Classifications

• • 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
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• • 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
  • A61P37/00—Drugs for immunological or allergic disorders
• • 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
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to the discovery of a new intercellular
• ICAM-3 ⁇ 15 adhesion molecule
• ICAM-3 mediates cellular interactions with other lymphocytes, macrophages and neutrophils at the sites of inflammation and sites of immune responses.
• the present invention further relates to the use of ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2, to inhibit intercellular adhesion of cells of granulocyte, lymphocyte, or macrophage lineage.
• ICAM-3 alone or in combination with ICAM-1 and/or ICAM-2
• the use of such molecules provides a method for the treatment of specific and non-specific inflammation.
• the invention also relates to therapeutic and prophylactic methods for suppressing the infection of leukocytes with HIV, and particularly with HIV-1, in an individual who is exposed to HIV or infected by HIV through the administration of ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV virus.
• AIDS Abrered Immunodeficiency Syndrome
• the invention also relates to a therapeutic method for suppressing the migration of HTV-1 infected cells from the circulatory system using ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2. It therefore provides a therapy for diseases, such a ⁇ , AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV-1 virus.
• diseases such as a ⁇ , AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV-1 virus.
• the invention also relates to a therapeutic method for suppressing T- cell death and "syncytia" formation in an individual infected with HIV using
• ICAM-3 alone or in combination with ICAM-1 and/or ICAM-2. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency
• the present invention relates to the use of ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2, in the treatment of asthma.
• the present invention additionally relates to molecules capable of binding to ICAM-3 (hereinafter anti-ICAM-3).
• anti-ICAM-3 molecules capable of binding to ICAM-3
• the binding of an anti-ICAM-3 molecule to ICAM-3 is intended to modulate the biological functions associated with ICAM-3.
• the binding molecules of the present invention can be an antibody, a peptide, or a carbohydrate which is capable of binding to ICAM-3. Such binding molecules are useful in modulating the biological functions of ICAM-3.
• the present invention also relates to the use of a anti-ICAM-3, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2, to inhibit intercellular adhesion of cells of granulocyte, lymphocyte, or macrophage lineage.
• a anti-ICAM-3 alone or in combination with anti-ICAM-1 and/or anti-ICAM-2, to inhibit intercellular adhesion of cells of granulocyte, lymphocyte, or macrophage lineage.
• the use of such molecules provides a method for the treatment of specific and non ⁇ specific inflammation.
• the invention also relates to therapeutic and prophylactic methods for suppressing the infection of leukocytes with HIV, and particularly with HIV-1, in an individual who is exposed to HIV or infected with HIV through the administration of a anti-ICAM-3, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV virus.
• the invention also relates to a therapeutic method for suppressing the migration of HIV-1 infected cells from the circulatory system using an anti- ICAM-3 agent, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2 agent.
• the present invention further relates to the use of ann anti-ICAM-3 agent, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2, in the treatment of asthma.
• Leukocytes must be able to attach to cellular substrates in order to properly defend the host against foreign invaders such as bacteria or viruses, see Eisen, H.W., (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418) for a review of these functions.
• Leukocytes must be able to attach to endothelial cells so that they can migrate from the circulatory system to sites of 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 the above attachment mediated functions were identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T-cells (Davignon, D. el al, Proc. Nad. Acad. Set USA 75:4535-4539 (1981)) and mouse spleen cells (Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979)) were identified which bound to leukocyte surfaces and inhibited the attachment related functions described above (Springer, T. et al, Fed. Proc. 44:2660-2663 (1985)). The molecules identified by those antibodies were called Mac-1 and Lymphocyte
• Mac-1 is a heterodimer found on macrophages, granulocytes aad large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, T.A. et al. Immunol. Rev. 65:111-135 (1982)). These two molecules, plus a third molecule, pl50,95 (which has a tissue distribution similar to Mac-1) play a role in cellular adhesion
• the above-described leukocyte molecules were found to be members of a related family of glycoproteins (Sanchez-Madrid, F. et al, J. Exper. Med. 255:1785-1803 (1983); Keizer, G.D. et al, Eur. J. Immunol. 25:1142-1147 (1985)), termed the "CD-18 family" of glycoproteins.
• This glycoprotein family is composed of heterodimers having one alpha chain and one beta chain. Although the alpha chain of each of the antigens differed from one another, the beta chain was found to be highly conserved (Sanchez-Madrid, F. et al, J. Exper. Med. 255:1785-1803 (1983)).
• the beta chain of the glycoprotein family (sometimes referred to as "CD18") was found to have a molecular weight of 95 kd whereas the alpha chains were found to vary from 150 kd to 180 kd (Springer, T., Fed. Proc. 44:2660-2663 (1985)).
• CD18 The beta chain of the glycoprotein family
• the alpha subunits of the membrane proteins do not share the extensive homology shared by the beta subunits, close analysis of the alpha subunits of the glycoproteins has revealed that there are substantial similarities between them. Reviews of the similarities between the alpha and beta subunits of the LFA-1 related glycoproteins are provided by Sanchez-Madrid, F. et al, (J. Exper. Med. 255:586-602 (1983); J. Exper. Med.
• Characteristic features of LAD patients include necrotic soft tissue lesions, impaired *pus formation and wound healing, as well as abnormalities of adhesion-dependent leukocyte functions in vitro, and susceptibility to chronic and recurring bacterial infections.
• Granulocytes from these LAD patients behave in the same defective manner in vitro as do their normal counterparts in the presence of anti-CD18 monoclonal antibody. That is, they are unable to perform adhesion related functions such as aggregation or attachment to endothelial cells. More importantly, however, is the observation that these patients are unable to mount a normal inflammatory response because of the inability of their granulocytes to attach to cellular substrates.
• HIV HTV infection B. Infection with HIV HTV infection is the cause of AIDS. Many variants of HIV have been described: the major two are HIV-1 and HIV-2. HIV-1 is prevalent in North
• HIV infection is believed to occur via the binding of a viral protein (termed “gpl20”) to a receptor molecule (termed “CD4") present on the surface of T4 ("T helper") lymphocytes (Schnittman, S. M. et al, J. Immunol. 141.4181-4186 (1988), which reference is incorporated herein by reference). After binding this receptor, the virus enters the cell and replicates, and in the process, kills the T cell. The destruction of an individual's T4 population is thus a direct result of HIV infection.
• gpl20 a viral protein
• CD4 receptor molecule
• T helper T helper lymphocytes
• T cells The destruction of the T cells results in an impairment in the ability of the infected patient to combat opportunistic infections.
• individuals afflicted with AIDS often develop cancers, the relationship between these cancers and HIV infection is, in most cases, uncertain.
• HIV infected cells can be destroyed through the action of cytotoxic, killer cells.
• Killer cells are normally present in humans, and serve to monitor the host and destroy any foreign cells (such as in mismatched blood transfusions or organ transplants, etc.) which may be encountered.
• T4 cells Upon infection with HIV, T4 cells display the gpl20 molecule on their cell surfaces. Killer cells recognize such T4 cells as foreign (rather than native cells), and accordingly, mediate their destruction.
• HIV infection can also lead to the destruction of non-infected healthy cells.
• Infected cells can secrete the gpl20 protein into the blood system.
• the free gpl20 molecules can then bind to the CD4 receptors of healthy, uninfected cells. Such binding causes the cells to take on the appearance of HIV infected cells.
• Cytotoxic, killer cells recognize the gpl20 bound to the uninfected T4 cells, conclude that the cell is foreign, and mediate the destruction of the cell.
• An additional mechanism, and one of special interest to the present invention, with which HIV can cause T4 cell death is through the formation of "syncytia."
• a "syncytium” is a multinucleated giant cell, formed from the fusion of as many as several hundred T4 cells.
• Infection with HIV causes the infected cell to gain the ability to fuse with other T4 cells, either HIV infected, or uninfected healthy cells.
• the syncytium cannot function and soon dies. Its death accomplishes the destruction of both HIV infected and HIV uninfected T4 cells.
• This process is of special interest to the present invention since it entails the direct cell-cell contact of T4 cells.
• the ability of HI V-infected cells to form syncytia indicates that such cells acquire a means for fusing with healthy cells.
• HIV infection and especially HIV-1 infection, appears to influence cell surface expression of the leukocyte integrins and cellular adherence reactions mediated by these heterodimers (Petit, A.J., et al, J. Clin. Invest. 79:188 (1987): Hildreth, J.E.K., et al, Science 244:1015 (1989); Valentin, A., et al, J. Immunology 144:934-931 (1990); Rossen, R.D., et al, Trans. Assoc. American Physicians 102:111-130 (1989), all of which references are incorporated herein by reference).
• HIV-1 infected U937 cells adhere to IL-1 stimulated endothelium in greater frequency than uninfected U937 cells; this behavior can be suppressed by treating the infected cells with anti-CD18 or anti- CDlla monoclonal antibodies or by treating endothelial substrates with anti-
• ICAM-1 antibodies Monoclonal antibodies to CD18 or CDlla have also been found to be able to inhibit formation of syncytia involving phytohemagglutinin (PHA)-stimulated lymphoblastoid cells and constitutively infected, CD4-negative T cells (Hildreth, J.E.K., et al, Science 244:1015 (1989)).
• PHA phytohemagglutinin
• HIV infection involves cell-cell interactions, and/or viral-cell interactions which mimic such cell-cell interactions.
• the cell-cell interactions ma ⁇ result in the transport of cell-free virus or the transport of virus infected cells across endothelial barriers.
• Viral-cell interactions which mimic the cell-cell interactions may facilitate or enable free virus to attach to and/or infect healthy cells.
• the present invention thus derives, in part, from the observation that HIV infection, and particularly HIV-1 infection, results in increased expression of the CDlla/CD18 heterodimer and its binding ligand.
• This increased expression is significant in that it enhances the ability of HIV-infected T cells to adhere or aggregate with one another (i.e. to undergo "homotypic aggregation"). Since such homotypic aggregation is not observed to occur among quiescent normal leukocytes, this discovery indicates that the expression of the CD11/CD18 receptors and/or ligands, such as ICAM-1, is required for such aggregation. LFA-1 must bind to ICAM-1 in order for homotypic aggregation to occur.
