WO1990003400A1 - Molecules d'adhesion intercellulaire et leurs ligands de liaison - Google Patents

Molecules d'adhesion intercellulaire et leurs ligands de liaison Download PDF

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Publication number
WO1990003400A1
WO1990003400A1 PCT/US1989/004242 US8904242W WO9003400A1 WO 1990003400 A1 WO1990003400 A1 WO 1990003400A1 US 8904242 W US8904242 W US 8904242W WO 9003400 A1 WO9003400 A1 WO 9003400A1
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Prior art keywords
icam
antibody
binding
cells
lfa
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PCT/US1989/004242
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English (en)
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Timothy A. Springer
Robert Rothlein
Steven D. Marlin
Michael L. Dustin
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Dana-Farber Cancer Institute
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Priority to RU92016375A priority Critical patent/RU2126449C1/ru
Priority to KR1019900701105A priority patent/KR900701846A/ko
Publication of WO1990003400A1 publication Critical patent/WO1990003400A1/fr
Priority to FI902490A priority patent/FI102181B/fi
Priority to DK199001289A priority patent/DK175362B1/da
Priority to NO90902311A priority patent/NO902311L/no
Priority to FI981097A priority patent/FI107451B/fi

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • 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
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K16/2821—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 ICAM molecules, e.g. CD50, CD54, CD102
    • 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
    • C07K16/2839—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 integrin superfamily
    • C07K16/2845—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 integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to intercellular adhesion molecule such as ICAM-1 which are involved in the process through whic populations of lymphocytes recognize and adhere to cellular substrate so that they may migrate to sites of inflammation and interact wit cells during inflammatory reactions.
  • the present inventio additionally relates to ligand molecules capable of binding to suc intercellular adhesion molecules, to a screening assay for thes ligands, and to uses for the intercellular adhesion molecule, th ligand molecules, and the screening assay.
  • Leukocytes must be able to attach to cellular substrates in order t properly defend the host against foreign invaders such as bacteria o viruses.
  • An excellent review of the defense system is provided b Eisen, H.W., fin: Microbiology. 3rd Ed., Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418). They must be able to attach t endothelial cells so that they can migrate from circulation to sites o ongoing inflammation. Furthermore, they must attach to antigen- presenting cells so that a normal specific immune response can occur, and finally, they must attach to appropriate target cells so that lysis of virally-infected or tumor cells can occur.
  • leukocyte surface molecules involved in mediating suc attachments were identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T-cells (Davignon, D. e al., Proc. Nat!. Acad. Sci . USA 78:4535-4539 (1981)) and mouse splee cells (Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979)) were identified which bound to leukocyte surfaces and inhibited the attach ment related functions described above (Springer, T. et al . , Fed. Proc. 44:2660-2663 (1985)). The molecules identified by those antibodie were called Mac-1 and Lymphocyte Function-associated Antigen-1 (LFA-1).
  • Mac-1 is a heterodimer found on macrophages, granulocytes and larg granular lymphocytes.
  • LFA-1 is a heterodimer found on most lymphocyte (Springer, T.A., et al. Immunol. Rev. 68:111-135 (1982)).
  • pl50,95 which has a tissu distribution similar to Mac-1 play a role in cellular adhesio (Keizer, G. et al.. Eur. J. Immunol. 15:1142-1147 (1985)).
  • the above-described leukocyte molecules were found to be members o a related family of glycoproteins (Sanchez-Madrid, F. et al ., J. Exper. Med. 158:1785-1803 (1983); Keizer, G.D. et al.. Eur. J. Immunol. 15:1142-1147 (1985)).
  • This glycoprotein family is composed o heterodimers having one alpha chain and one beta chain. Although th alpha chain of each of the antigens differed from one another, the bet chain was found to be highly conserved (Sanchez-Madrid, F. et al . , .L Exper. Med. 158:1785-1803 (1983)).
  • the beta chain of the glycoprotei family (sometimes referred to as "CD18") was found to have a molecula weight of 95 kd whereas the alpha chains were found to vary from 150 k to 180 kd (Springer, T., Fed. Proc. 44:2660-2663 (1985)).
  • th alpha subunits of the membrane proteins do not share the extensi ho ology shared by the beta subunits, close analysis of the alp subunits of the glycoproteins has revealed that there are substanti similarities between them. Reviews of the similarities between t alpha and beta subunits of the LFA-1 related glycoproteins are provid by Sanchez-Madrid, F. et al.. (J. Exper. Med. 158:586-602 (1983); v Exoer. Med. 158:1785-1803 (19831).
  • lymphocytes to maintain the healt and viability of an animal requires that they be capable of adhering t other cells (such as endothelial cells). This adherence has been foun to require cell-cell contacts which involve specific receptor molecule present on the cell surface of the lymphocytes. These receptors enabl a lymphocyte to adhere to other lymphocytes or to endothelial, an other non-vascular cells.
  • the cell surface receptor molecules hav been found to be highly related to one another. Humans whos lymphocytes lack these cell surface receptor molecules exhibit chroni and recurring infections, as well as other clinical symptoms includin defective antibody responses.
  • lymphocyte adhesion is involved in the process through whic foreign tissue is identified and rejected, an understanding of thi process is of significant value in the fields of organ transplantation, tissue grafting, allergy and oncology.
  • the present invention relates to Intercellular Adhesion Molecule-1 (ICAM-1) as well as to its functional derivatives.
  • the invention additionally pertains to antibodies and fragments of antibodies capable of inhibiting the function of ICAM-1, and to other inhibitors of ICAM-1 function; and to assays capable of identifying such inhibitors.
  • the invention additionally includes diagnostic and therapeutic uses for all of the above-described molecules.
  • the invention includes the intercellular adhesion molecule ICAM-1 or its functional derivatives, which are substantially free of natural contaminants.
  • the invention further pertains to such molecules which are additionally capable of binding to a molecule present on the surface of a lymphocyte.
  • the invention further pertains to the intercellular adhesion molecule ICAM-1, and its derivatives which are detectably labeled.
  • the invention additionally includes a recombinant DNA molecule capable of expressing ICAM-1 or a functional derivative thereof.
  • the invention also includes a method for recovering ICAM-1 in substantially pure form which comprises the steps:
  • the invention additionally includes an antibody capable of bindin to a molecule selected from the group consisting of ICAM-1 and functional derivative of ICAM-1.
  • the invention also includes hybridoma cell capable of producing such an antibody.
  • the invention further includes a hybridoma cell capable of producing the monoclonal antibody R6-5-D6.
  • the invention further includes a method for producing a desired hybridoma cell that produces an antibody which is capable of binding to ICAM-1, which comprises:
  • the invention includes as well the hybridoma cell, and the antibody produced by the hybridoma cell, obtained by the above method.
  • the invention is also directed to a method of identifying a non- immunoglobulin antagonist of intercellular adhesion which comprises:
  • the invention is also directed toward a method for treating inflammation resulting from a response of the specific defense system in a mammalian subject which comprises providing to a subject in need of such treatment an amount of an anti-inflammatory agent sufficient to suppress the inflammation; wherein the anti-inflammatory agent is selected from the group consisting of: an antibody capable of binding to ICAM-1; a fragment of an antibody, the fragment being capable of binding to ICAM-1; ICAM-1; a functional derivative of ICAM-1; and a non-immunoglobulin antagonist of ICAM-1.
  • the invention further includes the above-described method of treating inflammation wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulin antagonist of ICAM-1 other than LFA-1.
  • the invention is also directed to a method of suppressing the metastasis of a hematopoietic tumor cell, the cell requiring a functional member of the LFA-1 family for migration, which method comprises providing to a patient in need of such treatment an amount of an anti-inflammatory agent sufficient to suppress the metastasis; wherein the anti-inflammatory agent is selected from the group consisting of: an antibody capable of binding to ICAM-1; a fragment o an antibody, the fragment being capable of binding to ICAM-1; ICAM-1; ICAM-1; a functional derivative of ICAM-1; and a non-immunoglobulin antagonist of ICAM-1.
  • the invention further includes the above-described method o suppressing the metastasis of a hematopoietic tumor cell, wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulin antagonist of ICAM-1 other than LFA-1.
  • the invention also includes a method of suppressing the growth of an ICAM-1-expressing tumor cell which comprises providing to a patient in need of such treatment an amount of a toxin sufficient to suppress the growth, the toxin being selected from the group consisting of a toxin- derivatized antibody capable of binding to ICAM-1; a toxin-derivatized fragment of an antibody, the fragment being capable of binding to ICAM- 1; a toxin-derivatized member of the LFA-1 family of molecules; and toxin-derivatized functional derivative of a member of the LFA-1 family of molecules.
  • the invention is also directed to a method of suppressing the growth of an LFA-1-expressing tumor cell which comprises providing to patient in need of such treatment an amount of toxin sufficient to suppress such growth, the toxin being selected from the group consisting of a toxin-derivatized ICAM-1; and a toxin-derivatize functional derivative of ICAM-1.
  • the invention is further directed toward a method of diagnosing th presence and location of an inflammation resulting from a response o the specific defense system in a mammalian subject suspected of havin the inflammation which comprises:
  • the invention additionally provides a method of diagnosing th presence and location of an inflammation resulting from a response o the specific defense system in a mammalian subject suspected of havin the inflammation which comprises:
  • the invention also pertains to a method of diagnosing the presenc and location of an ICAM-1-expressing tumor cell in a mammalian subjec suspected of having such a cell, which comprises:
  • the invention also pertains to a method of diagnosing the presenc and location of an ICAM-1-expressing tumor cell in a mammalian subjec suspected of having such a cell, which comprises:
  • the invention also pertains to a method of diagnosing the presence and location of a tumor cell which expresses a member of the LFA-1 family of molecules in a subject suspected of having such a cell, which comprises:
  • composition containing a detectably labeled binding ligand capable of binding to a member of the LFA-1 family of molecules, the ligand being selected from the group consisting of ICAM-1 and a functional derivative of ICAM-1, and
  • the invention also pertains to a method of diagnosing the presence and location of a tumor cell which expresses a member of the LFA-1 family of molecules in a subject suspected of having such a cell, which comprises:
  • the invention additionally includes a phamaceutical composition comprising:
  • an anti-inflammatory agent selected from the group consisting of: an antibody capable of binding to ICAM-1; a fragment of an antibody, the fragment being capable of binding to ICAM-1; ICAM-1; a functional derivative of ICAM-1; and a non-immunoglobulin antagonist of ICAM-1, and
  • Figure 1 shows in diagrammatic form the adhesion between a norma and an LFA-1 deficient cell.
  • Figure 2 shows in diagrammatic form the process of normal/norma cel'l adhesion.
  • Figure 3 shows the kinetics of cellular aggregation in the absenc (X) or presence of 50 ng/ml of PMA (0).
  • Figure 4 shows coaggregation between LFA-1 " and LFA-1 + cells
  • Carboxyfluorescein diacetate labeled EBV-transformed cells (10 4 ) a designated in the figure were mixed with 10 ⁇ unlabeled autologous cell (solid bars) or JY cells (open bars) in the presence of PMA. After 1. h the labeled cells, in aggregates or free, were enumerated using th qualitative assay of Example 2. The percentage of labeled cells i aggregates is shown. One representative experiment of two is shown.
  • Figure 5 shows the immunoprecipitation of ICAM-1 and LFA-1 from J cells.
  • Triton X-100 lysates of JY cells (lanes 1 and 2) or control lysis buffer (lanes 3 and 4) were immunoprecipitated with antibod capable of binding to ICAM-1 (lanes 1 and 3) or antibodies capable o binding to LFA-1 (lanes 2 and 4).
  • Panel A shows results under reducing conditions;
  • Panel B shows results obtained under non-reducing conditions.
  • Molecular weight standards were run in lane S.
  • Figure 6 shows the kinetics of IL-1 and gamma interferon effects on ICAM-1 expression on human dermal fibroblasts.
  • Human dermal fibroblasts were grown to a density of 8 x 10 4 cells/0.32 cm 2 well.
  • IL-1 (10 U/ml, closed circles) or recombinant gamma interferon (10 U/ml, open squares) was added, and at the indicated time, the assay was cooled to 4°C and an indirect binding assay was performed. The standard deviation did not exceed 10%.
  • Figure 7 shows the concentration dependence of IL-1 and gamma interferon effects on ICAM-1.
  • Human dermal fibroblasts were grown to a density of 8 x 10 4 cells/0.32 cm 2 / ell .
  • IL-2 open circle
  • recombinant human IL-1 open square
  • recombinant mouse IL-1 solid square
  • recombinant human gamma interferon solid circles
  • beta interferon open triangle
  • Figure 8 shows the nucleotide and amino acid sequence of ICAM-1 cONA.
  • the first ATG is at position 58.
  • Translated sequences corresponding to ICAM-1 tryptic peptides are underlined.
  • the hydrophobic putative signal peptide and transmembrane sequences have a bold underline.
  • N-linked glycosylation sites are boxed.
  • the polyadenylation signal AATAAA at position 2976 is over-lined.
  • the sequence shown is for the HL-60 cONA clone.
  • the endothelial cell cDNA was sequenced over most of its length and showed only minor differences.
  • Figure 9 shows the ICAM-1 homologous domains and relationship to the immunoglobulin supergene family.
  • A Alignment of 5 homologous domains (Pl-5). Two or more identical residues which aligned are boxed. Residues conserved 2 or more times in NCAM domains, as well as resides conserved in domains of the sets C2 and Cl were aligned with the ICAM-1 internal repeats. The location of the predicted ⁇ strands in the ICAM- 1 domain is marked with bars and lower case letters above the alignments, and the known location of J-strands in immunoglobulin C domains is marked with bars and capital letters below the alignment. The position of the putative disulfide bridge within ICAM-1 domains is indicated by S-S.
  • Figure 10 shows a diagrammatic comparison of the secondary structures of ICAM-1 and MAG.
  • Figure 11 shows LFA-1-positive EBV-transfor ed B-lymphoblastoid cells binding to ICAM-1 in planar membranes.
  • Figure 12 shows LFA-1-positive T lymphoblasts and T lymphoma cell bind to ICAM-1 in plastic-bound vesicles.
  • Figure 13 shows the inhibition of binding of JY B-lymphoblastoi cell binding to ICAM-1 in plastic-bound vesicles by pretreatment o cells or vesicles with monoclonal antibodies.
  • Figure 14 shows the effect of temperature on binding of T lymphoblasts to ICAM-1 in plastic-bound vesicles.
  • Figure 15 shows divalent cation requirements for binding of T ly phoblasts to ICAM-1 in plastic-bound vesicles.
  • Figure 16 shows the effect of anti-adhesion antibodies on th ability of peripheral blood mononuclear cells to proliferate i response to the recognition of the T-cell associated antigen 0KT3 "0KT3" indicates the addition of antigen.
  • Figure 17 shows the effect of anti-adhesion antibodies on th ability of peripheral blood mononuclear cells to proliferate i response to the recognition of the non-specific T-cell mitogen concanavalin A.
  • CONA indicates the addition of concanavalin A.
  • Figure 18 shows the effect of anti-adhesion antibodies on th ability of peripheral blood mononuclear cells to proliferate i response to the recognition of the keyhole limpet hemocyanin antigen The addition of keyhole limpet hemocyanin to the cells is indicated b "KLH.”
  • Figure 19 shows the effect of anti-adhesion antibodies on th ability of peripheral blood mononuclear cells to proliferate i response to the recognition of the tetanus toxoid antigen. Th addition of tetanus toxoid antigen to the cells is indicated by "AGN.”
  • Figure 20 shows the binding of monoclonal antibodies RR1/1, R6.5, LB2, and CL203 to ICAM-1 deletion mutants.
  • Figure 21 shows the binding of ICAM-1 deletion mutants to LFA-1.
  • Figure 22 shows the epitopes recognized by anti-ICAM-1 monoclonal antibodies RR1/1, R6.5, LB2, and CL203.
  • Figure 23 shows binding capacity of ICAM-1 domain 2 mutants to LFA- 1.
  • Figure 24 shows binding capacity of ICAM-1 domain 3 mutants to LFA- 1.
  • Figure 25 shows binding capacity of ICAM-1 domain 1 mutants to LFA- 1.
  • Figure 26 shows the alignment of ICAM amino-terminal domains.
  • One aspect of the present invention relates to the discovery of a natural binding ligand to LFA-1.
  • Molecules such as those of LFA-1 family, which are involved in the process of cellular adhesion are referred to as "adhesion molecules.”
  • the natural binding ligand of the present invention is designated "Intercellular Aadhesion Molecule-1" or "ICAM-1.”
  • ICAM-1 is a 76-97 Kd glycoprotein. ICAM-1 is not a heterodimer.
  • the present invention is directed toward ICAM-1 and its "functional derivatives.”
