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WO1998051345A2 - Methode permettant de chasser les leucocytes infiltres dans un tissu - Google Patents

Methode permettant de chasser les leucocytes infiltres dans un tissu Download PDF

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Publication number
WO1998051345A2
WO1998051345A2 PCT/IB1998/000860 IB9800860W WO9851345A2 WO 1998051345 A2 WO1998051345 A2 WO 1998051345A2 IB 9800860 W IB9800860 W IB 9800860W WO 9851345 A2 WO9851345 A2 WO 9851345A2
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Prior art keywords
leukocytes
cell
mammal
tissue
compound
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PCT/IB1998/000860
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English (en)
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WO1998051345A3 (fr
Inventor
Bosco M. C. Chan
Bhagirath Singh
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The John P. Robarts Research Institute
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Priority to AU73487/98A priority Critical patent/AU7348798A/en
Publication of WO1998051345A2 publication Critical patent/WO1998051345A2/fr
Publication of WO1998051345A3 publication Critical patent/WO1998051345A3/fr

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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
    • C07K16/2803—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
    • C07K16/2806—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 CD2
    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to tissue infiltrating leukocytes.
  • the infiltration of tissues by leukocytes is one of the critical steps leading to an immune response.
  • the presence of tissue infiltrating leukocytes can be beneficial, as in the case of tumor infiltrating leukocytes which play a role in host defence against cancer.
  • the presence of infiltrating leukocytes in the tissue can cause severe damage.
  • tissue infiltrating leukocytes can lead to the destruction of targeted organs.
  • tissue infiltrating leukocytes can lead to graft rejection.
  • a means by which to prevent leukocytes from infiltrating tissues in non-cancerous patients with an autoimmune disease, or in patients who are recipients of allogeneic and xenogeneic organ transplants, would be of high therapeutic benefit.
  • this invention features a method of lowering the number of leukocytes in a target tissue in a mammal.
  • the method includes the step of administering a therapeutically effective amount of a composition capable of specifically binding at least two cell surface molecules expressed on leukocytes or on targeted tissue.
  • the number of leukocytes is lowered by at least 25%, more preferably by at least 50%, and most preferably by at least 75% relative to a mammal not administered the therapeutic composition.
  • the leukocytes are dislodged in a target tissue.
  • the composition may include a compound which is a chemical, drug, antibody (e.g., a monoclonal antibody which may be humanized, or a polyclonal antibody), or a protein.
  • the compound may be a fusion protein, or may include a first domain capable of specifically binding to a first cell surface molecule expressed on leukocytes and a second, non-specific domain, which may be the invariant portion of an antibody molecule which may engineered to be incapable of binding complement, or may be a toxin, such as ricin.
  • the composition may be in a non-solid state (e.g., a liquid or gaseous state).
  • at least one of the cell surface molecules on the leukocytes is an adhesion molecule (e.g., an integrin, a selectin, or a member of the Ig superfamily).
  • the mammal may be a human or a rodent
  • the target tissue may be a transplanted graft (e.g., an allograft or xenograft).
  • the transplanted graft may be a heart, kidney, liver, or skin graft, and may be from an adult or a fetus.
  • a transplanted xenograft may be, for example, from a swine or a non-human primate.
  • the graft may be cultured in vitro or may receive pre-treatment with the composition of the invention or another therapeutic composition prior to transplantation.
  • the methods of the first aspect of the invention may be used to prevent rejection of the graft, to prolong the function of the graft, or both.
  • a heart graft may be the target tissue and may be treated with a composition that includes a first compound capable of specifically binding to LFA- 1 and a second compound capable of specifically binding to NLA-4.
  • the mammal may have an autoimmune disease, e.g., autoimmune hemolytic anemia, autoimmune neutropenia, autoimmune lymphopenia, autoimmune thrombocytopenia, Goodpasture's diease, Myasthenia gravis, Graves' disease, multiple sclerosis, autoimmune pemphigus bullous skin disease, autoimmune pemphigoid bullous skin disease, system lupus erythematosus, rheumatoid arthritis, Sjogren's disease, scleroderma, polymyositis/dermatomyositis, mixed connective tissue disease, or insulin-dependent diabetes mellitus.
  • an autoimmune disease e.g., autoimmune hemolytic anemia, autoimmune neutropenia, autoimmune lymphopenia, autoimmune
  • the target tissue of the method is a tissue affected by leukocyte infiltration in the autoimmune disease.
  • the methods of the first aspect may be used to attain a favorable outcome for the autoimmune disease, may be used to alleviate symptoms of the autoimmune disease, or both.
  • a mammal with insulin-dependent diabetes mellitus and/or a target tissue that is the pancreas may be treated with a composition that includes a first compound capable of specifically binding to NLA-5 and a second compound capable of specifically binding to NLA-4.
  • the mammal is mammal having a predisposition to develop an autoimmune disease.
  • the predisposition may be a genetic or an environmental predisposition.
  • the invention features method of detecting a compound capable of modulating the number of leukocytes which have infiltrated a target tissue in a mammal.
  • the method includes the steps of administering to the mammal a therapeutically effective amount of a composition that includes at least one compound being tested for an ability to specifically bind a cell surface molecule expressed on the leukocytes or the targeted tissue, and measuring the number of leukocytes which have infiltrated the targeted tissue, where a change in the number of tissue infiltrating leukocytes relative to the number of tissue infiltrating leukocytes in a mammal not administered the composition indicates that the compound modulates the number of leukocytes which have infiltrated the target tissue in the mammal.
  • the mammal is a rodent or a human.
  • the antibody is attached to a solid-phase substance, such as a microtiter plate, and the leukocytes carry a marker, such as green fluorescent protein, which is easily quantitated.
  • a change that is a decrease in the number indicates the compound is capable of decreasing the number of leukocytes which have infiltrated the target tissue and a change that is an increase in the number indicates the compound is capable of increasing the number of leukocytes which have infiltrated the target tissue.
