wherein said method comprises providing to a subject in the need of such a treatment an effective amount of a specific autoantigen combined with an agent capable of bridging Fc ⁇ RII molecules with the above-described functional effects of preventing the essential co ⁇ stimulatory molecules B7-1 and -2 expression and/or by down-modulating the adhesion molecule ICAM-3 expression , wherein said Fc ⁇ RII-bridging agent is selected from the group consisting of: aggregated human IgG molecules; aggregated Fc fragments of human IgG molecules; a bivalent monoclonal antibody to the Fc ⁇ RII; a multivalent monoclonal antibody to the Fc ⁇ RII; functionally active fragments of the latter antibodies; a recombinant fusion protein of 2 or more human IgG Fc parts; liposomes containing any of the foregoing as detailed above, possibly in combination with an autoantigen and an immunosuppressive or immunomodulatory agent as detailed above.
  • Fc ⁇ RII bridging agents or compositions to be used according to this particular aspect of the present invention may comprise known FcR bridging agents or principles or in a preferred way specific Fc ⁇ RII bridging agents or compositions particularly aimed at in the present invention (resulting in the prevention of B7-1/2 molecules expression and/or the down-modulation of ICAM-3 molecules expression) as detailed above.
  • the present invention relates to a medicament comprising a composition as defined above.
  • the present invention relates to Fc ⁇ RII bridged professional APCs such as monocytes such as prepared by bridging professional APCs such as monocytes with any of the Fc ⁇ RII bridging compositions as defined above.
  • the invention also provides bridged professional APCs as defined above for use as a medicament, particularly for preventing a condition as detailed below.
  • the invention also relates to a method for preparing such bridged APCs as detailed above.
  • the invention also provides a method for preventing rejection of solid organs, tissues or cells after transplantation in humans, wherein said method comprises providing to a subject in the need of such a treatment an effective amount of alloantigen-expressing professional APCs such as monocytes from the graft donor on which several Fc ⁇ RII molecules have been bridged with the above-described functional effects of preventing the essential co-stimulatory molecules B7-1 and -2 expression and by down-modulating the adhesion molecule ICAM-3 expression, wherein said Fc ⁇ RII-bridging is accomplished by a method selected from the group consisting of: culturing the professional APCs (such as monocytes) in culture dishes coated with human IgG; culturing the APCs in culture dishes together with aggregated human IgG molecules or aggregated Fc fragments of human IgG molecules; culturing the APCs in culture dishes in the presence of soluble human IgG and an antibody to human IgG; culturing the APCs in culture dishes in the presence of
  • Fc ⁇ RII bridging agents or compositions to be used according to this particular aspect of the present invention may comprise known FcR bridging agents or principles or in a preferred way specific Fc ⁇ RII bridging compositions particularly aimed at in the present invention (resulting in the prevention of B7-1/2 molecules expression and/or the down- modulation of ICAM-3 molecules expression) as detailed above.
  • the present invention also contemplates a therapeutic composition comprising Fc ⁇ RII bridged professional APCs as defined above.
  • the present invention relates to Fc ⁇ RII bridged professional APCs as defined above, for use as a medicament, more particularly a medicament for treating or preventing any of the above-mentioned disease states.
  • the present invention relates to the use of Fc ⁇ RII bridged professional APCs as defined above for the preparation of a medicament for preventing rejection of solid organs, tissues or transplantations in humans.
  • the present invention also relates to an in vitro method for screening for or selecting a new Fc ⁇ RII bridging agent or composition, with said bridging agent or composition being characterized as preventing the B7 molecules expression on professional APCs and/or down modulating ICAM-3 expression on professional APCs.
  • a test system to screen for such molecules comprises an in vitro experimental set up in which professional APCs (for instance monocytes) are incubated in the absence or presence of the molecule(s) to be tested as having possible Fc ⁇ ll bridging capacities as detailed above by any of the techniques known in the art (such as ELISA or immunofluorescence (FACS) analysis as described in the Examples section) and measuring the amount of B7-1, B7-2 and/or ICAM-3 is measured.
  • professional APCs for instance monocytes
  • FACS immunofluorescence
  • Table 1 Modulation of cell surface antigens on monocytes after FcR bridging.
  • Cell surface expression of various antigens was determined after overnight culture on plates coated with human IgG or on un-coated plates as control. The relative expression is defined as the expression after overnight culture on human IgG-coated plates compared to the expression after culture in un-coated plates. Strong increase is indicated by + + + , modest increase by + + , slight increase by + , strong decrease by — , modest decrease by - and slight decrease by -. No change in expression is indicated by o in the Table.