  • a “functional derivative” of ICAM-1 is a compound which posesses a biological activity (either functional or structural) that is substantially similar to a biological activity of ICAM-1.
  • the term “functional derivatives” is intended to include the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule such as ICAM-1 is meant to refer to any polypeptide subset of the molecule.
  • Fragments of ICAM-1 which have ICAM-1 activity and which are soluble (i.e not membrane bound) are especially preferred.
  • a "variant" of a molecule such as ICAM-1 is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof.
  • a molecule is said to be "substantially similar” to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules not found in the other, or if the sequence of amino acid residues is not identical.
  • an “analog” of a molecule such as ICAM-1 is meant to refer to a molecul substantially similar in function to either the entire molecule or to fragment thereof.
  • a molecule is said to be a "chemica derivative" of another molecule when it contains additional chemica moieties not normally a part of the molecule. Such moieties ma improve the molecule's solubility, absorption, biological half life etc. The moieties may alternatively decrease the toxicity of th molecule, eliminate or attenuate any undesirable side effect of th molecule, etc. Moieties capable of mediating such effects ar disclosed in Remington's Pharmaceutical Sciences (1980).
  • Toxin derivatized molecules constitute a special class of "chemical deriva tives.”
  • a "toxin-derivatized” molecule is a molecule (such as ICAM-1 or an antibody) which contains a toxin moiety. The binding of such molecule to a cell brings the toxin moiety into close proximity wit the cell and thereby promotes cell death.
  • Any suitable toxin moiet may be employed; however, it is preferable to employ toxins such as, for example, the ricin toxin, the diphtheria toxin, radioisotopic toxins, membrane-channel-forming toxins, etc. Procedures for coupling such moieties to a molecule are well known in the art.
  • an antigenic molecule such as ICAM-1, or members of the LFA-1 family of molecules are naturally expressed on the surfaces of lymphocytes.
  • the introduction of such cells into an appropriate animal, as by intraperitoneal injection, etc. will result in the production of antibodies capable of binding to ICAM-1 or members of the LFA-1 family of molecules.
  • the serum of such an animal may be removed and used as a source of polyclonal antibodies capable of binding these molecules. It is, however, preferable to remove splenocytes from such animals, to fuse such spleen cells with a myeloma cell line and to permit such fusion cells to form a hybridoma cell which secretes monoclonal antibodies capable of binding ICAM-1 or members of the LFA-1 family of molecules.
  • the hybridoma cells obtained in the manner described above may be screened by a variety of methods to identify desired hybridoma cells that secrete antibody capable of binding either to ICAM-1 or to members of the LFA-1 family of molecules.
  • such molecules are identified by their ability to inhibit the aggregation of Epstein-Barr virus-transformed cells.
  • Antibodies capable of inhibiting such aggregation are then further screened to determine whether they inhibit such aggregation by binding to ICAM-1, or to a member of the LFA-1 family of molecules. Any means capable of distinguishing ICAM-1 from the LFA-1 family of molecules may be employed in such a screen.
  • the antigen bound by the antibody may be analyzed as by immunoprecipitation and polyacrylamide gel electrophoresis. If the bound antigen is a member of the LFA-1 family of molecules then the immunoprecipitated antigen will be found to be a dimer, whereas if the bound antigen is ICAM-1 a single molecular weight species will have been immunoprecipitated. Alternatively, it is possible to distinguish between those antibodies which bind to members of the LFA-1 family of molecules from those which bind ICAM-1 by screening for the ability of the antibody to bind to cells such as granulocytes, which express LFA- 1, but not ICAM-1.
  • an antibody (known to inhibit cellular aggregation) to bind to granulocytes indicates that the antibody is capable of binding LFA-1. The absence of such binding is indicative of an antibody capable of recognizing ICAM-1.
  • the ability of an antibody to bind to a cell such as a granulocyte may be detected by means commonly employed by those of ordinary skill. Such means include im unoassays, cellular agglutination, filter binding studies, antibody precipitation, etc.
  • the anti-aggregation antibodies of the present invention may alternatively be identified by measuring their ability to differen ⁇ tially bind to cells which express ICAM-1 (such as activated endothelial cells), and their inability to bind to cells which fail to express ICAM-1.
  • ICAM-1 such as activated endothelial cells
  • the above assays may be modified, or performed in a different sequential order to provide a variety of potential screening assays, each of which is capable of identifying and discriminating between antibodies capable of binding to ICAM-1 versus members of the LFA- family of molecules.
  • the anti-inflammatory agents of the present invention may b obtained by natural processes (such as, for example, by inducing a animal, plant, fungi, bacteria, etc., to produce a non-immunoglobuli antagonist of ICAM-1, or by inducing an animal to produce polyclona antibodies capable of binding to ICAM-1); by synthetic methods (suc as, for example, by using the Merrifield method for synthesizin polypeptides to synthesize ICAM-1, functional derivatives of ICAM-1, o protein antagonists of ICAM-1 (either immunoglobulin or non immunoglobulin)); by hybridoma technology (such as, for example, t produce monoclonal antibodies capable of binding to ICAM-1); or b recombinant technology (such as, for example, to produce the anti- inflammatory agents of the present invention in diverse hosts (i.e., yeast, bacteria, fungi, cultured mammalian cells, etc.), or fro recombinant plasmids or viral vectors).
  • Epstein-Barr virus-transformed cells exhibit aggregation. This aggregation can be enhanced in the presence of phorbol esters. Such homotypic aggregation (i.e., aggregation involving only one cell type) was found to be blocked by anti-LFA-1 antibodies (Rothlein, R. et al .. J. Exper. Med. 163:1132-1149 (1986)), which reference is incorporated herein by reference). Thus, the extent of LFA-1-dependent binding may be determined by assessing the extent of spontaneous or phorbol ester- dependent aggregate formation.
  • Epstein-Barr virus-transformed cells may be employed in such an assay as long as the cells are capable of expressing the LFA-1 receptor molecule.
  • Such cells may be prepared according to the technique of Springer, T.A. et al .. J. Exper. Med. 160:1901-1918 (1984), which reference is herein incorporated by reference. Although any such cell may be employed in the LFA-1 dependent binding assay of the present invention, it is preferable to employ cells of the JY cell line (Terhost, CT.
  • the cells may be cultivated in any suitable culture medium; however, it is most preferable to culture the cells in RMPI 1640 culture medium supplemented with 10% fetal calf serum and 50 ⁇ g/ml gentamycin (Gibco Laboratories, NY).
  • the cells should be cultured under conditions suitable for mammalian cell proliferation (i.e., at a temperature of generally 37 ⁇ C, in an atmosphere of 5% CO2, at a relative humidity of 95%, etc.).
  • LFA-1 receptor molecules Human individuals have been identified whose lymphocytes lack the family of LFA-1 receptor molecules (Anderson, O.C. et al .. Fed. Proc. 44:2671-2677 (1985); Anderson, D.C. et al.. J. Infect. Pis. 152:668-689 (1985)). Such individuals are said to suffer from Leukocyte Adhesion Peficiency (LAP). EBV-transformed cells of such individuals fail to aggregate either spontaneously or in the presence of phorbol esters in the above-described aggregation assay. When such cells are mixed with LFA-1-expressing cells aggregation was observed (Rothlein, R. et al .. J. Exoer. Med.
  • the novel intercellular adhesion molecule ICAM-1 was first identified and partially characterized according to the procedure of Rothlein, R. et al . (J. Immunol. 137:1270-1274 (1986)), which reference is herein incorporated by reference.
  • monoclonal antibodies were prepared from spleen cells of mice immunized with cells from individuals genetically deficient in LFA-1 expression. Resultant antibodies were screened for their ability to inhibit the aggregation of LFA-1-expressing cells ( Figure 2).
  • the ICAM- 1 molecule mice were immunized with EBV-transformed B cells from LAP patients which do not express the LFA-1 antigen.
  • the spleen, cells from these animals were subsequently removed, fused with myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • EBV- transformed B cells from normal individuals which express LFA-1 were then incubated in the presence of the monoclonal antibody of the hybridoma cell in order to identify any monoclonal antibody which was capable of inhibiting the phorbol ester mediated, LFA-1 dependent, spontaneous aggregation of the EBV-transformed B cells. Since the hybridoma cells were derived from cells which had never encountered the LFA-1 antigen no monoclonal antibody to LFA-1 was produced.
  • any antibody found to inhibit aggregation must be capable of binding to an antigen that, although not LFA-1, participated in the LFA-1 adhesion process.
  • any method of obtaining such monoclonal antibodies may be employed, it is preferable to obtain ICAM-1-binding monoclonal antibodies by immunizing BALB/C mice using the routes and schedules described by Rothlein, R. et al . (J. Immunol. 137:1270-1274 (1986)) with Epstein-Barr virus-transformed peripheral blood mononuclear cells from an LFA-1-deficient individuals. Such cells are disclosed by Springer, T.A., et al.. (J. Exoer. Med. 160:1901-1918 (1984)).
  • mice are immunized with either EBV- transformed B cells which express both ICAM-1 and LFA-1 or more preferably with TNF-activated endothelial cells which express ICAM-1 but not LFA-1.
  • EBV- transformed B cells which express both ICAM-1 and LFA-1
  • TNF-activated endothelial cells which express ICAM-1 but not LFA-1.
  • a Balb/C mouse was sequen ⁇ tially immunized with JY cells and with differentiated U937 cells (ATCC CRL-1593). The spleen cells from such animals are removed, fused with myeloma cells and permitted to develop into antibody-producing hybridoma cells.
  • the antibodies are screened for their ability to inhibit the LFA-1 dependent, phorbol ester induced aggregation of an EBV transformed cell line, such as JY cells, that expresses both the LFA-1 receptor and ICAM-1.
  • an EBV transformed cell line such as JY cells
  • antibodies capable of inhibiting such aggregation are then tested for their ability to inhibit the phorbol ester induced aggregation of a cell line, such as SKW3 (Oustin, M., et al., J. Exoer. Med.
  • antibodies that are capable of binding to ICAM-1 may be identified by screening for anti ⁇ bodies which are capable of inhibiting the LFA-1 dependent aggregation of LFA-expression cells (such as JY cells) but are incapable of binding to cells that express LFA-1 but little or no ICAM-1 (such as normal granulocytes) or are capable of binding to cells that express ICAM-1 but not LFA-1 (such as TNF-activated endothelial cells).
  • LFA-1 dependent aggregation of LFA-expression cells such as JY cells
  • ICAM-1 such as normal granulocytes
  • Another alternative is to immunoprecipitate from cells expressing ICAM-1, LFA 1, or both, using antibodies that inhibit the LFA-1 dependen aggregation of cells, such as JY cells, and through SPS-PAGE or a equivalent method determine some molecular characteristic of th molecule precipitated with the antibody. If the characteristic is th same as that of ICAM-1 then the antibody can be assumed to be an anti- ICAM-1 antibody.
  • the ICAM-1 cell surface molecule was purified, and characterized.
  • ICAM-1 was purified from human cells or tissue using monoclonal antibody affinity chromatography.
  • a monoclonal antibody reactive with ICAM-1 is coupled to an inert column matrix. Any method of accomplishing such coupling may be employed; however, it is preferable to use the method of Oettgen, H.C. et al .. J. Bio! . Chem. 259:12034 (1984)).
  • Oettgen H.C. et al .. J. Bio! . Chem. 259:12034 (1984)
  • the bound ICAM-1 molecules may be eluted from the column.
  • any suitable matrix can be employed, it is preferable to employ sepharose (Pharmacia) as the matrix material.
  • sepharose Pharmacia
  • the formation of column matrices, and their use in protein purification are well known in the art.
  • the above- described assays may be used to identify compounds capable of attenuating or inhibiting the rate or extent of cellular adhesion.
  • ICAM-1 is a cell surface glycoprotein expressed on non- hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal centered B cells and dendritic cells in tonsils, lymph nodes, and Peyer's patches.
  • ICAM-1 is highly expressed on vascular endothelial cells in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood lymphocytes.
  • Phorbol ester-stimulated differentiation of some myelomonocytic cell lines greatly increases ICAM-1 expression.
  • ICAM-1 is preferentially expressed at sites of inflammation, and is not generally expressed by quiescent cells.
  • ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either interleukin 1 or gamma interferon at levels of 10 U/ml over a period of 4 or 10 hours, respectively. The induction is dependent on protein and mRNA synthesis and is reversible.
  • ICAM-1 displays molecular weight heterogeneity in different cell types with a molecular weight of 97 kd on fibroblasts, 114 kd on the myelomonocytic cell line U937, and 90 kd on the B lymphoblastoid cell JY.
  • ICAM-1 biosynthesis has been found to involve an approximately 73 kd intracellular precursor.
  • the non-N-glycosylated form resulting from tunicamycin treatment (which inhibits glycosylation) has a molecular weight of 55 kd.
  • ICAM-1 isolated from phorbol ester stimulated U937 cells or from fibroblast cells yields an identical major product having a molecular weight of 60 kd after chemical deglycosylation.
  • ICAM-1 monoclonal antibodies interfere with the adhesion of phytohemagglutinin blasts to LFA-1 deficient cell lines.
  • Pretreatment of fibroblasts, but not lymphocytes, with monoclonal antibodies capable of binding ICAM-1 inhibits lymphocyte-fibroblast adhesion.
  • Pretreatment of lymphocytes, but not fibroblasts, with antibodies against LFA-1 has also been found to inhibit lymphocyte-fibroblast adhesion.
  • ICAM-1 is, thus, the binding ligand of the CO 18 complex on leukocytes. It is inducible on fibroblasts and endothelial cells vn vitro by inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor in a time frame consistent with the infiltration of lymphocytes into inflammatory lesions in vivo (Pustin, M.L., et. al.. J. Immunol 137:245-254. (1986); Prober, J.S., et. al.. J. Immunol 137:1893-1896, (1986)).
  • inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor
  • ICAM-1 is expressed on non- hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, other epithelial cells, and fibroblasts and on hematopoietic cells such as tissue macophages, mitogen-stimulated T lymphocyte blasts, and germinal center B-cells and dendritic cells in tonsils, lymph nodes and Peyer's patches (Dustin, M.L., et. al.. J Immunol 132:245-254, (1986)).
  • ICAM-1 is expressed on keratinocytes in benign inflammatory lesions such as allergic eczema, lichen planus, exanthema, urticaria and bullous diseases.
  • ICAM-1 is present on keratinocytes from biopsies of skin lesions from various dermatological disorders and ICAM-1 expression is induced on lesions from allergic patch tests while keratinocytes from toxic patch test lesions failed to express ICAM-1.
  • ICAM-1 is, therefore, a cellular substrate to which lymphocytes can attach, so that the lymphocytes may migrate to sites of inflammation and/or carry out various effector functions contributing to this inflammation. Such functions include the production of antibody, lysis of virally infected target cells, etc.
  • the term "inflammation,” as used herein, is meant to include reactions of the specific and non ⁇ specific defense systems.
  • the term "specific defense system” is intended to refer to that component of the immune system that reacts to the presence of specific antigens. Inflammation is said to result from a response of the specific defense system if the inflammation is caused by, mediated by, or associated with a reaction of the specific defense system.
  • inflammation resulting from a response of the specific defense system examples include the response to antigens such as rubella virus, autoimmune diseases, delayed type hypersensitivity response mediated by T-cells (as seen, for example in individuals who test "positive” in the Mantaux test), etc.
  • antigens such as rubella virus, autoimmune diseases, delayed type hypersensitivity response mediated by T-cells (as seen, for example in individuals who test "positive” in the Mantaux test), etc.
  • a "non-specific defense system reaction” is a response mediated by leukocytes incapable of im unological memory. Such cells include granulocytes and macrophages. As used herein, inflammation is said to result from a response of the non-specific defense system, if the inflammation is caused by, mediated by, or associated with a reaction of the non-specific defense system.
  • inflammation which result, at least in part, from a reaction of the non-specific defense system include inflammation associated with conditions such as: adult respiratory distress syndrome (ARDS) or multiple organ injury syndromes secondary to septicemia or trauma; reperfusion injury of myocardial or other tissues; acute glomerulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory disorders; thermal injury; hemodialysis; leukapheresis; ulcerative colitis; Crohn's disease; necrotizing enterocol tis; granulocyte trans ⁇ fusion associated syndromes; and cytokine- nduced toxicity.
  • ARDS adult respiratory distress syndrome
  • multiple organ injury syndromes secondary to septicemia or trauma reperfusion injury of myocardial or other tissues
  • acute glomerulonephritis reactive arthritis
  • dermatoses with acute inflammatory components acute purulent meningitis or other central nervous system inflammatory disorders
  • thermal injury hemodialysis
  • ICAM-1 functional derivatives and especially such derivatives which comprise fragments or mutant variants of ICAM-1 which possess domains 1, 2 and 3 can be used in the treatment or therapy of such reactions of the non-specific defense system. More preferred for such treatment or therapy are ICAM- 1 fragments or mutant variants which contain domain 2 of ICAM-1. Most preferred for such treatment or therapy are ICAM-1 fragments or mutant variants which contain domain 1 of ICAM-1.