  • the detection of a therapeutic compound capable of modulating leukocyte infiltration of a target tissue in a mammal is determined by a change in the number of leukocytes is at least 25%, more preferably at least 50%, and most preferably at least 75% relative to the number of leukocytes which have not been treated with the compound.
  • Compounds found to enhance leukocyte infiltration of a target tissue may be useful as anti-cancer therapeutics.
  • the mammal may have an autoimmune disease, e.g., autoimmune hemolytic anemia, autoimmune neutropenia, autoimmune lymphopenia, autoimmune thrombocytopenia, Goodpasture's diease, Myasthenia gravis, Graves' disease, multiple sclerosis, autoimmune pemphigus bullous skin disease, autoimmune pemphigoid bullous skin disease, system lupus erythematosus, rheumatoid arthritis, Sjogren's disease, scleroderma, polymyositis/dermatomyositis, mixed connective tissue disease or insulin-dependent diabetes mellitus.
  • an autoimmune disease e.g., autoimmune hemolytic anemia, autoimmune neutropenia, autoimmune lymphopenia, autoimmune thrombocytopenia, Goodpasture's diease, Myasthenia gravis, Graves' disease, multiple sclerosis, autoimmune pemphigus bullous skin disease, autoimmune pe
  • the target tissue of the method is a tissue affected by leukocyte infiltration in the autoimmune disease.
  • the mammal has been transplanted with a graft, and the graft is the target tissue.
  • the transplanted graft may be a heart, kidney, liver, or skin graft, and may be from an adult or a fetus.
  • the graft may be an allograft or xenograft (e.g., from a swine or a non-human primate), and may be cultured in vitro or may receive treatment prior to transplantation.
  • the compound may be in a non-solid state (e.g., a liquid or gaseous state).
  • the invention features a method of identifying a cell surface molecule important for tissue infiltration by leukocytes.
  • the method includes the steps of first, administering to the mammal with a targeted tissue a therapeutically effective amount of a composition that includes at least one compound being tested for ability to specifically bind a cell surface molecule, and measuring the number of leukocytes which have infiltrated the tissue, where a change in the number of leukocytes in the administered mammal relative to the number of leukocytes in a mammal not administered the composition indicates the compound modulates the number of the leukocytes which have infiltrated the tissue; and second, using the compound modulates the number of leukocytes which have infiltrated the tissue to identify a cell surface molecule important for tissue infiltrating leukocytes.
  • the compound is an antibody.
  • the targeted tissue may be a transplanted graft, a tumor, or an affected tissue in a mammal with an autoimmune disease.
  • the mammal may be a human or a rodent and the composition may include a compound which may be a chemical, drug, antibody (e.g., a monoclonal antibody which may be humanized, or a polyclonal antibody), or a protein.
  • the composition may be in a non-solid state (e.g., a liquid or gaseous state).
  • the invention features a cell which produces a monoclonal antibody that specifically binds murine NLA-5.
  • the invention features a cell which produces a monoclonal antibody that specifically binds human NLA-5.
  • the cell is a hybridoma cell of a first and second cell fused together, where the first cell is a rat spleen cell capable of producing the antibody, and said second cell is a mouse myeloma cell incapable of producing the antibody.
  • the antibody is most preferably a monoclonal antibody.
  • the invention features a composition for reducing the number of leukocytes in a targeted tissue in a mammal, where the composition specifically binds at least two cell surface molecules expressed on the leukocytes or on the targeted tissue.
  • the composition dislodges leukocytes in the targeted tissue.
  • the composition of the seventh aspect of the invention may include at least two compounds, the first compound specifically binds a first cell surface molecule expressed on a leukocyte and the second compound specifically binds a second cell surface molecule expressed on a leukocyte.
  • the first compound may specifically binds to LFA-1 and the second compound may specifically binds to NLA-4.
  • the first compound may specifically binds to NLA-
  • the second compound may specifically binds to NLA-4.
  • the composition may include compounds that are antibodies (e.g., monoclonal antibodies, polyclonal antibodies, humanized antibodies), fusion proteins, or may have at least two domains, where the first domain specifically binds the cell surface molecule and the second domain may be the invariant portion of an antibody molecule which may be unable to bind complement.
  • the second domain may also be a toxin, such as ricin.
  • the cell surface molecules may be integrins, selectins, or member of the Ig superfamily.
  • the composition may be attached to a solid-phase substance, such as a microtiter plate, or the composition may be in a non-solid state (e.g., the composition may be in an aqueous or gaseous state).
  • Specifically binds means a reagent, such as an antibody (also known as an immunoglobulin or immunoglobulin protein), that recognizes and binds a target molecule (e.g., a protein), but that does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, that naturally includes protein.
  • the reagent specifically binds a target molecule with a K D of less than 10 "6 .
  • Cell surface molecule means a molecule present on the cell surface at some point in its in vivo existence. All integral membrane molecules are cell surface molecules.
  • cell surface molecules molecules attached to the membrane by means other than a transmembrane domain, such a glycosyl phosphatidyl inositol (GPI) linkage (e.g. , cell surface molecule thy- 1).
  • GPI glycosyl phosphatidyl inositol
  • cell surface molecules are CD45, CD28, CD3, LFA-1,
  • ICAM-1 ICAM-1, L-selectin, and surface IgM.
  • Adhesion molecule means a molecule which mediates a specific interaction with a second molecule. Either the first, second, or both of these molecules may be on the surface of a cell. Two such interacting cell surface adhesion molecules may be the same molecule expressed on different cells.
  • the first adhesion molecule is a cell surface molecule
  • the second adhesion molecule is attached to the extracellular matrix (e.g., cell surface molecule NLA-4 binding to extracellular matrix molecule fibronectin), or is soluble (e.g., cell surface IgM binding to a soluble protein).
  • both molecules are soluble (e.g., a secreted antibody binding to a soluble protein).
  • adhesion molecules are immunoglobulins (e.g., antibodies), LFA-1, NLA-4, L-selectin, and the receptor for platelet-derived growth factor (PDGF). Interacting molecules are sometimes referred to as receptor/ligand pairs.