• ICAM-3 is the only member of the ICAM family of molecules which is expressed at a high level on resting T-cells. Only anti-ICAM-3 antibodies are capable of blocking the adhesion of T-cells to LFA-1 unless the T-cells are "activated”. Therefore anti-ICAM-3 antibodies can be used to suppress aggregation of T-cells.
• anti-ICAM-3 antibodies may be used to block the adhesion processes of infected T-cells which permits HIV-1 to be transmitted from an infected cell to a healthy cell of an individual, and also permits or facilitates infection of healthy cells with free virus.
• leukocytes The migration and dissemination of leukocytes is important in protecting an individual from the consequences of infection. These processes, however, are also responsible for the migration and dissemination of viral- infected leukocytes. Of particular concern is the migration and dissemination of leukocytes infected with HIV. The migration of such cells results in the formation of extravascular foca, and may cause tumors and other abnormalities. Histologic examination of affected organs reveals focal extravascular mononuclear cell infiltrates. Attempts to identify virus-infected cells in such infiltrates in the central nervous system have revealed the presence of HIV-1 infected cells. These studies have shown that the HIV-1 virus resides primarily in monocytes and macrophages, and other cells of this lineage (R.T. Johnson, et al. FASEB I. 2:2910 (1988); M.H. Stoler et al, I. Amer. Med. Assn. 256:2360 (1986); S. Gartner et al. Science 233:215 (19
• the mechanisms which stimulate formation of extravascular infiltrates of HIV-1-infected monocytoid cells have not previously been well defined.
• the mechanisms may involve either the transport of cell-free virus or the transport of virus across endothelial barriers within the cytoplasm of infected mononuclear cells. Since infection with HIV-1 stimulates cell surface expression of molecules which facilitate adherence of leukocytes to vascular endothelial cells and the translocation of leukocytes from the blood to extravascular tissue sites (C.W. Smith et al, J. Clin. Invest. 82:1146 (1988), herein incorporated by reference) it has been proposed to use antibodies which inhibit cellular migration to prevent the dissemination of HIV infected cells (WO 90/13316).
• Asthma is a heterogeneous family of diseases. It is characterized by a hyper-responsiveness of the tracheobronchi to stimuli (Kay, A.B., Allergy and Inflammation, Academic Press, NY (1987); which reference is incorporated herein by reference). Clinically, asthma is manifested by the extensive narrowing of the tracheobronchi, by thick tenacious secretions, by paroxysms of dyspnea, coughing, and wheezing. Although the relative contribution of each of these conditions is unknown, the net result is an increase in airway resistance, hyperinflation of the lungs and thorax and abnormal distribution of ventilation and pulmonary blood flow.
• Allergic asthma is usually associated with a heritable allergic disease, such as rhinitis, urticaria, eczema, etc.
• the condition is characterized by positive wheal-and-flare reactions to intradermal injections of airborne antigens (such as pollen, environmental or occupational pollutants, etc.), and increased serum levels of IgE.
• airborne antigens such as pollen, environmental or occupational pollutants, etc.
• AUergic asthma is believed to be dependent upon an IgE response controlled by T and B lymphocytes and activated by the interaction of airborne antigen with mast cell-bound pre-formed IgE molecules.
• the antigenic encounter must occur at concentrations sufficient to lead to IgE production for a prolonged period of time in order to sensitize an individual. Once sensitized, an asthma patient may exhibit symptoms in response to extremely low levels of antigen.
• Asthma symptoms may be exacerbated by the presence and amount of the triggering antigen, environmental factors, occupational factors, physical exertion, and emotional stress.
• Asthma may be treated with methylxanthines (such as theophylline), beta-adrenergic agonists (such as catecholamines, resorcinols,* saligenins, and ephedrine), glucocorticoids (such as hydrocortisone), inhibitors of mast cell degranulation (i.e. chromones such as cromolyn sodium) and anticholinergics (such as atropine).
• methylxanthines such as theophylline
• beta-adrenergic agonists such as catecholamines, resorcinols,* saligenins, and ephedrine
• glucocorticoids such as hydrocortisone
• inhibitors of mast cell degranulation i.e. chromones such as cromolyn sodium
• anticholinergics such as atropine
• eosinophilia eosinophils
• eosinophil hypothesis suggests that eosinophils are attracted to the bronchus in order to neutralize harmful mediators released by the mast cells of the lung. According to the hypothesis eosinophils are attracted to the bronchi where they degranulate to release cytotoxic molecules. Upon degranulation, eosinophils release enzymes such as histaminase, arylsulfatase and phospholipase D which enzymatically neutralize the harmful mediators of the mast cell.
• asthma involves the migration of cells, it has been proposed to use antibodies which inhibit this migration to mitigate the effects of allergens in a subject (WO 90/10453).
• the present invention is based on the discovery of a new cellular adhesion molecule, denoted Intercellular Adhesion Molecule-3 (ICAM-3).
• ICAM-3 Intercellular Adhesion Molecule-3
• the invention additionally pertains to functional derivatives of ICAM-3, anti-ICAM- 3 antibodies, fragments of said antibodies humanized anti-ICAM-3 antibodies, and other molecules capable of binding to and inhibiting the biological function of ICAM-3.
• the invention additionally includes diagnostic and therapeutic uses for all of the above-described molecules.
• the invention includes ICAM-3, or a functional derivative thereof, substantially free of natural contaminants.
• the invention provides a method of obtaining a recombinant or synthetic DNA molecule capable of encoding, or expressing ICAM-3, or a functional derivative thereof.
• the invention additionally provides an antibody, and especially a monoclonal antibody, capable of binding to a molecule selected from the group consisting of ICAM-3, and a functional derivative of ICAM-3.
• the invention also provides a hybridoma cell capable of producing the above-described monoclonal antibody.
• the invention includes a method for producing a desired hybridoma cell that produces an antibody which is capable of binding to ICAM-3, or a functional derivative thereof, which comprises the steps:
• ICAM-3 a membrane of a cell expressing ICAM-3, ICAM-3 bound to a carrier, a peptide fragment of ICAM-3, or a peptide fragment of ICAM-3 bound to a carrier,
• the invention also provides a method for modulating the ICAM-3 mediated biological functions of a cell wherein said method comprises providing to a subject in need of such a treatment an effective amount of an ICAM-3 modulating agent, wherein said ICAM-3 modulating agent is selected from the group consisting of: an antibody capable of binding to ICAM-3; a fragment of said antibody, said fragment being capable of binding to ICAM-3; ICAM-3; a functional derivative of ICAM-3; and a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1, ICAM-2, or a member of the CD-18 family of molecules.
• the invention also provides a method of treating specific inflammation in humans, and other mammals, wherein said method 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-
• the invention also provides a method for treating non-specific inflammation in humans, and other mammals, wherein said method 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-3; a fragment of said antibody; said fragment being capable of binding to ICAM-3; substantially pure ICAM-3; a functional derivative of ICAM-3; or a non-immunoglobulin antagonist of ICAM-3.
• the invention further includes the above-described method for treating inflammation wherein the inflammation is associated with a condition selected from the group consisting of: adult respiratory distress syndrome; multiple organ injury syndrome secondary to septicemia; multiple organ injury syndrome secondary to septicemia, hemorrhage, or trauma; reperfusion injury of myocardial or other tissues; acute glomerulonephritis; reactive arthritis; dermatosis with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory disorders, e.g. stroke; thermal injury; hemodialysis; leukapheresis; ulcerative colitis; Crohn's disease; necrotizing enterocolitis; granulocyte transfusion associated syndrome; and cytokine-induced toxicity.
• a condition selected from the group consisting of: adult respiratory distress syndrome; multiple organ injury syndrome secondary to septicemia; multiple organ injury syndrome secondary to septicemia, hemorrhage, or trauma; reperfusion injury of myocardial or other tissues; acute glomerulonephritis; reactive
• the invention also includes a method of suppressing the metastasis of a hematopoietic tumor cell, the cell utilizing a member of the CD-18 (especially LFA-1) for migration, wherein said method comprises providing to a patient in need of such treatment an amount of an agent sufficient to suppress the metastasis; wherein said agent is selected from the group consisting of: an antibody capable of binding to ICAM-3; a toxin-derivatization of said antibody; a fragment of said antibody, said fragment being capable of binding to ICAM-3; a toxin-derivatization of said fragment; substantially pure ICAM-3; a toxin- derivatized ICAM-3; a functional derivative of ICAM-3; a toxin-derivatization of said functional derivative of ICAM-3; or a non-immunoglobulin antagonist of ICAM-3 other than a member of the CD-18 family of molecules.
• the invention also includes a method of suppressing the growth of an ICAM-3-expressing tumor cell wherein said method comprises providing to a patient in need of such treatment an amount of an agent sufficient to suppress the growth, wherein said agent is selected from the group consisting of: an antibody capable of binding to ICAM-3; a toxin-derivatization of said antibody; a fragment of said antibody, said fragment being capable of binding to ICAM-3: a toxin-derivatization of said fragment; a toxin-derivatized member of the CD-18 family of molecules; or a toxin-derivatized functional derivative of a member of the CD-18 family of molecules.
• said agent is selected from the group consisting of: an antibody capable of binding to ICAM-3; a toxin-derivatization of said antibody; a fragment of said antibody, said fragment being capable of binding to ICAM-3: a toxin-derivatization of said fragment; a toxin-derivatized member of the CD-18 family of molecules; or a toxin
• the invention also provides a method for detecting the presence of a cell expressing ICAM-3 wherein said method comprises:
• the invention also provides a method for detecting the presence of ICAM-3 in a biological fluid sample wherein said method comprises:
• the invention also provides a pharmaceutical composition comprising:
• Adhesion of cell lines (A) and lymphocytes (B) to purified LFA-1 is accounted for by ICAM-1, ICAM-2, and ICAM-3. Binding of BCECF-labeled cells on LFA-1-coated microtitre wells in the presence of blocking MAb specific for LFA-1, ICAM-1, ICAM-2, and ICAM-3. Control wells were coated without
• FIG. 1 Flow cytometric analysis of cellular ICAM-1. ICAM-2 and ICAM-3 expresion.
• MAb RR1/1 anti-ICAM-1
• MAb CBR-IC2/1 anti-ICAM-2
• MAb CB-IC3/1 anti-ICAM-3
• ICAM-3 was immunoprecipitated from I 125 labeled lymphocyte and neutrophil lysates using mAb-coupled Sepharose. The immunoprecipatated
• ICAM-3 was resolved using polyacrylamide gel electrophoresis. Other mAb were used to immunoprecipitate other cell surface molecules. Human Ig
• COS cells were transfected with an LFA-1 expression vector as previously described. (deFougerolles et al, J. Exp. Med. 275:185-190 (1992)).