  • the present invention contemplates the use of ICAM-1 and its functional derivatives in the treatment of asthma.
  • ICAM-1 gene Any of a variety of procedures may be used to clone the ICAM-1 gene.
  • One such method entails analyzing a shuttle vector library of cDNA inserts (derived from an ICAM-1 expressing cell) for the presence of an insert which contains the ICAM-1 gene. Such an analysis may be conducted by transfecting cells with the vector and then assaying for ICAM-1 expression.
  • the preferred method for cloning this gene entails determining the amino acid sequence of the ICAM-1 molecule.
  • ICAM-1 protein may be purified and analyzed by automated sequenators. Alternatively, the molecule may be fragmente as with cyanogen bromide, or with proteases such as papain chymotrypsin or trypsin (Oike, Y.
  • amino acid residues in a peptide is designate herein either through the use of their commonly employed 3-lette designations or by their single-letter designations. A listing o these 3-letter and 1-letter designations may be found in textbooks suc as Biochemistry. Lehninger, A., Worth Publishers, New York, NY (1970). When such a sequence is listed vertically, the amino terminal residu is intended to be at the top of the list, and the carboxy terminal residue of the peptide Is intended to be at the bottom of the list. Similarly, when listed horizontally, the amino terminus is intended to be on the left end whereas the carboxy terminus is intended to be at the right end.
  • the residues of amino acids in a peptide may be separated by hyphens. Such hyphens are intended solely to facilitate the presentation of a sequence. As a purely illustrative example, the amino acid sequence designated:
  • -Gly-Ala-Ser-Phe- indicates that an Ala residue is linked to the carboxy group of Gly, and that a Ser residue is linked to the carboxy group of the Ala residue and to the amino group of a Phe residue.
  • the designation further indicates that the amino acid sequence contains the tetrapeptide Gly-Ala-Ser-Phe. The designation is not intended to limit the amino acid sequence to this one tetrapeptide, but is intended to include (1) the tetrapeptide having one or more amino acid residues linked to either its amino or carboxy end, (2) the tetrapeptide having one or more amino acid residues linked to both its amino and its carboxy ends, (3) the tetrapeptide having no additional amino acid residues.
  • the DNA sequences capable of encoding them are examined. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene. 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356- 357).
  • the peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
  • amino acid sequences may be encoded by only a single oligonucleotide, frequently the amino acid sequence can be encoded by any of a set of similar oligonucleotides.
  • all of the members of the set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same* nucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains a nucleotide sequence that is identical to the nucleotide sequence of this gene.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
  • oligonucleotide or set of oligonucleotides which have a nucleotide sequence that is complementary to the oligonucleotide sequence or set of sequences that is capable of encoding the peptide fragment.
  • a suitable oligonucleotide, or set of oligonucleotides which is capable of encoding a fragment of the ICAM-1 gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the above-described procedure), synthesized, and hybridized, by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from human cells which are capable of expressing ICAM-1 gene sequences. Techniques of nucleic acid hybridization are disclosed by Maniatis, T. et al . , In: Molecular Cloning, a Laboratory Manual, Coldspring Harbor, NY (1982), and by
  • the source of DNA or cDNA used will preferably have been enriched for ICAM-1 sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells cultured under conditions which induce ICAM-1 synthesis (such as U937 grown in the presence of phorbol esters, etc.).
  • a library of expression vectors is prepared by cloning DNA or, more preferably cDNA, from a cell capable of expressing ICAM-1 into an expression vector.
  • the library is then screened for members capable of expressing a protein which binds to anti-ICAM-1 antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as ICAM-1 or fragments of ICAM-1.
  • the cloned ICAM-1 gene obtained through the methods described above, may be operably linked to an expression vector, and introduced into bacterial, or eukaryotic cells to produce ICAM-1 protein. Techniques for such manipulations are disclosed by Maniatis, T. et al .. supra, and are well known in the art. D. Uses of Assays of LFA-1 Dependent Aggregation
  • the above-described assay capable of measuring LFA-1 dependent aggregation, may be employed to identify agents which act as antagonists to inhibit the extent of LFA-1 dependent aggregation.
  • agents which act as antagonists to inhibit the extent of LFA-1 dependent aggregation.
  • Such antagonists may act by impairing the ability of LFA-1 or of ICAM-1 to mediate aggregation.
  • agents include immunoglobulins such as an antibody capable of binding to either LFA-1 or ICAM-1.
  • non-immunoglobulin (i.e., chemical) agents may be examined, using the above-described assay, to determine whether they are antagonists of LFA-1 aggregation.
  • Monoclonal antibodies to members of the CD 18 complex inhibit many adhesion dependent functions of leukocytes including binding to endothelium (Haskard, D., et al.. J. Immunol. 137:2901-2906 (1986)), homotypic adhesions (Rothlein, R., et al .. J. Exp. Med. 163:1132-1149 (1986)), antigen and mitogen induced proliferation of lymphocytes (Davignon, D., et al .. Proc. Nat! . Acad. Sci.. USA 78:4535-4539 (1981)), antibody formation (Fischer, A., et al.. J. Immunol.
  • lymphocytes are capable o continually monitoring an animal for the presence of foreign antigens
  • lymphocytes are normally desirable, they are also th cause of organ transplant rejection, tissue graft rejection and man autoimmune diseases.
  • any means capable of attenuating o inhibiting cellular adhesion would be highly desirable in recipients o organ transplants, tissue grafts or autoimmune patients.
  • Monoclonal antibodies capable of binding to ICAM-1 are highl suitable as anti-inflammatory agents in a mammalian subject
  • such agents differ from general anti-inflammatory agent in that they are capable of selectively inhibiting adhesion, and do no offer other side effects such as nephrotoxicity which are found wit conventional agents.
  • Monoclonal antibodies capable of binding to ICAM 1 can therefore be used to prevent organ or tissue rejection, or modif autoimmune responses without the fear of such side effects, in th • mammalian subject.
  • monoclonal antibodies capable of recognizin ICAM-1 may permit one to perform organ transplants even betwee individuals having HLA mismatch.
  • a composition containing a monoclonal antibody against ICAM-1 may be administered to a pati ⁇ nt experiencing delayed type hyper ⁇ sensitivity reaction.
  • such compositions might be provided to a individual who had been in contact with antigens such as poison ivy, poison oak, etc.
  • the monoclonal antibody capable of binding to ICAM-1 is administered to a patient in conjunction with an antigen in order to prevent a subsequent inflammatory reaction.
  • the additional administration of an antigen with an ICAM-1-binding monoclonal antibody may temporarily tolerize an individual to subsequent presentation of that antigen.
  • ICAM-1 Since LAD patients that lack LFA-1 do not mount an inflammatory response, it is believed that antagonism of LFA-1's natural ligand. ICAM-1, will also inhibit an inflammatory response.
  • the ability of antibodies against ICAM-1 to inhibit inflammation provides the basis for their therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes Reynaud's syndrome, rheumatoid arthritis, etc.
  • EAE experimental allergic encephalomyelitis
  • Such antibodies may also be employed as a therapy in the treatment of psoriasis.
  • the monoclonal antibodies capable of binding to ICAM-1 may be employed in the treatment of those diseases currently treatable through steroid therapy.
  • monoclonal antibodies capable of binding to ICAM-1 may be employed as a means of imaging or visualizing the sites of infection and inflammation in a patient.
  • the monoclonal antibodies are detectably labeled, through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc. Procedures for accomplishing such labeling are well known to the art.
  • Clinical application of antibodies in diagnostic imaging are reviewed by Grossman, H.B., Urol . Clin. North Amer. 13:465-474 (1986)), Unger, E.C. et al.. Invest. Radio!. 20:693-700 (1985)), and Khaw, B.A. et al.. Science 209:295-297 (1980)).
  • the presence of inflammation may also be detected through the use of binding ligands, such as mRNA, cDNA, or DNA which bind to ICAM-1 gene sequences, or to ICAM-1 mRNA sequences, of cells which express ICAM-1.
  • binding ligands such as mRNA, cDNA, or DNA which bind to ICAM-1 gene sequences, or to ICAM-1 mRNA sequences, of cells which express ICAM-1.
  • the detection of foci of such detectably labeled antibodies is indicative of a site of inflammation or tumor development.
  • this examination for inflammation is done by removing samples of tissue or blood and incubating such samples in the presence of the detectably labeled antibodies.
  • this technique is done in a non-invasive manner through the use of magnetic imaging, fluorography, etc.
  • Such a diagnostic test may be employed in monitoring organ transplant recipients for early signs of potential tissue rejection.
  • Such assays may also be conducted in efforts to determine an individual's predilection to rheumatoid arthritis or other chronic inflammatory diseases.
  • Immune responses to therapeutic or diagnostic agents such as, for example, bovine insulin, interferon, tissue-type plasminogen activator or murine monoclonal antibodies substantially impair the therapeutic or diagnostic value of such agents, and can, in fact, causes diseases such as serum sickness.
  • therapeutic or diagnostic agents such as, for example, bovine insulin, interferon, tissue-type plasminogen activator or murine monoclonal antibodies
  • Such a situation can be remedied through the use of the antibodies of the present invention.
  • such antibodies would be administered in combination with the therapeutic or diagnostic agent.
  • the addition of the antibodies prevents the recipient from recognizing the agent, and therefore prevents the recipient from initiating an immune response against it.
  • the absence of such an immune response results in the ability of the patient to receive additional administrations of the therapeutic or diagnostic agent.
  • ICAM-1 is a binding partner of LFA-1.
  • ICAM-1 or its functional derivatives may be employed interchangeably with antibodies capable of binding to LFA-1 in the treatment of disease.
  • such molecules may be employed to inhibit inflamma ⁇ tion, organ rejection, graft rejection, etc.
  • ICAM-1, or its functional derivatives may be used in the same manner as anti-ICAM antibodies to decrease the immunogenicity of therapeutic or diagnostic agents.
  • ICAM-1 may be used to block the metastasis or proliferation of tumor cells which express either ICAM-1 or LFA-1 on their surfaces.
  • a variety of methods may be used to accomplish such a goal. For example, the migration of hematopoietic cells requires LFA-l-ICAM-1 binding. Antagonists of such binding therefore suppress this migration and block the metastasis of tumor cells of leukocyte lineage.
  • toxin-derivatized molecules capable of binding either ICAM-1 or a member of the LFA-1 family of molecules, may be administered to a patient. When such toxin-derivatized molecules bind to tumor cells that express ICAM-1 or a member of the LFA-1 family of molecules, the presence of the toxin kills the tumor cell thereby inhibiting the proliferation of the tumor.
  • ICAM-1-dependent adhesion can be inhibited by non-immunoglobulin antagonists which are capable of binding to either ICAM-1 or to LFA-1.
  • a non-immunoglobulin antagonist of ICAM-1 is LFA-1.
  • An example of a non-immunoglobulin antagonist which binds to LFA-1 is ICAM-1.
  • additional non- immunoglobulin antagonists can be identified and purified.
  • Non- immunoglobulin antagonists of ICAM-1 dependent adhesion may be used for the same purpose as antibodies to LFA-1 or antibodies to ICAM-1.
  • the therapeutic effects of ICAM-1 may be obtained by providing to a patient the entire ICAM-1 molecule, or any therapeutically active peptide fragments thereof.
  • ICAM-1 and its functional derivatives may be obtained either synthetically, through the use of recombinant DNA technology, or by proteolysis.
  • the therapeutic advantages of ICAM-1 may be augmented through the use of functional derivatives of ICAM-1 possessing addi ⁇ tional amino acid residues added to enhance coupling to carrier or to enhance the activity of the ICAM-1.
  • the scope of the present invention is further intended to include functional derivatives of ICAM-1 which lack certain amino acid residues, or which contain altered amino acid residues, so long as such derivatives exhibit the capacity to affect cellular adhesion.
  • Both the antibodies of the present invention and the ICAM-1 molecule disclosed herein are said to be "substantially free of natural contaminants” if preparations which contain them are substantially free of materials with which these products are normally and naturally found.
  • the present invention extends to antibodies, and biologically active fragments thereof, (whether polyclonal or monoclonal) which are capable of binding to ICAM-1.
  • Such antibodies may be produced either by an animal, or by tissue culture, or recombinant DNA means.
  • the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of antibody which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient), although a lower or higher dosage may be administered.
  • ICAM-1 molecules or their functional derivatives When providing ICAM-1 molecules or their functional derivatives to a patient, it is preferable to administer such molecules in a dosage which also ranges from about 1 pg/kg to 10 mg/kg (body weight of patient) although a lower or higher dosage may also be administered.
  • the therapeutically effective dose can be lowered if the anti-ICAM-1 antibody is additionally administered with an anti-LFA-1 antibody.
  • one compound is said to be additionally administered with a second compound when the administration of the two compounds is in such proximity of time that both compounds can be detected at the same time in the patient's serum.
  • Both the antibody capable of binding to ICAM-1 and ICAM-1 itself ma be administered to patients intravenously, intramuscularly, subcutaneously, enterally, or parenterally.
  • the administration may be b continuous infusion, or by single or multiple boluses.
  • the anti-inflammatory agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to suppress inflammation.
  • An amount is said to be sufficient to "suppress" inflammation if the dosage, route of administration, etc. o the agent are sufficient to attenuate or prevent inflammation.
  • Anti-ICAM-1 antibody, or a fragment thereof may be administered either alone or in combination with one or more additional immunosuppressive agents (especially to a recipient of an organ o tissue transplant).
  • the administration of such compound(s) may be fo either a "prophylactic" or "therapeutic" purpose.
  • the immunosuppressive compound(s) are provided i advance of any inflammatory response or symptom (for example, prior to, at, or shortly after) the time of an organ or tissue transplant but in advance of any symptoms of organ rejection).
  • the prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent inflammatory response (such as, for example, rejection of a transplanted organ or tissue, etc.).
  • the immunosuppressive compound(s) When provided therapeutically, the immunosuppressive compound(s) is provided at (or shortly after) the onset of a symptom of actual inflammation (such as, for example, organ or tissue rejection).
  • the therapeutic administration of the compound(s) serves to attenuate any actual inflammation (such as, for example, the rejection of a transplanted organ or tissue).
  • the anti-inflammatory agents of the present invention may, thus, be provided either prior to the onset of inflammation (so as to suppress an anticipated inflammation) or after the initiation of inflammation.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
  • Such an agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • the antibody and ICAM-1 molecules of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • a pharmaceutically acceptable carrier vehicle e.g., a pharmaceutically acceptable carrier vehicle.
  • suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol , A., Ed., Mack, Easton PA (1980)).
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of anti-ICAM antibody or ICAM-1 molecule, or their functional derivatives, together with a suitable amount of carrier vehicle.
  • Control release preparations may be achieved through the use of polymers to complex or absorb anti-ICAM-1 antibody or ICAM-1, or their functional derivatives.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinyl- acetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate anti-ICAM-1 antibody or ICAM-1 molecules, or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly- (methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • coacervation techniques for example, hydroxymethylcellulose or gelatine-microcapsules and poly- (methylmethacylate) microcapsules, respectively
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • the EBV-transformed and hybridoma cells of the present invention were maintained in RMPI 1640 culture medium, supplemented with 20 mM L-glutamine, 50 ⁇ g/ml gentamicin, and 10% fetal bovine (or fetal calf) sera.
  • Cells were cultured at 37 ⁇ C in a 5% CO , 95% air humidity atmosphere.
  • EBV Epstein-Barr virus
  • 10 6 T cel depleted peripheral blood mononuclear cells/ml in RPMI 1640 maxim supplemented with 20% fetal calf serum (FCS), and 50 ⁇ g/ml gentamici were incubated for 16 hours with EBV-containing supernatant of B95- cells (Thorley-Lawson, D.A. et al.. J. Exper. Med. 146:495 (1977)) Cells in 0.2 ml aliquot were placed in 10 microtiter wells. Medium wa replaced with RPMI 1640 medium (supplemented with 20% fetal calf seru and 50 ⁇ g/ml gentamicin) until cell growth was noted.
  • FCS fetal calf serum
  • PHA Phytohemagglutini
  • aggregatio assays were employed. Cell lines used in such assays were washed tw times with RPMI 1640 medium containing 5 mM Hepes buffer (Sigm Chemical Co., St. Louis) and resuspended to a concentration of 2 x 10 cells/ml. Added to flat-bottomed, 96-well microtiter plates (No.