  • the cell surface molecule ICAM-1 is a ligand for the LFA-1 receptor, which means that LFA-1 binds to ICAM-1.
  • PDGF is a ligand for the PDGF receptor, which means that the PDGF receptor binds to PDGF.
  • An adhesion molecule may be defined as such even if its ligand is not known.
  • the cell surface molecule CD2 was referred to as an adhesion molecule years before its ligand, LFA-3, was identified.
  • Immunoglobulin (Ig) superfamily member means a molecule characterized by the presence of at least one domain (70- 110 amino acid residues in length) which exhibits structural homology with either the variable (N) or constant (C) domain of an immunoglobulin protein (also known as an antibody). Most of the identified members of the Ig superfamily are integral membrane proteins with the Ig domains in the extracellular portion, although there are exceptions. Some examples of integral membrane Ig superfamily members are the cell surface adhesion molecules CD2, CD3, CD4, CD8, CD28, and ICAM-1.
  • “Integrin” means a member of the integrin family of cell surface adhesion molecules. All integrins are heterodimeric cell surface proteins composed of two noncovalently linked polypeptide chains, ⁇ and ⁇ . Examples of integrins include LFA-1 (leukocyte function-associated antigen- 1), Mac-1, NLA-1, NLA-2, NLA-3, NLA-4, NLA-5, NLA-6, CR3, CR4, ⁇ 4 ⁇ 7, and Glycoprotein Ilb/IIIa. These molecules may be designated by their assigned Cluster of Differentiation (CD) number, or by the and ⁇ chains of which they are comprised, or merely by the chain of which they are comprised.
  • CD Cluster of Differentiation
  • LFA-1 is referred to as CD1 la/CD18, ⁇ L ⁇ 2, CD1 la, or ⁇ L.
  • NLA-4 is referred to as CD49d/CD29, ⁇ 4 ⁇ l, CD49d, or ⁇ 4.
  • Selectin means a member of the selectin family of cell surface adhesion molecules. The extracellular portion of a selectin includes an ⁇ - terminal carbohydrate-binding lectin-related segment, an epidermal growth factor receptor related repeat, and multiple complement regulatory protein motifs.
  • L-selectin also referred to as MEL- 14 and Leu8, among others
  • E-selectin also referred to as ELAM-1, among others
  • P-selectin also referred to as PADGEM and GMP-140, among others.
  • Leukocyte means a white blood cell. Leukocytes are derived from pluripotent precursors which, in the adult, reside in the bone marrow. Some examples of leukocytes are T lymphocytes, B lymphocytes, monocytes, neutrophils, eosinophils, basophils, megakaryocytes, and Natural Killer (NK) cells. Also included among leukocytes are cells which no longer reside in the blood, but normally reside in the tissues. Some examples of tissue-localized leukocytes are macrophages and mast cells.
  • “Graft” means the transplantation of a tissue or organ from a first mammal (donor) to a second mammal (host or recipient). Included in the description of a graft is modification of the graft prior to or during transplantation. Hence, the graft may be sustained in vitro prior to transplantation. The graft may also be treated (e.g. , perfused with host blood) prior to or during transplantation. In addition, the definition of a graft includes tissue from a donor mammal at any stage of development to a host mammal at any stage of development. Hence, donor fetal tissue may be used as a graft to an adult host mammal.
  • a graft where the purpose of the tissue by the host animal differs from the purpose in the donor.
  • a donor kidney may be used a host liver.
  • the modification of the location of the tissue in the donor and host mammals Hence, a donor liver may be used as a host bone marrow.
  • Allograft means a graft between two genetically dissimilar animals of the same species.
  • One example of an allograft is a kidney donated by one human to an unrelated human recipient. Included in examples of allografts is an organ donated to a non-identical twin sibling, because the two siblings, although genetically related, are not genetically identical.
  • Xenograft means a graft between two animals of differing species.
  • a xenograft is a kidney donated by non-human primate (e.g., baboon) to an unrelated human recipient.
  • tissue infiltrating leukocyte means a leukocyte which has already left the blood stream and has entered and has remained in a tissue for an abnormally long amount of time. Included in the definition is a leukocyte which has formed adhesions to the tissue. A leukocyte which has been present in the tissue for many days is included in the definition. Also included in the definition is a leukocyte which has just entered the tissue, but which will remain in the tissue for an abnormally long amount of time.
  • the definition includes the reference to "tumor infiltrating leukocytes.” If the tissue is a tumor, the definition includes the reference to "tumor infiltrating leukocytes.” If the tissue is a graft, the definition includes the reference to "graft infiltrating leukocytes.”
  • the definition does not include a leukocyte which has left the bloodstream and is part of the normal migration of blood-borne leukocytes into surrounding tissues. These leukocytes, although entering the tissue, do not remain over-long in the tissue, and quickly depart for other tissues or the lymphatic system, or return to the blood. The definition also does not include such leukocytes as mast cells and macrophages which, in the normal individual, are localized to tissues.
  • Leukocyte extravasation means the event of a leukocyte leaving the blood stream and entering the tissue. This is accomplished by the adherence of a leukocyte to the vascular endothelium via numerous cell surface adhesion molecules, followed by the passage of the leukocyte through vascular endothelial cell junctions and into the underlying tissue.
  • “Lowering the number of leukocytes” means a decrease in the number of leukocytes in a tissue determined using an assay which measures the number of leukocytes present in a tissue (e.g., the assays described in Example N and Example NI). This decrease in the number of leukocytes is preferably at least 25%, more preferably at least 50%, and most preferably at least 75% lower than the number of leukocytes otherwise present.
  • Figs. 1 A and IB are graph demonstrating the fibronectin-binding ability of splenic T cells isolated from NOD (Fig. 1A) and Balb/c (Fig. IB) mice of various ages in the presence or absence of PMA.
  • Splenic T cells from NOD mice, but not from Balb/c mice, between 5 weeks and 10 weeks of age exhibited increased adhesive function for fibronectin.