• transfected cells were tested for their ability to bind to immobilized ICAM- 1 and ICAM-3 in the presence and absence of an anti-LFA-1 antibody (TS1/22).
• TS1/22 anti-LFA-1 antibody
• ICAM-3 was tested at 4, 16, 22, and 37°C as previously described. (Marlin et al, Cell 52:813-819 (1987)). Assay done in presence or absence of an LFA-1 mAb (TS1/22). One representative experiment is shown and error bars indicate one SD.
• FIG. 10 Effects of anti-ICAM antibodies on the mixed lymphocyte reaction.
• Figure 11 Gas chromatograph of peptide fragments of ICAM-3.
• ICAM-3 was purified and subjected to digestion with Lys-C. Peptide fragments were resolved using high performance liquid chromatography.
• FIG. 12 Diagrammatic representation of clone 11.2.
• FIG. 13 Sequence comparison of the NK-10 primed sequence with human ICAM-1.
• the GAP program was used to identify sequence homology of the NK-
• the GAP program was used to identify sequence homology of the RM13 primed sequence with * human ICAM-1.
• the GAP program was used to identify sequence homology of the
• the GAP program was used to identify sequence homology of the T7 primed sequence and human ICAM-1.
• Figure 17 Sequence comparison of the T7 primed sequence with human ICAM-2.
• the GAP program was used to identify sequence homology of the T7 primed sequence and human ICAM-2.
• FIG. 1 Partial sequence of ICAM-3.
• the DNA sequence of clone 11.2 was determined using standard procedures.
• A Sequences obtained from the 5' end of ICAM-3. These sequences were obtained by priming clone 11.2 with the T7 primer.
• B Sequences obtained within the ICAM-3 gene. These sequences were obtained by priming clone 11.2 with the NK-10 probe.
• the present invention is based on the discovery of a previously unidentified binding ligand to LFA-1.
• Molecules such as those of CD-18 family, which are involved in the process of cellular adhesion are referred to as "adhesion molecules.”
• LFA-1 The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation (Springer, T.A. et al, Ann. Rev. Immunol. 5:223-252 (1987)).
• LFA-1 is a receptor for ICAM-1, ICAM-2, and the newly identified ICAM-3 (which is disclosed herein). These surface molecules are constitutively expressed on some tissues and induced on others in inflammation (Marlin, S.D. et al, Cell 52:813-819 (1987); Dustin, M.L. et al, J. Immunol. 137:245-254 (1986); Dustin, M.L. et al, Immunol Today 9:213-215 (1988); U.S. Patent
• LFA-1 functions in both antigen-specific and antigen-independent T cytotoxic, T helper, natural killer, granulocyte, and monocyte interactions with other cell types (Springer, T.A. et al, Ann. Rev. Immunol 5:223-252 (1987): Kishimoto, T.K. et al, Adv. Immunol (1988, in press)).
• ICAM-1 is a single chain glycoprotein varying in mass on different cell types from 76-114 kD, and is a member of the Ig superfamily with five C-like domains (Dustin, ML. et al, Immunol. Today 9:213-215 (1988); Staunton, D.E. et al, Cell 52:925-933 (1988); Simmons, D. et al, Nature 552:624-627 (1988)). ICAM-1 is highly inducible with cytokines including IFN-g, TNF, and IL-1 on a wide range of cell types (Dustin, M.L. et al, Immunol. Today 9:213-215 (1988)).
• lymphocytes Induction of ICAM-1 on epithelial cells, endothelial cells, and fibroblasts mediates LFA-1 dependent adhesion of lymphocytes (Dustin, M.L. et al, J. Immunol. 137:245-254 (1986); Dustin, M.L. et al, J. Cell. Biol. 107:321- 331 (1988); Dustin, M.L. et al, J. Exp. Med. 167:1323-1340 (1988)). Adhesion is blocked by pretreatment of lymphocytes with LFA-1 MAb or pretreatment of the other cell with ICAM-1 MAb (Dustin, M.L et al, J. Immunol. 257:245-
• LFA-1 ligands distinct from ICAM-1, have been postulated (Rothlein, R. et al, J. Immunol. 257:1270-1274 (1986); Makgoba, M.W. et al, Eur. J. Immunol 25:637-640 (1988); Dustin, M.L. et al, J. Cell. Biol. 107:321-331
• the second LFA-1 ligand identified is designated "ICAM-2".
• ICAM-2 differs from ICAM-1 in cell distribution and in a lack of cytokine induction. ICAM-2 is an integral membrane protein with 2 Ig-like domains, whereas ICAM-1 has 5 Ig-like domains (Staunton, D.E. et al, Cell 52:925-933 (1988); Simmons, D. et al, Nature 552:624-627 (1988)). Remarkably,
• ICAM-2 is much more closely related to the two most N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other members of the Ig superfamily, demonstrating a sub-family of Ig-like ligands which bind the same integrin family receptor. Further description of ICAM-2 is provided in U.S. patent application serial No. 07/454,294, herein incorporated by reference.
• the present invention concerns the discovery of a third LFA-1 ligand, designated "ICAM-3". Development of MAb to ICAM-2 allowed several previously
• LFA-1-dependent, ICAM-1-independent phenomena to be analyzed, and suggested that a third ligand for LFA-1 existed. Binding of several cell types such as epithelial and endothelial cells to purified LFA-1 could be completely blocked with a combination of ICAM-1 and ICAM-2 MAb, whereas an ICAM-1, ICAM-2-independent pathway of adhesion to LFA-1 existed on many lymphoid cell lines, including the T cell lymphoma cell line, SKW3 ( Figure 1).
• the adhesion of SKW3 to purified LFA-1 was mediated largely by ICAM-3 and also, in part, by ICAM-2.
• each of the four cell lines utilized the three ICAMs to different degrees, as demonstrated by the different- ⁇ atterns of MAb inhibition.
• the adhesion of B lymphoblastoid cell line, JY occurred primarily through an ICAM-1 pathway with slight contributions through the ICAM-2 and ICAM-3 pathways.
• Another B lymphoblastoid cell line, SLA utilized both ICAM-1 and ICAM-3, since adhesion was not inhibited by either ICAM-1 or ICAM-3 MAb alone and almost completely by the MAbs together.
• Adhesion of both resting T lymphocytes and those activated with phytohemagglutinin (PHA) was also investigated (Figure IB). Resting lymphocytes were previously shown to bind strongly to purified LFA-1 (Dustin et al, Nature 341:619-624 (1989),and this binding was found to be largely ICAM-3-dependent.
• PHA phytohemagglutinin
• the ICAM-2 component of adhesion in both the resting and activated T cells was detectable, though masked by the presence of ICAM-1 and ICAM-3. There is considerable redundancy in use of ICAMs since for each cell, MAb's to at least two ICAMs were required to achieve substantial inhibition. The notable exception to this is the adhesion of resting lymphocytes to LFA-1, which was largely ICAM-3-mediated. In all cases, the combination of all three anti-ICAM- MAb eliminated binding to LFA-1 to levels comparable to those seen in the presence of anti-LFA-1 MAb.
• the relative affinity for three ICAMs for LFA-1 can be examined by comparing their contributions to binding LFA-1 as measured above to their cell surface expression as measured by immunofluorescence flow cytometry ( Figure 2).
• the comparison of surface expression of the ICAMs and their contribution to LFA-1 adhesion revealed that ICAM-1 has the greatest affinity for LFA-1, and that ICAM-2 and ICAM-3 have similar, but lower, affinities.
• Jurkat cells expressed ICAM-2 and ICAM-3 at similar levels, and each contributed to binding to LFA-1. Where ICAM-2 expression was greater than that of ICAM-3, such as on JY cells, the ICAM-2 pathway of LFA-1 adhesion prevailed over ICAM-3.
• SLA and SKW3 expressed 3-5 fold more ICAM-3 than ICAM-2 and, not surprisingly, the ICAM-3 pathway of adhesion predominated over the ICAM-2 pathway.
• ICAM-3 was well expressed, and ICAM-1 absent, adhesion to LFA-1 was mediated largely through ICAM-3.
• ICAM-1 was well expressed, as was the case with SLA, JY, and the PHA-activated T cells, this was the primary pathway of adhesion.
• the pattern of distribution of ICAM-3 differed from that of ICAM-1 and ICAM-2 in several ways. Unlike ICAM-1 and ICAM-2, ICAM-3 was not expressed on either resting or stimulated endothelium (data not shown). This is in agreement with the finding that LFA-1-dependent binding of cells to both resting and stimulated endothelium was an ICAM-1 and ICAM-2-dependent phenomenon.
• ICAM-3 was restricted to the hematopoietic lineage, being highly expressed on lymphoid and monocytic cell lines, with a few exceptions. In all cases, however, expression of ICAM-3 on cell lines was coordinate with LFA- 1-dependent, ICAM-1, ICAM-2-independent pathway of adhesion. Cell lines (HUVEC, Raji) binding LFA-1 solely through ICAM-1 and ICAM-2 showed no ICAM-3 expression, while cell lines (JY, U937, Sup T) which showed weak binding through this third pathway of adhesion had correspondingly low ICAM- 3 surface expression (data not shown).
• ICAM-3 differed markedly from ICAM-1 and ICAM-2 in its expression on leukocytes (Figure 2B). ICAM-3 was expressed at high levels on resting lymphocytes, monocytes, and neutrophils, while ICAM-1 and ICAM-2 were expressed much more weakly or absent. By comparison, ICAM-1 and ICAM-2 were weakly expressed on monocytes, and only ICAM-2 was present on resting lymphocytes. Neither ICAM-1 nor ICAM-2 were expressed on neutrophils. Upon activation of lymphocytes with PHA, ICAM-3 expression increased 2-3 fold, whereas ICAM-1 expression was greatly induced (Dustin et al, J. Immunol. 137:245-254 (1986)) (Figure 2B).
• Immunoprecipitates of ICAM-3 from various 125 I labeled cell lines revealed a sharp band of 124,000 M r under reducing conditions with only slightly increased mobility under nonreducing condition (Figure 3A).