  • 3596; Costar, Cambridge, MA were 50 ⁇ l of appropriate monoclonal antibody supernatant or 50 ⁇ l of complete medium with or without purified monoclonal antibodies, 50 ⁇ l of complete medium containing 200 ng/ml of the phorbol ester phorbol myristate acetate (PMA) and 100 ⁇ l of cells at a concentration of 2 x 10 6 cells/ml in complete medium. This yielded a final concentration of 50 ng/ml PMA and 2 x 10 5 cells/well. Cells were allowed to settle spontaneously, and the degree of aggrega ⁇ tion was scored at various time points.
  • PMA phorbol ester phorbol myristate acetate
  • Scores ranged from 0 to 5+, where 0 indicated that essentially no cells were in clusters; 1+ indicated that less than 10% of the cells were in aggregates; 2+ indicated that less than 50% of the cells were aggregated; 3+ indicated that up to 100% of the cells were in small, loose clusters; 4+ indicated that up to 100% of the cells were aggregated in larger clusters; and 5+ indicated that 100% of the cells were in large, very compact aggregates.
  • reagents and cells were added to 5 ml polystyrene tubes in the same order as above. Tubes were placed in a rack on a gyratory shaker at 37 ⁇ C. After 1 hour at approximately 200 rp , 10 ⁇ l of the cell suspension was placed in a hemocytometer and the number of free cells was quantitated. Percent aggregation was determined by the following equation:
  • the number of input cells in the above formula is the number of cells per ml in a control tube containing only cells and complete medium that had not been incubated.
  • the number of free cells in the above equation equals the number of non-aggregated cells per ml from experimental tubes. The above procedures were described by Rothlein, R., et al .. jL. Exoer. Med. 163:1132-1149 (1986).
  • Example 2 The qualitative aggregation assay described in Example 2 was performed using the Epstein-Barr transformed cell line JY. Upon addition of PMA to the culture medium in the microtiter plates aggregation of cells was observed. Time lapse video recordings showe that the JY cells on the bottom of the microtiter wells were motile an exhibited active membrane ruffling and pseudopodia movement. Contac between the pseudopodia of neighboring cells often resulted in cell cell adherence. If adherence was sustained, the region of cell contac moved to the uropod. Contact could be maintained despite vigorous cel movements and the tugging of the cells in opposite directions. Th primary difference between PMA-treated and untreated cells appeared t be in the stability of these contacts once they were formed. With PMA clusters of cells developed, growing in size as additional cell adhered at their periphery.
  • EBV-transformed ly phoblastoid cells were prepared from patients in the manner described in Example 1. Such cells were screened against monoclonal antibodies capable of recognizing LFA-1 and the cells were found to be LFA-1 deficient.
  • Example 2 The qualitative aggregation assay described in Example 2 was employed, using the LFA-1 deficient cells described above. Such cells failed to spontaneously aggregate, even in the presence of PMA.
  • Example 5 The LFA-1 deficient cells of Example 5 were labeled with carboxyfluorescein diacetate (Patarroyo, M. et al., Cell. Immunol.
  • the labeled cells were mixed in a ratio of 1:10 with autologous or JY cells and the percentage of fluorescein-labeled cells in aggregates was determined according to the procedure of Rothlein, R. et al.. J. Exoer. Med. 163:1132-1149 (1986).
  • the LFA-1 deficient cells were found to be capable of coaggregating with LFA-1 expressing cells ( Figure 4).
  • Monoclonal antibodies capable of binding to ICAM-1 were isolated according to the method of Rothlein, R. et al ., J. Immunol. 137:1270 1274 (1986), which reference has been incorporated by reference herein.
  • 3 BALB/C mice were immunized intraperitoneally with EBV- transformed peripheral blood mononuclear cells from an LFA-1-deficien individual (Springer, T.A. et al .. J. Exoer. Med. 160:1901 (1984)).
  • Approximately 10 ⁇ cells in 1 ml RPMI 1640 medium was used for eac immunization.
  • mice were given an additional 10? cells in 0.15 m medium (intravenously).
  • Isolated spleen cells from the above-described animals were fuse with P3X73Ag8.653 myeloma cells at a ratio of 4:1 according to th protocol of Galfre, G. et al.. Nature 266:550 (1977). Aliquots of th resulting hybridoma cells were introduced into 96-well microtite plates. The hybridoma supernatants were screened for inhibition o aggregation, and one inhibitory hybridoma (of over 600 wells tested was cloned and subcloned by limiting dilution. This subclone wa designated RRl/1.1.1 (hereinafter designated "RR1/1").
  • Monoclonal antibody RR1/1 was consistently found to inhibit PMA stimulated aggregation of the LFA-1 expressing cell line JY.
  • the RR1/ monoclonal antibody inhibited aggregation equivalently, or slightl less than some monoclonal antibodies to the LFA-1 alpha or bet subunits.
  • control monoclonal antibody against HLA, whic is abundantly expressed on JY cells did not inhibit aggregation.
  • Th antigen bound by monoclonal antibody RR1/1 is defined as th intercellular adhesion molecule-1 (ICAM-1).
  • Lysates were centrifuged at 10,000 x g for 10 minutes and precleared with 50 ⁇ l of a 50% suspension of CNBr-activated, glycine-quenched Sepharose C1-4B for 1 hour at 4 * C.
  • One illiliter of lysate was immunoprecipitated with 20 ⁇ l of a 50% suspension of monoclonal antibody RR1/1 coupled to Sepharose C1-4B (1 mg/ml) overnight at 4 ⁇ C (Springer, T.A. et al.. J. Exoer. Med. 160:1901 (1984)).
  • Sepharose- bound monoclonal antibody was prepared using CNBr-activation of Sepharose CL-4B in carbonate buffer according to the- method of March, S.
  • Example 2 In order to determine the effect of monoclonal antibody RR1/1 on PHA-lymphoblast aggregation, the quantitative aggregation assay described in Example 2 was employed. Thus, T cell blast cells were stimulated for 4 days with PHA, thoroughly washed, then cultured for 6 days in the presence of IL-2 conditioned medium. PHA was found to be internalized during this 6-day culture, and did not contribute to the aggregation assay. In three different assays with different T cell blast preparations, ICAM-1 monoclonal antibodies consistently inhibited aggregation (Table 2). TABLE 2
  • ⁇ Percent inhibition relative to cells treated with PMA and X63 monoclonal antibody aggregation was measured 1 hr after the simultaneous addition of monoclonal antibody and PMA. Cells were shaken at 175 rpm. d A negative number indicates percent enhancement of aggregation. aggregation was measured 1 hr after the simultaneous addition of monoclonal antibody and PMA. Cells were pelleted at 200 x G for 1 min. incubated at 37°C for 15 min. gently resuspended, and shaken for 45 min. at 100 rpm. f Cells were pretreated with PMA for 4 hr at 37°C. After monoclonal antibody was added, the tubes were incubated at 37°C stationary for 20 min.
  • LFA-1 monoclonal antibodies were consistently more inhibitory than ICAM-1 monoclonal antibodies, whereas HLA-A, B and LFA-3 monoclonal antibodies were without effect. These results indicate that of the monoclonal antibodies tested, only those capable of binding to LFA-1 o ICAM-1 were capable of inhibiting cellular adhesion.
  • mice were immunized intraperitoneally (i.p.) with 0.5 mi of 2 x 10 7 JY cells in RPMI medium 103 days and 24 days prior t fusion. On day 4 and 3 prior to fusion, mice were immunized i.p. wit 10 7 cells of PMA differentiated U937 cells in 0.5 ml of RPMI medium.
  • U937 cells (ATCC CRL-1593) were differentiated by incubating them a 5 x 10 5 /ml in RPMI with 10% Fetal Bovine Serum, 1% gluta ine and 5 ⁇ g/ml gentamyin (complete medium) containing 2 ng/ml phorbol-12 myristate acetate (PMA) in a sterile polypropylene container. On th third day of this incubation, one-half of the volume of medium wa withdrawn and replaced with fresh complete medium containing PMA. O day 4, cells were removed, washed and prepared for immunization.
  • PMA phorbol-12 myristate acetate
  • Spleen cells from the immunized mice were fused with P3x63 Ag8-65 myeloma cells at a 4:1 ratio according to Galfre et al.. (Natur 266:550 (1977)). After the fusion, cells were plated in a 96 well fla bottomed microtiter plates at 10 ⁇ spleen cells/well.
  • a radioimmune assay was developed.
  • purified RRl/1 was iodinated using iodogen to a specific activity of 10 ⁇ Ci/ ⁇ g.
  • Endothelial cells were grown in 96 well plates and treated as described for each experiment. The plates were cooled at 4 ⁇ C by placing in a cold room for 0.5-1 hr, not immediately on ice. The onolayers were washed 3x with cold complete media and then incubated 30 at 4 * C with ⁇ i RRl/1. The monolayers were then washed 3x with complete media. The bound - ⁇ ⁇ l was released using 0.1 N NaOH and counted.
  • the specific activity of the * 25 I RRl/1 was adjusted using unlabeled RRl/1 to obtain a linear signal over the range of antigen densities encountered in this study.
  • Non-specific binding was determined in the presence of a thousand fold excess of unlabeled RRl/1 and was subtracted from total binding to yield the specific binding.
  • ICAM-1 expression measured using the above described radioimmune assay, is increased on human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) by IL-1, TNF, LPS and IFN gamma (Table 3).
  • Saphenous vein endothelial cells were used in this study to confirm the results from umbilical vein endothelial cells in cultured large vein endothelial cells derived from adult tissue.
  • the basal expression of ICAM-1 is 2 fold higher on saphenous vein endothelial cells than on umbilical vein endothelial cells.
  • IL-1 alpha, TNF and LPS were the most potent inducers and IL-1 was less potent on a weight basis and also at saturating concentrations for the respons (Table 3).
  • IL-1 beta at 100 ng/ml increased ICAM-1 expression by fold on HUVEC and 7.3 fid on HSVEC with half-maximal increase occurin at 15 ng/ml.
  • rTNF at 50 ng/ml increased ICAM-1 expression 16 fold o HUVEC and 11 fold on HSVEC with half maximal effects at 0.5 ng/ml
  • Interferon-gamma produced a significant increase in ICAM-1 expressio of 5.2 fold on HUVEC or 3.5 fold on HSVEC at 10,000 U/ml.
  • the effec of LPS at 10 ⁇ g/ml was similar in magnitude to that of rTNF. Pairwis combinations of these mediators resulted in additive or slightly les than additive effects on ICAM-1 expression (Table 3).
  • Cross-titratio of rTNF with rIL-1 beta or rIFN gamma showed no synergis between thes at subopti al or optimal concentrations.
  • Upregulation of ICAM-1 expression on HVEC and HSVEC- HUVEC or HSVEC were seeded into 96 well plates at 1:3 from a confluent monolayer and allowed to grow to confluence. Cells were then treated with the indicated mater or media for 16 hr and the RIA done as in methods. All points were done in quadruplicate.
  • human dermal fibroblasts were grown in a 96-well microtiter plate to a density of 2-8 x 10 4 cells/well (0.32 cm 2 ). The cells were washed twice with RPMI 1640 medium supplemented as described in Example 1. The cells were additionally washed once with Hanks Balanced Salt Solution (HBSS), 10 mM HEPES, 0.05% NaN3 and 10% heat-inactivated fetal bovine serum. Washing with this binding buffer was done at 4 ⁇ C.
  • HBSS Hanks Balanced Salt Solution
  • 10 mM HEPES 10 mM HEPES
  • NaN3 heat-inactivated fetal bovine serum
  • the dose response curves for induction of ICAM-1 by recombina mouse and human interleukin 1, and for recombinant human gam interferon, are shown in Figure 7.
  • Gamma interferon and interleukin were found to have similar concentration dependencies with nearl identical effects at 1 ng/ml.
  • the human and mouse recombinan interleukin 1 also have similar curves, but are much less effectiv than human interleukin 1 preparations in inducing ICAM-1 expression.
  • Cyclohexamide an inhibitor of protein synthesis, and actinomycin D an inhibitor of mRNA synthesis, abolish the effects of both interleuki 1 and gamma interferon on ICAM-1 expression on fibroblasts (Table 4)
  • tunicamycin an inhibitor of N-linked glycosylation, onl inhibited the interleukin 1 effect by 43%.
  • a Human fibroblasts were grown to a density of 8 x 10 4 cells/0.32 cm 2 well. Treatments were carried out in a final volume, of 50 ⁇ l containing the indicated reagents. Cycloheximide, actinomycin D, and tunicamycin were added at 20 ⁇ g/ml, 10 ⁇ M, and 2 ⁇ g/ml, respectively, at the same time as the cytokines. All points are means of quadruplicate wells ⁇ SD.
  • the sections were sequentially incubated with biotinylated horse anti-mouse IgG and avidin-biotinylated peroxidase complexes.
  • the sections were finally dipped in a solution containin 3-amino-9-ethyl-carbazole (Aldrich Chemical Co., Inc., Milwaukee, WI to develop a color reaction.
  • the sections were then fixed in 4 formaldehyde for 5 minutes and were counterstained with hematoxylin. Controls included sections incubated with unrelated monoclonal antibodies instead of the RRl/1 antibody.
  • ICAM-1 was found to have a distribution most simil-ar to that of th major histocompatibility complex (MHC) Class II antigens.
  • MHC major histocompatibility complex
  • the vascular endothelial staining was more intense in the interfollicular (paracortical) area in lymph nodes, tonsils, and Peyer's patches as compared with vessels in kidney, liver, and normal skin. In the liver, the staining was mostly restricted to sinusoidal lining cells; the hepatocytes and the endothelial cells lining most of the portal veins and arteries were not stained.
  • the staining pattern was focal and predominantly dendritic. Thymocytes were not stained. In the peripheral lymphoid tissue, the germinal center cells of the secondary lymphoid follicles were intensely stained. In some lymphoid follicles, the staining pattern was mostly dendritic, with no recognizable staining of lymphocytes. Faint staining of cells in the mantle zone was also observed. In addition, dendritic cells with cytoplas ic extensions (interdigitating reticulum cells) and a small number of lymphocytes in the interfollicular or paracortical areas stained with the ICAM-1 binding antibody.
  • cytoplas ic extensions interdigitating reticulum cells
  • epithelial cells The staining of epithelial cells was consistently seen in the mucosa of the tonsils. Although hepatocytes, bile duct epithelium, intestinal epithelial cells, and tubular epithelial cells in kidney did not stain in most circumstances, sections of normal kidney tissue obtained from a nephrectomy specimen with renal cell carcinoma showed staining of many proximal tubular cells for ICAM-1. These tubular epithelial cells also stained with an anti-HLA-DR binding antibody.
  • ICAM-I is expressed on non-hematopoietic cells such as vascular endothelial cells and on hematopoietic cells such as tissu macrophages and mitogen-stimulated T lymphocyte blasts. ICAM-1 was found to be expressed in low amounts on peripheral blood lymphocytes.
  • ICAM-1 was purified from human cells or tissue using monoclonal antibody affinity chromatography. Monoclonal antibody, RRl/1 ' , reactiv with ICAM-1 was first purified, and coupled to an inert column matrix. This antibody is described by Rothlein, R. et al. J. Immunol. 137:1270- 1274 (1986), and Dustin, M.L. et al . (J. Immunol. 137:245 (1986). ICAM-1 was solubilized from cell membranes by lysing the cells in non-ionic detergent, Triton X-100, at a near neutral pH.
  • non-ionic detergent Triton X-100
  • the cell lysate containing solubilized ICAM-1 was then passed through pre columns designed to remove materials that bind nonspecifically to th column matrix material, and then through the monoclonal antibody colum matrix, allowing the ICAM-1 to bind to the antibody.
  • the antibod column was then washed with a series of detergent wash buffers o increasing pH up to pH 11.0. During these washes ICAM-1 remained boun to the antibody matrix, while non-binding and weakly bindin contaminants were removed.
  • the bound ICAM-1 was then specificall eluted from the column by applying a detergent buffer of pH 12.5.
  • the anti-ICAM-1 monoclonal antibody RRl/1 was purified from th ascites fluid of hybrido a-bearing mice, or from hybridoma cultur supernates by standard techniques of ammonium sulfate precipitation an protein A affinity chromatography (Ey et al., Immunochem. 15:42
  • Sepharose CL-4B Sepharose CL-4B (Pharmacia, Upsala Sweden) was covalently coupled to Sepharose CL-4B (Pharmacia, Upsala Sweden) using a modification of the method of March et al . (Anal . Biochem. 60:149 (1974)). Briefly, Sepharose CL-4B was washed i distilled water, activated with 40 mg/ l CNBr in 5 M K 2 HP0 (p approximately 12) for 5 minutes, and then washed extensively with 0. mM HC1 at 4*C.
  • the filtered, activated Sepharose was resuspended wit an equal volume of purified antibody (2-10 mg/ml in 0.1 M NaHC03, 0.1 NaCl). The suspension was incubated for 18 hours at 4°C with gentl end-over-end rotation. The supernatant was then monitored for unboun antibody by absorbance at 280 nm, and remaining reactive sites on th activated Sepharose were saturated by adding glycine to 0.05 M. Coupling efficiency was usually greater than 90%.