  • Cell surface expression was measured by FACS analysis using specific monoclonal antibodies to the cell surface molecules as described in the specific Examples. (*) designates that B7-1/2 expression was determined with the CTLA-4Ig fusion protein as described in the specific Examples.
  • Table 2 FcR bridging inhibits antigen-specific proliferation of T cells.
  • PBMC 10 6 cells/ml
  • HSA human serum albumin
  • HGG human IgG
  • the following antigens were added to the culture : Tetanus toxoid (0.5Lfu/ml), Varidase (lOOIU/ml), Tuberculin (5IU/ml),
  • Cytomegalovirus antigen (CMV) (O.OlIU/ml), Herpes simplex virus antigen (0.02IU/ml), Varicella antigen (O.lIU/ml), Mumps antigen (1/2000), Influenza virus antigen (1/1000), Candida albicans (0.5 ⁇ g/ml).
  • CMV Cytomegalovirus antigen
  • Herpes simplex virus antigen 0.02IU/ml
  • Varicella antigen O.lIU/ml
  • Mumps antigen 1/2000
  • Influenza virus antigen (1/1000
  • Candida albicans 0.5 ⁇ g/ml
  • l ⁇ Ci( 3 H)-Thymidine l ⁇ Ci( 3 H)-Thymidine
  • Table 3 Release of soluble immunosuppressive mediators after FcR bridging.
  • Purified T cells (10 6 cells/ml) were cultured in the presence of syngeneic or allogeneic monocytes (10 6 cells/ml) as stimulator cells. Cultures were set up in normal plates, or plates precoated with human IgG or F(ab') 2 fragments of human IgG. MLR supernatants were taken after 24h culture and tested for the presence of various cytokines. The results are representative of one experiment out of three. (+ + ): TNF- ⁇ was determined by ELISA with a detection limit of 10 pg/ml.
  • TGF- ⁇ was measured in a bioassay using the MV1 Lu cell line. This bioassay has a detection limit of 50 pg/ml. ( ⁇ ) means below the detection limit of the specific bioassay.
  • Table 4 PGE2 production by human monocytes after FcR bridging.
  • Purified T cells (lOVml) were cultured in the presence of syngeneic or allogeneic monocytes (10 6 /ml) as stimulator cells. After 3 days of culture, cells were pulsed for 16h with 0.5 ⁇ Ci( 3 H)-Thymidine. Proliferative values represent the average of triplicate wells. (+ + ): After 24h, supernatants were recovered and analyzed for PGE2 content using an ELISA system. ( s ): Cells were cultured in 96- well round bottom culture plates. (
  • Table 5 FcR bridging on monocytes from an Fc ⁇ RII-deficient individual results in inhibition of antigen-specific proliferation of T cells.
  • PBMC 10 6 cells/ml
  • HSA human serum albumin
  • IgG human IgG
  • FIG. 1 Modulation of B7-1 expression on human monocytes.
  • Monocytes (10 6 /ml) were cultured on human IgG-coated dishes or with various cytokines for 24h and analyzed by FACS for B7-1 expression using mAb B7-24.
  • d monocytes cultured 24h with IFN- ⁇ ;
  • e monocytes cultured 24h with TNF- ⁇ ;
  • f monocytes cultured 24h with GM-CSF;
  • g monocytes cultured 24h with IL-2;
  • h monocytes cultured 24h with IL-4. Staining with isotype-matched antibody is shown as control.
  • FIG. 1 Modulation of B7-1 expression on human monocytes by Fc ⁇ RH bridging.
  • Monocytes (lOVml) were cultured for 24h in the presence of soluble human IgG (dotted line), rabbit anti-human IgG (solid line) or soluble human IgG and rabbit anti-human IgG (dashed line).
  • B7-1 expression was analyzed by FACS using mAB B7-24.
  • FIG. 3 Effects of FcR bridging on B7-1, CD40, HLA-DR and ICAM-1 expression on monocytes when activated by IFN- ⁇ or GM-CSF.
  • Monocytes (lOVml) were cultured for 24h in medium; in medium on human IgG-coated dishes; in the presence of GM-CSF; in the presence of IFN- ⁇ ; on human IgG-coated dishes in the presence of IFN- ⁇ . After a 24h culture period, cells were recovered and analyzed by FACS. Staining with isotype-matched antibody is shown as control.
  • the monoclonal antibodies used are as detailed in the Materials and Methods.
  • A monocytes cultured 24h in medium; b: monocytes cultured 24h on human IgG-coated dishes; c: monocytes cultured 24h with GM-CSF; d: monocytes cultured 24h with GM-CSF on human IgG-coated dishes; e: monocytes cultured 24h with IFN-; f: monocytes cultured 24h with IFN- on human IgG-coated dishes.