• Treatment with N-glycanase resulted in reduction of the ICAM-3 band to M r 87,000, indicating that ICAM-3, like ICAM-1 and ICAM-2, is a highly glycosylated protein (Figure 3B).
• the biochemical characteristics, patterns of expression, and functional properties of ICAM-3 distinguish it from previously described adhesion molecules, including the human homing receptor LAM-1 (Tedder et al, J.
• VLA family of matrix receptors (Hemler, M.E., Ann. Rev. Immunol. 5:365-400 (1990)), and no MAbs with similar cell distributions were found in the fourth leukocyte workshop databases (Gilks, W.R., et al. Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England, (1990)).
• ICAM-1 is basally expressed on endothelium and many epithelial cell types, and is strongly induced in inflammation and immunity where it regulates cell localization and facilitates specific antigen recognition (Wawryk et al, Immunol. Rev. 108:135- 161 (1989)). Since ICAM-2 is the predominant LFA-1 ligand on resting endothelium, this pathway of adhesion may have important consequences for normal recirculation of LFA-1-bearing lymphocytes through tissue endothelium (Hamann et al, I. Immunol 140:693-699 (1988); Mackay et al, J. Exp.
• ICAM-1 LFA-1 ligand(s) other than ICAM-1 in both allogeneic and autologous mixed lymphocyte reactions (Bagnasco et al, Cell Immunol 225:362-369 (1990)).
• ICAM-3 would be predicted to be important in antigen-specific interactions between T and B lymphocytes, where one of these cells has not yet been activated.
• ICAM- 3 may also be important in lysis of certain targets by T cells that is dependent on LFA-1 but not ICAM-1 (Makgoba et al, Eur. I. Immunol. 25:637-640 (1988)).
• the existence of multiple ICAMs has important implications for clinical treatment with MAb to ICAMs or ICAM analogues.
• ICAM-1 MAb is efficacious in vivo in prolonging renal (Cosimi et al, I. Immunol. 144:4604-4612 (1990)) and cardiac (Flavin et al, Transplant. Proc. 25:533-534 (1991)) allografts.
• ICAM-3 MAb inhibits a distinctive and overlapping number of immune responses in vivo, since it inhibits LFA-1-dependent adhesive interactions of a distinct subset of cell types.
• Any of a variety of procedures may be utilized to clone the ICAM-3 gene.
• One such method entails analyzing a shuttle vector library of cDNA inserts (derived from an ICAM-3 expressing cell) for the presence of an insert which contains the ICAM-3 gene. Such an analysis may be conducted by transfecring cells with the vector and then assaying for ICAM-3 expression.
• ICAM-3 cDNA is preferably identified using a modification of the procedure of Aruffo and Seed (Seed, B. et al, Proc. Natl Acad. Sci. USA 54:3365-3369 (1987)) to identify ligands of adhesion molecules.
• a cDNA library is prepared from cells which express ICAM-3 (such as SLA, Jurkat, or SKW3 lymphoblastoid cell lines).
• the cDNA library is prepared from T-cells. This library is used to transfect cells which do not normally express ICAM-3 (such as Cos or HeLa cells). The transfected cells are introduced into a petri dish which has been previously coated with either
• LFA-1 or anti-ICAM-3 antibodies The transfected cells containing ICAM-3 encoding sequences, and which express this ligand on their cell surfaces, will adhere to the LFA-1 or anti-ICAM-3 antibodies on the surface of the petri dish. Non-adherent cells are washed away, and the adherent cells are then removed from the petri dish and cultured. The recombinant ICAM-3 expressing sequences in these cells is then removed is sequenced. If LFA-1 is used to coat the petri plate, anti-ICAM-1 and anti-ICAM-2 specific antibodies are added to the petri dish in order to prevent the adherence of ICAM-1 or ICAM-2 expressing cells.
• Adherence of ICAM-1 + or ICAM-2 + transfectants to LFA-1 coated plastic may thus be inhibited with antibodies such as RR1/1, an anti-ICAM-1 MAb and CBR-IC2/2, an anti-ICAM-2 MAb.
• Binding of ICAM-3 transfected cells to LFA-1 coated petri plates is inhibited by EDTA and anti-LFA-1 MAbs, but is not inhibited by anti-ICAM-1 or anti- ICAM-2 MAbs. Therefore the ICAM-1 or ICAM-2 expressing cells are unable to adhere to the petri dish and are therefore mostly washed away with all of the other non-adherent cells. This will enrich for cells expressing ICAM-3.
• One such method for obtaining a gene sequence which encodes ICAM- 3 is to use an oligonucleotide probe to screen a cDNA or genomic DNA library.
• the ICAM-3 protein is purified, preferably using immuno- purification procedures known in the art, and the terminal amino acid sequence is determined using one of the methods known in the art.
• ICAM- 3 enzymatically cleaned, as with trypsin or Lys-C, peptides are purified, and the amino acid sequence of one of the internal fragments is determined.
• an oligonucleotide probe is made based on the codon preference displayed by the organism, a degenerate probe is made based on all possible codon combinations, or a combination of codon preference, homology with known ICAM-1 or ICAM-2 DNA sequences, and codon degeneracy is used to contruct probes.
• This probe is then used to screen either a genomic or cDNA library for sequences which hybridize to the probe.
• an expression library is prepared by cloning genomic DNA or, more preferably cDNA, from a cell which expresses ICAM-3. The library is then screened for members capable of expressing a protein which binds to an anti-ICAM-3 antibody.
• the cloned ICAM-3 gene obtained through the use of any of the methods described above, may be operably linked to an expression vector, and introduced into bacterial, or eukaryotic cells to produce the ICAM-3 protein. Techniques for such manipulations are disclosed in Maniatis, T. et al, supra, and are well known in the art. In an alternative method utilizing PCR to clone to ICAM-3 gene, an assumption that ICAM-3 is homologous to ICAM-1 and/or ICAM-2 is made.
• oligonucleotide primers based on sequences conserved between ICAM-1 and ICAM-2 are used amplify by the polymerase chain reaction cDNA or mRNA, from cells known to express ICAM-3 protein, such as SKW3 or tonsil (deFougerolles et al, J. Exp. Med., 275:185-190 (1992)).
• Clones are sequenced, and those distinct from ICAM-1 and ICAM-2 are used to obtain full length cDNA clones. In any of the methods described above, the authenticity of clones can be confirmed by expressing full length clones, for example in COS cells, and testing for reactivity with ICAM-3 mAb.
• the present invention is further directed toward ICAM-3, its "functional derivatives,” its “agonists” and “antagonists.”
• a “functional derivative” of ICAM-3 is a compound which possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of ICAM-3.
• the term “functional derivative” includes
• a “fragment” of ICAM-3 is meant to refer to any polypeptide subset of the molecule. Fragments of ICAM-3 which have ICAM-3 activity and which are soluble (i.e not membrane bound) are especially preferred. A soluble fragment is preferably generated by deleting the membrane spanning region of the parent molecule or by deleting or substituting hydrophilic amino acid residues for hydrophobic residues. Identification of such residues is well known in the art. Fragments containing any number of whole Ig-like domain are preferred, such as domain 1 (DI), DI and D2, Dl-3, Dl-4, and Dl-5.
• a “variant" of ICAM-3 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.
• two molecules possess a similar activity they are considered variants, as that term is used herein, even if one of the molecules possesses a structure not found in the other molecule, or if the sequence of amino acid residues is not identical.
• a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties which are not normally a part of the naturally occurring molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, or eliminate or attenuate any undesirable side effect of the molecule. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). "Toxin-derivatized" molecules constitute a special class of "chemical derivatives.”
• a "toxin-derivatized” molecule is a molecule (such as ICAM-3 or an anti-ICAM-3 antibody) which is covalently attached to a toxin moiety.
• Functional derivatives of ICAM-3 having up to about 100 residues may be conveniently prepared by in vitro synthesis. If desired, such fragments may be modified using methods known in the art by reacting targeted amino acid residues of the purified or crude protein with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The resulting covalent derivatives may be used to identify residues important for biological activity.
• ICAM-3 many methods may be employed to generate and isolate fragments of ICAM-3. Derivatization with bifunctional agents can be used to crosslink ICAM-3 to a water-insoluble support matrix. Alternatively, reactive water- insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
• Functional derivatives of ICAM-3 having altered amino acid sequences can also be prepared by mutating the DNA encoding ICAM-3. Such functional derivatives include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence of ICAM-3.
• any- combination of deletion, insertion, and substitution may be employed to generate the final construct, provided that the final construct possesses the desired activity.
• the mutations that will be made in the DNA encoding the functional derivative must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see EP Patent Application Publication No. 75,444).
• functional derivatives can be prepared by site- directed mutagenesis of the DNA encoding ICAM-3, thereby producing DNA encoding the functional derivative, and thereafter expressing the DNA in recombinant cell culture.
• the mutation perse need not be predetermined.
• random mutagenesis such as linker scanning mutagenesis, may be conducted at a target codon or target region to create a large number of derivative which could then be expressed and screened for the optimal combination of desired activity.
• Techniques for making mutations at predetermined sites in a DNA known sequence are well known, for example, site-directed mutagenesis.
• Preparation of functional derivative of ICAM-3 in accordance herewith is preferably achieved by site-directed mutagenesis of the DNA that encodes ICAM-3 or an earlier prepared functional derivative of ICAM-3.
• site-directed mutagenesis is well known in the art, as exemplified by publications such as Maniatis, T. et al, In: Molecular Cloning a Laboratory Manual, Coldspring Harbor, NY (1982), the disclosure of which is incorporated herein by reference.
• Site-directed mutagenesis allows the production of ICAM-3 functional derivatives through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation.
• Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably 1 to 10 residues, and typically are con- tiguous. The most preferred deletions are those which are performed to generate a soluble form of ICAM-3. Soluble forms of ICAM-3 are most preferably generated by deleting either the membrane spanning region of ICAM-3 or hydrophobic residues within ICAM-3.
• Amino acid insertions include insertions of single or multiple amino acid residues within the ICAM-3 coding sequences as well as terminal fusions with polypeptides of essentially unrestricted length.
• Intrasequence insertions i.e., insertions within the complete ICAM-3 molecule sequence
• An example of a terminal insertion includes a fusion of a signal sequence, whether heterologous or homologous to the host cell, to the N-terminus of the molecule to facilitate the secretion of the derivative from recombinant hosts.