  • Fig. 2 is a graph of the fibronectin-binding ability of splenic T cells isolated from NOD mice in the presence of indicated antibodies. Adhesion of splenic NOD T cells to fibronectin is mediated by cc4 and 5 integrins such that adhesion was inhibited in the presence of monoclonal antibodies to both integrins.
  • Figs. 3A-3F is a graph showing the percentages of ⁇ 4 integrin and ⁇ 5 integrin expressing splenic CD4 + and CD8 + T lymphocytes from female NOD and Balb/c mice of varying ages.
  • Fig. 3 A shows the expression levels of NLA- 4 on CD4 + T cells.
  • Fig. 3B shows the expression levels of NLA-5 on CD4 + T cells.
  • Fig. 3C shows the expression levels of NLA- ⁇ l on CD4 + T cells.
  • Fig. 3D shows the expression levels of NLA-4 on CD8 + T cells.
  • Fig. 3E shows the expression levels of NLA-5 on CD8 + T cells.
  • Fig. 3F shows the expression levels of NLA- ⁇ l on CD8 + T cells.
  • Fig. 4 is a graph showing the effects of indicated antibodies on insulitis in 10 week old NOD mice.
  • Monoclonal antibody PS/2 recognizes ⁇ 4 integrins
  • monoclonal antibody BMA5 recognizes ⁇ 5 integrins.
  • NRIg is a control antibody.
  • Fig. 5A-5D is a photograph of a histopathological analysis of pancreas tissue from 10 week old NOD mice which had received indicated antibodies.
  • Fig. 5 A shows the effects of control antibody (NRI9);
  • Fig. 5B shows the effects of mAb PS/2, which blocks ⁇ 4 integrin;
  • Fig. 5C shows mAb BMA5, which blocks NLA-5;
  • Fig. 5D shows the effects of both PS/2 and BMA5.
  • Fig. 6 is a graph showing the effects of indicated antibodies on insulitis in cyclophosphamide treated NOD mice.
  • Monoclonal antibody PS/2 recognizes ⁇ 4 integrins
  • monoclonal antibody BMA5 recognizes ⁇ 5 integrins.
  • the control antibody is an isotype matched monoclonal antibody.
  • Host rejection of a graft from a unrelated donor involves the infiltration of the graft with leukocytes. The similar situation also occurs in the case of tissue specific autoimmune disease; for example, Insulin-Dependent Diabetes
  • IDDM Mellitus
  • IDDM Mellitus
  • the invention described herein is a method to remove leukocytes which have already infiltrated into the tissue.
  • the invention features a method for dislodging leukocytes which have infiltrated tissue and, thus, are eliminated from the tissue and returned to either the lymphatic or circulatory system.
  • the method involves the utilization of a combination of reagents that block the ligand binding function of at least two different cell surface adhesion molecules. Administration of a combination of these reagents will result in an elimination of the leukocyte infiltrate and attenuate tissue damage.
  • tissue infiltrating leukocytes are harmful.
  • an important mechanism in host defense to cancer is the generation and maintenance of tumor infiltrating leukocytes.
  • the cell surface molecules which mediate the generation and maintenance of tumor infiltrating leukocytes on both the leukocytes and on the tumor cells are poorly defined.
  • another aspect of the invention is an assay for the identification of reagents capable of enhancing leukocyte infiltration into tumors in cancer patients.
  • the tumor infiltrating leukocyte-dislodging reagents of the first aspect of the invention can then be used to identify the cell surface molecules involved in infiltration. Once identified, cell surface molecules involved in tumor infiltrating leukocytes may be used to develop highly beneficial anti- cancer therapies.
  • the infiltration of engrafted tissue by recipient leukocytes is a critical step leading to both acute and chronic graft rejection.
  • the migration of leukocytes across the vascular endothelium and into the underlying tissue is mediated by cell surface molecules from several gene families.
  • the first family of cell surface molecules involved in generating and maintaining tissue infiltrating leukocytes is the selectin family.
  • Selectins are integral membrane adhesion proteins.
  • the extracellular portion of a selectin includes an N- terminal carbohydrate-binding lectin-related segment, an epidermal growth factor receptor related repeat, and a varying number of short consensus repeats such as those generally found in proteins regulating complement activation (Springer et al., Nature 349: 196-197, 1991).
  • L-selectin is expressed on the endothelium, while L-selectin is found on leukocytes.
  • P-selectin is found on platelets, and can also be induced on endothelial cells.
  • integrin gene family A second family of cell surface adhesion molecules involved in tissue infiltrating leukocytes is the integrin gene family. All integrins are heterodimeric cell surface proteins composed of two noncovalently linked polypeptide chains, cc and ⁇ . The extracellular domains of the two chains bind to various ligands, including extracellular matrix glycoproteins, complement components, and proteins on the surface of other cells. The integrins are sometimes divided into subfamilies, based upon which ⁇ subunit was used to form the heterodimer since, with exceptions, each ⁇ chain pairs with a distinct set of chains.
  • Ig immunoglobulin
  • Reagents that block cell surface adhesion molecule binding to ligands One type of reagent which may be used to block the binding function of an adhesion molecule expressed on the cell surface is an antibody. Many antibodies directed toward cell surface adhesion molecules are commercially available. Further antibodies may be prepared by a variety of methods. For example, the adhesion molecules, or antigenic fragments thereof, can be administered to an animal in order to induce the production of polyclonal antibodies. Alternatively, antibodies may be monoclonal antibodies, which are prepared using hybridoma technology well known in the art of immunology (see, e.g., Ausubel et al, Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY, 1994).
  • the invention features the use of antibodies that specifically bind cell surface molecules, or fragments thereof.
  • the invention features the use of "neutralizing” antibodies.
  • neutralizing antibodies is meant antibodies that interfere with any of the biological activities of cell surface molecules, particularly the ability of cell surface molecules to participate in adhesion.
  • the neutralizing antibody may reduce the ability of cell surface molecules to participate in adhesion by, preferably 50%, more preferably by 70%, and most preferably by 90% or more. Any standard assay of adhesion, including those described herein, may be used to assess potentially neutralizing antibodies.