  • FIG. 5A Effect of FcR bridging on monocytes on the activation of T cells in MLR.
  • T cells (10 6 /ml) were cultured in 96-well round bottom tissue culture plates (circles) or plates pre-coated with human IgG (triangles) in the presence of various numbers of syngeneic (closed symbols) or allogeneic (open symbols) monocytes as stimulator cells.
  • Figure 5B Effect of FcR bridging on monocytes on the activation of T cells in MLR.
  • T cells (10 6 /ml) were cultured in 96-well round bottom tissue culture plates (circles) or plates pre-coated with human IgG (triangles) in the presence of various numbers of syngeneic (closed symbols) or allogeneic (open symbols) monocytes as stimulator cells.
  • IL-2 production by the T cells was measured after 24 hours and is expressed as the proliferative response of the CTLL bioassay.
  • FIG. 6A Blocking the B7-1/CD28 interaction is not sufficient to block the activation of T cells in MLR.
  • Purified T cells (10 6 /ml) were cultured in the presence of various numbers of monocytes as stimulator cells. Monocytes were added directly to the T cells (circles) or after pre-incubation with an anti-B7 mAb (triangles). After 3 days of culture T- cell proliferation was measured by [ 3 H]-Thymidine incorporation and is expressed as the mean of three different experiments +. SD.
  • FIG. 6B Blocking the B7-1/CD28 interaction is not sufficient to block the activation of T cells in MLR.
  • Purified T cells (10 6 /ml) were cultured in the presence of various numbers of allogeneic monocytes as stimulator cells. Monocytes were added directly to the
  • T cells (circles) or after pre-incubation with an anti-B7 mAb (triangles).
  • IL-2 production by the T cells was measured after 24 hours and is expressed as the proliferative response of the CTLL bioassay.
  • FIG. 7A Impairement of T-cell stimulatory capacity of monocytes after FcR bridging with intact human IgG is specifically mediated by Fc ⁇ RII.
  • Purified T cells (lOVml) were cultured in 96-well flat bottom tissue culture plates pre-coated with F(ab') 2 fragments of human IgG (light bars) or plates pre-coated with intact human IgG (dark bars) in the presence of syngeneic monocytes which were pre-incubated with monoclonal antibodies to different Fc-receptors (anti-Fc ⁇ RI mAb 197, anti-Fc ⁇ RII mAb IV.3).
  • T-cell proliferation was measured after 6 days by ( 3 H)-Thymidine incorporation.
  • T-cell stimulatory capacity of monocytes after FcR bridging with intact human IgG is specifically mediated by Fc ⁇ RII.
  • Purified T cells (lOVml) were cultured in 96-well flat bottom tissue culture plates pre-coated with F(ab') 2 fragments of human IgG (light bars) or plates pre-coated with intact human IgG (dark bars) in the presence of syngeneic monocytes which were pre-incubated with monoclonal antibodies to different Fc-receptors (anti-Fc ⁇ RI mAb 197, anti-Fc ⁇ RII mAb IV.3).
  • T-cell proliferation was measured after 6 days by ( 3 H)-Thymidine incorporation. T-cell proliferation specific for the Varidase antigen.
  • FIG. 7C Impairement of T-cell stimulatory capacity of monocytes after FcR bridging with intact human IgG is specifically mediated by Fc ⁇ RII.
  • Purified T cells (10 6 /ml) were cultured in 96-well flat bottom tissue culture plates pre-coated with F(ab') 2 fragments of human IgG (light bars) or plates pre-coated with intact human IgG (dark bars) in the presence of syngeneic monocytes which were pre-incubated with monoclonal antibodies to different Fc-receptors (anti-Fc ⁇ RI mAb 197, anti-Fc ⁇ RII mAb IV.3).
  • T-cell proliferation was measured after 6 days by ( 3 H)-Thymidine incorporation. T-cell proliferation specific for the
  • FIG. 8 The impaired capacity of monocytes to stimulate antigen-specific T-cell activation mediated by FcR bridging with intact human IgG can be prevented by mAb to Fc ⁇ RII.
  • Purified T cells were cultured in 96-well flat bottom tissue culture plates pre- coated with human serum albumin (black bars and dotted bars) or plates pre-coated with intact human IgG (squared bars and hatched bars) with syngeneic monocytes in the absence (black bars and squared bars) or presence (dotted bars and hatched bars) of anti-FcRII mAb IV.3. T-cell proliferation was measured after 6 days by 3 (H)-Thymidine incorporation.
  • the present invention is based on the new finding that the bridging of low affinity IgG receptor Fc ⁇ RII (CD32) molecules on monocytes specifically prevents the up-regulation of the essential co-stimulatory molecules B7-1/2 expression and results in the down modulation of the adhesion molecule ICAM-3 expression, with the functional consequence of an impaired capacity of the monocytes to co-stimulate the activation of antigen-specific T cells.