  • Tween 40 polyoxyethylene sorbitan monopalmitate
  • the homogenate was extracted using three strokes of a Dounce or, more preferably, a Teflon Potter Elvejhem homogenizer, and then centrifuged at 1000 x g for 15 minutes. The supernatant was retained and the pellet was re-extracted with 200 ml of 2.5% Tween 40 in Tris-saline. After centrifugation at 1000 x g for 15 minutes, the supernatants from both extractions were combined and centrifuged at 150,000 x g for 1 hour to pellet the membranes. The membranes were washed by resuspending in 200 ml Tris-saline, centrifuged at 150,000 x g for 1 hour.
  • the membrane pellet was resuspended in 200 ml Tris-saline and was homogenized with a motorized homogenizer and Teflon pestle until the suspension was uniformly turbid. The volume was then brought up to 900 ml with Tris-saline, and N-lauroyl sarcosine was added to a final concentration of 1%. After stirring at 4"C for 30 minutes, insoluble material in the detergent lysate was removed by centrifugation at 150,000 x g for 1 hour. Triton X-100 was then added to the supernatant to a final concentration of 2%, and the lysate was stirred at 4 * C for 1 hour.
  • the EBV-transformed B-lymphoblastoid cell line JY was grown in RPMI- 1640 containing 10% fetal calf serum (FCS) and 10 mM HEPES to an approximate density of 0.8 - 1.0 x 10- * cells/ml.
  • FCS fetal calf serum
  • HEPES fetal calf serum
  • PMA phorbol 12-myristate 13-acetate
  • Sodiu vanadate 50 ⁇ M was also added to the cultures during this time.
  • the cells were pelleted by centrifugation at 500 x g for 10 minutes, and washed twice in Hank's Balanced Salt Solution (HBSS) by resuspension and centrifugation.
  • HBSS Hank's Balanced Salt Solution
  • the cells (approximately 5 g per 5 liters o culture) were lysed in 50 ml of lysis buffer (0.14 M NaCl, 50 M Tris pH 8.0, 1% Triton X-100, 0.2 U/ml aprotinin, 1 mM PMSF, 50 ⁇ M sodiu vanadate) by stirring at 4 ⁇ C for 30 minutes. Unlysed nuclei and insoluble debris were removed by centrifugation at 10,000 x g for 1 minutes, followed by centrifugation of the supernatant at 150,000 x g for 1 hour, and filtration of the supernatant through Whatman 3m filter paper.
  • the column of RRl/1 -Sepharose and bound ICAM-1 wa washed sequentially at a flow rate of 1 ml/minute with a minimum of column volumes each of the following: 1) lysis buffer, 2) 20 mM Tri pH 8.0/0.14 M NaCl/0.1% Triton X-100, 3) 20 mM glycine pH 10.0/0.1 Triton X-100, and 4) 50 mM triethyl amine pH 11.0/0:1% Triton X-100. All wash buffers contained 1 mM PMSF and 0.2 U/ml aprotinin.
  • the remaining bound ICAM-1 was eluted with 5 column volumes o elution buffer (50 mM triethyl ami ne/0.1% Triton X-100/pH 12.5 at 4"C) at a flow rate of 1 ml/3 minutes.
  • the eluted ICAM-1 was collected in ml fractions and immediately neutralized by the addition of 0.1 ml of 1 M Tris, pH 6.7. Fractions containing ICAM-1 were identified by SDS- polyacryl amide electrophoresis of 10 ⁇ l aliquots (Springer et al . , « Exp. Med.
  • the bul of the ICAM-1 eluted in approximately 1 column volume and was greate than 90% pure as judged from silver-stained electropherograms (a small amount of IgG, leeched from the affinity matrix, was the majo contaminant).
  • the fractions containing ICAM-1 were pooled and concentrated approximately 20-fold using Centricon-30 microconcentrators (Amicon, Danvers, MA).
  • the purified ICAM-1 was quantitated by Lowry protein assay of an ethanol -precipitated aliquot of the pool: approximately 500 ⁇ g of pure ICAM-1 were produced from the 200 g of human spleen.
  • ICAM-1 Approximately 200 ⁇ g of purified ICAM-1 was subjected to a second stage purification by preparative SDS-polyacryl amide gel electrophoresis. The band representing ICAM-1 was visualized by soaking the gel in 1 M KC1. The gel region which contained ICAM-1 was then excised and electroeluted according to the method of Hunkapiller et al.. Meth. Enzvmol . £1:227-236 (1983). The purified protein was greater than 98% pure as judged by SDS-PAGE and silver staining.
  • ICAM-1 for use in functional studies was purified from detergent lysates of JY cells as described above, but on a smaller scale (a 1 ml column of RRl/1-Sepharose), and with the following modifications. All solutions contained 50 ⁇ M sodium vanadate. After washing the colum with pH 11.0 buffer containing 0.1% Triton X-100, the column was washe again with five column volumes of the same buffer containing 1% n- octyl-beta-D-glucopyranoside (octylglucoside) in place of 0.1% Trito X-100.
  • the octylglucoside detergent displaces the Triton X-100 boun to the ICAM-1, and unlike Triton X-100, can be subsequently removed b dialysis.
  • the ICAM-1 was then eluted with pH 12.5 buffer containing 1 octylglucoside in place of 0.1% Triton X-100, and was analyzed an concentrated as described above.
  • ICAM-1 purified from human spleen migrates in SDS-polyacrylamid gels as a broad band of M r of 72,000 to 91,000.
  • ICAM-1 purified fro JY cells also migrates as a broad band of M r of 76,500 to 97,000.
  • Th non-glycosylated precursor has a M r of 55,000 (Dustin et al.).
  • Th protein purified from either JY cells or human spleen retains it antigenic activity as evidenced by its ability to re-bind to th original affinity column, and by immunoprecipitation with RR1/1 Sepharose and SPS-polyacrylamide electrophoresis.
  • peptide fragments of ICAM-1 approximately 200 ⁇ g wa reduced with 2 mM dithiothreitol/2% SDS, followed by alkylation with mM iodoacetic acid.
  • the protein was precipitated with ethanol, an redissolved in 0.1 M NH4CO3/O.I mM CaCl 2 /0.1% zwittergent 3-1 (Calbiochem), and digested with 1% w/w trypsin at 37 ⁇ C for 4 hours, followed by an additional digestion with 1% trypsin for 12 hours a 37"C.
  • the tryptic peptides were purified by reverse-phase HPLC using 0.4 x 15 cm C4 column (Vydac).
  • the peptides were eluted with a linea gradient of 0% to 60% acetonitrile in 0.1% trifluoroacetic acid. Selected peptides were subjected to sequence analysis on a gas phas icrosequenator (Applied Biosystems). The sequence informatio obtained from this study is shown in Table 5.
  • the gene for ICAM-1 may be cloned using any of a variety of procedures. For example, the amino acid sequence information obtained through the sequencing of the tryptic fragments of ICAM-1 (Table 5) can be used to identify an oligonucleotide sequence which would correspond to the ICAM-1 gene. Alternatively, the ICAM-1 gene can be cloned using anti-ICAM-1 antibody to detect clones which produce ICAM-1.
  • oligonucleotides can be identified, each of which would be capable of encoding the ICAM-1 tryptic peptides.
  • the probability that a particular oligonucleotide will, in fact, constitute the actual ICAM- 1 encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
  • Such "codon usage rules" are disclosed by Lathe, R., et al . , J___ Molec.
  • the oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the ICAM-1 fragments is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probably" sequence, or set of sequences.
  • An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the ICAM-1 gene (Maniatis, T., et al . , Molecular Cloning A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1982).
  • ICAM-1 gene As described in Section C, supra, it is possible to clone the ICAM-1 gene from eukaryotic DNA preparations suspected of containing this gene.
  • a DNA library is screened for its ability to hybridize with the oligonucleotide probes described above. Because it is likely that there will be only two copies of the gene for ICAM-1 in a normal diploid cell, and because it is possible that the ICAM-1 gene may have large non-transcribed intervening sequences (introns) whose cloning is not desired, it is preferable to isolate ICAM-1-encoding sequences from a cDNA library prepared from the mRNA of an ICAM-1-producing cell, rather than from genomic DNA.
  • Suitable DNA, or cDNA preparations are enzymatically cleaved, or randomly sheared, and ligated into recombinant vectors. The ability of these recombinant vectors to hybridize to the above-described oligonucleotide probes is then measured. Procedures for hybridization are disclosed, for example, in Maniatis, T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982) or in Haymes, B.T., et al . , Nucleic Acid Hybridization a Practical Approach. IRL Press, Oxford, England (1985). Vectors found capable of such hybridization are then analyzed to determine the extent and nature of the ICAM-1 sequences which they contain. Based purely on statistical considerations, a gene such as that which encodes the ICAM-1 molecule could be unambiguously identified (via hybridization screening) using an oligonucleotide probe having only 18 nucleotides.
  • the actual identification of ICAM-1 peptide sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
  • a DNA molecule or set of DNA molecules, capable of function ⁇ ing as a probe to identify and isolate the ICAM-1 gene.
  • ICAM-1 peptide sequences of Table 5 the sequence of the "most probable" sequence of an oligonucleotide capable of encoding the AA and J peptides was determined (Tables 6 and 7, respectively). Oligonucleotides complementary to these sequences were synthesized and purified for use as probes to isolate ICAM-1 gene sequences. Suitable size-selected cDNA libraries were generated from the poly(A) + RNA of both PMA-induced HL-60 cells and from PS-stimulated umbilical vein endothelial cells. A size-selected cDNA library was prepared using poly(A) + RNA from PMA-induced HL-60 cells according to the method of Gubler, U., et al. ((Gene 25:263-269 (1983)) and Corbi, A., et al . (EMBO J. 6:4023-4028 (1987)), which references are herein incorporated by reference.
  • a size-selected cDNA library was prepared using poly(A) + RNA from umbilical vein endothelial cells which had been stimulated for 4 hours with PS 5 ⁇ g/ml.
  • the RNA was extracted by homogenizing the cells in 4 M guanidinium isothiocyanate and subjecting the supernatant to ultracentrifugation through a CsCl gradient (Chirgwin, J.M., et al . , Biochem. 18:5294-5299 (1979)).
  • Poly(A) + RNA was isolated from the mixture of total RNA species through the use of oligo (dT)-cellulose chromatography (Type 3, Collaborative Research) (Aviv, H., et al., Proc. Natl. Acad. Sci. (USA) 69:1408-1412 (1972).
  • 3' 5' First strand cDNA was synthesized using 8 ⁇ g of poly(A) + RNA, avian myeloblastosis virus reverse transcriptase (Life Sciences) and an oligo(dT) primer.
  • the DNA-RNA hybrid was digested with RNase H (BRL) and the second strand was synthesized using DNA polymerase I (New England Biolabs).
  • the product was methylated with Eco Rl methylase (New England Biolabs), blunt end ligated to Eco Rl linkers (New England Biolabs), digested with Eco Rl and size selected on a low melting point agarose gel.
  • cDNA greater than 500bp were ligated to ⁇ gtlO which had previously been Eco Rl digested and dephosphorylated (Stratagene) The product of the ligation was then packaged (Stratagene gold).
  • the umbilical vein endothelial cell and HL-60 cDNA libraries were then plated at 20,000 PFU/150mm plate. Recombinant DNA was transferred in duplicate to nitrocellulose filters, denatured in 0.5 M Na0H/1.5M NaCl, neutralized in 1M Tris, pH7.5/1.5M NaCl and baked at 80°C for 2 hours (Benton, W.D., et al.. Science 196:180-182 (1977)). Filters were prehybridized and hybridized in 5X SSC containing 5X Denhardt's solution, 50 mM NaP04 and 1 ⁇ g/ml salmon sperm DNA. Prehybridization was carried out at 45 * for 1 hour.
  • Hybridization was carried out using 32bp ('5-TTGGGCTGGTCACAG- GAGGTGGAGCAGGTGAC) or 47bp (5'-GAGGTGTTCTCAAACAGCTCCAGGCCCTGG GGCCGCAGGTCCAGCTC) anti-sense oligonucleotides based, in the manner discussed above, on the ICAM-I tryptic peptides J and AA, respectively
  • Oligonucleotides were end labeled with ⁇ -( P)ATP using T4 polynucleotide kinase and conditions recommended by the manufacturer
  • the gene for ICAM-1 may alternatively be cloned through the use o anti-ICAM-1 antibody.
  • DNA, or more preferably cDNA is extracted an purified from a cell which is capable of expressing ICAM-1. Th purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to prduct a pool of DNA or cDNA fragments. DNA or cDNA fragment from this pool are then cloned into an expression vector in order t produce a genomic library of expression vectors whose members eac contain a unique cloned DNA or cDNA fragment.
  • Phage DNA from positive clones were digested with Eco Rl and examined by Southern analysis using a cDNA from one clone as a probe. Maximal size cDNA inserts which cross-hybridized were subcloned into the Eco Rl site of plasmid vector pGEM 4Z (Promega). HL-60 subclones containing the cDNA in both orientations were deleted by exonuclease III digestion (Henikoff, S., Gene 28:351-359 (1984)) according to the manufacturers recommendations (Erase-a-Base, Promega). Progressively deleted cDNAs were then cloned and subjected to dideoxynucleotide chain termination sequencing (Sanger, F.
  • HL-60 cDNA 5' and coding regions were sequenced completely on both strands and the 3' region was sequenced approximately 70% on both strands.
  • a representative endothelial cDNA was sequenced over most of its length by shotgun cloning of 4bp-recognition restriction enzyme fragments.
  • the cDNA sequence of one HL-60 and one endothelial cell cDNA was established (Figure 8).
  • the 3023 bp sequence contains a short 5' untranslated region and a 1.3 kb 3' untranslated region with a consensus polyadenylation signal at position 2966.
  • the longest open reading frame begins with the first ATG at position 58 and ends with a TGA terminating triplet at position 1653.
  • Identity between the translated amino acid sequence and sequences determined from 8 different tryptic peptides totaling 91 amino acids confirmed that authentic ICAM-1 cONA clones had been isolated.
  • the amino acid sequences of ICAM-1 tryptic peptides are shown in Table 8.
  • Hydrophobicity analysis suggests the presence of a 27 residue signal sequence.
  • the assignment of the +1 glutamine is consistent with our inability to obtain N-terminal sequence on 3 different ICAM-1 protein preparations; glutamine may cyclize to pyroglumatic acid, resulting in a blocked N- terminus.
  • the translated sequence from 1 to 453 is predominantly hydrophilic followed by a 24 residue hydrophobic putative transmembrane domain. The transmembrane domain is immediately followed by severa charged residues contained within a 27 residue putative cytoplasmi domain.
  • the predicted size of the mature polypeptide chain is 55,21 daltons, in excellent agreement with the observed size of 55,000 fo deglycosylated ICAM-1 (Oustin, M.L., et al .. J. Immunol. 137:245-25 (1986)).
  • Eight N-linked glycosylation sites are predicted. Absence o asparagine in the tryptic peptide sequences of 2 of these sites confir their glycosylation and their extracellular orientation. Assumin 2,500 daltons per high mannose N-linked carbohydrate, a size of 75,00 daltons is predicted for the ICAM-1 precursor, compared to the observe six of 73,000 daltons (Oustin, M.L., et al .. J.
  • ICAM-1 i a heavily glycosylated but otherwise typical integral membrane protein.
  • ICAM-1 is an Integrin-Binding Member of the Immunoglobulin
  • ICAM-1 is a ligand of an integrin, it was unexpected that it would be a member of the immunoglobulin supergene family. However, inspection of the ICAM-1 sequence shows that it fulfills all criteria proposed for membership in the immunoglobulin supergene family. These criteria are discussed in turn below.
  • ICAM-1 The entire extracellular domain of ICAM-1 is constructed from 5 homologous immunoglobulin-like domains which are shown aligned in Figure 9A. Domains 1-4 are 88, 97, 99, and 99 residues long, respectively and thus are of typical Ig domain size; domain 5 is truncated within 68 residues. Searches of the NBRF data base using the FASTP program revealed significant homologies with members of the immunoglobulin supergene family including IgM and IgG C domains, T cell receptor a subunit variable domain, and alpha 1 beta glycoprotein (Fig. 9B-D).
  • the amino acid sequence of ICAM-1 was compared with the amino acid sequences of other members of the i mununoglobulin supergene family.
  • V and C2 Three types of Ig superfamily domains, V, Cl, and C2 have been differentiated. Both V and C domains are constructed from 2 5-sheets linked together by the intradomain disulfide bond; V domains contain 9 anti-parallel ⁇ -strands while C domains have 7. Constant domains were divided into the Cl- and C2- sets based on characteristic residues shown in Figure 9A.
