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WO2000009151A1 - Regulation de l'activite de cellules dendritiques - Google Patents

Regulation de l'activite de cellules dendritiques Download PDF

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Publication number
WO2000009151A1
WO2000009151A1 PCT/US1999/016715 US9916715W WO0009151A1 WO 2000009151 A1 WO2000009151 A1 WO 2000009151A1 US 9916715 W US9916715 W US 9916715W WO 0009151 A1 WO0009151 A1 WO 0009151A1
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cell
mip
cells
ligand
dendritic cell
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PCT/US1999/016715
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English (en)
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Sirid-Aimee Kellermann
Leslie M. Mcevoy
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Schering Corporation
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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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • 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 generally to the use of mammalian chemokines in the treatment of clinical disease states, including cancer.
  • Administered chemokines direct the migration of dendritic cells, particularly the mature subset.
  • antagonists will block the effects on trafficking at specific locations or compartments.
  • the circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid and myeloid cell lineages. See, e.g., Rapaport (1987) Introduction to
  • Dendritic cells are antigen-presenting cells, and are found in all tissues of the body. They can be classified into various categories, including: interstitial dendritic cells of the heart, kidney, gut, and lung; Langerhans cells in the skin and mucous membranes; interdigitating dendritic cells in the thymic medulla and secondary lymphoid tissue; and blood and lymph dendritic cells. Although dendritic cells in each of these compartments are CD45+ leukocytes that apparently arise from bone marrow, they may exhibit differences that relate to maturation state and microenvironment .
  • Antigen presentation refers to the cellular events in which a proteinaceous antigen is taken up, processed by antigen presenting cells (APC) , and then recognized to initiate an immune response. The most active antigen presenting cells have been characterized as the macrophages (which are direct developmental products from monocytes) , dendritic cells, and certain B cells.
  • the chemokines are a large and diverse superfamily of small soluble proteins. The superfamily is subdivided into two classical branches, based upon whether the first two cysteines in the chemokine motif are adjacent (termed the "C-C” branch) , or spaced by an intervening residue ("C-X-C"). A more recently identified branch of chemokines lacks two cysteines in the corresponding motif, and is represented by the chemokines known as lymphotactins. Another recently identified branch has three intervening residues between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int.
  • dendritic cells are poorly characterized, both in terms of responses to soluble factors, and many of their functions and mechanisms of action. Signals regulating the traffic pattern of DC are complex and not fully understood. The absence of knowledge about the physiological properties and responses of these cells limits their understanding. Thus, medical conditions where regulation, development, or physiology of dendritic cells is unusual remain unmanageable.
  • the present invention is based, in part, upon the identification of specific chemokines which modulate dendritic cell physiology.
  • chemokines have been demonstrated to have roles in chemoattracting various cell types.
  • the present invention provides methods of modulating the attraction, to a chemokine, of a dendritic cell expressing a CCR7 receptor, comprising contacting the cell with a modulating amount of: an agonist ligand for the CCR7 receptor; a mutein antagonist of the ligand for the receptor; an antibody to CCR7 which antagonizes the ligand; or an antibody to the ligand which antagonizes binding of the ligand to the receptor.
  • the dendritic cell is a mature dendritic cell; the dendritic cell is an MHC class II ni B7-2 ni cell; the ligand is a primate 6Ckine and/or MIP-3 ⁇ .
  • the modulating is blocking of the attraction, and the antibody is a: neutralizing antibody against CCR7 ; neutralizing antibody against 6Ckine; neutralizing antibody against MIP-3 ⁇ ; or a combination of the neutralizing antibodies.
  • the contacting is with a combination of 6Ckine and MIP-3 ⁇ .
  • the amount is from 30 ng/ml to 300 ⁇ g/ml; or the antibody is a monoclonal antibody.
  • the dendritic cell is in skin, e.g., wherein the skin is being prepared for a graft, or the skin exhibits the symptoms of a condition selected from the group consisting of: systemic lupus erythematosus; spondyloarthropathies ; sclerodermas; acute or chronic inflammation; or atopic or contact dermatitis.
  • the MHC class II ni B7-2 ni cell is: a bone marrow derived dendritic cell; a langerhans cell; a tissue derived cell; or a lymphoid organ dendritic cell.
  • Perferred embodiments include wherein the modulating is inducing attraction, and the dendritic cell is an MHC class II n ⁇ , B7-2h-*-: cell from skin; or from lymph node.
  • the contacting is with an agonist selected from the group of 6Ckine and MIP-3 ⁇ .
  • the invention provides a mutein variant of a ⁇ Ckine or MIP-3 ⁇ chemokine comprising a substitution corresponding to a residue in human MIP-3 ⁇ at position: 19 or 46; 17, 18, 20, or 21; 43, 44, or 45; or the a helical portion of the chemokine structure.
  • the substitution is a non- conservative substitution.
  • Others include a sterile composition comprising the variant, and: neutralizing antibody against CCR7 ; neutralizing antibody against 6Ckine; neutralizing antibody against MIP-3 ⁇ ; or a combination of the neutralizing antibodies.
  • Additional embodiments include methods of modulating mobility of a dendritic cell, comprising contacting the cell with a modulating amount of: a ligand for the receptor; a mutein antagonist of the ligand for the receptor; an antibody to CCR7 which antagonizes the ligand; or an antibody to the ligand which antagonizes binding of the ligand to the receptor.