  • impaired co-stimulatory capacity of monocytes presenting specific antigen to T cells may lead to T-cell unresponsiveness or T-cell tolerance (Harding et al. Nature 356:607 (1992); Vassiliki et al. J. Exp. Med. 178: 1753 (1993)).
  • T-cell tolerance toward specific antigens is valuable for certain clinical usages in treating T-cell mediated diseases.
  • B7 molecules in co-stimulation of T cells and the prevention of T-cell tolerance is known in the art and although it is also known that bridging of Fc ⁇ RII results in the activation of monocytes resulting in the release of cytokines and other soluble mediators, nothing in the art relates to the finding that the specific bridging of the low affinity IgG receptor Fc ⁇ RII (CD32) on monocytes very selectively prevents the up- regulation of the essential co-stimulatory molecules B7-1/2 expression and down-modulates the ICAM-3 adhesion molecule expression.
  • CD32 low affinity IgG receptor Fc ⁇ RII
  • the observed modulation of the B7 molecules and the ICAM-3 molecule expression is a specific process in the cells which is mediated by specific mediators of intercellular signal transduction. This is demonstrated by the fact that treatment of monocytes with IFN- ⁇ or GM-CSF normally up-regulates the expression of such molecules as B7, HLA-DR, ICAM-1 and CD40. In contrast, treatment of monocytes with IFN- ⁇ or GM-CSF with concomitant bridging of the Fc ⁇ RII on monocytes prevents the up- regulation of the B7 molecules without affecting the up-regulation of HLA-DR and ICAM-1.
  • Monocytes are professional antigen presenting cells and Fc ⁇ RII bridging does not affect the up-regulation of HLA-DR on these cells and should therefore not affect their capacity to present antigenic peptides in the context of the HLA-DR molecules. Furthermore, it has been demonstrated that Fc ⁇ RII is capable of mediating phagocytosis of IgG-opsonized erythrocytes (Indik et al., J. Clin. Invest. 88: 1766 (1991); Tuijnman et al. , Blood 79: 1651 (1992)) and has been shown to be active in endocytosis of human IgG immune complexes (Engelhardt et al., Eur. J. Immunol. 21:2227 (1991)).
  • the anti-B7 mAb B7-24 (IgG2a) and anti-CD40 mAb 5D12 (IgG2b) have been previously described (De Boer et al. Eur. J. Immunol. 22:3071 (1992)).
  • the anti-ICAM-1 mAb B-C14 was kindly provided by Dr. J. Wijdenes (Innotherapie Bessancon, France).
  • the mAbs to HLA-DR (PE-labeled), CD20 (PE- labeled), CD3 (FITC-labeled),CD14 (FITC-labeled) and the isotype control antibodies were purchased from Becton and Dickinson (Erembodegem, Belgium).
  • the CTLA-4 human IgG fusion protein was purified from the supernatant of a cell line stably transfected with a cDNA encoding the fusion protein. This cell line was a gift of Dr. A. Lanzavecchia (Basel Institute for Immunology, Basel, Switzerland).
  • Goat anti-human (affinity isolated polyclonal goat F(ab') 2 anti-human IgG FITC-labeled, Tago, CA, USA) was used as second reagent to reveal CTLA-4 IgG staining.
  • monocytes Buffy coats obtained after cytophoresis of healthy donors were used to prepare monocyte cultures.
  • Mononuclear cell suspensions were obtained after buoyant density centrifugation of buffy coats on Lymphoprep (Nycomed, Oslo, Norway).
  • the monocyte- enriched, E-negative, fraction was separated from T lymphocytes by standard roset formation with SRBC followed by Lymphoprep sedimentation.
  • Monocytes were further enriched by the cold aggregation technique. Briefly the cell suspension was allowed to clump by low speed rotation at 4 °C. Cell clumps were separated from the rest of the cells by centrifugation, this population was > 89% CD14 + .
  • monocytes were isolated by adherence to plastic dishes for 30-40 min at 37 °C in a 5 % C0 2 atmosphere. After incubation, the non- adherent cells were removed by repeated vigorous washing. The resulting cultures contained at least 80% monocytes and less than 20% of cells as revealed by phenotype analysis (FACS).
  • FACS phenotype analysis
  • Monocytes (lxl0 6 /ml) were cultured in RPMI 1640 supplemented with 10 % heat- inactivated fetal calf serum, non-essential amino acids, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, 10 mM L-glutamine, 2 mM sodium pyruvate and, 50 ⁇ M 2-mercaptoethanol, in the absence or presence of various stimuli or human recombinant cytokines.