• the third group of functional derivatives are those in which at one or more amino acid residue in the ICAM-3 molecule has been removed and a different residue inserted in its place. Such substitutions preferably are made in accordance with the following Table when it is desired to modulate finely the characteristics of the ICAM-3 molecule.
• Substantial changes in functional or immunological identity are made by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining
• substitutions that are in general less conservative are those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted;
• a hydrophilic residue is substituted for a hydrophobic residue;
• a cysteine residue is substituted for (or by) any other residue;
• a residue having an electropositive side chain is substituted for (or by) a residue having an electronegative charge; or
• a residue having a bulky side chain is substituted for (or by) one not having such a side chain.
• substitution which effect the solubility of ICAM-3.
• Mutations designed to increase the affinity of ICAM-3 may be guided by the introduction of the amino acid residues which are present at homologous positions in ICAM-1 or ICAM-2. Similarly, such mutant ICAM-3 molecules maybe prepared which lack N-linked CHO at homologous positions in ICAM-1 or ICAM-2.
• a derivative typically is made by linker scanning site-directed mutagenesis of the DNA encoding the native ICAM-3 molecule.
• the derivative is then expressed in a recombinant host, and, optionally, purified from the cell culture, for example, by immunoaffinity chromatography.
• the activity of the cell lysate or the purified derivative is then screened in a suitable screening assay for the desired characteristic. For example, a change in the immunological character of the functional derivative, such as affinity for a given antibody, is measured by a competitive type immunoassay. Changes in immunomodulation activity are measured by the appropriate assay.
• An "agonist" of ICAM-3 is a compound which enhances or increases the ability of ICAM-3 to carry out any of its biological functions.
• An example of such an agonist is an agent which increases the ability of ICAM-3 to bind to a cellular receptor.
• an "antagonist" of ICAM-3 is a compound which diminishes or prevents the ability of ICAM-3 to carry out any of its biological functions.
• antagonists include ICAM-1 and ICAM-2, a functional derivative of ICAM-1 and ICAM-2, an anti-ICAM-3 antibody, an anti-LFA-1 antibody, etc.
• Serial Nos. 07/045,963; 07/115,798; 07/155,943; 07/189,815 or 07/250,446, all of which applications have been herein incorporated by reference in their entirety, are capable of measuring LFA-1 dependent aggregation, and may thus be employed to identify agents which affect the extent of ICAM-3/LFA-1 aggrega- tion.
• assays may be employed to identify agonists and antagonists of ICAM-3.
• Antagonists may act by impairing the ability of LFA-1 or of ICAM-3 to mediated aggregation.
• non-immunoglobulin (i.e., chemical) agents may be examined, using the above-described assay, to determine whether they are agonists or antagonists of ICAM-3/LFA-1 mediated aggregation.
• Such aggregation assays are typically performed using peripheral blood cells
• T-cells stimulated with PMA.
• LFA-1 binding of peripheral blood cells to LFA-1 is used to assay ICAM-3 mediated aggregation.
• the preferred immunoglobulin antagonist of the present invention is an antibody to ICAM-3 such as CBR-IC3/1, disclosed herein.
• Other suitable anti-ICAM-3 such as CBR-IC3/1, disclosed herein.
• ICAM-3 antibodies can be obtained in any of a variety of ways.
• the antibodies to ICAM-3 may be either polyclonal or monoclonal. Additionally, the antibodies of the present application may be humanized by procedures known in the art or by the procedures disclosed in PCT Application Nos. PCT/US91/02942 and PCT/US91/02946 filed at the U.S. Receiving Office on April 22, 1991.
• Anti-ICAM-3 antibodies may be made by introducing ICAM-3, or peptide fragments thereof, into an appropriate animal. The immunized animal will produce polyclonal antibody in response to such exposure. The use of peptide fragments of ICAM-3 permits one to obtain region specific antibodies which are reactive only with the epitope(s) contained in the peptide fragments used to immunize the animal.
• anti-ICAM-3 antibodies may be made using the ICAM-3 which is naturally expressed on the surfaces of lymphocytes.
• the introduction of such cells into an appropriate animal, such as by intraperitoneal injection, will result in the production of antibodies capable of binding to ICAM-3 or members of the CD-18 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.
• anti-ICAM-3 antibodies may be produced by adaptation of the method of Selden, R.F. (European Patent Application Publication No.
• the cells of a suitable animal are transfected with a vector capable of expressing either the intact ICAM-3 molecule, or a fragment thereof.
• a suitable animal for example a mouse
• the production of ICAM-3 in the transfected cells of the animal will elicit an immune response in the animal, and lead to the production of anti-ICAM-3 antibodies by the animal.
• the hybridoma cells obtained in the manner described above, may be screened by a variety of methods to identify a hybridoma cell that secretes an antibody capable of binding to ICAM-3. In a preferred screening assay, such molecules are identified by their ability to inhibit the aggregation of ICAM-3- expressing, ICAM-1 and ICAM-2 non-expressing cells. Antibodies capable of inhibiting such aggregation are then further screened to determine whether they inhibit such aggregation by binding to ICAM-3, or to a member of the CD-18 family of molecules.
• any means capable of distinguishing ICAM-3 from the CD-18 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. It is possible to distinguish between those antibodies which bind to members of the CD-18 family of molecules from those which bind ICAM-3 by screening for the ability of the antibody to bind to cells which express LFA-1, but not ICAM-3 (or vice versa). The ability of an antibody to bind to a cell expressing LFA-1 but not
• ICAM-3 may be detected by means commonly employed by those of ordinary skill. Such means include immunoassays (especially those using immunofluorescence), cellular agglutination, filter binding studies, antibody precipitation, etc. In the preferred method, an antibody is selected for its ability to bind to cells expressing ICAM-3, but not to cells which do not express ICAM-3.
• agents which may be used in accordance of the present invention in the treatment of inflammation, HIV infection, asthma, etc. include anti-idiotypic antibodies to anti-ICAM-3 antibodies, and receptor molecules, or fragments of such molecules, which are capable of binding to ICAM-3.
• the anti-idiotypic antibodies of interest to the present invention are capable of binding in competition with (or to the exclusion of) ICAM-3.
• Such antibodies can be obtained, for example, by raising an antibody to an anti- ICAM-3 antibody, and then screening the antibody for the ability to bind to one of the natural binding ligands of ICAM-3.
• molecules of the CD-18 family are able to bind to ICAM-3
• administration of such molecules will compete with cells for the binding to ICAM-3 present on a cell.
• the anti-aggregation antibodies of the present invention may be identified and tittered in any of a variety of ways. For example, one can measure the ability of the antibodies to differentially bind to cells which express ICAM-3
• Suitable assays of cellular aggregation are those described in U.S. Patent Ap ⁇ plications Serial Nos. 07/045,963; 07/115,798; 07/155,943; 07/189,815 or 07 250,446, all of which applications have been herein incorporated by reference in their entirety.
• the above assays may be modified, or per ⁇ formed in a different sequential order to provide a variety of potential screening assays, each of which is capable of identifying and discriminating between anti- bodies capable of binding to ICAM-3 versus members of the CD-18 family of molecules.
• the agents of the present invention may be obtained by: natural processes
• the agents of the present invention can be used to moderate the various biological activities which are mediated by ICAM-3.
• One aspect of the present invention derives from the ability of ICAM-3 and its functional derivatives to interact with receptors of the CD-18 family of molecules, especially LFA-1.
• ICAM-3 By virtue of the ability of ICAM-3 to interact with members of the CD-18 family of glycoproteins, it may be used to suppress (i.e. to prevent, or attenuate) inflammation.
• inflammation is meant to include both the reactions of the specific defense system, and the reactions of the non-specific defense system.
• 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. Examples of inflammation resulting from a response of the specific defense system include the response to antigens such as rubella virus, autoimmune diseases, and delayed type hypersensitivity response mediated by T-cells (as seen, for example in individuals who test "positive" in the Mantaux test). Chronic inflammatory diseases and the rejection of solid transplanted tissue and organs, e.g. kidney and bone marrow transplants, are further examples of inflammatory reactions of the specific defense system.
• a reaction of the "non-specific defense system” is intended to refer to a reaction mediated by leukocytes which are incapable of im- munological memory. Such cells include granulocytes and macrophages.
• 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, trauma or hemorrhage; 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 such as stroke; thermal injury; hemodialysis; leukapheresis; ulcerative colitis; Crohn's disease; necrotizing enterocolitis: granulocyte transfusion associated syndromes; and cytokine-induced toxicity.
• ARDS adult respiratory distress syndrome
• multiple organ injury syndromes secondary to septicemia, trauma or hemorrhage 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 such as stroke; thermal injury
• lymphocytes are capable of continually monitoring an animal for the presence of foreign antigens.
• solid organ trans ⁇ plant rejection e.g. kidney
• non-solid organ transplant rejection e.g. bone marrow
• tissue graft rejection e.g. a malignant neoplasm graft rejection
• any means capable of attenuating or inhibiting cellular adhesion would be highly desirable in recipients of solid organ transplants (especially kidney transplants), non-solid organ transplants (especially bone marrow transplants), tissue grafts, or for autoimmune patients.
• Monoclonal antibodies to members of the CD-18 family inhibit many adhesion dependent functions of leukocytes including binding to endothelium (Haskard, D. et al, J. Immunol. 257:2901-2906 (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. Natl. Acad. Set, USA 75:4535-4539 (1981)), antibody formation (Fischer, A. et al, J. Immunol.
• ICAM-3 Such functions, to the extent that they involve ICAM-3/LFA-1 interac ⁇ tions, can be suppressed with anti-ICAM-3 antibodies.
• monoclonal antibodies capable of binding to ICAM-3 can be employed as an anti-inflammatory agent in a mammalian subject.
• such agents differ from general anti-inflammatory agents in that they are capable of selectively inhibiting adhesion, and do not offer other side effects, such as nephrotoxicity, which are found with conventional agents.
• ICAM-3 particularly in soluble form is capable of acting in the same manner as an antibody to members of the CD-18 family, it may be used to suppress inflammation.
• the functional derivatives and antagonists of ICAM-3 may also be employed to suppress inflammation.
• ICAM-3 mediates, in part, adhesion events necessary to mount inflammatory reactions such as delayed type hypersensitivity reactions.