  • the Cl-set includes proteins involved in antigen recognition.
  • the C2-set includes several Fc receptors and proteins involved in call adhesion including CD2, LFA-3, MAG, and NCAM.
  • ICAM-1 domains were found to be most strongly homologous with domains of the C2-set placing ICAM-1 in this set; this is reflected in stronger similarity to conserved residues in C2 than Cl domains as shown for ⁇ - strands B-F in Figure 9. Also, ICAM-1 domains aligned much better with ⁇ -strands A and G of C2 domains than with these strands in V and Cl domains, allowing good alignments across the entire C2 domain strength. Alignments with C2 domains from NCAM, MAG, and alpha 1-/3 glycoprotein are shown in Figures 9B and 9C; identity ranged from 28 to 33%. Alignments with a T cell receptor V ⁇ 27% identity and IgM C domain 334% identity are also shown ( Figures 9B, 9D).
  • Endothelial cell ICAM-1 was used because i shows less glycosylation heterogeneity than JY or hairy cell spleni ICAM-1 and allows greater sensitivity to shifts in M r .
  • ICAM-1 was therefore, purified from 16 hour LPS (5 ⁇ g/ml) stimulated umbilica vein endothelial cell cultures by immunoaffinity chromatography a described above.
  • Acetone precipitated ICAM-1 was resuspended in sampl buffer (Laemmli, U.K., Nature 227:680-685 (1970)) with 0.25% 2-mer captoethanol or 25 mM iodoacetamide and brought to 100°C for 5 min.
  • Endothelial cell ICAM-1 had an apparent M r of 100 Kd under reducin conditions and 96 Kd under non-reducing conditions strongly suggestin the presence of intrachain disulfides in native ICAM-1.
  • ICAM-1 was found to be most strongly homologous with the NCAM an MAG glycoproteins of the C2 set. This is of particular interest sinc both NCAM and MAG mediate cell-cell adhesion.
  • NCAM is important i neuron-neuron and neuro-muscular interactions (Cunningham, B.A., e al ..
  • MAG Surzer, J.L., et al.. J. Cell. Biol. 104:957-96 (1987)
  • sinc each is an integral membrane glycoprotein constructed from 5 C2 domain forming the N-terminal extracellular region, although in NCAM som additional non-Ig-like sequence is present between the last C2 domai and the transmembrane domain.
  • ICAM-1 aligns over its entire lengt including the transmembrane and cytoplasmic domains with MAG with 21 identity; the same % identity is found comparing the 5 domains of ICAM 1 and NCAM-1.
  • FIG. 10 A diagrammatic comparison of the secondary structures o ICAM-1 and MAG is shown in Figure 10. Domain by domain comparison show that the level of homology between domains within the ICAM-1 an NCAM molecules (x + s.d. 21 + 2.8% and 18.6 + 3.8%, respectively) i the same as the level of homology comparing ICAM-1 domains to NCAM an MAG domains (20.4 + 3.7 and 21.9 + 2.7, respectively). Although ther is evidence for alternative splicing in the C-terminal regions of NCA (Cunningham, B.A., et al.. Science 236:799-806 (1987); Barthels, D., e al.. EMBO J.
  • ICAM-1 functions as a ligand for LFA-1 in lymphocyte interactions with a number of different cell types.
  • Lymphocytes bind to ICAM-1 incorporated in artificial membrane bilayers, and this requires LFA-1 on the lymphocyte, directly demonstrating LFA-1 interaction with ICAM-1 (Marlin, S.D., et al.. cell 51:813-819 (1987)).
  • LFA-1 is a leukocyte integrin and has no immunoglobulin-like features.
  • Leukocyte integrins comprise one integrin subfamily. The other two subfamilies mediate cell-matrix interactions and recognize the sequence RGD within their ligands which include fibronectin, vitronectin, collagen, and fibrinogen (Hynes, R.O., Cell 48:549-554 (1987); Ruoslahti, E., et al .
  • the leukocyte integrins are only expressed on leukocytes, are involved in cell-cell interactions, and the only known ligands are ICAM-1 and iC3b, a fragment of the complement component C3 which shows no immunoglobulin-like features and is recognized by Mac-1 (Kishimoto, T.K., et al .. In: Leukocyte Typing III. McMichael, M. (ed.), Springer-Verlag, New York (1987); Springer, T.A., et al.. Ann. Rev. Immunol. 5:223-252 (1987); Anderson, O.C., et al ., Ann. Rev. Med. 38:175-194 (1987)). Based upon sequence analysis, possible peptides within the ICAM-1 sequence recognized by LFA-1 are shown in Table 9.
  • -D-L-R-P-Q-G-L-E- ICAM-1 is the first example of a member of the immunoglobuli supergene family which binds to an integrin. Although both of the families play an important role in cell adhesion, interaction betwe them had not previously been expected. In contrast, interactio within the immunoglobulin gene superfamily are quite common. It i quite possible that further examples of interactions between t integrin and immunoglobulin families will be found. LFA-1 recognizes ligand distinct from ICAM-1 (Springer, T.A., et al .. Ann. Rev.
  • NCAM's role in neural-neural and neural-muscular cell interactio has been suggested to be due to homophilic NCAM-NCAM interactio (Cunningham, B.A., et al .. Science 236:799-806 (1987)).
  • the importa role of MAG in interactions between adjacent turning loops of Schwa cells enveloping axons during myelin sheath formation might be due interaction with a distinct receptor, or due to homophilic MAG-M interactions.
  • homologous MAG molecule contains an RGD sequence between domains 1 and 2 (Poltorak, M., et al .. J. Cell Biol. 105:1893-1899 (1987); Salzer, J-L., et al., J. Cell. Biol. 104:957-965 (1987)).
  • Southern blots were performed using a 5 ⁇ g of genomic DNA extracted from three cell lines: BL2, a Burkitt lymphoma cell line (a gift from Dr. Gilbert Lenoir); JY and Er-LCL, EBV transformed B-lymphoblastoid cell lines.
  • the DNAs were digested with 5X the manufacturers recommended quantity of Bam HI and Eco Rl endonucleases (New England Biolabs). Following electrophoresis through a 0.8% agarose gel, the DNAs were transferred to a nylon membrane (Zeta Probe, BioRad). The filter was prehybridized and hybridized following standard procedures using ICAM
  • an "expression vector” is a vector which (due to the presence o appropriate transcriptional and/or translational control sequences) i capable of expressing a DNA (or cDNA) molecule which has been clone into the vector and of thereby producing a polypeptide or protein. Expression of the cloned sequences occurs when the expression vector i introduced into an appropriate host cell. If a prokaryotic expressio vector is employed, then the appropriate host cell would be an prokaryotic cell capable of expressing the cloned sequences Similarly, if a eukaryotic expression vector is employed, then th appropriate host cell would be any eukaryotic cell capable o expressing the cloned sequences.
  • eukaryotic DNA ma contain intervening sequences, and since such sequences cannot b correctly processed in prokaryotic cells, it is preferable to emplo cDNA from a cell which is capable of expressing ICAM-1 in order t produce a prokaryotic genomic expression vector library.
  • Procedure for preparing cDNA and for producing a genomic library are disclosed b Maniatis, T., et al . (Molecular Cloning: A Laboratory Manual, Col Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • the above-described expression vector genomic library is used t create a bank of host cells (each of which contains one member of th library).
  • the expression vector may be introduced into the host cell by any of a variety of means (i.e., transformation, transfection, protoplast fusion, electroporation, etc.).
  • the bank of expression vector-containing cells is clonally propagated, and its members are individually assayed (using an immunoassay) to determine whether they produce a protein capable of binding to anti-ICAM-1 antibody.
  • the expression vectors of those cells which produce a protein capable of binding to anti-ICAM-1 antibody are then further analyzed to determine whether they express (and thus contain) the entire ICAM-1 gene, whether they express (and contain) only a fragment of the ICAM-1 gene, or whether they express (and contain) a gene whose product, though immunologically related to ICAM-1, is not ICAM-1.
  • an analysis may be performed by any convenient means, it is preferable to determine the nucleotide sequence of the DNA or cDNA fragment which had been cloned into the expression vector. Such nucleotide sequences are then examined to determine whether they are capable of encoding polypeptides having the same amino acid sequence as the tryptic digestion fragments of ICAM-1 (Table 5).
  • An expression vector which contains a DNA or cDNA molecule which encodes the ICAM-1 gene may, thus, be recognized by: (i) the ability to direct the expression of a protein which is capable of binding to anti-ICAM-1 antibody; and (ii) the presence of a nucleotide sequence which is capable of encoding each of the tryptic fragments of ICAM-1.
  • the cloned DNA molecule of such an expression vector may be removed from the expression vector and isolated in pure form.
  • ICAM-1 In cells, ICAM-1 normally functions as a surface protein associated with the cell membrane. Therefore, the function of purified ICAM-1 was tested after the molecule was reconstituted into artificial lipid membranes (liposomes, or vesicles) by dissolving the protein in detergent-solubilized lipids, followed by the removal of the detergent by dialysis. ICAM-1 purified from JY cells and eluted in the detergent octylglucoside as described above was reconstituted into vesicles, and the ICAM-1 containing vesicles were fused to glass coverslips or plastic culture wells to allow the detection of cells binding to the protein.
  • Vesicles were prepared by the method of Gay et al . (J. Immunol. 136:2026 (1986)). Briefly, egg phosphatidylcholine and cholesterol were dissolved in chloroform and mixed in a molar ratio of 7:2. The lipid mixture was dried to a thin film while rotating under a stream of nitrogen gas, and was then lyophilized for 1 hour to remove all traces of chloroform. The lipid film was then dissolved in 1% octylgluco- side/0.14 M NaCl/20 mM Tris (pH 7.2) to a final concentration of phosphatidylcholine of 0.1 mM.
  • Planar membranes were prepared by the method of Brian et al . , Proc. Nat! . Acad. Sci. 81:6159 (1984). Glass coverslips (11 mm in diameter) were boiled for 15 minutes in a 1:6 dilution of 7X detergent (Linbro), washed overnight in distilled water, soaked in 70% ethanol, and air dried. An 80 ⁇ l drop of vesicle suspension containing either ICAM-1 or glycophorin was placed in the bottom of wells in a 24-well cluster plate, and the prepared glass coverslips were gently floated on top. After 20-30 minutes incubation at room temperature, the wells were filled with HBSS, and the coverslips were inverted to bring the planar membrane face up.
  • vesicles containing ICAM-1 were found to bind directly to th plastic surface of multi-well tissue culture plates, and retai functional activity as evidenced by specific cell binding.
  • Suc vesicles are hereinafter referred to as "plastic-bound vesicles" (PBV since the nature of the lipid vesicles bound to the plastic has no been determined.
  • Plastic-bound vesicles were prepared by adding 30 ⁇ of vesicle suspension directly to the bottom of wells in 96-well tissu culture trays (Falcon), followed by incubation and washing as describe for planar membranes.
  • T-lymphocytes from normal controls and a Leukocyte Adhesio Oeficiency (LAP) patient whose cells fail to express LFA-1 were prepared by culturing peripheral blood mononuclear cells with 1 ⁇ g/ml Concanavalin-A (Con-A) in RPMI-1640 plus 20% FCS at 5 x 10 5 cells/ml for 3 days. The cells were then washed twice with RPMI and once with 5 mM methyl-alpha-0- mannopyranoside to remove residual lectin from the cell surface. The cells were grown in RPMI/20% FCS containing 1 ng/ml recombinant IL-2, and were used between 10 and 22 days after the initiation of culture.
  • LAP Leukocyte Adhesio Oeficiency
  • ⁇ - • ' • ⁇ Cr-labeled cells (5 x 10> EBV-transformants in planar membrane assays; 1 x 10 5 EBV- transformants or SKW-3 cells, 2 x 10 5 Con-A blasts in PBV assays) were centrifuged for 2 minutes at 25 x g onto planar membranes or PBV, followed by incubation at 4'C, 22"C, or 37°C for one hour. After incubation, unbound cells were removed by eight cycles of filling and aspiration with buffer pre-equilibrated to the appropriate temperature.
  • Bound cells were quantitated by solubilization of well contents with 0.1 N NaOH/1% Triton X-100 and counting in a gamma counter. Percent cell binding was determined by dividing cpm from bound cells by input cell-associated cpm. In planar membrane assays, input cpm were corrected for the ratio of the surface area of coverslips compared to the surface area of the culture wells.
  • EBV-transformed B-lymphoblastoid cells, SKW-3 T- lymphoma cells, and Con-A T-lymphoblasts bound specifically to ICAM-1 in artificial membranes ( Figures 11 and 12).
  • the binding was specific since the cells bound very poorly to control planar membranes or vesicles containing equivalent amounts of another human cell surface glycoprotein, glycophorin.
  • LFA-1 positive EBV- transformants and Con-A blasts bound, while their LFA-1 negative counterparts failed to bind to any significant extent, demonstrating that the binding was dependent on the presence of LFA-1 on the cells.
  • ICAM-1 is a specific ligand for the LFA- 1-dependent adhesion system.
  • HLA-DR The expression of HLA-DR on keratinocytes in the allergic ski lesions was less frequent than that of ICAM-1. None of the subject studied had lesions with keratinocytes that stained positively for HLA DR up to 24 hours after the application of the hapten. In fact, onl four biopsy samples had keratinocytes that expressed HLA-DR and n biopsy had keratinocytes that was positive for HLA-DR and not ICAM- (Table 10).
  • the toxic patch In contrast to the allergic patch test lesion, the toxic patch tes lesion induced with croton oil or sodium lauryl sulfate ha keratinocytes that displayed little if any ICAM-1 on their surfaces a all time points tested (Table 11). In fact, at 48 hours after th patch application, which was the optimum time point for ICAM- expression in the allergic patch test subjects, only one of the 1 toxic patch test subjects had keratinocytes expressing ICAM-1 in thei lesions. Also in contrast to the allergic patch test biopsies, ther was no HLA-DR expressed on keratinocytes of toxic patch test lesions.
  • ICAM-1 is expressed in immune-base inflammation and not in toxic based inflammation, and thus th expression of ICAM-1 may be used to distinguish between immuno based and toxic based inflammation, such as acute renal failure in kidney transplant patients where it is difficult to determine whether failure is due to rejection or nephrotoxicity of the immuno-suppressive therapeutic agent. Renal biopsy and assessment of upregulation of ICAM-1 expression will allow differentiation of immune based rejection and non-immune based toxicity reaction.
  • ICAM-1 The expression of ICAM-1 on keratinocytes from skin biopsies o patients with exanthema and urticaria was less pronounced. Only fou out of the seven patients tested with these diseases had keratinocytes that expressed ICAM-1 at the site of the lesion. HLA-DR expression was only found on one patient and this was in conjunction with ICAM-1.
  • ICAM-1 ICAM-1 in skin biopsies from 5 patients with psoriasis were studied before the initiation and periodically during a course of PUVA treatment. Biopsies were obtained from 5 patients with classical psoriasis confirmed by histology. Biopsies were taken sequentially before and during indicated time of PUVA treatment. PUVA was given 3 to 4 times weekly. Biopsies were taken from the periphery of the psoriatic plaques in five patients and, in addition biopsies were taken from clinically normal skin in four of the patients.
  • Fresh skin biopsy specimens were frozen and stored in liquid nitrogen. Six micron cryostat sections were air dried overnight at room temperature, fixed in acetone for 10 minutes and either stained immediately or wrapped in aluminum foil and stored at -80°C until staining.
  • Staining was accomplished in the following manner. Sections were incubated with monoclonal antibodies and stained by a three stage immunoperoxidase method (Stein, H., et. al.. Adv. Cancer Res 42:67- 147, (1984)), using a diaminobenzidine H2O2, substrate. Tonsils and lymph nodes were used as positive control for anti-ICAM-1 and HLA-PR staining. Tissue stained in the absence of primary antibody were negative controls.
  • the monoclonal antibodies against HLA-PR were purchased from Becton Pickinson (Mountainview, California).
  • the monoclonal anti-ICAM-1 antibody was R6-5-P6.
  • Peroxidase-conjugated rabbit anti-mouse Ig and peroxidase-conjugated swine anti-rabbit Ig were purchased from PAKAPATTS, Copenhagen, Penmark.
  • Diaminobenzidine-tetrahydrochloride were obtained from Sigma (St. Louis, Mo.).
  • Patients 1, 4 and 5 had decreases an increases of ICAM-1 expression during the PUVA treatment whic correlated to clinical remissions and relapses, respectively. Ther was no ICAM-1 expression on keratinocytes from normal skin before o after PUVA treatment. This indicates that PUVA does not induce ICAM- on keratinocytes from normal skin.