  • the dendritic cell is a mature dendritic cell; the dendritic cell is an MHC class II ni B7-2 hi cell; or the ligand is a primate 6Ckine or MIP-3 ⁇ ; alternatively, the modulating is blocking of the mobility, and the antibody is a: neutralizing antibody against CCR7 ; neutralizing antibody against ⁇ Ckine; or neutralizing antibody against MIP-3 ⁇ .
  • the modulating is inducing attraction
  • the mature dendritic cell is a class II n ⁇ , B7-2 n ⁇ or resident skin dendritic cell.
  • CCR7 ligands ⁇ Ckine and MIP-3 ⁇ are potent selective chemoattractants for class II n ⁇ , B7-2 nl bone marrow-derived dendritic cells, as well as freshly isolated lymph node dendritic cells. Furthermore, these chemokines stimulate the egress of resident skin dendritic cells ex vivo.
  • 6Ckine and MIP-3 ⁇ exhibit chemoattractant activity for dendritic cells over a broad concentration range (10 -* -- to 10 _11 M) .
  • 6Ckine and MIP-3 ⁇ are expressed in lymphoid organs, and ⁇ Ckine has been localized to high endothelial venules and lymphatic endothelium. Based on its expression pattern and potent chemoattractant capability, ⁇ Ckine, in particular, should play an important role in the homing of dendritic cells to lymphoid tissues.
  • T cell dependent immunity Efficient initiation of T cell dependent immunity is a complex sequence of events that ultimately depends upon simultaneous co-localization of antigen-presenting dendritic cells and antigen-specific CD4 + or CD8 + T cells in the T cell areas of lymphoid organs (Ingulli, et al . (1997) J . Exp . Med . 185:2133-41), leading to activation and expansion of rare T cell clones. Satisfaction of the rather demanding requirements of this rendezvous is facilitated by (1) the constant recirculation of na ⁇ ve T cells through the lymphoid organs (Butcher and Picker (1996) Science 272:60-66) together with (2) the migration into lymphoid organs of antigen-presenting dendritic cells.
  • Dendritic cells are bone-marrow derived cells that serve a sentinel role in vivo. Steinman (1991) Ann . Rev . Immunol . 9:271-96; and Banchereau and Steinman (1998) Nature 392:245-52).
  • Immature DCs are distributed throughout many tissues. They are specialized at antigen uptake and processing, but generally express low levels of costimulatory molecules and MHC-peptide complexes on their cell surface. Cella, et al. (1997) Nature 388:782-7; and Pierre, et al . (1997) Nature 388:787-92. Upon stimulation, e.g., by inflammatory signals or CD40 triggering, dendritic cells upregulate costimulatory molecules such as CD86 (B7-2), CD80 (B7-1), and CD40 (Hart (1997) Blood 90:3245-87). Furthermore, their capability to take up antigen is supplanted by a heightened ability to present antigen (Cella, et al .
  • chemokines a large family of low molecular weight chemoattractant cytokines, are also being illuminated. Baggiolini (1998) Nature 392:565-8.
  • ⁇ Ckine is a recently discovered chemokine that features an unusually long carboxy-terminal tail containing two additional cysteines. Nagira, et al . (1997) J. Biol . Chem. 272:19518-24; Hromas, et al . (1997) J. Immunol. 159:2554-8; and Hedrick and Zlotnik (1997) J. Immunol. 159:1589-93.
  • ⁇ Ckine is also expressed by the endothelial cells lining lymphatic venules (or vessels; Willimann, et al . (1998) and S. Lira) suggesting a role for this chemokine in dendritic cell trafficking to lymph nodes. This distribution pattern suggests that ⁇ Ckine may play an important role in homing of dendritic cells to secondary lymphoid organs.
  • the present invention is based, in part, upon the observation that 6Ckine, as well as MIP-3 ⁇ , is an extremely potent inducer of in vitro, as well as in vivo, derived MHC class ll n -i- B7-2 n i dendritic cell migration.
  • the ability of ⁇ Ckine and MIP-3 ⁇ to attract dendritic cells, combined with the limited expression pattern of these chemokines (Nagira, et al . (1997) J. Biol . Chem. 272:19518-24; Hromas , et al . (1997) J. Immunol. 159:2554- 8; Hedrick and Zlotnik (1997) J. Immunol.
  • Dendritic cells are key players in both initiation and modulation of the immune response. Central to their role as immune sentinels is their ability to capture, process, and transport antigen to secondary lymphoid tissues where they serve as potent antigen presenting cells capable of stimulating T cells in the T cell area of lymphoid tissues. Trafficking of both T cells and DC to lymphoid organs, followed by precise microenvironmental localization within the T cell area, is critical for efficient immune surveillance and is thought to be directed by chemokines (Baggiolini (1998) Nature 392:565- 8). ⁇ Ckine, a recently discovered CC chemokine (Nagira, et al. (1997) J. Biol. Chem. 272:19518-24; Hromas, et al .
  • 6Ckine is also expressed by endothelial cells in lymphatic venules (Gunn, et al . (1998) Proc. Nat ' 1 Acad. Sci . USA 95:258-63.), the major route of dendritic cell entry into lymph nodes, further suggesting a role in directing dendritic cell trafficking.
  • 6Ckine is a potent chemoattractant for bone marrow dendritic cells in vitro and for class II + CDllc + skin and lymph node dendritic cells ex vivo.