  • the invention features various genetically engineered antibodies, humanized antibodies, and antibody fragments, including F(ab')2, Fab', Fab, Fv and sFv fragments.
  • Antibodies can be humanized by methods known in the art, e.g., monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA). Fully human antibodies, such as those expressed in transgenic animals, are also features of the invention (Green et al. , Nature Genetics 7:13-21, 1994).
  • Ladner (U.S. Patent 4,946,778 and 4,704,692) describes methods for preparing single polypeptide chain antibodies.
  • Ward et al. (Nature 341 :544- 546, 1989) describe the preparation of heavy chain variable domains, termed "single domain antibodies," which have high antigen-binding affinities.
  • McCafferty et al. (Nature 348:552-554, 1990) show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen, and that rare phage (one in a million) can be isolated after affinity chromatography.
  • Boss et al. (U.S.
  • Patent 4,816,397) describe various methods for producing immunoglobulins, and immunologically functional fragments thereof, which include at least the variable domains of the heavy and light chain in a single host cell.
  • Cabilly et al. U.S. Patent 4,816,567) describe methods for preparing chimeric antibodies.
  • other reagents that can be used to bind cell surface proteins are readily attainable.
  • One such reagent is soluble form of a ligand that specifically binds to a cell surface adhesion molecule.
  • Full length or truncated forms of such ligands can be engineered from known sequences and used to compete with the physiological ligand for binding to a cell surface adhesion molecule.
  • a reagent that specifically binds to a cell surface adhesion molecule may be a fusion protein, comprising a domain capable of specifically binding to a cell surface adhesion molecule bonded to a second, non-specific domain.
  • This second non-specific domain may be the constant domain of an immunoglobulin heavy chain.
  • the constant domain of the immunoglobulin heavy chain may be engineered such that it is unable to bind complement.
  • the second, non-specific domain of the fusion protein may also be a toxin, such as ricin, which will kill the cell to which the fusion protein binds.
  • reagents that specifically bind to a cell surface adhesion molecule may be chemicals or drugs.
  • selectins bind to carbohydrate determinants
  • both chemical and other non-protein reagents encompassing these carbohydrate determinants may be used to compete with the phsyiological ligand for binding to a selectin.
  • administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, infraspinal, infracistemal, intraperitoneal, infranasal, aerosol, by suppositories, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for infranasal formulations, in the form of powders, nasal drops, or aerosols. Methods well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences 18 th edition), ed.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for a combination of these anti- adhesion molecule reagents include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • treatment with a combination of anti-adhesion molecule reagents may be combined with more traditional therapies for the disease such as surgery, steroid therapy, or chemotherapy for cancer; and immunosuppressive therapies for tissue transplantation.
  • Certain autoimmune diseases target specific tissues.
  • the resulting symptoms of the disease are, hence, due to the malfunction of the targeted tissue.
  • IDM Insulin-Dependent Diabetes Mellitus
  • patients with rheumatoid arthritis exhibit arthritic symptoms due to the destruction of the joint cartilage and inflammation of the synovium by infiltrating leukocytes.
  • mice Although many autoimmune diseases result from unknown causes, animal models of these diseases often suggest the symptoms are due to infiltrating leukocytes.
  • Experimental Allergic Encephalomyelitis an animal model for the human disease, Multiple Sclerosis, is characterized by destruction of myelin basic protein by central nervous system (C ⁇ S) infiltrating leukocytes.
  • C ⁇ S central nervous system
  • a composition which can eliminate leukocytes infiltrating the targeted tissue can be initiated immediately and continued as required to maintain the absence of targeted tissue infiltrating leukocytes and attain a favorable outcome for the autoimmune disease.
  • Certain individuals have a greater propensity to develop certain autoimmune disease.
  • individuals bearing the HLA-DR4 haplotype have an increased susceptibility to rheumatoid arthritis.
  • patients with a predisposition to develop an autoimmune disease whether the predisposition is genetic, environmental, or both, may be screened for the presence of targeted tissue infiltrating leukocytes while still a-symptomatic.
  • treatment with the administration of a composition which can eliminate leukocytes infiltrating the targeted tissue can be initiated immediately, and continued as required to maintain the absence of targeted tissue infiltrating leukocytes.
  • Treatment to remove leukocytes which have infiltrated a transplanted graft is the presence of graft- infiltrating leukocytes.
  • treatment of the host with the adminisfration of a composition which can eliminate leukocytes infiltrating the graft can be initiated immediately, and continued, as required, to maintain the absence of graft infiltrating leukocytes and prevent graft rejection.
  • This treatment may be administered in conjunction with other therapies to prevent graft rejection, including immunosupressive agents such as cyclosporin and FK506.
  • immunosupressive agents such as cyclosporin and FK506.
  • leukocytes also infiltrate solid tumors.
  • TILs tumor-infiltrating leukocytes
  • cytotoxic T lymphocytes which have the capacity to specifically recognize and lyse cells from the tumor they have infiltrated.
  • the presence of these tumor-specific lymphocytes in the population tumor infiltrating leukocytes is part of the host defense against cancer. Identifying cell surface molecules, expressed either on the tumor cells or on the leukocytes which have infiltrated the tumor, can lead to the development of reagents which block the adhesive properties of these molecules. Such reagents find use as anti-cancer therapeutics.
  • the invention described herein also defines a method to identify cell surface molecules, expressed on either leukocytes or the tissues they invade (e.g., tumor cells where the tissue infiltrating leukocytes are TILs), which mediate the generation and maintenance of leukocytes which infiltrate tissues.
  • a pool of reagents capable of binding cell surface molecules may be administered to a mammal with a solid tumor in an attempt to reduce the number of leukocytes infiltrating the tumor.
  • a limited number of reagents capable of binding to cell surface molecules involved in the maintenance of tumor infiltrating leukocytes may be attained.
  • reagents can then be used to identify and clone cell surface molecules involved in the generation and maintenance of tumor infiltrating leukocytes. Strategies may then be employed to increase expression of these molecules on tumor cells, thereby increasing the number of tumor infiltrating leukocytes.