  • IFN- ⁇ 100 U/ml
  • GM-CSF 0.8 ⁇ g/ml
  • TNF- ⁇ 1000 U/ml
  • IL-2 50 U/ml
  • PBMC Peripheral blood mononuclear cells
  • T cells were cultured in 96-well round-bottom tissue culture plates (Falcon 3072, Becton Dickinson, Oxnard, CA) in the presence of various numbers of syngeneic or allogeneic monocytes as stimulator cells. After 3 days of culture, cells were pulsed for 16 h with 0.5 ⁇ Ci [ 3 H]-Thymidine (specific activity 5 Ci/mMol, Isotopchim, Ganagobie-Peyruis, France), after which the cells were harvested using an automated cell harvester. [ 3 H]-Thymidine incorporation was determined with a liquid scintillation counter. Proliferation of T cells were performed in triplicate wells.
  • monocytes were pre-incubated with mAb B7-24 (2 ⁇ g/ml per 10 6 cells) or an isotype control mAb in complete RPMI for 30 min at 4 °C before they were dispensed in 96 well round- bottom plates. Bridging the FcR of the monocytes during the MLR was achieved by using 96-wells round-bottom culture plates that had been pre-coated with human IgG, 1 mg/ml, in PBS for 30 min at 37 °C and washed three times with cold PBS. Human IgG F(ab'), fragment coated plates were used as control.
  • PBMC Peripheral blood mononuclear cells isolated on a Ficoll-Hypaque gradient (density 1.077) (Pharmacia LKB, Uppsala, Sweden) were washed and resuspended in complete medium, consisting of RPMI 1640 (Gibco, Paisley, Scotland) supplemented with penicillin, streptomycin, glutamine and 5 % autologous plasma. They were cultured at a concentration of lxlO 6 cells per ml, in 96 well flat bottom culture plates (Falcon, Labware,
  • HSA human serum albumin
  • PBS phosphate buffered saline
  • Tetanous toxoid (Wyeth, USA) 0.5 Lfu/ml; Varidase (Lederle, Belgium) lOOIU/ml; Tuberculin (Statens Serum Institute, Copenhagen, Danmark) 5IU/ml; Cytomegalovirus antigen (Behringwerke) O.OlIU/ml; Herpes simplex virus antigen (Behringwerke) 0.02IU/ml; Varicella antigen (Behringwerke) 0.1 U/ml; Mumps antigen (Behringwerke) 1/2000; Influenza virus antigen (Duphar, Belgium) 1/1000; Candida albicans
  • PHA Phytohemaggglutinin
  • ConA Concanavalin A
  • PWM Pokweed Mitogen
  • Cells were cultured in a 5 % CO2 humidified atmosphere for 3 days (PHA, ConA) or 6 days (PWM, antigens). Eight hours after a 1 ⁇ Ci ( 3 H)-thymidine pulse (Amersham, Buckinghamshire, England), cells were harvested and processed for determination of ( 3 H)-thymidine incorporation in a liquid scintillation counter.
  • IL-2 production was measured by bioassay using a subclone of the murine CTLL cell line, using recombinant human IL-2 as standard and is expressed as ( 3 H)-thymidine incorporation of the CTLL cells.
  • TNF- ⁇ was determined by ELISA (Innotest hTNF- ⁇ , Innogenetics, Belgium) with a detection limit of 10 pg/ml.
  • IL-10 was measured by ELISA using two mAbs: B-N10 as coating antibody and biotinylated BT10 as detecting antibody.
  • Recombinant IL-10 was used as a standard, the detection limit of this ELISA is 5 pg/ml.
  • TGF- ⁇ was measured in a bioassay using the MVl Lu cells line . This bioassay has a detection limit of 50 pg/ml.
  • Prostaglandin E2 measurement Prostaglandin E2 produced by monocytes during the MLR under various culture conditions was measured using an ELISA system (Boehringer Manheim, Brussels, Belgium). To test the effect of the prostaglandin synthesis on T cell activation, the cyclooxygenase inhibitor Indomethacin (Sigma) (100 ⁇ M) was added at the beginning of the culture. FACS analysis
  • purified monocytes (lxlO 6 ) were resuspended in PBS containing 1 % FCS, 0.1 % NaN 3 and 10 % normal rabbit serum. The cells were incubated at 4 °C for 30 min to block FcR-binding sites. The cells were subsequently incubated for 30 min at 4 °C with primary mAbs of the appropriate specificity, washed in PBS containing 1 % FCS and 0.1 % NaN 3 and, incubated for an additional 30 min at 4 °C with FITC-labeled goat anti-mouse Ig (Sigma). The cells were analysis on a FACScan * instrument (Becton Dickinson & Co, Mountain View, CA).