  • Previous reports (Dieu, et al . (1998) J ⁇ Exp . Med . 188:373-86; and Sozzani, et al . (1998) J ⁇ Immunol . 161:1083-6) are confirmed that MIP-3 ⁇ , an alternative CCR7 ligand, is a chemoattractant for dendritic cells in vitro. Furthermore, these findings are extended to show that MIP3 ⁇ is active on class II + CDllc + skin and lymph node dendritic cells ex vivo.
  • dendritic cells within the tissues efficiently capture and process antigen and, following activation, upregulate cell surface expression of MHC class II and costimulatory molecules such as B7-2, features associated with mature dendritic cells (Banchereau and Steinman (1998) Nature 392:245-52). These cells then migrate to lymphoid tissues and become potent antigen presenting cells capable of T cell priming.
  • ⁇ Ckine (and MIP-3 ⁇ ) preferentially chemoattracted dendritic cells expressing high levels of MHC class II and B7-2, but were relatively ineffective at attracting MHC class II 10 B7-2 10 dendritic cells.
  • Chemokine responsiveness and chemokine receptor expression have often been studied using dendritic cells generated in vitro from precursors or isolated from blood or other tissue and expanded in vitro.
  • chemokine receptor expression can be achieved by culturing dendritic cells in the presence of cytokines . Delgado, et al . (1998) Immunobiology 198:490-500; Val, et al . (1998) J. Exp . Med. 188:373-86; and Sozzani, et al . (1998) J ⁇ Immunol . 161:1083-6.
  • lymph node dendritic cells responded to 6Ckine and MIP-3 ⁇ in transwell chemotaxis assays .
  • the percentage of lymph node dendritic cells that respond in these assays was lower than that observed for cultured bone marrow-derived dendritic cells.
  • evidence suggests that the lymph node dendritic cell population is heterogeneous with respect to lineage (Salomon, et al . (1998) J . Immunol . 160:708-17; and Steinman, et al . (1997) Immunol. Rev.
  • modulating of chemotaxis or chemokinetic movement will be a statistically significant effect on the movement.
  • the effect will be at the half- maximum concentration of the chemokine, but certain effects may be desired at lesser amounts of the chemokine. Effects may be measured at, e.g., 3x over backgound amounts of chemokine.
  • the amounts of, or effects of, agonists or antagonists may be more easily detected.
  • the pit mutation also results in reduced numbers of interdigitating dendritic cells in lymph nodes (M.D. Gunn, personal communication). This observation further supports the hypothesis that CCR7 ligands, and perhaps 6Ckine in particular, influence dendritic cell trafficking to lymph nodes in vivo.
  • Dendritic cells are represented by a diverse population of morphologically similar cell types distributed widely throughout the body in a variety of lymphoid and non-lymphoid tissues. See Caux, et al . (1995) Immunology Today 16:2; and Steinman (1991) Ann. Rev. Immunol. 9:271-296. These cells include lymphoid DC of the spleen, Langerhans cells of the epidermis, and veiled cells in the blood circulation.
  • DC are collectively classified as a group based on their morphology, high levels of surface MHC-class II expression as well as several accessory molecules (B7-1[CD80] and B7- 2[CD86]) that mediate T cell binding and costimulation (Inaba, et al . (1990) Intern. Rev. Immunol. 6:197-206; Frendenthal, et al. (1990) Proc. Natl. Acad. Sci. USA 87:769-xxx), and absence of certain other surface markers expressed on T cells, B cells, monocytes, and natural killer cells.
  • DC are specialized antigen-presenting cells which efficiently process and present antigens to, e.g., T cells. They stimulate responses from naive and memory T cells in the paracortical area of secondary lymphoid organs. There is also some evidence for a role in induction of tolerance.
  • DC are typically bone marrow-derived and migrate as precursors through blood stream to tissues, where they become resident cells such as Langerhans cells in the epidermis.
  • immature DC e.g., fresh Langerhans cells
  • immature DC e.g., fresh Langerhans cells
  • they capture and process antigens Inaba, et al . (1986) J. Exp. Med. 164:605-613; Streilein, et al. (1989) J. Immunol.
  • Antigen-loaded DC migrate from the peripheral tissue via the lymphatic system to the T cell rich area of lymph nodes, where the mature DC are called interdigitating cells (IDC; Austyn, et al . (1988) J . Exp . Med. 167:646-651; Kupiec-Weglinski , et al . (1988) J. Exp . Med. 167:632-645; Larsen, et al . (1990) J . Exp . Med . 172:1483-1494; Fossum (1988) Scand. J. Immunol. 27:97-105; Macatonia, et al . (1987) J . Exp . Med .
  • the primary and secondary B-cell follicles contain follicular dendritic cells that trap and retain intact antigen as immune complexes for long periods of time. These dendritic cells present native antigen to B cells and are likely to be involved being the affinity maturation of antibodies, the generation of immune memory, and the maintenance of humoral immune responses .
  • chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes.
  • These small secreted molecules are a growing superfamily of 8-14 kDa proteins originally characterized by a conserved four cysteine motif. See, e.g., Schall (1991) Cytokine 3:165-183; and The Cytokine Handbook Academic Press, NY.
  • the chemokines are secreted by activated leukocytes, and by stromal cells including endothelial cells and epithelial cells upon inflammatory stimuli (Oppenheim
  • chemokines have been shown to induce other biological responses, e.g., modulation of second messenger levels such as Ca ++ ; inositol phosphate pool changes, see, e.g., Berridge (1993) Nature 361:315- 325 or Billah and Anthes (1990) Bioche . J.