• antibodies especially monoclonal antibodies capable of binding to ICAM-3 have therapeutic potential in the attenuation or elimination of such reactions.
• ICAM-3 is an antagonist of the ICAM-l/LFA-1 interaction, ICAM-3 (particularly in solubilized form), or its functional derivatives can be used to suppress delayed type hypersensitivity reactions.
• a composition containing an anti-ICAM-3 monoclonal antibody or soluble derivative of ICAM-3 may be administered to a patient experiencing delayed type hypersensitivity reactions.
• such compositions might be provided to a individual who had been in contact with antigens such as poison ivy, poison oak, etc.
• a monoclonal antibody capable of binding to ICAM-3 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 anti-ICAM-3 antibody can act to temporarily tolerize an individual to subsequent presentation of that antigen.
• LAD patients that lack LFA-1 do not mount an inflammatory response, it is believed that antagonism of LFA-l's natural ligands, will also inhibit an inflammatory response.
• the ability of antibodies against ICAM-3 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 anti-ICAM-3 antibodies of the present invention may be administered alone or in combination with anti-ICAM-1 and/or anti-ICAM-2 antibodies in the treatment of those diseases currently treatable through steroid therapy.
• such inflammatory and immune rejection responses may be suppressed (i.e. either prevented or attenuated) by providing to a subject in need of such treatment an amount of an anti- inflammatory agent sufficient to suppress said inflammation.
• Suitable anti- inflammatory agents include: an antibody capable of binding to ICAM-3; a fragment of said antibody, said fragment being capable of binding to ICAM-3; substantially pure ICAM-3; a functional derivative of ICAM-3; a non-immuno ⁇ globulin antagonist of ICAM-3, or a non-immunoglobulin antagonist of ICAM-3 other than LFA-1.
• anti-inflammatory agents composed of a soluble functional derivative of ICAM-3.
• Such anti-inflammatory treatment can also include the additional administration of an agent selected from the group consisting of: an antibody capable of binding to LFA-1; a non-im ⁇ munoglobulin antagonist of LFA-1; substantially pure ICAM-1 and/or ICAM-2, or derivatives thereof, or an anti-ICAM-1 and/or anti-ICAM-2 antibody or fragment thereof.
• an agent selected from the group consisting of: an antibody capable of binding to LFA-1; a non-im ⁇ munoglobulin antagonist of LFA-1; substantially pure ICAM-1 and/or ICAM-2, or derivatives thereof, or an anti-ICAM-1 and/or anti-ICAM-2 antibody or fragment thereof.
• the invention further includes the above-described methods for suppressing an inflammatory response of the specific defense system in which an immunosuppressive agent is additionally provided to the subject.
• an immunosuppressive agent is additionally provided to the subject.
• Such an agent is preferably provided at a dose lower (i.e. a "sub-optimal" dose) than that at which it would normally be required.
• a sub-optimal dose is possible because of the synergistic effect of the agents of the present invention.
• suitable immunosuppressive agents include but are not limited to dexamethasone, azathioprine, ICAM-1, ICAM-2, or cyclosporin A.
• 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, trauma or hemorrhage; 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 such as stroke; thermal injury; hemodialysis; leukapheresis; ulcerative colitis; Crohn's disease; necrotizing enterocolitis: granulocyte transfusion associated syndromes; and cytokine-induced toxicity.
• ARDS adult respiratory distress syndrome
• multiple organ injury syndromes secondary to septicemia, trauma or hemorrhage 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 such as stroke; thermal injury
• the anti-inflammatory agents of the present invention are compounds capable of specifically antagonizing the interaction of the CD-18 complex on granulocytes with endothelial cells.
• Such antagonists comprise: ICAM-3: a functional derivative of ICAM-3; a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1, ICAM-2, or a member of the CD-18 family of molecules.
• ICAM-3 since ICAM-3, particularly in soluble form, is capable of acting in the same manner as an antibody to members of the CD-18 family, it may be used to suppress organ or tissue rejection caused by any of the cellular adhesion- dependent functions. Moreover, an anti-ICAM-3 antibody, functional deriva ⁇ tives of ICAM-3, and antagonists of ICAM-3 may also be employed to suppress such rejection.
• ICAM-3 and anti-ICAM-3 antibodies can be used to prevent solid organ or tissue rejection, e.g. kidney, non-solid organ rejection, e.g. bone marrow, or modify autoimmune responses, in the mammalian subject.
• solid organ or tissue rejection e.g. kidney
• non-solid organ rejection e.g. bone marrow
• 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 in some instances, 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
• anti-ICAM-3 antibodies would be administered in combination with the therapeutic or diagnostic agent.
• the addition of the antibody 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-3 (particularly in solubilized form) or its functional derivatives may be employed alone or in combination with ICAM-1 or ICAM-2, or with antibodies capable of binding to LFA-1, in the treatment of disease. Thus, in solubilized form, such molecules may be employed to inhibit organ or graft rejection. ICAM-3, or its functional derivatives may be used in the same manner as anti-ICAM-3 antibodies to decrease the immunogenicity of therapeutic or diagnostic agents.
• the agents of the present invention may also be employed to suppress the metastasis of a hematopoietic tumor cell, which requires a functional member of the CD-18 family for migration.
• a patient in need of such treatment is provided with an amount of an agent (such as an antibody capable of binding to ICAM-3; a toxin-derivatization of said antibody; a fragment of an antibody, said fragment being capable of binding to ICAM-3; a toxin-derivatization or said fragment; substantially pure ICAM-3; a functional derivative of ICAM-3; or a non- immunoglobulin antagonist of ICAM-3 other than ICAM-1 or ICAM-2) sufficient to suppress said metastasis.
• an agent such as an antibody capable of binding to ICAM-3; a toxin-derivatization of said antibody; a fragment of an antibody, said fragment being capable of binding to ICAM-3; a toxin-derivatization or said fragment; substantially pure ICAM-3; a functional derivative of ICAM-3; or a non- immunoglobul
• a method for suppressing the growth of an ICAM-3-expressing tumor cell is provided.
• said method comprises providing to a patient in need of such treatment an amount of an agent sufficient to suppress said growth.
• Suitable agents include an antibody capable of binding to ICAM-3; a toxin-derivatization of said antibody; a fragment of an antibody, said fragment being capable of binding to ICAM-3; a toxin-derivatization of said fragment; a toxin-derivatized member of the CD-18 family of molecules; or a toxin-derivatized functional derivative of a member of the CD-18 family of molecules.
• a method of suppressing the growth of an LFA-1-expressing tumor cell is provide.
• said method comprises providing to a patient in need of such treatment an amount of a toxin sufficient to suppress said growth.
• Suitable toxins include a toxin- derivatized ICAM-3, or a toxin-derivatized functional derivative of ICAM-3.
• a method for suppressing the infection of HIV comprises administering to an HIV-infected individual an effective amount of an HIV infection suppression agent.
• an HIV infection suppression agent such as, for example, HIV- 2
• the method may be applied to any HIV variant (such as, for example, HIV- 2) which may infect cells in a way which may be suppressed by the agents of the present invention.
• HIV variants are the equivalents of HIV-1 for the purposes of the present invention.
• One aspect of the present invention derives from the recognition that expression of LFA-1 and, in some cases, LFA-l's binding ligand, stimulated by HIV infection, promotes cell-to-cell adherence reactions that can increase the contact time of infected with uninfected cells, facilitating transfer of virus from infected to uninfected cells.
• agents of the present invention which are capable of modulating LFA-1/ligand interactions are able to suppress infection by HIV, and, in particular, by HIV-1.
• One means through which molecules which inhibit LFA-1/ligand interactions may suppress HIV infection is by impairing the ability of the LFA-1 ligand expressed by HIV-infected cells to bind to the CD11/CD18 receptors of a healthy T cell.
• molecules which inhibit LFA-1/ligand interactions may impair the ability of the CD11/CD18 receptors expressed by HIV-infected cells to bind to LFA-1 of a healthy T cell.
• molecules which inhibit LFA-1/ligand interactions may impair the ability of the CD11/CD18 receptors expressed by HIV-infected cells to bind to LFA-1 of a healthy T cell.
• ICAM-3 a fragment of ICAM-3, a functional derivative of ICAM-3, or anti- ICAM-3 antibodies.
• the agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to achieve a suppression of HIV infection.
• An amount is said to be sufficient to "suppress" HIV infection if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent such HIV infection.
• the agents are to be provided to patients who are exposed to, or effected by HIV infection.
• the agents of the present invention may be for either a "prophylactic" or "therapeutic" purpose in the treatment of HIV infection.
• the agent When provided prophylacticalry, the agent is provided in advance of any symptom of viral infection (for example, prior to, at, or shortly after) the time of such infection, but in advance of any symptoms of such infection).
• the prophylactic administration of the agent selves to prevent or attenuate any subsequent HIV infection.
• the agent is provided at (or shortly after) the detection of virally infected cells.
• the therapeutic administration of the agent serves to attenuate any additional HIV infection.
• the agents of the present invention may, thus, be provided either prior to the onset of viral infection (so as to suppress the anticipated HIV infection) or after the actual detection of such virally infected cells (to suppress further infection).
• the invention provides an improved therapy for AIDS, and an enhanced means for suppressing HIV infection, and particularly HIV-1 infection, which comprises the co-administration of:
• ICAM-3 ICAM-3, a soluble ICAM-3 derivative, CD11 (either CDlla, CDllb, or CDllc), a soluble CD11 derivative, CD18, a soluble CD18 derivative, or a CD11/CD18 heterodimer, or a soluble derivative of a CD11/CD18 heterodimer and/or
• a method for suppressing the migration of HIV-infected cells comprises administering an effective amount of an anti-migration agent to an HIV-infected individual.
• the anti-migration agents of the present invention include ICAM-3, a fragment of ICAM-3, a functional derivative of ICAM-3, or anti-ICAM-3 antibodies which are capable of impairing the ability of an HIV-infected T cell to bind to a LFA-1 ligand.
• Antibodies which bind to ICAM-3 will suppress migration by impairing the ability of the ICAM-3 expressed by HIV-infected T cells to bind to cells expressing a CD11/CD18 receptor. In order to impair the ability of a cell to bind to the CDlla/CD18 receptor it is possible to employ an antibody capable of binding to ICAM-3.
• the agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to suppress the migration of HIV (or other virally) infected T cells. An amount is said to be sufficient to "suppress" migration of T cells if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent such migration.
