NO317658B1 - Intercellular adhesion molecule-3 and its binding ligands - Google Patents

Abstract

The present invention relates to intercellular adhesion molecules (ICAM-3) which are involved in the process by which leukocytes recognize and migrate to sites of inflammation as well as attach to cellular substrates during inflammation. The invention relates to such molecules, screening assays for identifying such molecules and antibodies which can bind such molecules. The invention also encompasses therapeutic and diagnostic uses for adhesion molecules and the antibodies that can bind them.

Description

The present invention relates to the use of an ICAM-3 modulating agent for the production of a pharmaceutical preparation.

The invention also relates to a method for identifying agents capable of modulating ICAM-3 binding to LFA-1.

A new intercellular adhesion molecule called ICAM-3 has been found which is involved in the process by which populations of leukocytes recognize and attach to cellular substrates. ICAM-3 mediates cellular interactions with other lymphocytes, macrophages and neutrophils at sites of inflammation and sites of immune responses.

ICAM-3 can be used alone or in combination with ICAM-1 and/or lCAM-2 to inhibit intercellular adhesion of cells of the granulocyte, lymphocyte or macrophage type. The use of such molecules provides methods for the treatment of specific and non-specific inflammations.

Therapeutic and prophylactic methods for suppressing the infection of leukocytes with HIV, and particularly with HIV-1, in an individual exposed to HIV or infected with HIV can be achieved through the administration of ICAM-3 alone or in combination with ICAM-1 and/or ICAM-2.

A therapeutic method to suppress the migration of HIV-1 infected cells from the circulatory system can be achieved by using ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2.

A therapeutic method for suppressing T-cell death and <n>syncytia" formation in an individual infected with HIV can be achieved by using ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2.

ICAM-3, alone or in combination with ICAM-1 and/or ICAM-2 can be used in the treatment of asthma.

Molecules that can bind to ICAM-3 are termed anti-ICAM-3. The binding of an anti-ICAM-3 molecule to ICAM-3 aims to modulate the biological functions associated with ICAM-3. The binding molecules can be an antibody, a peptide or a carbohydrate that can bind to ICAM-3. Such binding molecules can be used to modulate the biological functions of ICAM-3.

An anti-ICAM-3, alone or in combination with anti-ICAM-1 and/or anti-ICAM-2 can be used to inhibit intercellular adhesion of cells of the granulocyte, lymphocyte or macrophage type. The use of such molecules provides a method for treating specific or non-specific inflammation.

Therapeutic and prophylactic methods for suppressing the infection of leukocytes with HIV and particularly with HIV-1, in an individual exposed to HIV or infected with HIV can be achieved through the administration of an anti-ICAM-3, alone or in combination with anti- ICAM-1 and/or anti-ICAM-2.

A therapeutic method of suppressing the migration of HIV-1 infected cells from the circulatory system can be achieved by using an anti-ICAM-3 agent alone or in combination with an anti-ICAM-1 and/or anti-ICAM-2 agent .

An anti-ICAM-3 agent alone or in combination with anti-ICAM-1 and/or anti-ICAM-2 can be used in the treatment of asthma.

A. Leukocyte attachment and functions

Leukocytes must be able to adhere to cellular substrates in order to fully defend the host against foreign invaders such as bacteria or viruses, see Eisen, H.W. (In: Microbiology, 3rd ed., Harper & Row, Philadelphia, PA (1980), pages 290-295 and 381-418) for an overview 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 virus-infected cells or tumor cells can occur.

Recently, leukocyte surface molecules involved in mediating the above attachment-mediated functions have been identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T cells (Davignon, D. et al., Proe. Nat. Acad. Sei. USA 78:4535-4539

(1981)) and mouse spleen cells (Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979)) identified as bound to leukocyte surfaces and inhibited the attachment-related functions described above (Springer, T. et al., Fed. Proe. 44:2660-2663

(1985)). The molecules identified by these antibodies were called Mac-1 and "Lymphocyte Function-associated Antigen-1" (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, T.A. et al. Immunol. Rev. 68:111-135 (1982)). These two molecules plus a third molecule, pages 150-195 (which has a tissue distribution similar to Mac-1) play a role in cellular adhesion (Keizer, G. et al., Eur. J. Immunol. 15:1142-1147 (1985 )).

The above leukocyte molecules were found to be members of a related family of glycoproteins (Sanchez-Madrid, F. et al., J. Exper. Med. 158:1785-1803 (1983), Keizer, G.D. et al., Eur. J. Immunol. 15:1142-1147 (1985)), termed the "CD-18 family" of glycoproteins. This glycoprotein family consists of heterodimers with an alpha chain and a beta chain. Although the alpha chain of each of the antigens differs from one another, the beta chain was found to be largely conserved (Sachez-Madrid, F. et al., J. Exper. Med. 158:1785-1803 (1983) ). The beta chain of the glycoprotein family (sometimes referred to as "CD18") was found to have a molecular weight of 95 kd while the alpha chain was found to range from 150 kd to 180 kd (Springer, T., Fed. Proe 44:2660-2663 (1985)). Although the alpha subunits of the membrane proteins do not share the extensive homology shared by the beta subunits, closer analysis of the alpha subunits of the glycoproteins has shown that there are significant similarities between them. Accounts of similarities between the alpha and beta subunits of the LFA-1 related glycoproteins are provided by Sanchez-Madrid, F. et al., (J. Exper. Med. 158:586-602

(1983); J. Exper. With. 158:1785-1803 (1983)).