  • chemokines may, alone or in combination with other therapeutic reagents, have similar advantageous combination effects.
  • chemokines may have effects on other cell types, e.g., attraction or activation of monocytes, dendritic cells, T cells, eosinophils, and/or perhaps on basophils and/or neutrophils. They may also have chemoattractive effects on various neural cells including, e.g., dorsal root ganglia neurons in the peripheral nervous system and/or central nervous system neurons.
  • the chemokine superfamily was originally divided into two main groups exhibiting characteristic structural motifs, the Cys-X-Cys (C-X-C) and Cys-Cys (C-C) families. These are distinguished on the basis of a single amino acid insertion between the NH-proximal pair of cysteine residues and sequence similarity.
  • C-X-C chemokines e.g., IL-8 and MGSA/Gro- ⁇
  • the C-C chemokines e.g., MlP-l ⁇ and RANTES, are potent chemoattractants for monocytes and lymphocytes but not neutrophils.
  • lymphotactin does not belong to either group and may constitute a first member of a third chemokine family, the C family. Lymphotactin does not have a characteristic CC or CXC motif, and acts on lymphocytes but not neutrophils and monocytes. See, e.g., Kelner et al . (1994) Science 266:1395-1399; Schall (1994) "The Chemokines” in The Cytokine Handbook (2d ed. ) Academic Press; and Schall and Bacon (1994) Current Opinion in Immunology 6:865-873.
  • 6Ckine and MIP-3 ⁇ are ligands for CCR7.
  • a comparison of the chemotaxis titrations of human and mouse MIP-3 ⁇ and mouse ⁇ Ckine on mouse T lymphocytes reveals that human MIP-3 ⁇ and mouse ⁇ Ckine have very similar chemotaxis curves whereas mouse MIP-3 ⁇ has a very different curve.
  • Mouse MIP-3 ⁇ provides chemotactic signals at lower concentrations than human MIP-3 ⁇ ; however, at optimal concentrations it is a less potent chemoattractant.
  • the titration curve of mouse 6Ckine is very similar to human MIP-3 ⁇ .
  • Residue 19 is serine in mouse ⁇ Ckine, tyrosine in human MIP-3 ⁇ , and asparagine in mouse MIP-3 ⁇ . Tyrosine and serine have polar, uncharged side chains whereas asparagine has a nonpolar acidic side chain.
  • Residue 46 is proline in mouse 6Ckine, arginine in human MIP-3 ⁇ , and tyrosine in mouse MIP-3 ⁇ .
  • Proline and arginine have nonpolar side chains and, additionally, arginine has a very basic side chain, whereas tyrosine has an uncharged, polar side chain.
  • This residue is predicted to be in the receptor binding face of these ligand molecules and the dramatic difference in the character of mouse ⁇ Ckine and human MIP-3 ⁇ vs mouse MIP-3 ⁇ suggest that this structure may be important in receptor binding. Significant changes, e.g., to aspartic acid, would likely affect agonist effects.
  • the structural model of the receptor and ligand interaction suggests that the interaction surface of the chemokine would probably include the residues, corresponding to mouse MIP3- ⁇ : 19 and 46; additionally, residues 17, 18, 20, 21, 43, 44, 45, and the ⁇ helical region of the chemokine, e.g., corresponding to WVD... to ...NST near the carboxy terminus.
  • agonists and antagonists may be developed by appropriate substitution of residues at those positions.
  • chemokines e.g., monocyte chemotactic protein (MCP)-3, MCP-4, macrophage inflammatory protein (M ⁇ P)-l , MlP-l ⁇ , RANTES (regulated on activation, normal T cell expressed and secreted) , SDF- 1, TECK (thymus expressed chemokine) and MDC (marcrophage derived chemokine)
  • MCP monocyte chemotactic protein
  • M ⁇ P macrophage inflammatory protein
  • MlP-l ⁇ RANTES (regulated on activation, normal T cell expressed and secreted)
  • SDF- 1, TECK thymus expressed chemokine
  • MDC macrophage derived chemokine
  • chemokine receptors have been identified on dendritic cells, the effects on them by chemokines have not been well investigated. Chemokines may have been proposed to attract naive dendritic cells to antigen, e.g., tumor associated antigens. However, the role of chemokines in attracting dendritic cells into the secondary immune sites, e.g., lymph nodes, spleen, and collections of mucosa-associated lymphoid tissues, has not been fully established. It is proposed that the means by which skin resident, or other, dendritic cells migrate specifically towards those secondary immune sites, may be mediated by, or supplemented by, chemokines. Those chemokines will be those whose receptors are found on the mature, e.g., antigen specific, dendritic cells. The primary chemokines for such are the ⁇ Ckine and MIP-3 ⁇ .
  • antibody antagonists to the chemokines may find use in preventing further responses.
  • the antagonists may be neutralizing antibodies to the soluble ligands, e.g., ⁇ Ckine or MIP-3 ⁇ , which can block interaction with the receptors, thereby preventing cellular response.
  • the antagonists may be neutralizing antibodies to the receptor, e.g., CCR7, which block chemokine binding or attraction.
  • Such antagonists should also exhibit the ability to prevent certain chemokinetic movement of the cells.
  • the present invention provides reagents and methods which will find use in therapeutic applications.
  • Antibodies and other binding agents directed towards ⁇ Ckine or MIP3 ⁇ chemokine proteins or nucleic acids will possess significant therapeutic value.