  • the density of the mononuclear cel infiltrate correlated with the amount of ICAM-1 expression o keratinocytes. This pertained to both a decreased number o mononuclear cells in lesions during PUVA treatment when ICAM- expression also waned and an increased number of mononuclear cell during PUVA treatment when ICAM-1 expression on keratinocytes was mor prominent. Endothelial cells and dermal mononuclear cells are als ICAM-1-positive. In clinically normal skin, ICAM-1 expression wa confined to endothelial cells with no labelling of keratinocytes.
  • HLA-DR HLA-DR on keratinocytes was variable. There wa no HLA-DR positive biopsy that was not also ICAM-1 positive.
  • ICAM-1 on keratinocytes of psoriatic lesions correlates with the clinical severity of the lesion as well as with the size of the dermal infiltrate.
  • ICAM-1 plays a central role in psoriasis and inhibition of its expression and/or inhibition of its interaction with the CD 18 complex on mononuclear cells will be an effective treatment of the disease.
  • monitoring ICAM-1 expression on keratinocytes will be an effective tool for diagnosis and prognosis, as well as evaluating the course of therapy of psoriasis.
  • ICAM-1 Unlike lesions from benign cutaneous conditions, the expression of ICAM-1 on keratinocytes from malignant skin lesions was much more variable (Table 14). Of the 23 cutaneous T-cell lymphomas studied, ICAM-1 positive keratinocytes were identified in only 14 cases. There was a tendency for keratinocytes from biopsies of mycosis fungoides lesions to lose their ICAM-1 expression with progression of the disease to more advanced stages. However, ICAM-1 expression was observed on a varying proportion of the mononuclear cell infiltrate from most of the cutaneous T cell lymphoma lesions. Among the remaining lymphomas studied, four of eight had keratinocytes that expressed ICAM-1. Of the 29 patients with malignant cutaneous diseases examined, 5 had keratinocytes that expressed HLA-DR without expressing ICAM-1 (Table 14).
  • Human peripheral blood mononuclear cells are induced to proliferate by the presence and recognition of antigens or mitogens. Certain molecules, such as the mitogen, concanavalin A, or the T-cell-binding antibody 0KT3, cause a non-specific proliferation of peripheral bloo mononuclear cells to occur.
  • Human peripheral blood mononuclear cells are heterogeneous in tha they are composed of subpopulations of cells which are capable o recognizing specific antigens. When a peripheral blood mononuclea cell which is capable of recognizing a particular specific antigen, encounters the antigen, the proliferation of that subpopulation o mononuclear cell is induced. Tetanus toxoid and keyhole limpet hemocyanin are examples of antigens which are recognized by subpopulations of peripheral mononuclear cells but are not recognized by all peripheral mononuclear cells in sensitized individuals.
  • anti-ICAM-1 monoclonal antibody R6-5-D6 to inhibit proliferative responses of human peripheral blood mononuclear cells in systems known to require cell-cell adhesions was tested.
  • Peripheral blood mononuclear cells were purified on Ficoll-Paque (Pharmacia) gradients as per the manufacturer's instructions. Following collection of the interface, the cells were washed three times with RPMI 1640 medium, and cultured in flat-bottomed 96-well microtiter plates at a concentration of 10 ⁇ cells/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM glutamine, and gentamicin (50 ⁇ g/ml).
  • Antigen either the T-cell mitogen, concanavalin A (0.25 ⁇ g/ml); the T-cell-binding antibody, 0KT3 (0.001 ⁇ g/ml); keyhole limpet hemocyanin (10 g/ml) or tetanus toxoid (1:100 dilution from source) were added to cells which were cultured as described above in either the presence or absence of anti-ICAM antibody (R6-5-06; final concentration of 5 g/ml). Cells were cultured for 3.5 days (concanavalin A experiment), 2.5 days (0KT3 experiment), or 5.5 days (keyhole limpet hemocyanin and tetanus toxoid experiments) before the assays were terminated.
  • anti-ICAM-1 antibody inhibits proliferative responses to the non-specific T-cell mitogen, ConA; the non-specific T- cell associated antigen, OKT-3; and the specific antigens, keyhole limpet hemocyanin and tetanus toxoid, in mononuclear cells.
  • the inhibition by anti-ICAM-1 antibody was comparable to that of anti-LFA-1 antibody suggesting that ICAM-1 is a functional ligand of LFA-1 and that antagonism of ICAM-1 will inhibit specific defense system responses.
  • ICAM-1 is necessary for effective cellular interactions during an immune response mediated through LFA-1-dependent cell adhesion.
  • the induction of ICAM-1 during immune responses or inflammatory disease allows for the interaction of leukocytes with each other and with endothelial cells.
  • lymphocytes from two unrelated indivduals are cultured in each others presence, blast transformation and cell proliferation of the lymphocytes are observed.
  • This response, of one population of lymphocytes to the presence of a second population of lymphocytes is known as a mixed lymphocyte reaction (MLR), and is analogous to the response of lymphocytes to the addition of togens (Immunology The Science of Self-Nonself Piscrimination. Klein, J., John Wiley & Sons, NY (1982), pp 453-458).
  • MLR mixed lymphocyte reaction
  • RPMI-1640 culture medium containing 0.5% of gentamicin, 1 mM L-glutamine (GIBCO) and 5% heat inactivated (56 ⁇ C, 30 min.) human AB sera (Flow Laboratories) (hereafter referred to as RPMI-culture medium).
  • Mouse anti-ICAM-1 (R6-5-06) was used in these experiments. All monoclonal antibodies (prepared from ascites by Jackson ImmunoResearch Laboratories, Boston, MA) were used as purified IgG preparations.
  • PBMC Peripheral blood mononuclear cells
  • Stimulator cells from a separate donor were irradiated at 1000 R and cultured with the responder cells at the same concentration. The total volume per culture was 0.2 ml. Controls included responder cells alone as well as stimulator cells alone.
  • the culture plates were incubated at 37 * C in a 5% C ⁇ 2-humidified air atmosphere for 5 days. The wells were pulsed with 0.5 ⁇ Ci of tritiated thymidine ( 3 HT) (New England Nuclear) for the last 18 hours of culture. In some cases a two-way MLR was performed. The protocol was the same except that the second donor's cells were not inactivated by irradiation.
  • 3 HT tritiated thymidine
  • the cells were harvested onto glass fiber filters using an automated multiple sample harvester (Skatron, Norway), rinsing with water and methanol .
  • the filters were oven dried and counted in Aquasol in a Beckman (LS-3801) liquid scintillation counter. Results are reported as the Mean CPM ⁇ standard error of 6 individual cultures.
  • Table 15 shows that purified anti-ICAM-1 monoclonal antibodies inhibited the MLR in a dose dependent manner with significant suppression apparent at 20 ng/ml. Purified mouse IgG had little or no suppressive effect. Suppression of the MLR by the anti-ICAM-1 monoclonal antibody occurs when the antibody is added within the first 24 hours of cultures (Table 16). TABLE 15
  • ICAM-1 monoclonal antibodies have therapeutic utility i acute graft rejection.
  • ICAM-1 monoclonal antibodies also hav therapeutic utility in related immune mediated disorders dependent o LFA-l/ICAM-1 regulated cell to cell interactions.
  • ICAM-1 is not expressed on resting human peripheral blood lymphocytes or monocytes.
  • ICAM-1 is up regulated on the monocytes of cultured cells alone or cells co-cultured with unrelated donor cells in a mixed lymphocyte reaction using conventional flow cyto etric analyses. This up regulation of ICAM-1 on monocytes can be used as an indicator of inflammation, particularly if ICAM-1 is expressed on fresh monocytes of individuals with acute or chronic inflammation.
  • the MLR is inhibited by anti-ICAM-1 antibody.
  • the MLR can also be inhibited by the anti-LFA-1 antibody.
  • an MLR assay (performed as described in Example 27) wa conducted in the presence of various concentrations of the tw antibodies.
  • the MLR is inhibited by combinations of anti-ICAM-1 and anti-LFA-1 antibodies.
  • anti-ICAM-1 and anti-LFA-1 antibodies include anti-ICAM-1 and anti-LFA-1 antibodies.
  • immunosi-ppressive agents such as dexamethasone, azathioprine, cyclosporin A or steroids (such as, for example, prednisone, etc.) would also have enhanced effects.
  • MLR assays were performed using sub-optimal concentrations (i.e concentrations which would be lower than the optimal concentration at which the agent alone would be provided to a subject) of R6-5-D6 in conjunction with other immunosuppressive agents as per the protocol in Example 27.
  • Stimulators 101 Responders (R) 4,461 R x S 34,199
  • Dex Dexamethasone
  • CyA Cyclosporin A
  • kidney transplantation was performed essentially as follows. Heterotropic renal allografts were performed in 3-5 kg Cynomolgus monkeys, essentially as described by Marquet (Marquet e al .. Medical Primatology. Part II, Basel, Karger, p. 125 (1972)) after induction of anesthesia with valium and ketamine. End-to-side anastomoses of donor renal vessels on a patch of aorta or vena cav were constructed using 7-0 Prolene suture. The donor ureter wa spatulated and implanted into the bladder by the extravesical approac (Taguchi, Y., et al .. in Dausset et al .
  • Renal function was evaluated by weekly or biweekly serum creatinin determinations.
  • frequent allograft biopsies were obtaine for histopathologic examination and complete autopsies were performe on all nonsurviving recipients.
  • bilateral nephrectomy was performed at the time of transplantation and subsequen uremic death was considered the end point of allograft survival.
  • unilateral native nephrectomy and contra!ateral ureteral ligation were performed at the time of transplantation.
  • R6-5-D6 was also tested in a therapeuti or acute kidney rejection model.
  • monkey kidneys were transplanted (using the protocol described in Example 30) and give perioperatively 15 mg/kg cyclosporin A (CyA) i.m. until stable renal function was achieved.
  • the dose of CyA was then reduced biweekly in 2.5 mg/kg increments until rejection occurred as indicated by a rise in blood creatinine levels.
  • R6-5-D6 was administered fo 10 days and the length of survival was monitored. It is important to note that in this protocol, the dose of CyA remains suboptimal since it does not change once the acute rejection episode occurs.
  • Days of survival/post treatment represents days of survival once creatinine levels started to rise. Died while under anesthesia. Creatinine levels were low. e Died of CyA toxicity. Creatinine levels were low. f Still living as of August 15, 1988.
  • ICAM-1 is a cell membrane-bound protein containing an extracellular region of 5 immunoglobulin-li e domains, a transmembrane domain, and a cytoplasmic domain. It was desirable to construct functional derivatives of ICAM-1 lacking the transmembrane domain and/or the cytoplasmic domain in that a soluble, secreted form of ICAM-1 could be generated. These functional derivatives we constructed by oligonucleotide-directed mutagenesis of the ICAM-1 gen followed by expression in monkey cells after transfection with t mutant gene.
  • ICAM-1 cD prepared as described above was digested with restriction endonucleas Sal 1 and Kpn 1, and the resulting 1.8 kb DNA fragment was subclon into the plasmid vector CDM8 (Seed, B. et al .. Proc. Natl. Acad. Sci (U.S.A.) 84:3365-3369 (1987)).
  • CDM8 Seed, B. et al .. Proc. Natl. Acad. Sci (U.S.A.) 84:3365-3369 (1987)).
  • the mutant ICAM-1 protein was expressed b transfection of Cos-7 cells with the mutant DNA in the eukaryoti expression vector CDM8 using standard DEAE-Dextran procedures (Selden R.F. et al .. In: Current Protocols in Molecular Biology (Ausubel, F.M et al., eds.) pages 9.2.1-9.2.6 (1987)).
  • a truncated functional derivative of ICAM-1 lacking th transmembrane and cytoplasmic domains, but containing the extracellula region possessing all 5 immunoglobulin-like domains was prepared.
  • the mutant was isolated by it unique Xba 1 restriction site, and was designated Y ⁇ 2 E/F,TAG.
  • COS cells were transfected wit three mutuant subclones (#2, #7, and #8).
  • ICAM-1 was precipitated fro the culture supernates of cells transfected with mutant subclones #2 and #8, but not from detergent lysates of those cells.
  • the molecula weight of ICAM-1 found in the culture supernate was reduced approximately 6 kd relative to the membrane form of ICAM-1, which is consistent with the size predicted from the mutant DNA.
  • ICAM-1 is excreted as a soluble protein.
  • ICAM-1 was not immunoprecipitated from control culture supernates of cells transfected with native ICAM-1, demonstrating that the membrane form of ICAM-1 is not shed from Cos cells. Futhermore, no ICAM-1 was immunoprecipitated from either culture supernates or cell lysates from negative control mock-transfected cells.
  • the truncated ICAM-1 secreted from transfected cells was purifie by immunoaffinity chromatography with an ICAM-1 specific antibody (R6- 5-D6) and tested for functional activity in a cell binding assay. After purification in the presence of the detergent octylglucoside, preparations containing native ICAM-1 or the truncated, secreted for were diluted to a final concentration of 0.25% octylglucoside ( concentration below the critical micelle concentration of th detergent). These preparations of ICAM-1 were allowed to bind to th surfaces of plastic 96-well plates (Nunc), to produce ICAM-1 bound to solid-phase.
  • a functional derivative of ICAM-1 lacking only the cytoplasmi domain was prepared by similar methods.
  • a 25 bp oligonucleotide (T AGC ACG TAC CTC TAG AAC CGC CA) was used to alter the codon for amin acid 476 (Y) to a TAG trans!ational stop codon.
  • the mutant wa designated Y 476 /TAG.
  • Immunoprecipitation analysis and SDS-PAGE of Co cells transfected with the mutant detected a membrane bound form o ICAM-1 with a molecular weight approximately 3 kd less than nativ ICAM-1.
  • ICAM-1 possesses 7 domains. Five of these domains are extracellular (domain 5 being closest to the cell surface, domain 1 being furthest from the cell surface), one domain is a transmembrane domain, and one domain is cytoplasmic (i.e. lies within the cell).
  • epitope mapping studies may be used. To conduct such studies, different deletion mutants are prepared and characterized for their capacity to bind to LFA-1. Alternatively, the studies may be accomplished using anti-ICAM antibody known to interfere with the capacity of ICAM-1 to bind LFA-1. Examples of such suitable antibody include RRl/1
  • Peletion mutants of ICAM-1 can be created by any of a variety of means. It is, however, preferable to produce such mutants via site directed mutagenesis, or by other recombinant means (such as by constructing ICAM-1 expressing gene sequences in which sequences that, encode particular protein regions have been deleted. Procedures which may be adapted to produce such mutants are well known in the art. Using such procedures, three ICAM-1 deletion mutants were prepared. The first mutant lacks amino acid residues F185 through P284 (i.e. deletion of domain 3). The second mutant lacks amino acid residues P284 through R451 (i.e. deletion of domains 4 and 5). The third mutant lacks amino acid residues after Y476 (i.e. deletion of cytoplasmic domain). The results of such studies indicate that domains 1, 2, and 3 are predominantly involved in ICAM-1 interactions with anti-ICAM-1 antibody and LFA-1.
  • ICAM-1 amino acid residues which are present in domains 1 of the ICAM-1 molecule are present in domains 1 of the ICAM-1 molecule ( Figures 8, 9 and 10). Such interactions are assisted, however, by contributions from amino acids present in domains 2 and 3 of ICAM-1.
  • soluble fragments of the ICAM-1 molecule which contain domains 1, 2, and 3 of ICAM-1. More preferred are soluble fragments of the ICAM-1 molecule which contain domains 1 and 2 of ICAM 1. Most preferred are soluble fragments of the ICAM-1 molecule whic contain domain 1 of ICAM-1.
  • Glycosylation sites in the second domain are also involved in LFA-1 binding ( Figure 23).
  • replacement of the glycosylation site N175 with A did not appear to substantially effect the capacity of the mutant ICAM-1 to bind LFA-1.
  • Mutations in the third ICAM-1 domain did not appreciably alter ICAM- 1 - LFA-1 binding ( Figure 24) .
  • Chimeric molecules are constructed in which domains 1 and 2 of ICAM- 1 are attached to the hinge region of the immunoglobulin heavy chain.
  • Preferred constructs attach the C-terminus of ICAM-1 domain 2 to a segment of the immunoglobulin heavy chain gene just N-terminal to the hinge region, allowing the segmental flexibility conferred by the hinge region.
  • the ICAM-1 domains 1 and 2 will thus replace the Fab fragment of an antibody. Attachment to heavy chains of the IgG class and production of animal cells will result in the production of a chimeric molecule. Production of molecules containing heavy chains derived from IgA or IgM will result in production of molecules of higher multimericy containing from 2 to 12 ICAM-1 molecules.
  • IgA and IgM multimers resulting predominantly in IgA molecules containing 4 to 6 ICAM-1 molecules and in the case of IgM containing approximately 10 ICAM-1 molecules.
  • Ig molecules may have several advantages. First, Ig molecules are designed to be long lasting in the circulation and this may improve biological half-life.