  • a similar strategy may be employed by administering a mammal transplanted with an allogeneic graft.
  • Reagents capable of binding to cell surface molecules involved in the maintenance of graft infiltrating leukocytes may be used to identify and clone cell surface molecules involved in the generation and maintenance of these leukocytes.
  • Strategies may then be employed to decrease and/or block expression of these molecules on graft cells, thereby enhancing graft survival by decreasing the number of graft infiltrating leukocytes.
  • Any of a variety of procedures may be utilized to clone cell surface molecules involved in the generation and maintenance of tissue infiltrating leukocytes using the reagents found to bind such cell surface molecules that were generated by the methods of this invention.
  • One such method entails analyzing shuttle vector libraries of cD ⁇ A inserts derived from the various cell types in the leukocyte infiltrate in tissue (e.g., graft cells or tumor cells), as well as from the tissue cells. Such an analysis may be conducted by fransfecting cells with the vector and then assaying for expression of a cell surface molecule involved in the generation and maintenance of tissue infiltrating leukocytes using as probes the reagents found to bind such cell surface molecules.
  • cD ⁇ As encoding cell surface molecules involved in the generation and maintenance of tissue infiltrating leukocytes are preferably identified using a modification of the procedure of Aruffo and Seed (Seed et al., Proc. ⁇ atl. Acad. Sci. USA 84:3365-33691987) to identify ligands of adhesion molecules.
  • cDNA libraries are prepared from cells which express the cell surface molecules which are involved in the generation and maintenance of tissue infiltrating leukocytes.
  • the cDNA libraries are prepared from the various cell types in the leukocyte infiltrate in tissue, as well as from the tissue cells.
  • transfect cells which do not normally bind the reagents which bind to cell surface molecules which mediate infiltration of tissue by leukocytes (such as COS or HeLa cells).
  • the transfected cells are introduced into a petri dish which has been previously coated with the reagents generated by the methods of the invention which specifically bind to cell surface molecules which are involved in the generation and maintenance of tissue infiltrating leukocytes.
  • the transfected cells containing sequences encoding cell surface molecules which mediate infiltration of tissues by leukocytes, and which express these ligands on their cell surfaces, will adhere to the surface of the petri dish. Non-adherent cells are washed away, and the adherent cells are then removed from the petri dish and cultured.
  • the cDNAs encoding the recombinant cell surface molecules which mediate infiltration of tissues by leukocytes in these cells are then removed and sequenced.
  • One such method for obtaining a gene sequence which encodes cell surface molecules which mediate infiltration of tissues by leukocytes is to use an oligonucleotide probe to screen a cDNA or genomic DNA library.
  • the cell surface molecules which mediate infiltration of tissues by leukocytes are purified, preferably using immunopurification procedures known in the art using the reagents known to bind to cell surface molecules which mediate infiltration of tissues by leukocytes.
  • the terminal amino acid sequences are determined using one of the methods known in the art.
  • cell surface molecules which are involved in the generation and maintenance of tissue infiltrating leukocytes are enzymatically cleaved, as with trypsin or Lys-C, peptides are purified, and the amino acid sequence of one of the internal fragments is determined.
  • an oligonucleotide probe is made based on the codon preference displayed by the organism, a degenerate probe is made based on all possible codon combinations, or a probe is made using a combination of codon preference and codon degeneracy. This probe is then used to screen either a genomic or cDNA library to identify sequences which hybridize to the probe.
  • the cloned genes obtained through the use of any of the methods described above, may be operably linked to an expression vector, and introduced into bacterial, or eukaryotic cells to produce the cell surface molecules which mediate infiltration of tissues by leukocytes. Techniques for such manipulations are disclosed in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.). CSH Press, 1989, and are well known in the art.
  • the authenticity of clones can be confirmed by expressing full length clones, for example, in COS cells, and testing for reactivity with the reagents capable of specifically binding to cell surface molecules which mediate infiltration of tissues by leukocytes.
  • a Fischer rat was immunized three times at 3-week intervals with 1 x 10 8 NLA-5 expressing SSC-NII cells, which are commercially available from the American Type Culture Collection (ATCC; Rockville, MD). Rats received immunization with SSC-NII cells via intraperitoneal (i.p.) injections, and were boosted 4 days before the fusion. Fusion of rat spleen cells with the mouse myeloma P3X63Ag8.653 (ATCC) was carried out with polyethylene glycol according to an established method (Gefter et al, Somatic Cell Genet. 3: 231- 236, 1977).
  • HAT hypoxanthine, aminopterine, and thymidine
  • SSC-NII cells (10 7 ) were 125 I surface labeled by lactoperoxidase- catalyzed reaction and lysed in 0.5% ⁇ onidet P-40 containing the protease inhibitors leupeptin, aprotinin, and phenylmethylsulphomyl fluoride. The lysates were pre-cleared with normal rat serum. Immunoprecipitation was carried out with supematants from positive hybridoma cultures, and solid-phase immobilized on protein A agarose. The immunoprecipitated material was eluted from the protein A agarose with SDS-Page sample buffer and analyzed by SDS-Page under non-reducing conditions, followed by visualization by autoradiography. Results
  • a murine NLA-5 specific positive hybridoma clone designated BMA5 was obtained after repeated limiting dilution of SSC-NII binding hybridoma cells.
  • the BMA5 antibody was isotyped as IgG2b/ ⁇ .
  • the non-obese diabetic (NOD) strain of mice develop insulitis at the age of 10 weeks.
  • NOD non-obese diabetic
  • T lymphocytes were harvested from spleens of Balb/c and NOD mice of various ages by standard procedures known in the art. Briefly, spleens were removed from sacrificed mice and minced in culture medium RPMI 1640.
  • Macrophage/monocyte cells were removed by adherence to a plastic surface
  • fibronectin was found to be optimal for cell binding.
  • Cells were labeled with the fluorogenic compound (2',7')-bis(carboxyethyl)- (5,6)-carboxyfluorescein (BCECF-AM) (Sigma, USA). The labeled cells were then washed and resuspended in RPMI/1%> BSA/1 mM MnCl 2 .