  • the CCR7 chemokine ligands naturally occurring or recombinant
  • fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to a the ligands or receptor, are useful in the treatment of conditions associated with abnormal physiology or development, including immunological reaction or tissue rejection conditions .
  • Various skin associated medical conditions e.g., overly active immunological conditions, such as systemic lupus erythematosus, rheumatoid arthritis, spondyloarthropathies, sclerodermas, acute or chronic inflammation, atopic or contact dermatitis, skin grafts or transplants, allergic conditions, or dermatological conditions, may involve the skin or mucosal resident dendritic cells. See, particularly, Frank, et al . (eds.) Samter ' s Immunological Diseases Little, Brown, Boston; Kay (ed. ) Allergy and Allergic Diseases Blackwell, Oxford; Stone (ed. ) Dermatologic Immunology and Allergy Mosby, St.
  • a disease or disorder associated with chemoattractive or chemokinetic signaling by a ⁇ Ckine or MIP-3 ⁇ chemokine is a target for an agonist or antagonist of the protein.
  • the proteins likely play a role in regulation or attraction of dendritic cells.
  • Mutein or ligand sequence based antagonists might be created by N-terminal modification, e.g., either truncation of addition of N-terminal extensions, or ligand sequence variants.
  • Other abnormal medical conditions are known to be mediated by abnormal dendritic cell function. See Reid
  • chemokines especially in dendritic cells or in T cells, along with the ligand expression patterns, suggest roles in immune functions.
  • the ⁇ Ckine and MIP-3 ⁇ chemokines are likely to recruit these cell types in vivo, thereby enhancing the immune response mediated by these cell types.
  • the expression patterns appear consistent with a functional importance of the ligands in initiation of the immune response, particularly in recruiting or mobilizing dendritic cells to the secondary lymph tissues.
  • antagonists will have the opposite effects, and will be useful, e.g., in minimizing autoimmune or suppressing responses in desired contexts, e.g., in a tissue rejection situation.
  • Antagonists may be muteins of the chemokine ligands, antibodies which block binding to receptor, antibodies to the CCR7 receptor, or small drugs which interfere with the ligand-receptor interaction.
  • Recombinant ⁇ Ckine or MIP-3 ⁇ chemokine or ⁇ Ckine or MIP-3 ⁇ chemokine antibodies can be purified and then administered to a patient.
  • These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients .
  • additional active or inert ingredients e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients .
  • These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
  • This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
  • antibodies to the CCR7 may be used to block signaling by its identified ligands, ⁇ Ckine and MIP- 3 ⁇ .
  • antibody antagonists of 6Ckine and MIP-3 ⁇ are now available in the form of antibodies to the corresponding receptor .
  • Drug screening using antibodies or receptor or fragments thereof can identify compounds having binding affinity to ⁇ Ckine or MIP-3 ⁇ chemokine. Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the protein. This invention further contemplates the therapeutic use of antibodies to ⁇ Ckine or MIP-3 ⁇ chemokine as antagonists.
  • reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al . Goodman and Gilman' s: The Pharmacological Bases of Therapeutics (current ed. ) Pergamon Press; and Remington ' s Pharmaceutical Sciences (latest ed. ) Mack Publishing Co., Easton, PA.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway, NJ. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar) , and most preferably less than about 1 fM (femtomolar) , with an appropriate carrier.
  • ⁇ Ckine or MIP-3 ⁇ chemokines, fragments thereof, and antibodies to it or its fragments, antagonists, and agonists may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in many conventional dosage formulations, including sterile formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typically comprise at least one active ingredient, as described above, together with one or more acceptable carriers thereof.
  • Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al . (eds. 1990) Goodman and Gilman' s: The Pharmacological Bases of Therapeutics (8th ed. ) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences
  • compositions Tablets Dekker, NY; and Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms : Disperse Systems Dekker, NY.
  • the therapy of this invention may be combined with or used in association with other therapeutic agents, e.g., with other similar reagents directed to the other MIPs, particularly the MIP- Is or MIP-3s.
  • antagonists can normally be found once the protein has been structurally defined. Testing of potential protein analogs is now possible upon the development of highly automated assay methods using a purified receptor. In particular, new agonists and antagonists will be discovered by using screening techniques described herein.
  • EXAMPLE 2 PCR analysis. cDNA libraries prepared from resting bone-marrow derived dendritic cells or bone marrow-derived dendritic cells stimulated overnight with anti-CD40 mAb were subjected to PCR amplification. Plasmid DNA encoding mCCR ⁇ , mCCR7 , or mCXCR3 served as controls. Appropriate primers used for mCCR ⁇ ; mCCR7 ; and mCXCR3. ⁇ -actin was amplified as an internal control. PCR products were resolved on a 1.2% agarose gel.
  • chemokines and receptors can be found, e.g., in the GenBank or NCBI databases. See also, R&D Systems, Minneapolis, MN.
  • Bone marrow-derived dendritic cells were generated by culturing bone marrow cell suspensions (Inaba, et al . (1992) J . Exp . Med . 176:1693-702; and Talmor, et al . (1998) Eur. J. Immunol.
  • Freshly isolated lymph node cells were prepared by homogenizing lymph nodes from Balb/c mice. The resulting cell suspension was washed and incubated in RPMI containing 1% FCS for 60 minutes at 37° C prior to assaying. Occasionally, B cells were removed by magnetic depletion, which did not affect the results of chemotaxis assays.