  • IgA and IgM are highly glycosylated molecules normally present in secretions in mucosal sites as in the nose. Their highly hydrophilic nature helps to keep bacteria and viruses to which they bind out in the mucosa, preventing attachment to cells and preventing crossing of the epithelial cell membrane barrier. Thus, they may have increased therapeutic efficacy.
  • IgM an in particularly IgA are stable in mucosal environments and they ma increase the stability of the ICAM-1 constructs.
  • ICAM- functional derivative may als increase biological half-life.
  • IgA does not fix complement and thu would be ideal for applications in which this would be deleterious.
  • I IgG H chain chimerics are desired, it would be possible to mutat regions involved in attachment to complement as well as in interaction with Fc receptors.
  • E. coli strain XS127 was transformed with pCDl.8. Single colonies were grown in one ml of Luria Broth (LB) medium (Difco) with 13 ⁇ g/ml ampicillin and 8 ⁇ g/ml tetracycline until near saturation. 100 ⁇ l of the culture was infected with R408 helper phage (Strategene) at a multiplicity of infection (MOI) of 10, and 10 ml of LB medium with ampicillin and tetracycline was added for a 16 hr culture at 37°C. Following centrifugation at 10,000 rpm for one minute, and 0.22 ⁇ m filtration of the supernatant, the phage suspension was used to infect E.
  • LB Luria Broth
  • MOI multiplicity of infection
  • coli BW313/P3 which was then plated on LB agar (Difco) plates supplemented with ampicillin a d tetracycline. Colonies were picked, grown in 1 ml LB medium • ⁇ th ampicillin and tetracycline to near saturation and infected with helper phage at MOI of 10. Culture volume was then increased to 250 ml and the cells were cultured overnight. Single strand DNA was isolated by standard phage extraction.
  • Mutant oligonucleotides were phosphorylated and utilized with the pCDl.8 template in a second strand synthesis reaction (Staunton, D.E. et al.. Cell 52:925-933 (1988)).
  • COS cells were seeded into 10 cm tissue culture plates such that they would be 50% confluent by 16-24 hrs. COS cells were then washed once with TBS and incubated for 4 hrs with 4 ml RPMI containing 10% Nu sera (Collaborative) 5 ⁇ g/ml chloroquine, 3 ⁇ g of mutant plasmid and 200 ⁇ g/ml DEAE-dextran sulfate. Cells were then washed wit 10% DMSO/PBS followed by PBS and cultured 16 hrs in culture media.
  • OS cells were suspended by trypsin/EDTA (Gibco) treatment and divided into 2, 10 cm plates as well as 24 well tissue culture plates for HRV binding. At 72 hrs cells were harvested from 10 cm plates with 5 mM EDTA/HBSS and processed for adhesion to LFA-1 coated plastic and immunofluorescence.
  • LFA-1 was purified from SKW-3 lysates by immunoaffinity chromatography on TS2/4 LFA-1 mAb Sepahrose and eluted at pH 11.5 in the presence of 2 mM MgCl2 and 1% octylgucoside.
  • LFA-1 (10 ⁇ g per 200 ⁇ l per 6-cm plate) was bound to bacteriological Petri dishes by diluting octylglucoside to 0.1% in PBS (phosphate buffered saline) with 2 mM MgCl2 and overnight incubation at 4°C. Plates were blocked with 1% BSA (bovine serum albumin) and stored in PBS containing 2mM MgCl2, 0.2% BSA, 0.025% azide, and 50 ⁇ g/ml gentamycin.
  • BSA bovine serum albumin
  • Anti-ICAM-1 antibodies such as RRl/1, R6.5, LB-2, or CL203 have been identified. If these antibodies are capable of inhibiting ICAM-1 function, they must be capable of binding to a particular site in the ICAM-1 molecule which is also important to the ICAM-1 function. Thus, by preparing the above-described deletion mutants of ICAM-1, and determining the extent to which the anti-ICAM-1 antibodies can bind to the deletion, it is possible to determine whether the deleted domains are important for function.
  • ICAM-1 is an integral membrane protein, of which the extracellular domain is predicted to be composed of 5 Ig-like C-domains.
  • domain 3 and domains 4 and 5 were deleted by oligonucleotide-directed mutagenesis and tested functionally following expression in COS cells.
  • the entire cytoplasmic domain was deleted to ascertain its potential influence on ICAM-1 interactions.
  • Y476/* demonstrated no loss of RRl/1, R6.5, LB-2 and CL203 reactivity
  • deletion of domain 3, F185- R451 resulted in a decrease and loss of CL203 reactivity, respectively ( Figure 20).
  • the CL203 epitope appears to be located in domain 4 whereas RRl/1, R6.5 and LB-2 appear to be located in the 2 amino- terminal domains.
  • LFA-1 and HRV binding appears to be a function of the amino terminal Ig-like domain of ICAM-1.
  • Figure 26 shows an alignment o ICAM amino terminal domains.

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Abstract

L'invention concerne les molécules d'adhésion intercellulaire (ICAM-1) impliquées dans le processus de reconnaissance par des lymphocytes de sites d'inflammation, de migration des lymphocytes aux sites d'inflammation et de fixation des lymphocytes à des supports cellulaires lors d'une inflammation. L'invention concerne ces molécules, des essais d'analyse et d'identification de ces molécules et d'anticorps capables de se lier à de tels molécules, ainsi que des utilisations diverses de ces molécules d'adhésion et des anticorps capables de se lier à celles-ci.
PCT/US1989/004242 1988-09-28 1989-09-28 Molecules d'adhesion intercellulaire et leurs ligands de liaison WO1990003400A1 (fr)

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RU92016375A RU2126449C1 (ru) 1988-09-28 1989-09-28 Производное вещества межклеточной адгезии icam-1, последовательность днк
KR1019900701105A KR900701846A (ko) 1988-09-28 1989-09-28 세포간 점착분자 및 이의 결합 리간드
FI902490A FI102181B (fi) 1988-09-28 1990-05-21 Menetelmä ICAM-1 (solujen välisen kiinnikemolekyylin) saamiseksi oleel lisesti puhtaassa muodossa
DK199001289A DK175362B1 (da) 1988-09-28 1990-05-25 Oplöseligt funktionelt derivat af ICAM-1, rekombinant DNA-molekyle og fremgangsmåde til fremstilling af nævnte derivat, farmaceutisk præparat indeholdende et sådant derivat og derivatets anvendelse til fremstilling af et sådant præparat, samt....
NO90902311A NO902311L (no) 1988-09-28 1990-05-25 Intracellulaere adhesjonsmolekyler og deres bindingsligander.
FI981097A FI107451B (fi) 1988-09-28 1998-05-18 Menetelmä ICAM-1 (solujen välisen kiinnikemolekyylin) saamiseksi oleellisesti puhtaassa muodossa

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EP0391088A2 (fr) * 1989-03-16 1990-10-10 Center For Blood Research Laboratories, Inc. Utilisation de dérivés fonctionnels de la molécule d'adhésion intercellulaire ICAM-1 dans une thérapie anti-virale
WO1992000751A1 (fr) * 1990-07-06 1992-01-23 Novo Nordisk A/S Composition pharmaceutique comprenant une molecule d'adherence cellulaire
WO1993025218A1 (fr) * 1992-06-11 1993-12-23 The Scripps Research Institute Procedes et compositions d'inhibition d'inflammations provoquees par des cellules endotheliales et par le fibrinogene
WO1994008620A1 (fr) * 1992-10-09 1994-04-28 Center For Blood Research, Inc. SOUS-POPULATION DE MOLECULES Mac-1 (CD11B/CD 18) QUI INDUISENT L'ADHESION NEUTROPHILE A ICAM-1 ET AU FIBRINOGENE
WO1995010533A1 (fr) * 1993-10-15 1995-04-20 KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG Peptides pour therapie tumorale
US5589453A (en) * 1988-09-01 1996-12-31 Molecular Therapeutics, Inc. Human rhinovirus receptor protein (ICAM-1) that inhibits rhinovirus attachment and infectivity
WO1997000956A1 (fr) * 1995-06-20 1997-01-09 Trustees Of Boston University Molecules d'adherence reagissant a l'hypoxemie, anticorps specifiques et leurs utilisations
US5599790A (en) * 1992-06-11 1997-02-04 The Scripps Research Institute Fibrinogen γ chain polypeptide and compositions thereof
EP0762886A1 (fr) * 1994-04-19 1997-03-19 The University Of Kansas Peptides a chaine courte a base d'icam-1/lfa-1 et leur methode d'utilisation
US5674982A (en) * 1990-07-20 1997-10-07 Bayer Corporation Multimeric form of human rhinovirus receptor protein
US5686582A (en) * 1990-07-20 1997-11-11 Bayer Corporation Multimeric forms of human rhinovirus receptor protein
US5932214A (en) * 1994-08-11 1999-08-03 Biogen, Inc. Treatment for inflammatory bowel disease with VLA-4 blockers
US6107461A (en) * 1990-07-20 2000-08-22 Bayer Corporation Multimeric forms of human rhinovirus receptor and fragments thereof, and method of use
US6143298A (en) * 1988-09-01 2000-11-07 Bayer Corporation Soluble truncated forms of ICAM-1
WO2001015733A2 (fr) * 1999-09-01 2001-03-08 Boehringer Ingelheim Pharmaceuticals, Inc. Methodes et compositions destinees au traitement des maladies auto-immunes
US6844011B1 (en) 1990-08-30 2005-01-18 The General Hospital Corporation Methods for inhibiting rejection of transplanted tissue
US7314938B2 (en) 2003-11-05 2008-01-01 Sunesis Pharmaceuticals, Inc. Modulators of cellular adhesion
US8080562B2 (en) 2008-04-15 2011-12-20 Sarcode Bioscience Inc. Crystalline pharmaceutical and methods of preparation and use thereof
US8084047B2 (en) 2005-05-17 2011-12-27 Sarcode Bioscience Inc. Compositions and methods for treatment of eye disorders
US8378105B2 (en) 2009-10-21 2013-02-19 Sarcode Bioscience Inc. Crystalline pharmaceutical and methods of preparation and use thereof
US9085553B2 (en) 2012-07-25 2015-07-21 SARcode Bioscience, Inc. LFA-1 inhibitor and methods of preparation and polymorph thereof
WO2019105864A1 (fr) 2017-11-30 2019-06-06 F. Hoffmann-La Roche Ag Procédé de culture de lymphocytes b
US10960087B2 (en) 2007-10-19 2021-03-30 Novartis Ag Compositions and methods for treatment of diabetic retinopathy

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WO1991018010A1 (fr) * 1990-05-15 1991-11-28 Swinburne Limited Inhibition d'infection virale a l'aide de peptides analogues a la molecule-1 d'adhesion intercellulaire et/ou d'analogues de ces peptides
WO1991018011A1 (fr) * 1990-05-15 1991-11-28 Swinburne Limited Inhibition d'adhesion cellulaire a l'aide de peptides analogues a la molecule-1-d'adhesion intercellulaire et/ou d'analogues de ces peptides
CA2056143A1 (fr) * 1990-11-28 1992-05-29 Michael S. Diamond Le site de liaison mac-1 de l'icam-1
US9175091B2 (en) * 2007-11-15 2015-11-03 Chugai Seiyaku Kabushiki Kaisha Monoclonal antibody capable of binding to anexelekto, and use thereof

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Cited By (59)

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US5589453A (en) * 1988-09-01 1996-12-31 Molecular Therapeutics, Inc. Human rhinovirus receptor protein (ICAM-1) that inhibits rhinovirus attachment and infectivity
US6143298A (en) * 1988-09-01 2000-11-07 Bayer Corporation Soluble truncated forms of ICAM-1
EP0391088A2 (fr) * 1989-03-16 1990-10-10 Center For Blood Research Laboratories, Inc. Utilisation de dérivés fonctionnels de la molécule d'adhésion intercellulaire ICAM-1 dans une thérapie anti-virale
EP0391088A3 (fr) * 1989-03-16 1991-10-02 Center For Blood Research Laboratories, Inc. Utilisation de dérivés fonctionnels de la molécule d'adhésion intercellulaire ICAM-1 dans une thérapie anti-virale
WO1992000751A1 (fr) * 1990-07-06 1992-01-23 Novo Nordisk A/S Composition pharmaceutique comprenant une molecule d'adherence cellulaire
US6107461A (en) * 1990-07-20 2000-08-22 Bayer Corporation Multimeric forms of human rhinovirus receptor and fragments thereof, and method of use
US5674982A (en) * 1990-07-20 1997-10-07 Bayer Corporation Multimeric form of human rhinovirus receptor protein
US5686582A (en) * 1990-07-20 1997-11-11 Bayer Corporation Multimeric forms of human rhinovirus receptor protein
US5686581A (en) * 1990-07-20 1997-11-11 Bayer Corporation Multimeric form of human rhinovirus receptor protein
US5871733A (en) * 1990-07-20 1999-02-16 Bayer Corporation Multimeric forms of human rhinovirus receptor protein
US6844011B1 (en) 1990-08-30 2005-01-18 The General Hospital Corporation Methods for inhibiting rejection of transplanted tissue
US7176184B2 (en) 1992-02-12 2007-02-13 Biogen Idec Ma Inc. Treatment for inflammatory bowel disease with a fibronectin polypeptide
US6265549B1 (en) 1992-06-11 2001-07-24 The Scripps Research Institute Methods and compositions for inhibiting endothelial cell and fibrinogen mediated inflammation
US6737058B2 (en) 1992-06-11 2004-05-18 The Scripps Research Institute Methods for inhibiting endothelial cell and fibrinogen mediated inflammation using fibrinogen specific antibodies
US5919754A (en) * 1992-06-11 1999-07-06 The Scripps Research Institute Method of inhibiting fibrinogen binding to endothelial cells with fibrinogen gamma chain peptides
WO1993025218A1 (fr) * 1992-06-11 1993-12-23 The Scripps Research Institute Procedes et compositions d'inhibition d'inflammations provoquees par des cellules endotheliales et par le fibrinogene
US5599790A (en) * 1992-06-11 1997-02-04 The Scripps Research Institute Fibrinogen γ chain polypeptide and compositions thereof
US6676940B2 (en) 1992-06-11 2004-01-13 The Scripps Research Institute Methods and compositions for inhibiting endothelial cell and fibrinogen mediated inflammation
WO1994008620A1 (fr) * 1992-10-09 1994-04-28 Center For Blood Research, Inc. SOUS-POPULATION DE MOLECULES Mac-1 (CD11B/CD 18) QUI INDUISENT L'ADHESION NEUTROPHILE A ICAM-1 ET AU FIBRINOGENE
DE4335273A1 (de) * 1993-10-15 1995-04-20 Univ Ludwigs Albert Peptide zur Tumortherapie
WO1995010533A1 (fr) * 1993-10-15 1995-04-20 KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG Peptides pour therapie tumorale
EP0762886A4 (fr) * 1994-04-19 1999-03-31 Univ Kansas Peptides a chaine courte a base d'icam-1/lfa-1 et leur methode d'utilisation
EP0762886A1 (fr) * 1994-04-19 1997-03-19 The University Of Kansas Peptides a chaine courte a base d'icam-1/lfa-1 et leur methode d'utilisation
US5932214A (en) * 1994-08-11 1999-08-03 Biogen, Inc. Treatment for inflammatory bowel disease with VLA-4 blockers
US6482409B1 (en) 1994-08-11 2002-11-19 Biogen, Inc. Treatment for inflammatory bowel disease with a vcam-1/1gG fusion protein
WO1997000956A1 (fr) * 1995-06-20 1997-01-09 Trustees Of Boston University Molecules d'adherence reagissant a l'hypoxemie, anticorps specifiques et leurs utilisations
WO2001015733A3 (fr) * 1999-09-01 2001-09-13 Boehringer Ingelheim Pharma Methodes et compositions destinees au traitement des maladies auto-immunes
WO2001015733A2 (fr) * 1999-09-01 2001-03-08 Boehringer Ingelheim Pharmaceuticals, Inc. Methodes et compositions destinees au traitement des maladies auto-immunes
US7314938B2 (en) 2003-11-05 2008-01-01 Sunesis Pharmaceuticals, Inc. Modulators of cellular adhesion
US7745460B2 (en) 2003-11-05 2010-06-29 Sarcode Corporation Modulators of cellular adhesion
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US8367701B2 (en) 2008-04-15 2013-02-05 Sarcode Bioscience Inc. Crystalline pharmaceutical and methods of preparation and use thereof
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US10214517B2 (en) 2012-07-25 2019-02-26 Sarcode Bioscience Inc. LFA-1 inhibitor and methods of preparation and polymorph thereof
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US11952586B2 (en) 2017-11-30 2024-04-09 Hoffmann-La Roche Inc. B-cell cultivation method

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