A group of individuals has been identified who are unable to express normal amounts of any member of this adhesion protein family on their leukocyte cell surface (Anderson, D.C. et al., Fed. Proe. 44:2671-2677

(1985), Anderson, D.C. et al., J. Infect. Haze. 152:668-689

(1985)). Such individuals are said to suffer from "leukocyte adherence deficiency disease" ("LAD") (Anderson, D.C. et al., Fed. Proe. 44:2671-2677 (1985), Anderson, D.C. et al., J. Infect. Dis 152:668-689 (1985)). Characteristic features of LAD patients include soft tissue necrotic lesions, impaired pus formation and wound healing, as well as abnormalities in adhesion-dependent leukocyte functions in vitro, and susceptibility to chronic and recurrent bacterial infections. Granulocytes from these LAD patients behave in the same defective manner in vitro as their normal counterparts in the presence of anti-CD 18 monoclonal antibody. That is that they are unable to perform adhesion-related functions such as aggregation or attachment to endothelial cells. More important, however, is the observation that these patients are unable to initiate a normal inflammatory response due to the granulocytes' inability to attach to cellular substrates. Most notable is the observation that granulocytes from these LAD patients are unable to arrive at sites of inflammation such as skin infections due to their inability to attach to the endothelial cells in the blood vessels near the inflammatory lesions. Such attachment is an emergency step for extravasation.

Lymphocytes from these patients exhibit in vitro defects similar to those of normal counterparts whose CD-18 molecular family had been antagonized by antibodies. Furthermore, these individuals were unable to mount a normal immune response due to the cells' inability to adhere to cellular substrates (Anderson, D.C. et al., Fed. Proe. 44:2671-2677 (1985), Anderson, D.C. et al. , J. Infect. Dis 152:668-689 (1985)). These data show that immune responses are dampened when lymphocytes are unable to adhere normally due to the lack of functional adhesion molecules of the CD-18 family.

Thus, in brief, the ability of leukocytes to maintain health and viability in an animal requires that they be able to attach to other cells (such as endothelial cells). This attachment has been found to require cell-to-cell contact involving specific receptor molecules present on the cell surface of the leukocytes. These receptors enable a leukocyte to attach to other leukocytes, to endothelial cells and to other non-vascular cells. The cell surface receptor molecules, LFA-1, Mac-1 and p150,95 have been found to be highly related to each other. Humans whose leukocytes lack these cell surface receptor molecules exhibit chronic and recurrent infections as well as other clinical symptoms including deficient antibody responses.

In addition, as leukocyte adhesion is involved in the process by which foreign tissue is identified and eliminated, an understanding of this process is of great value in the fields of solid organ transplantation such as kidney, non-solid organ transplantation such as bone marrow, tissue transplantation, allergy and oncology.

B. Infection with HIV

HIV infection is the cause of AIDS. Many variants of HIV have been described: the two most important are HIV-1 and HIV-2. HIV-1 is most prominent in North America and Europe and HIV-2 is most prominent in Africa. The viruses have similar structures and code for proteins with similar functions. HIV infection is thought to occur via the binding of a viral protein (designated "gpl20") to a receptor molecule (designated "CD4") present on the surface of T4 ("T helper") lymphocytes (Schnittman, S.M. et al., J. Immunol 141:4181-4186 (1988)). After binding to this receptor, the virus enters the cell and replicates, killing the T-cell in the process. Thus, the destruction of an individual's T4 population is a direct result of HIV infection.

The destruction of the T cells results in a weakening of the ability of the infected patient to fight opportunistic infections. Although individuals afflicted by AIDS often develop cancer, the connection between these cancers and HIV infection is in most cases uncertain.

Although the replication of the HIV virus alone is lethal to infected cells, such replication typically only affects a small fraction of the T cells in an infected individual. New results suggest that there is more viremia than previously estimated and that the frequency of T-cell infection may be as high as 1%.

A number of research areas have investigated other mechanisms through which the HIV virus mediates the destruction of the T4 population.

Apart from HIV replication, 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 cell (as in blood transfusions or mismatched organ transplants, etc.) encountered. Upon infection with HIV, T4 cells display the gp120 molecule on their cell surfaces. Killer cells recognize such T4 cells as foreign (rather than native cells) and consequently mediate their destruction.