• the administration of such compound(s) may be for either a "prophylactic" or "therapeutic" purpose.
• the agents of the present invention is provided in advance of any symptom of viral infection (for example, prior to, at, or shortly after) the time of such infection, but in advance of any symptoms of such infection).
• the prophylactic administration of the agents serves to prevent or attenuate any subsequent migration of virally infected T cells.
• the agent is provided at (or shortly after) the detection of virally infected T cells.
• the therapeutic administration of the agent serves to attenuate any additional migration of such T cells.
• the agents of the present invention may, thus, be provided either prior to the onset of viral infection (so as to suppress the anticipated migration of infected T cells) or after the actual detection of such virally infected cells.
• an agent capable of modulating LFA-l/ICAM-3 interactions is used in the treatment of asthma.
• said method comprises administering an effective amount of an anti-asthma agent to an individual in need of such treatment.
• the anti-asthma agents of the present invention include ICAM-3, a fragment of ICAM-3, a functional derivative of ICAM-3, or anti-ICAM-3 antibodies which are capable of impairing the ability of a cell to bind to a LFA- 1.
• Antibodies which bind to ICAM-3 will suppress the migration of eosinophils by impairing the ability of the ICAM-3 expressed on these cells to bind to cells expressing a CD11/CD18 receptor.
• the anti-asthma agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to lessen or attenuate the severity, extent or duration of the asthma symptoms.
• the agents of the present invention may be administered either alone or in combination with one or more additional anti-asthma agents (such as methykanthines (such as theophylline), beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins, and ephedrine), glucocorticoids (such as choir 5 hydrocortisone), chromones (such as cromolyn sodium) and anticholinergics
• additional anti-asthma agents such as methykanthines (such as theophylline), beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins, and ephedrine), glucocorticoids (such as consult 5 hydrocortisone), chromones (such as cromolyn sodium) and anticholinergics
• additional anti-asthma agents such as methykanthines (such as theophylline
• the administration of the agents of the present invention may be for either a "prophylactic" or "therapeutic" purpose.
• the agent When provided 10 prophylactically, the agent is provided in advance of any asthma symptom.
• the prophylactic administration of the agent serves to prevent or attenuate any subsequent asthmatic response.
• the agent When provided therapeutically, the agent is provided at (or shortly after) the onset of a symptom of asthma.
• the therapeutic administration of the agent serves to attenuate any actual asthmatic 15 episode.
• the agents of the present invention may, thus, be provided either prior to the onset of an anticipated asthmatic episode (so as to attenuate the anticipated severity, duration or extent of the episode) or after the initiation of the episode.
• Monoclonal antibodies capable of binding to ICAM-3 may be employed as a means of imaging or visualizing the sites of ICAM-3 expression and inflammation in a patient.
• anti-ICAM-3 monoclonal antibodies are detectably labeled, through the use of radioisotopes, affinity labels (such as biotin, avidin. etc.), fluorescent labels, or paramagnetic atoms. Procedures for , 25 accomplishing such labeling are well known to the art.
• the labeled antibodies can then be used in diagnostic imaging. Clinical application of antibodies in diagnostic imaging are reviewed by Grossman, H.B., Urol Clin. North Amer. 25:465-474 (1986)), Unger, E.C et al, Invest. Radiol. 20:693-100 (1985)), and Khaw, B.A. et al, Science 209:295-291 (1980)).
• the presence of ICAM-3 expression may also be detected through the use of hybridization probes, such as mRNA, cDNA, genomic DNA, or synthetic oligonucleotide probes which bind to ICAM-3 gene sequences, or to ICAM-3 mRNA sequences, present in a cell which expresses ICAM-3.
• hybridization probes such as mRNA, cDNA, genomic DNA, or synthetic oligonucleotide probes which bind to ICAM-3 gene sequences, or to ICAM-3 mRNA sequences, present in a cell which expresses ICAM-3.
• the detection of foci of ICAM-3 expression can be used in the diagnosis of tumor development
• samples of tissue or blood are removed from a subject and are incubated in the presence of antibodies of which are or which can be detectably labeled.
• 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, e.g. kidney, for early signs of potential tissue rejection.
• assays may also be conducted in efforts to determine an individual's predilection to rheumatoid arthritis or other chronic inflammatory diseases.
• radioimmune assays A good description of a radioimmune assay (RLA) may be found in
• the antibody can be labeled with a fluorescent compound, an enzyme, or other suitable labels known in the art.
• antibodies capable of binding to ICAM-3 can be employed to assay biological fluids for the presence of circulating ICAM-3 using any of the commonly employed mediums for fluid assays.
• the presence of circulating ICAM-3 in a fluid sample is indicative of an inflammatory response or other ICAM-3 mediated biological functions.
• the presence of ICAM-3 in amniotic fluid is associated with complicating arising during pregnancy.
• Any biological fluid can be used for the assay.
• the preferred biological fluids are; blood, serum, plasma synovial fluid, amniotic fluid, spinal fluid or urine.
• ICAM-3 The therapeutic effects of ICAM-3 may be obtained by providing to a patient the entire ICAM-3 molecule, or any therapeutically active peptide fragments thereof. Of special interest are therapeutically active peptide fragments of ICAM-3 which are soluble.
• ICAM-3 and its functional derivatives may be obtained synthetically, through the use of recombinant DNA technology, by proteolysis, or by a combination of such methods.
• the therapeutic advantages of ICAM-3 may be augmented through the use of functional derivatives of ICAM-3 possessing addi ⁇ tional amino acid residues added to enhance coupling to carrier or to enhance the activity of the ICAM-3.
• the scope of the present invention is further intended to include functional derivatives of ICAM-3 which lack certain amino acid residues, or which contain altered amino acid residues, so long as such derivatives possess or affect a biological or pharmacological activity of ICAM-3.
• Both the antibodies of the present invention and the ICAM-3 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-3.
• Such antibodies may be produced either by an animal, or by tissue culture, or by recombinant DNA means.
• ICAM-3 or molecules derived from ICAM-3
• ICAM-1 and or ICAM-2 can be done alone or in combination with ICAM-1 and or ICAM-2.
• Administration of anti-ICAM-3 antibodies, or other molecules capable of binding to ICAM-3 or to molecules derived from ICAM-3 can be done alone or in combination with anti-ICAM-1 antibodies and/or anti-ICAM-2 antibodies.
• 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.
• 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 admin ⁇ istered.
• ICAM-3 molecules or their functional derivatives 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-3 antibody is additionally administered with an anti-LFA-1 antibody, an anti-ICAM-1 antibody and/or an anti-ICAM-2 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-3 and ICAM-3 itself may be administered to patients intravenously, intramuscularly, subcutaneously, enterally, topically or parenterally.
• the administration may be by continuous injections, or by single or multiple boluses.
• the agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to "physiologically effective.”
• An amount is said to be physiologically effective if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent the physiological effect associated with ICAM-3.
• one of the agents of the present invention is provided to a patient for the intention of suppressing inflammation is said to be physiologically effective if it is provided in sufficient dosage to "suppress" inflammation.
• anti-ICAM-3 antibodies may be administered either alone or in combination with one or more additional immunosuppressive agents (especially to a recipient of an organ or tissue transplant).
• the administration of such compound(s) may be for either a "prophylactic” or "therapeutic” purpose.
• the immunosuppressive compound(s) are provided in 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 compound(s) When provided therapeutically, the compound(s) is provided at (or shortly after) the onset of a symptom of actual inflammation (such as, for example, organ or tissue rejection).
• 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 solid organ or tissue, say kidney or non-solid organs, say bone marrow).
• 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 antibodies and the ICAM-3 molecules of the present invention can be formulated according to known methods of preparing pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined with 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 an agent of the present invention together with a suitable amount of carrier.
• the antibodies of the present invention may be humanized, through chimerization or CDR grafting, to become more "pharmacologically acceptable" to a patient.
• Such methods for chimerization of antibodies, specifically anti-ICAM-1 antibodies are described elsewhere, British Patent application nos. 9009548.0,
• Control release preparations may be achieved through the use of polymers to complex or absorb the agents of the present invention.
• the rate and duration of the controlled delivery may be regulated to a certain extent by selecting an appropriate macromolecule matrix, by varying the concentration of macromolecules incorporated, as well as the methods of incorporation.
• Another possible method to control the duration of action by controlled release preparations is to incorporate the agents of the present invention into particles of a polymeric material, such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers.
• a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers.
• the invention further includes a pharmaceutical composition
• a pharmaceutical composition comprising: (a) an anti-inflammatory agent (such as an antibody capable of binding to ICAM-3; a fragment of said antibody capable of binding to ICAM-3; ICAM-3; a functional derivative of ICAM-3; or a non-immunoglobulin antagonist of
• ICAM-3 other than ICAM-1 and ICAM-2 and (b) at least one immunosuppressive agent.
• suitable immunosuppressive agents include: dexamethasone, azathioprine and cyclosporin A.
• 96-well microtitre plates (Linbro-titertek) at 1100 sites/ ⁇ m 2 . Non-specific binding sites were blocked with 1% BSA and washed with PBS/5% FBS/2mM MgCl 2 /0.5% HSA (assay media). Specific inhibition of LFA-1 was achieved by incubation for 30 min at RT of the microtitre wells with a 1/200 dilution of TS1/22 (anti-LFA-ltt) ascites. Resting T cells were isolated from whole blood by plastic adherence and nylon wool filtration and were 91% CD2 + , while PHA-blasts were generated by culturing the cells in the presence of lO ⁇ g/ml phytohemagglutinin (PHA). Cells were labeled with the fluorochrome BCECF (Molecular Probes Inc.), washed and resuspended in assay medium. MAb pretreatment of cells consisted of incubation with 1/200 dilution for ascites for
• CBR-IC3/1 was derived from the fusion of the murine myeloma P3X63Ag8.653 with spleen cells from SKW3-injected Balb/c mice (Gefter et al, Som. Cell Gen. 5:231-236 (1977)), and 600 hybridomas were screened for the ability to inhibit
• PBMC Peripheral blood mononuclear cells
• Neutrophils were recovered from the cell pellet and contaminating erythrocytes removed by hypotonic lysis. Lymphocytes and monocytes were separated by cytometric analysis using forward and perpendicular light scatter, and was confirmed by monocyte and T cell specific MAb.