  • splenic T cells from 5 to 10 week old NOD mice showed a vastly enhanced fibronectin-binding ability when compared to age- matched Balb/c mice (Fig. IB).
  • This enhanced fibronectin-binding ability of splenic T cells from 5 to 10 week old NOD mice was apparent, even in the absence of PMA stimulation, although splenic T lymphocytes of mice of all ages from either strain showed enhanced fibronectin-binding ability following stimulation with PMA.
  • Adhesion of splenic NOD T cells to fibronectin is mediated by ⁇ 4 and 0.5 integrins such that adhesion was inhibited in the presence of monoclonal antibodies (mAbs) to both integrins
  • NLA-4 ⁇ 4
  • NLA-5 o_5
  • Splenic T cells were isolated as described in Example II from NOD mice of three age groups: non-diabetic 3-4 week old, insulitic 5-10 week old, and diabetic 11-25 week old. T cell adhesion to fibronectin was carried out as described in Example II, with the addition of either control rat antibody (NRIg), rat monoclonal (mAb) anti- 4 antibody (PS/2), rat monoclonal (mAb) anti- ⁇ 5 antibody (BMA5), or a combination of rat mAb anti- ⁇ 4 and rat mAb anti- 5 antibody (PS/2 + BMA5) at a concentration of 15 ⁇ g per ml.
  • the PS/2 antibody is commercially available from the ATCC.
  • the BMA5 antibody was generated as described in Example I.
  • the T cells were then equally divided, with half the population incubated with phycoerythrin labeled antibody directed toward the CD4 co-receptor and half the population incubated with phcoerythrin labeled antibody directed toward the CD8 co-receptor.
  • Each of the CD4 or CD8 stained population was then further divided into three, and each of the three groups stained with fluorescein labeled rat mAb directed towards either ⁇ 4 (NLA-4), ⁇ 5 (NLA-5), or the ⁇ l integrin chain (NLA- ⁇ l).
  • the rat monoclonal antibody directed toward the ⁇ l integrin chain binds to all the NLA molecules (e.g., NLA-1, -2, -3, -4, -5, and -6) present on the cell, and was generated according to the methods described in Example I, except that the resulting clone recognized all integrin molecules incorporating the ⁇ l chain. All six populations were analyzed by flow cytometry on a Becton-Dickinson® FACScan with the fluorescein binding cells measured on the gated population of phycoerythrin binding cells. The number of fluorescein bound cells was determined as a percentage of the total phycoerythrin bound cell population in the sample.
  • NLA molecules e.g., NLA-1, -2, -3, -4, -5, and -6
  • the CD4 + T cells from all age groups of NOD female mice showed higher levels of expression of NLA-4, NLA-5, and NLA- ⁇ l than CD4 + T cells from Balb/c female mice of the same age.
  • CD4 + T cells from 5-10 week old NOD female mice showed the highest expression of NLA-4, NLA-5, and NLA- ⁇ l compared to older or younger mice.
  • Figs. 3D, 3E, and 3F demonstrate that CD8 + T cells from all age groups of NOD female mice showed higher levels of expression of NLA-4, NLA-5, and NLA- ⁇ l than CD8 + T cells from Balb/c female mice of the same age, although this is less dramatic in mice of ages 3-4 weeks than in mice older than 5-10 weeks of age.
  • CD8 + T cells from NOD female mice aged 20 weeks or older showed the highest expression of NLA-4, NLA-5, and NLA- ⁇ l compared to both groups of younger NOD mice.
  • the treatment protocol involved injection of 100 ⁇ g intraperitoneally and 100 ⁇ g intravenously per mouse on both Day 0 and Day 2. Animals were sacrificed daily and the pancreas histologically examined and subjected to hematoxylin and eosin (H and E) staining as according to standard protocols.
  • the degree of insulitis was scored by number of pancreas infiltrating leukocytes which targeted the ⁇ islets of Langerhans, and was scored as follows: 0 indicated a normal number of ⁇ -islet targeting pancreas infiltrating leukocytes; 1 indicated a mild increase in the number of ⁇ -islet targeting pancreas infiltrating leukocytes (i.e., peripheral-islet leukocytes without islet infiltration); 2 indicated a moderate increase in the number of ⁇ -islet targeting pancreas infiltrating leukocytes (i.e., less than 25 % islet area infiltrated by leukocytes); 3 indicated a marked increase in the number of ⁇ -islet targeting pancreas infiltrating leukocytes (25 to 75 % islet area infiltrated); and 4 indicated a substantial increase in the number of ⁇ -islet targeting pancreas infiltrating leukocytes (i.e., greater than 75 % islet
  • mice receiving a combination of mAbs directed toward ⁇ 4 and ⁇ 5 integrins had a dramatically reduced msulitis score by Day 3 when compared to untreated insulitic NOD mice. This reduction lasted for up to 7 days.
  • Histopathological analysis, as shown on Fig. 5D revealed that injection of combination of mAbs directed toward ⁇ 4 and ⁇ 5 integrins resulted in the removal of pancreas infiltrating leukocytes. A similar removal was not seen in the pancreas of mice which had received control antibody (Fig. 5A), or antibody directed toward 0.4 or ⁇ 5 separately (Figs. 5B and 5C, respectively).
  • IDDM insulin-dependent diabetes mellitus
  • NOD mice with recent onset of IDDM were then treated with monoclonal antibodies (mAbs) at 200 ⁇ g i.p injection, and another 200 ⁇ g intravenous (i.v.) injection. After 48 hours, the antibody treatment was repeated. The mice were sacrificed 24 hours after the second antibody treatment and evaluated for the degree of leukocyte infilfrate (i.e., evaluated for an insulitic score).
  • mAbs monoclonal antibodies
  • Treatment groups were: untreated; treatment with an isotype matched control antibody; treatment with mAb PS/2 ( 4 integrin-specific); treatment with mAb (NRIg), treatment with mAb BMA5 ( ⁇ 5 integrin specific); or treatment with a combination of PS/2 and BMA5.