  • Example 1 Modulation of B7-1 expression on human monocytes
  • B7-1 expression on monocytes can be up-regulated by culture in the presence of IFN- ⁇ (Freedman et al. Cell. Immunol. 137:429 (1991)).
  • IFN- ⁇ Freedman et al. Cell. Immunol. 137:429 (1991)
  • Several cytokines were tested for their capacity to induce B7-1 expression on monocytes.
  • B7-1 expression could be induced by FcR bridging on monocytes.
  • Primary human monocytes were isolated by the cold aggregation technique and examined for cell surface expression of B7-1 using flow cytometry.
  • Figure 1 shows that freshly isolated monocytes lack detectable B7-1 cell surface protein, when stained with the anti-B7-l mAb B7-24.
  • B7-1 and B7-2 Freeman et al., Science 262:909 (1993)
  • the experiments described above were performed with a monoclonal antibody specific for the B7-1 molecule.
  • the CTLA-4-Ig fusion protein as described in the section Materials and Methods above, is known to recognize both B7-1 and B7-2 on monocytes and other antigen presenting cells (Freeman et al., Science 262:909 (1993)). It was therefore also tested, as described in the legend of Figure 4 and in the section Materials and Methods above, whether the expression of the B7-2 molecule on monocytes was influenced by FcR bridging.
  • Figure 4 shows that when monocytes cultured on IgG-coated plates were analyzed for expression of both B7 molecules using a CTLA-4-Ig fusion protein, it was found that this treatment down-modulated both B7- 1 and B7-2.
  • Example 5 Blocking the B7-1/CD28 interaction is not sufficient to block the activation of T cells in Mixed Lymphocyte Cultures
  • Example 6 FcR bridging inhibits antigen-specific proliferation of T cells.
  • Example 7 The impaired capacity of APC to co-stimulate T cells after FcR bridging is not mediated by the induction of soluble immunosuppressive mediators
  • FcR bridging on monocytes is known to deliver a very strong activation signal for the release of soluble mediators. It is also known that monocytes can produce potent soluble immunosuppressive factors (Valitutti et al. , Immunology 67:44 (1989); Paswell et al., J. Immunol. 123: 115 (1979)).
  • the content of the MLR supernatants were analyzed by specific immuno assays or bioassays as described in detail in the section Materials and Methods above, for the presence of the well known cytokines IL-10, TGF- ⁇ and TNF- ⁇ .
  • Table 3 there was no significant release of TGF- ⁇ in MLR supernatants when using IgG-treated monocytes as APC, whereas only small amounts of IL10 where found. In contrast, very large amounts of TNF-c. were secreted in the MLR cultures following the FcR bridging.
  • TNF-cv was mainly produced by the monocytes, since control cultures of monocytes only on IgG-coated plates gave about the same TNF-c. production as in the presence of T cells.
  • TNF- ⁇ as immunosuppressive agent in our experimental system, we added recombinant human TNF- ⁇ to a MLR using untreated monocytes as APC. Under those culture conditions, we found no inhibition of the T-cell proliferation or IL-2 release (data not shown). This demonstrates that TNF- ⁇ is not involved in the inhibition of T-cell responses induced by the FcR bridging on monocytes.
  • Lip id mediators derived from cell membranes are known to be produced during T cell-macrophages interactions (Coquette et al., Eur. J. Pharmacol. 226: 1 (1992)).
  • Prostaglandins particularly prostaglandin E2 (PGE2), modulates the function of immunocompetent cells by suppressing T-cell and macrophage function (Wong et al., J. Immunol. 148:2118 (1992); Gallay et al. , J. Immunol. 150:5086 (1993)).
  • PGE2 biosynthesis can be initiated by cytokines such as IL-1 or TNF- ⁇ (Elliott et al., Growth-factors 6: 15 (1992)). Furthermore, it has been demonstrated that FcR bridging on monocytes can result in the secretion of PGE2 (Finbloom et al. , J. Immunol. 150:2382 (1993); Singh et al., J. Immunol. 151:2786 (1993)). Therefore the PGE2 content in supernatants of MLR cultures was determined using a specific immuno assay as described in the section Materials and Methods described above. Table 4 shows that PGE2 is released in large amounts only in the cultures on IgG-coated plates.