  • Magnetic depletions were performed using anti-CD3 (KT3; Serotec, Kidlington, UK), anti-B220 (RA3-6B2; PharMingen), and anti-Gr-1 (RB6-8C5; kindly provided by B. Coffman, DNAX) , followed by incubation with anti-rat immunoglobulin Dynabeads (Dynal, Oslo, Norway) .
  • CD40 stimulation of dendritic cells was achieved using mAb 1C10 (Heath, et al. (1994) Eur . J . Immunol . 24:1828-34).
  • Murine ⁇ Ckine, MIP- 3 ⁇ , and MlP-l ⁇ as well as human MIP-3 ⁇ and SDF-l ⁇ were from R&D Systems (Minneapolis, MN) .
  • EXAMPLE 4 Chemotaxis assays Chemokine proteins are produced, e.g., in COS cells transfected with a plasmid carrying the chemokine cDNA by electroporation. See, Hara, et al . (1992) EMBO J. 10:1875-1884. Physical analytical methods may be applied, e.g., CD analysis, to compare tertiary structure to other chemokines to evaluate whether the protein has likely folded into an active conformation. After transfection, a culture supernatant is collected and subjected to bioassays. A mock control, e.g., a plasmid carrying the luciferase cDNA, is used.
  • a positive control e.g., recombinant murine MlP-l ⁇ from R&D Systems (Minneapolis, MN) , is typically used.
  • antibodies may be used to block the biological activities, e.g., as a control.
  • Lymphocyte migration assays are performed as previously described, e.g., in Bacon, et al . (1988) Br . J. Pharmacol . 95:966-974. Other trafficking assays are also available. See, e.g., Quidling-Jarbrink, et al . (1995) Eur . J . Immunol . 25:322-327; Koch, et al . (1994) J. Clin. Inv. 93:921-928; and Antony, et al . (1993) J. Immunol.
  • Murine Th2 T cell clones CDC-25 (see Tony, et al . (1985) J . Exp . Med . 161:223-241) and HDK-1 (see Cherwinski, et al . (1987) J . Exp . Med . 166:1229-1244), made available from R. Coffman and A. 0 ' Garra (DNAX, Palo Alto, CA) , respectively, are used as controls .
  • Ca2+ flux upon chemokine stimulation is measured according to the published procedure described in Bacon, et al. (1995) J. Immunol. 154:3654-3666.
  • EXAMPLE 5 Biological activities, direct and indirect
  • a robust and sensitive assay is selected as described above, e.g., on immune cells, neuronal cells, or stem cells.
  • Chemokine is added to the assay in increasing doses to see if a dose response is detected.
  • a proliferation assay cells are plated out in plates. Appropriate culture medium is provided, and chemokine is added to the cells in varying amounts. Growth is monitored over a period of time which will detect either a direct effect on the cells, or an indirect effect of the chemokine .
  • an activation assay or attraction assay is used.
  • An appropriate cell type is selected, e.g., hematopoietic cells, myeloid (macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (T cell, B cell, or NK cells), neural cells (neurons, neuroglia, oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitor cells which differentiate to other cell types, e.g., gut crypt cells and undifferentiated cell types .
  • Retroviral infection assays have also been described using, e.g., the CCR1 , CCR3 , and CCR5 receptors. While the human CCR7 has not been established to be a receptor, the possibility that it might has not been completely eliminated. Recent description of these other chemokine receptors in retroviral infection processes, and the effects by the related RANTES and MIP-1 chemokines, suggest similar effects could exist with the ⁇ Ckine or MIP-3 ⁇ . See, e.g., Baiter (1996) Science 272:1740 (describing evidence for chemokine receptors as coreceptors for HIV); and Deng, et al . (1996) Nature 381:661-666.
  • Chemokines may also be assayed for activity in hemopoietic assays as described, e.g., by H. Broxmeyer. See Bellido, et al . (1995) J. Clin. Inv. 95:2886-2895; and Jilka, et al . (1995) Expt ' 1 Hem. 23:500-506. They may be assayed for angiogenic activities as described, e.g., by Streiter, et al . (1992) Am. J. Pathol . 141:1279-1284. Or for a role in inflammation. See, e.g., Wakefield, et al . (1996) J. Surgical Res. 64:26-31.
  • Ears from BALB/c mice were aseptically removed and split into dorsal and ventral halves. Both halves were cultured in individual wells of a 24 well plate in medium lacking cytokines, in the absence or presence of 10 ⁇ to 10 -7 M ⁇ Ckine or MIP-3 ⁇ . After 18 to 48 hours of culture at 37° C, emigrated cells from each ear were stained for CDllc and I-A d /I-E d , and analyzed by FACS . Cells were quantitated by adding a defined quantity of Dynosphere beads to each sample, and emigrated cell numbers were corrected for variations in ear weight. The ultrastructure of emigrated cells was examined in cytospin preparations .
  • EXAMPLE 7 ⁇ Ckine selectively attracts dendritic cells
  • Bone marrow cells were not subjected to any depletion regimen in advance of assaying chemotactic responses, and the population assayed contained a mixture of several cell types. Dendritic cells were identified by their distinct morphology in cytospin preparations and by CDllc expression (typically about 25-30% of the total cell population as assessed by FACS) , but polymorphonuclear cells expressing B220 and/or Gr-1 (Lin + ) were also present.
  • Cytospin preparations from bone marrow-derived cells cultured in GM-CSF and IL-4 were then tested in transwell chemotaxis assays for their response to ⁇ Ckine.