• Plastic-adherence was used to enrich lymphocytes from PBMC and cells were cultured in the presence of 10 ⁇ g/ml phytohemagglutinin (PHA). Samples were analyzed using an EPICS V flow cytometer, and fluorescence quantitated using EPICS Immune-Brite fluorescent beads (Coulter) to calibrate the cytometer. Expression of ICAM-3 on resting lymphocytes was 2-3 fold greater than either CD3 or LFA-1, while monocytes expressed 3-4 fold more ICAM-3 than LFA-1. Levels of ICAM-3 expression on neutrophils was equivalent to that of Mac-1 (CD lib/CD 18). Treatment of cells with phospholipase C revealed no Pi-linked form of ICAM-3 existed.
• PHA phytohemagglutinin
• Membrane expression determined by immunofluorescence flow cytometiy as outlined in materials and methods. Values are determinative of at least two experiments. Fluorescent beads were used to calibrate the cytometer such that one unit was approximately 10 3 fluorescein equivalents (Coulte Diagnostics, Hialeah, FL).
• Miscellaneous cell lines include: Human umbilical vein endothelial cells, huvec;; human breast carcinoma, Hep G2; human epithelioid carcinoma cell line, HeLa; human rhabdomyosarcoma, RD 3/5; human fibrosarcoma, and FS 1,2,3; human glioblastomas.
• the anti-ICAM-3 antibodies of the present invention were used to further determine the tissue distribution of the ICAM-3 protein using flow cytometiy.
• Table 3 presents a summary of cell types which display surface expression of ICAM-3. ICAM-3's expression appears to be restricted to hemopoietic cells.
• the flow cytometiy data was confirmed using immunohistochemical staining of tissue sections using labeled anti- ICAM-3 antibodies (data not shown). As with the flow cytometry data, immunohistochemical studies have shown that ICAM-3 expression appears to be restricted to hemopoietic cells.
• Membrane expression determined by immunofluorescence flow cytometry as outlined in materials and methods. Values are determinative of at least two experiments. Fluorescent beads were used to calibrate the cytometer such that one unit was approximately 10 3 fluorescein equivalents (Coulte Diagnostics, Hialeah, FL).
• Miscellaneous cell lines include: Human umbihcal vein endothelial cells, HUVEC; human breast carcinoma, Hep G2; human epitheloid carcinoma cell line, HeLa; human rhabdomyosarcoma, RD 3/5; human fibrosarcoma, and FS 1,2,3; human glioblastoma.
• ICAM-3 was purified using immunoaffinity chromatography in which an anti-ICAM-3 antibody CBR-IC3/1 was immobilized on a matrix using methods known in the art. Numerous sources have been used as starting material for isolating ICAM-3. These include, but are not limited to SKW3, a human thymoma cell line, and human tonsil. The methods utilized in purifying ICAM-3 are essentially those described by deFougerolles et al in/. Exp. Med. 275:185-190 (1992). One skilled in the art will recognize that washing and elution regimes (reagent vary slightly with each antigen to be purified and with each antibody to be used in immunoaffinity chromatography.
• ICAM-3 purified from SKW3 cells or human tonsilar cells appears to have a molecular weight from about 120 to 124 kD, while ICAM-3 purified from neutrophil derived-lysates yielded a broad band displaying a molecular weight from about 120 to 150 kD.
• ICAM-3 purified in this fashion has been shown to maintain its ability to bind anti-ICAM-3 antibodies, and LFA-1 on a variety of cells ( Figure 6 and 7).
• Immunoprecipitation and analysis by polyacrylamide gel electrophoresis was used to determined the molecular weight of ICAM-3 on various cell types.
• the molecular weight of ICAM-3 immunoprecipitated from neutrophils was found to be different from that immunoprecipitated from lymphocytes.
• ICAM-3 isolated from lymphocyte and lymphoid cell lines appears as a band with a molecular weight from about 120 to 124 kD.
• the molecular weight of ICAM-3 isolated from neutrophils is sUghtly higher than that expressed by lymphoid cells, appearing as a diffuse band from about 120 to 150 kD.
• ICAM-1 demonstrates a similar variation in molecular weight among different cell types.
• the variation in molecular weight seen for ICAM-1 has been shown to be caused by variations in the extent of glycosylation. Therefore, the variations in the molecular weight of ICAM-3 is most likely caused by differences in glycosylation.
• One skilled in the art can readily confirm this by subjecting ICAM-3 isolated from neutrophils to various glycosidases and other enzymatic treatments and looking at the effect this has on the molecular weight
• ICAM-1 MAC-1 (CDllb/CD18) binding. Therefore, by varying the extent of glycosylation of ICAM-3, it is possible to generate ICAM-3 derivatives that have modified affinities for binding to the various ligands of ICAM-3, for example, members of the CD11/CD18 family of glycoproteins.
• mice were injected with a combination of adjuvant and ICAM-3 protein which was purified from SKW3 or tonsil cells as described above.
• Monoclonal antibodies from immunized animals were generated using procedures known in the art. Table 4 presents a summary of the various anti-ICAM-3 antibodies obtained. The various antibodies were identified on the basis of their ability to react by ELISA to purified immobilize ICAM-3. Positive mAb were then screened on various cell lines known to be ICAM-3 positive and then immunoprecipitated from radiolabeled ICAM-3 positive cell lysates. The mAbs were also tested for their ability to block PMA- induced SKW3 cell aggregation in the presence of anti-ICAM-1 and anti- ICAM-2 antibodies. Alternatively, anti-ICAM-3 antibodies can be identified by their ability to inhibit binding of SKW3 cells to immobilized purified ICAM-3.
• An anti-ICAM-1 antibody (RRl/1), an anti-ICAM-2 antibody (CBR-IC2/2) and anti-ICAM-3 antibodies (a combination of CBR-IC3/1 and CBR-IC3/2) were tested for their ability to block (1) PMA-stimulated
• Figure 5 demonstrates that a similar activation mechanism is present in LFA-l/ICAM-3 binding.
• Figure 9 shows a summary of the effects of various antibodies on PHA stimulation of cell division.
• PHA stimulates cell proliferation in a manner that is inhibitable with anti-LFA-1 in anti-ICAM-1 antibodies, anti-ICAM-2 antibodies, or a combination of anti-ICAM-1 and anti- ICAM-2 antibodies had little effect on PHA stimulated cell division.
• anti-ICAM-1, anti-ICAM-2, and anti-ICAM-3 antibodies were effective at blocking PHA stimulated cell division.
• the MLR assay is used to assess immunologic reactivity.
• the assay basically involves adding chemically fixed foreign cells (the stimulator cells) to a solution containing PBL's from a different individual (responder cells) and measuring the level of responsiveness, using the proliferation of the responder cells as an index and has been described previously. (Krensky et al, J. Immunol 252:611-616 (1983)).
• This assay is the first step in identifying composition useful for treating graft rejection.
• the effects of various antibodies and antibody combinations on the MLR reactions is presented in Figure 10.
• anti-ICAM-3 alone displayed a low level effect.
• a greater level of effectiveness at blocking the MLR was found to occur with a combination of anti-ICAM-1 and anti-ICAM-3 antibodies.
• the highest response obtained was with the combination of anti-ICAM-1, anti-ICAM-2, and anti-ICAM-3 antibodies.
• ICAM-3 was purified from human tonsil cells by immunoaffinity chromatography as described in Example 5.
• the purified ICAM-3 was enzymatically digested with the Lys-C enzyme, using known methods, and peptide fragments were resolved by HPLC.
• Figure 11 presents a gas chromatograph of the various protein peaks observed in a typical digestion. Peaks 10 and 17 were identified as containing peptide fragments of sufficient size and structure to be sequenced (hereinafter the NK-10 and NK-17 peptide respectively).
• the amino acid sequence of the NK-17 and NK-10 peptide was determined using standard procedures.
• the sequence of the NK-10 protein was found to be KIDRATCPQHLK (SEQ. ID NO. 1).
• the first lysine (K) residue depicted in the sequence was inferred to be present since Lys-C cleaves proteins after lysine residues.
• the sequence of the NK-17 peptide was found to be KIALETSLSK (SEQ. ID No. 2). As with the NK-10 protein, the first lysine residue was inferred and later confirmed using DNA sequence analysis. A sequence comparison of the NK-10 and NK-17 proteins with known ICAM-1 and ICAM-2 sequences revealed a high degree of homology. The NK-17 peptide shows significant homology to sequences contained in the first Ig domain of ICAM-2. The NK-10 peptide shows weak homology to sequences within domain 4 of ICAM-1.
• Example XI cDNA Cloning of ICAM-3 Degenerate oligonucleotide probes were generated based on the peptide sequences obtained above. The probes were further designed to include previously identified codon preferences observed within the ICAM-1 and ICAM-2 nucleotide sequences. The nucleotide sequences of the probe based on the amino acid sequence of the NK-17 and NK-10 proteins, is as follows;
• NK-10 probe 25 mer with 8 fold degeneracy containing 5 N's (inosine).
• a cDNA expression library was generated from tonsil RNA as previously described. (Wong et al, Proc. Natl Acad. Sci. USA 82:7711- 7716 (1985)). The library was screened using the NK-17 probe and plaques displaying positive hybridization were rescreened using the NK-10 probe. One clone, clone 11.2, was found to have an insert from about 1.6 to 1.8 kb in length. A diagrammatic representation of clone 11.2 and the location of the NK-10 and NK-17 peptides is depicted in Figure 12.
• ICAM-3 shows a high level of homology to both ICAM-1 and ICAM-2. Sequence homologies between ICAM-1 and ICAM-3 are found within the first ( Figure 16) and the fourth/fifth domains of ICAM-1 as well as the transmembrane domain and some of the cytoplasmic domain of ICAM-1 ( Figures 13 and 14). Additional searches of the gene bank database reveal no other sequence which is identical or similar to that obtained for ICAM-3. Significant homology were also seen between ICAM-3 and the first domains of ICAM-2 ( Figure 17).
• Clone 11.2 was used to further screen libraries. Additional clones were found to obtain cDNA inserts from about 2 to 2.4 kD. In all likelihood these represent full-length cDNA clones since nothing of larger size have thus far been identified. SEQUENCE LISTING
• ADDRESSEE Sterne, Kessler, Goldstein & Fox

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