  • Example 10 The impaired capacity of APC to co-stimulate T cells after FcR bridging can be blocked by specific monoclonal antibodies to Fc ⁇ R ⁇

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Abstract

La présente invention se rapporte à de nouvelles compositions de pontage de FcηRII qui se caractérisent en ce qu'elles altèrent la capacité des cellules présentant un antigène à stimuler l'activation des lymphocytes T spécifiques d'un antigène, ce qui entraîne une modulation de la réaction des lymphocytes T spécifiques d'un antigène. Plus spécifiquement, ces agents de pontage de FcηRII sont choisis parmi le groupe constitué par: des molécules agrégées de l'immunoglobuline (IgG) humaine; des fragments agrégées du récepteur Fc des molécules d'IgG humaines; un anticorps monoclonal bivalent contre FcηRII; un anticorps monoclonal multivalent contre FcηRII; un fragment fonctionnellement actif de cet anticorps monoclonal bivalent ou multivalent; une protéine de fusion de recombinaison d'au moins deux parties du récepteur Fc d'IgG humaine; des vésicules de liposomes comprenant n'importe lequel des éléments précédents, à condition que la composition FcηRII inhibe l'expression des molécules costimulatrices B7-1/2 et/ou module négativement l'expression de la molécule ICAM-3 par les cellules présentant un antigène spécialisées. La présente invention se rapporte également à des procédés prophylactiques et thérapeutiques ainsi qu'à des compositions utilisées pour prévenir ou traiter le rejet d'organes solides, de tissus et de cellules après une transplantation, pour induire l'anergie des lymphocytes T ou leur tolérance, pour traiter les maladies allergiques ou pour traiter les maladies auto-immunes. La présente invention se rapporte également à des cellules présentant un antigène spécialisées, pontées par FcηRII, à l'aide d'un agent de pontage de FcηRII.
PCT/EP1995/002012 1994-05-26 1995-05-26 Nouveaux procedes et composes destines a la modulation selective de la reaction des lymphocytes t specifiques d'un l'antigene WO1995032734A1 (fr)

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WO1997007218A1 (fr) * 1995-08-16 1997-02-27 Novartis Ag Proteines de fusion entre cd32 et des allergenes
EP0856314A1 (fr) * 1995-04-07 1998-08-05 Chugai Seiyaku Kabushiki Kaisha Immunodepresseur
US6077833A (en) * 1996-12-31 2000-06-20 Isis Pharmaceuticals, Inc. Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
WO2001029192A2 (fr) * 1999-10-15 2001-04-26 Baylor Research Institute Utilisation de lignees cellulaires allogeniques pour charger des cellules presentant des antigenes afin de declencher ou d'eliminer des reponses immunes
EP1118331A1 (fr) * 2000-01-21 2001-07-25 I.D.M. Immuno-Designed Molecules Méthode pour augmenter la présentation d'antigène exogène par les cellules humaines présentatrices d'antigène et complexes de microparticules opsonisées pour appliquer cette méthode
US6319906B1 (en) 1996-12-31 2001-11-20 Isis Pharmaceuticals Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
WO2003066095A2 (fr) * 2002-02-07 2003-08-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Modulation de tolerance par modulation de signalisation de recepteur fc$g(g)riib
WO2003101427A1 (fr) * 2002-05-31 2003-12-11 Imperial College Innovations Limited Immunoliposomes formes avec un anticorps agglutine
EP1462111A1 (fr) * 2003-03-28 2004-09-29 Universiteit Utrecht Holding B.V. Composition pour l'induction d'immunotolérance
WO2004099374A2 (fr) * 2003-04-30 2004-11-18 The Research Foundation Of State University Of New York Methodes de traitement a l'aide d'immunoglobuline de recombinaison
EP1516881A2 (fr) 1999-04-19 2005-03-23 Katholieke Universiteit Nijmegen Composition et méthode pour moduler l'interaction des cellules dendritiques et les cellules T
US7235653B2 (en) 1996-12-31 2007-06-26 Isis Pharmaceuticals, Inc. Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
US7541032B2 (en) 2002-09-20 2009-06-02 Stichting Katholieke Universiteit Antigen uptake receptor for Candida albicans on dendritic cells
US7691591B2 (en) 2002-09-20 2010-04-06 Stichting Katholieke Universiteit Methods of identifying and isolating cells expressing DC-sign
EP2241331A2 (fr) 2003-12-15 2010-10-20 Alexion Pharmaceuticals, Inc. Nouveaux anticorps anti-signe DC
US7897582B2 (en) 2003-05-23 2011-03-01 Isis Pharmaceuticals, Inc. Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
US7960355B2 (en) 2003-05-23 2011-06-14 Isis Pharmaceuticals, Inc. Compositions and methods for the modulation of the expression of B7 protein