  • Dendritic cells are greatly enriched in the population.
  • the classll n ⁇ B7-2 n i subpopulation of dendritic cells is enriched upon migration toward ⁇ Ckine.
  • Bone marrow- derived cells were gated on CDllc + Lin- (B220 ⁇ , Gr-1 ⁇ ) cells and analyzed for their expression of class II and B7-2 before and after chemotaxis in response to ⁇ Ckine.
  • a classll hi B7-2 ni and classll 10 B7-2 lc ⁇ dendritic cell population is detected; CDllc + classll " B7-2 lo / ⁇ cells are also detected, and likely represent myeloid progenitors.
  • Bone marrow-derived cells or freshly isolated lymph node cells were tested in parallel in transwell chemotaxis assays in which an increasing concentration of ⁇ Ckine was added to the bottom well. The transmigrated populations were stained to identify dendritic cells or Lin + cells, or na ⁇ ve CD4 + T cells, and analyzed by FACS.
  • CC chemokine ⁇ Ckine (Nagira, et al . (1997) J. Biol. Chem. 272:19518-24; Hromas, et al . (1997) J. Immunol. 159:2554-8; Hedrick and Zlotnik (1997) J. Immunol . 159:1589-93) was able to efficiently and selectively chemoattract a significant proportion of bone marrow-derived cells.
  • the population that responded to 1 ng/ml ⁇ Ckine was enriched in dendritic cells, and were 70- 75% CDllc + Lin ⁇ .
  • the dendritic cells generated from bone marrow progenitors were heterogeneous for their expression of MHC class II and B7-2, which are upregulated on activated or mature dendritic cells (Steinman (1991) Ann. Rev. Immunol. 9:271-96).
  • the starting population of CDllc + Lin- cells were typically about 25% class ll n:L B7-2 hi and 15% class II 10 B7-2 1Q , with the remaining CDllc + cells likely representing myeloid progenitors
  • the CDllc + cells responding to 6Ckine were dramatically enriched for class II n ⁇ B7-2 n ⁇ cells, which were typically 90% or more of the total CDllc + Lin- population.
  • ⁇ Ckine was a potent chemoattractant for class II n i B7-2 n; *- dendritic cells over a broad concentration range, routinely attracting 60-90% of this subset of dendritic cells at peak concentrations. In contrast, only a small percentage of class II ⁇ 0 B7-2-*- 0 dendritic cells were found in the responding population, and the effective concentration range was much more limited.
  • the apparent specificity of ⁇ Ckine for class II n: *- B7-2 n i dendritic cells was further underscored by the unresponsiveness of contaminating Lin + cells except at high concentrations of 6Ckine .
  • CD4 + T cells responded to 6Ckine in the range of 10 -6 to
  • Bone marrow-derived dendritic cells exhibit a robust chemotactic response to CCR7 ligands ⁇ Ckine and MIP-3 ⁇ .
  • Bone marrow-derived cells were tested in transwell chemotaxis assays in which increasing concentrations of individual chemokines were added to the lower well. The transmigrated populations were stained to identify dendritic cells, and analyzed by FACS.
  • chemoattract dendritic cells such as SDF-l ⁇ (Sozzani, et al. (1997) J. Immunol. 159:1993-2000; and Delgado, et al . (1998) Immunobiology 198:490-500), MIP-3CC (Power, et al. (1997) J. Exp. Med. 186:825-35; and Val, et al . (1998) J. Exp . Med . 188:373-86, and MlP-l ⁇ (Sozzani, et al . (1997) J. Immunol. 159:1993-2000; Gro, et al . (1998) J . Exp . Med.
  • Both SDF-l ⁇ and MlP-l ⁇ were capable of attracting a small percentage of class Ill° B7-2-*- 0 dendritic cells; the dose-dependence of the response to SDF-l ⁇ mirrored that of the class II n ⁇ B7- 2 n ⁇ dendritic cells, while MlP-l ⁇ was effective at 10 ng/ml (data not shown) .
  • Murine MIP-3 ⁇ and SDF-l ⁇ are not currently available, and the low activity of human MIP-3 ⁇ in particular could be due, at least in part, to species differences.
  • CD40 ligation leads to upregulation of costimulatory molecules and activation of dendritic cells.
  • Cella, et al. (1996) J . Exp . Med . 184:747-52.
  • CD40-mediated activation altered dendritic cell responsiveness to ⁇ Ckine
  • five-day bone marrow-derived cultures were depleted of CD3 + , B220 + , and most Gr-1 + cells and incubated overnight in the presence or absence of anti-CD40 Ab. While CD40 stimulation increased the proportion of dendritic cells that were class II-- 1 --- B7-2 n ⁇ , the percentage of class Ilhi B7-2 n ⁇ dendritic cells responding to ⁇ Ckine was unchanged.
  • MIP-3 ⁇ Like ⁇ Ckine, and in contrast to SDF-l ⁇ , MIP-3 ⁇ , and MlP-l ⁇ , MIP-3 ⁇ attracted dendritic cells over a broad range of concentrations, and was similar to ⁇ Ckine with respect to its specificity for class II n ⁇ - B7-2 n ⁇ dendritic cells.
  • each chemokine elicited migration of dendritic cells in a uniform field (i.e., chemokine was present in both upper and lower chambers) , but not in the presence of a negative gradient (chemokine present only in the upper chamber) .