  • animals receiving treatment with a combination of mAbs PS/2 and BMA5 received an i.p. injection of 100 ⁇ g PS/2 and 100 ⁇ g BMA5 and an i.v. injection of 100 ⁇ g PS/2 and 100 ⁇ g BMA5.
  • the degree of insulitis was scored by number of pancreas infiltrating leukocytes which targeted the ⁇ islets of Langerhans, and was scored as follows: 0 indicated a normal number of ⁇ -islet targeting pancreas infiltrating leukocytes; 1 indicated a mild increase in the number of ⁇ - islet targeting pancreas infiltrating leukocytes (i.e., peripheral-islet leukocytes without islet infiltration); 2 indicated a moderate increase in the number of ⁇ - islet targeting pancreas infiltrating leukocytes (i.e., less than 25 % islet area infiltrated by leukocytes); 3 indicated a marked increase in the number of ⁇ - islet targeting pancreas infiltrating leukocytes (25 to 75 % islet area infiltrated); and 4 indicated a substantial increase in the number of ⁇ -islet targeting pancreas infilfrating leukocytes (i.e., greater than 75 % is
  • a combination of mAbs directed toward c.4 and KL integrins reduced the number of allograft infiltrating leukocytes and prolonged allograft survival
  • mice Eight week old C57BL/6 mice were heterotopically transplanted with allogeneic Balb/c mouse heart grafts on Day 0. Starting on the fourth day following the transplantation (Post-operative Day 4, or POD 4), transplanted mice received an intravenous (i.v.) injection of 200 ⁇ g per mouse per day of non-specific rat antibody, rat mAb directed toward ⁇ 4 (NLA-4) integrin, rat mAb directed toward o.L (LFA-1) integrin (commercially available from the
  • IL-2 dependent CTLL-2 cells (available from ATCC) were washed three times in IL-2 deficient media, resuspended at 5 x 10 4 cells per ml in IL-2 deficient media, and plated in triplicate onto microtiter plates. Serial dilutions of supernatant from the MLR were added to the CTLL-2 cells, and the cells were incubated at 37°C for 24 hours. The cells were then pulsed with 3 H-thymidine and returned to culture for an additional 6 hour incubation. Cell were then harvested onto filter mats. The level of inco ⁇ oration of radioactivity was then assessed by counting using a micro-beta counter.
  • Table II demonstrates that injection on POD 4, 5, and 7 of antibodies directed toward either ⁇ 4 or ⁇ L, when administrated individually, showed a higher mean graft survival time than rat IgG alone.
  • the group of mice showing the highest mean graft survival time received an injection of a combination of antibodies directed toward both ⁇ 4 and ⁇ L integrins on POD 4, 5, 7, 9, and 11.
  • continued administration of a combination of ⁇ 4 and ⁇ L antibodies can clear leukocytes which have infiltrated allograft tissue for up to 3 weeks.
  • MLR and production of IL-2 by the MLR cells were both significantly reduced in POD 8 splenocytes from transplanted C57BL/6 mice which had received injections of anti- ⁇ L antibody compared to splenocytes from transplanted mice receiving control antibodies.
  • treatment by injection of a combination of anti- ⁇ L and anti- ⁇ 4 mAbs resulted in MLR and IL-2 production at levels approximately equal to the levels observed from splenocytes from transplanted mice treated with control antibodies.
  • treatment of allograft recipients with a combination of anti- ⁇ L and anti- ⁇ 4 antibodies appears to be particularly useful in preventing graft rejection without reducing immune responsiveness.
  • anti- ⁇ 5 antibodies had not effect, alone or in combination with anti- ⁇ L and/or anti- ⁇ 4 antibodies, on allograft rejection, despite the beneficial effects of this antibody in reducing insulitis (see Example N above).
  • tissue e.g., the heart or the pancreas
  • different cell surface molecules are involves in the generation and maintenance of leukocytes that infiltrate that tissue.
  • EXAMPLE NIII A combination of mAbs directed toward ⁇ 4. ⁇ 5. and ⁇ 6 integrins reduces the number of tumor infiltrating leukocytes and enhances growth of the tumor Methods 10 5 B16F1 melanoma tumor cells are injected into the lateral tail veins of C57BL/6 mice.
  • mice are intravenously (i.v.) injected with 200 ⁇ g per mouse per day of non-specific rat antibody, or rat mAb directed toward either ⁇ 4 integrin, ⁇ 5 integrin, ⁇ 6 integrin (Fehlner-Gardiner et al, Allergy 51 : 650-656, 1996), or a combination of rat mAbs directed toward ⁇ 4, ⁇ 5, and ⁇ 6 integrins. Further injections are administered to all groups of mice on Day 25 and Day 27.
  • mice from the group of mice receiving a combination of rat mAbs directed against ⁇ 4, ⁇ 5, and ⁇ 6 integrins are administered two additional injections of the combination of rat mAbs on Day 29 and Day 31.
  • Animals from each of the groups are sacrificed on Day 25 and Day 29 for an in vitro mixed leukocyte reaction against irradiated B16F10 melanoma tumor cells, as well as histological examination of the lung. Animals from all groups are sacrificed on Day 40, and lung nodules are histologically examined.
  • mice freated with a combination of antibodies directed against ⁇ 4, ⁇ 5, and ⁇ 6 integrins is reduced compared to the number found in mice which had received each of the antibodies individually.
  • all antibody treated mice show a decreased number of leukocytes in lung nodules compared to untreated mice.

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Abstract

L'invention concerne une méthode qui permet de diminuer le nombre de leucocytes infiltrant les tissus. Cette méthode peut être utilisée pour diminuer le nombre de leucocytes qui infiltrent un greffon après une greffe ou bien un tissu cible chez un individu atteint d'une maladie auto-immune. Elle peut également être employée pour identifier des molécules de la surface cellulaire impliquées dans la production et la persistance des leucocytes infiltrant les tissus.
PCT/IB1998/000860 1997-05-09 1998-05-08 Methode permettant de chasser les leucocytes infiltres dans un tissu WO1998051345A2 (fr)

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