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EP0856314A1 (fr) * 1995-04-07 1998-08-05 Chugai Seiyaku Kabushiki Kaisha Immunodepresseur
EP0856314A4 (fr) * 1995-04-07 2000-05-24 Chugai Pharmaceutical Co Ltd Immunodepresseur
WO1997007218A1 (fr) * 1995-08-16 1997-02-27 Novartis Ag Proteines de fusion entre cd32 et des allergenes
US6077833A (en) * 1996-12-31 2000-06-20 Isis Pharmaceuticals, Inc. Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
US7235653B2 (en) 1996-12-31 2007-06-26 Isis Pharmaceuticals, Inc. Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
US6319906B1 (en) 1996-12-31 2001-11-20 Isis Pharmaceuticals Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
EP1516881A2 (fr) 1999-04-19 2005-03-23 Katholieke Universiteit Nijmegen Composition et méthode pour moduler l'interaction des cellules dendritiques et les cellules T
US8105599B2 (en) 1999-04-19 2012-01-31 Katholieke Universiteit Nijmegen Composition and method for modulating dendritic cell-T cell interaction
US8058400B2 (en) 1999-04-19 2011-11-15 Katholieke Universiteit Nijmegen Composition and method for modulating dendritic cell-t cell interaction
WO2001029192A3 (fr) * 1999-10-15 2002-01-17 Baylor Res Inst Utilisation de lignees cellulaires allogeniques pour charger des cellules presentant des antigenes afin de declencher ou d'eliminer des reponses immunes
WO2001029192A2 (fr) * 1999-10-15 2001-04-26 Baylor Research Institute Utilisation de lignees cellulaires allogeniques pour charger des cellules presentant des antigenes afin de declencher ou d'eliminer des reponses immunes
US7988963B1 (en) 1999-10-15 2011-08-02 Baylor Research Institute Use of allogeneic cell lines to load antigen presenting cells to elicit or eliminate immune responses
EP2302038A1 (fr) * 1999-10-15 2011-03-30 Baylor Research Institute Utilisation de lignées cellulaires allogeniques pour charger des cellules présentant des antigenes afin de déclencher ou d'éliminer des réponses immunes
EP2151245A1 (fr) * 1999-10-15 2010-02-10 Baylor Research Institute Utilisation de lignées cellulaires allogéniques pour charger des cellules présentant des antigènes afin de déclencher ou d'éliminer des réponses immunes
EP1118331A1 (fr) * 2000-01-21 2001-07-25 I.D.M. Immuno-Designed Molecules Méthode pour augmenter la présentation d'antigène exogène par les cellules humaines présentatrices d'antigène et complexes de microparticules opsonisées pour appliquer cette méthode
WO2001052884A1 (fr) * 2000-01-21 2001-07-26 I.D.M. Immuno-Designed Molecules Procede d'amelioration de la presentation de l'antigene exogene par des cellules humaines et complexes de microparticules opsonisees utilises dans l'application de ce procede
WO2003066095A3 (fr) * 2002-02-07 2003-12-31 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Modulation de tolerance par modulation de signalisation de recepteur fc$g(g)riib
WO2003066095A2 (fr) * 2002-02-07 2003-08-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Modulation de tolerance par modulation de signalisation de recepteur fc$g(g)riib
WO2003101427A1 (fr) * 2002-05-31 2003-12-11 Imperial College Innovations Limited Immunoliposomes formes avec un anticorps agglutine
US7691591B2 (en) 2002-09-20 2010-04-06 Stichting Katholieke Universiteit Methods of identifying and isolating cells expressing DC-sign
US7541032B2 (en) 2002-09-20 2009-06-02 Stichting Katholieke Universiteit Antigen uptake receptor for Candida albicans on dendritic cells
EP1842550A2 (fr) * 2003-03-28 2007-10-10 Universiteit Utrecht Holding B.V. Compositions pour induire une immunotolérance
EP1772152A3 (fr) * 2003-03-28 2007-06-27 Universiteit Utrecht Holding B.V. Composition pour l'induction d'immunotolérance
EP1842550A3 (fr) * 2003-03-28 2008-12-10 Universiteit Utrecht Holding B.V. Compositions pour induire une immunotolérance
EP1772152A2 (fr) * 2003-03-28 2007-04-11 Universiteit Utrecht Holding B.V. Composition pour l'induction d'immunotolérance
WO2004084927A3 (fr) * 2003-03-28 2005-01-27 Univ Utrecht Holding Bv Procedes d'induction d'immunotolerance et compositions utilisees dans ces procedes
WO2004084927A2 (fr) * 2003-03-28 2004-10-07 Universiteit Utrecht Holding B.V. Procedes d'induction d'immunotolerance et compositions utilisees dans ces procedes
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WO2004099374A2 (fr) * 2003-04-30 2004-11-18 The Research Foundation Of State University Of New York Methodes de traitement a l'aide d'immunoglobuline de recombinaison
US7897582B2 (en) 2003-05-23 2011-03-01 Isis Pharmaceuticals, Inc. Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
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