  • the number of dendritic cells that migrated through transwells in a uniform field was never equivalent to that achieved in the presence of a positive gradient, suggesting that the response of dendritic cells to all chemokines tested was primarily chemotactic.
  • T cells exhibited some degree of chemokinetic activity in the presence of 10 ⁇ 6 M ⁇ Ckine.
  • cDNA libraries prepared from bone marrow-derived dendritic cells cultured in the absence or presence of anti-CD40 mAb were subjected to PCR analysis using probes specific for CCR7 or CXCR3. Control reactions were carried out without template or using plasmids expressing the relevant chemokine receptor sequence. ⁇ -actin served as an internal control. This analysis revealed that both dendritic cell populations express CCR7 , but not CXCR3 , suggesting that dendritic cell chemotaxis in response to ⁇ Ckine and MIP-3 ⁇ is mediated through their common receptor CCR7.
  • EXAMPLE 10 Lymph node dendritic cells These results indicated that ⁇ Ckine and MIP-3 ⁇ are effective chemoattractants for bone marrow-derived dendritic cells cultured in vitro. However, to determine whether these CCR7 ligands were physiologically relevant chemoattractants for dendritic cells, the chemotactic response of freshly isolated lymph node cells to ⁇ Ckine and MIP-3 ⁇ was assessed in transwell chemotaxis assays.
  • MIP-3 ⁇ was a consistent, but less potent chemoattractant for lymph node dendritic cells. The differential response to 6Ckine and MIP-3 ⁇ was also observed when CD4 + T cells were analyzed. There was no response to MIP-3 ⁇ .
  • Cytospins were prepared from cells obtained from ears cultured overnight in ⁇ Ckine and stained with Gie sa. ⁇ Ckine stimulates the egress of dendritic cells from skin. The emigration of class II + CDllc + cells, which had a distinctly dendritic morphology, was consistently augmented by ⁇ Ckine three- to twelvefold. Similar results were obtained using MIP-3 ⁇ .

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Abstract

Les cellules dendritiques jouent un rôle essentiel dans les réponses immunitaires spécifiques de l'antigène. La présente invention concerne des matériaux et des procédés qui permettent de traiter certains états pathologiques, notamment des maladies en rapport avec la peau et des maladies auto-immunes, en facilitant ou en inhibant la migration ou l'activation de cellules dendritiques. En particulier, on utilise des chimiokines pour déclencher, amplifier ou moduler une réponse immunitaire.
PCT/US1999/016715 1998-08-17 1999-08-16 Regulation de l'activite de cellules dendritiques WO2000009151A1 (fr)

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WO2000046248A1 (fr) * 1999-02-03 2000-08-10 Schering Corporation Utilisation d'agonistes ou d'antagonistes de la chimiokine mip-3a a des fins therapeutiques
WO2000038706A3 (fr) * 1998-12-31 2000-11-23 Chiron Corp Methodes de traitement du cancer et de mediation de la chimiotaxie des cellules dendritiques
EP1255112A3 (fr) * 2001-04-30 2003-07-23 Pfizer Products Inc. Traitement de maladies médiées par les cellules T
US6645491B1 (en) 1999-02-03 2003-11-11 Schering Corporation Method for treating inflammatory conditions using an antibody to MIP-3α
US7030228B1 (en) 1999-11-15 2006-04-18 Miltenyi Biotec Gmbh Antigen-binding fragments specific for dendritic cells, compositions and methods of use thereof antigens recognized thereby and cells obtained thereby
US7217700B2 (en) 1998-07-16 2007-05-15 Schering Corporation Chemokines as adjuvants of immune response
USRE43952E1 (en) 1989-10-05 2013-01-29 Medtronic Navigation, Inc. Interactive system for local intervention inside a non-homogeneous structure
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE43952E1 (en) 1989-10-05 2013-01-29 Medtronic Navigation, Inc. Interactive system for local intervention inside a non-homogeneous structure
US7217700B2 (en) 1998-07-16 2007-05-15 Schering Corporation Chemokines as adjuvants of immune response
WO2000038706A3 (fr) * 1998-12-31 2000-11-23 Chiron Corp Methodes de traitement du cancer et de mediation de la chimiotaxie des cellules dendritiques
WO2000046248A1 (fr) * 1999-02-03 2000-08-10 Schering Corporation Utilisation d'agonistes ou d'antagonistes de la chimiokine mip-3a a des fins therapeutiques
US6645491B1 (en) 1999-02-03 2003-11-11 Schering Corporation Method for treating inflammatory conditions using an antibody to MIP-3α
US7030228B1 (en) 1999-11-15 2006-04-18 Miltenyi Biotec Gmbh Antigen-binding fragments specific for dendritic cells, compositions and methods of use thereof antigens recognized thereby and cells obtained thereby
US8183039B2 (en) 1999-11-15 2012-05-22 Miltenyi Biotec Gmbh Method for isolating BDCA-4+ dendritic cells
US10407486B2 (en) 1999-11-15 2019-09-10 Miltenyi Biotech Gmbh Ligating BDCA-2 protein with BDCA-2 specific antibody for detecting or modulating dendritic cells
EP1255112A3 (fr) * 2001-04-30 2003-07-23 Pfizer Products Inc. Traitement de maladies médiées par les cellules T
US11634497B2 (en) 2017-02-03 2023-04-25 Novartis Ag Anti-CCR7 antibody drug conjugates
US12180291B2 (en) 2017-02-03 2024-12-31 Novartis Ag Anti-CCR7 antibody drug conjugates

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