HIV infection can also lead to the destruction of uninfected healthy cells. Infected cells can secrete the gpl20 protein into the bloodstream. The free gp120 molecules can then bind to the CD4 receptors in healthy, uninfected cells. Such binding causes the cells to appear as HIV-infected cells. Cytotoxic killer cells recognize gp120 bound to the uninfected T4 cells and conclude that the cell is foreign and cause destruction of the cell. A further mechanism of particular interest in connection with the present invention by which HIV can cause T4 cell death is through the formation of syncytia. A 11 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 acquire the ability to fuse with other T4 cells, either HIV-infected or uninfected healthy cells . The syncytium cannot function and soon dies. Its death initiates the destruction of both HIV-infected and HIV-uninfected T4 cells. This process is of particular interest in the context of the invention as it involves the direct cell-to-cell contact of T4 cells The ability of HIV-infected cells to form syncytia indicates that such cells acquire a means of fusion with healthy cells.

HIV infection and especially HIV-1 infection is shown to affect cell surface expression of 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 244:1075 (2989), Valentin, A., et al., J. Immunology 144:934-937 (1990), Rossen, R. D., et al., Trans. Assoc. American Physicians 102: 117-130

(1989)). After infection with HIV-1, homotypic aggregation of U937 cells is increased, as is cell surface expression of CD18 and CD11b (Petit, A.J., et al., J. Clin. Invest 79:188

(1987)). HIV-1-infected U937 cells adhere to IL-1-stimulated endothelium to a greater extent than uninfected U937 cells. This behavior can be suppressed by treating the infected cells with anti-CD18 or anti-CD11a monoclonal antibodies or by treating endothelial substrates with anti-ICAM-1 antibodies (Rossen, R.D., et al., Trans. Assoc. American Physicians 102:117 -130 (1989)). Monoclonal antibodies against CD18 or CDlla have also been found to inhibit formation of syncytia involving phytohemagglutinin (PHA)-stimulated lymphoblastoid cells and substantially infected CD-4-negative T cells (Hildreth, J.E.K., et al., Science 244:1075 ( 1989))..Treatment of only the virus-infected cells with anti-CDl8 or anti-CDlla monoclonal antibodies was found to have little effect on syncytium formation, suggesting that these antibodies mainly protect uninfected target cells from infection (Hildreth, J.E.K., et al., Science 244:1075 (1989), Valentin, A., et al., J. Immunology 144:934-937 (1990)). Valentin et al. (Valentin, A. et al., J. Immunology 144:934-937 (1990)) have recently confirmed these observations by showing that monoclonal antibodies specific for CD18 inhibit syncytia formed when continuous T cell lines are cocultured with HIV-1 infected U937 cells.

Although the mechanism by which monoclonal antibodies specific for CD18 or CDlla protect susceptible cells from fusing HIV-infected cells remains unknown, and is not necessary for an understanding of the present invention, studies with radiolabeled gpl20 suggest that heterodimers containing CD18 do not provide a binding site for the virus (Valentin, A., et al., J. Immunology 144:934-937

(1990)). Thus, the HIV infection involves cell-cell interactions and/or virus-cell interactions that mimic such cell-cell interactions. The cell-cell interactions can provide transport of cell-free virus or transport of virus-infected cells across the endothelial barriers. Virus-cell interactions that mimic cell-cell interactions may facilitate or enable free virus to attach to and/or infect healthy cells.

The present invention is thus partly derived from the observation that HIV infection and particularly HIV-1 infection results in increased expression of the CD11a/CD18 heterodimer and its binding ligand. This increased expression is significant in that it increases the ability of the HIV-infected T cells to adhere or aggregate with each other (ie, to undergo "homotypic aggregation"). As such homotypic aggregation has not been observed to occur among quiescent normal leukocytes, this finding 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 for homotypic aggregation to occur. As noted herein, ICAM-3 is the only member of the ICAM family of molecules that is expressed in high abundance on resting T cells. Only anti-ICAM-3 antibodies can block adhesion of T cells to LFA-1 as long as the T cells are not "activated". Therefore, anti-ICAM-3 antibodies can be used to suppress aggregation of T cells.

Anti-ICAM-3 antibodies can further be used to block the adhesion processes to infected T cells that allow HIV-1 to be transferred from the infected cell to a healthy cell in an individual and also allow or facilitate infection of healthy cells with free virus .

C. Migration of HIV-infected cells

The migration and spread of leukocytes is important to protect an individual from the consequences of infection. These processes are. however, also responsible for the migration and spread of virus-infected leukocytes. Of particular interest is the migration and spread of leukocytes infected with HIV. The migration of such cells results in the formation of extravascular foci and can produce tumors and other abnormalities.

Histological examination of affected organs shows focal extravascular mononuclear cell infiltrates. Attempts to identify virus-infected cells in such infiltrates in the central nervous system have shown the presence of HIV-1-infected cells. These studies have shown that the HIV virus is primarily in monocytes and macrophages and other cells of this genus (R.T. Johnson et al., FASEB J. 2:2970 (1988); M.H. Stoler et al., J. Amer. Med Assn. 256:2360 (1986), S. Gartner et al. Science 233:215 (1986)).

The mechanisms that stimulate the formation of extravascular filtrates by 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 that facilitate the adherence of leukocytes to vascular endothelial cells and the migration of leukocytes from the blood to extravascular tissue sites (C.W. Smith et al., J. Clin. Invest. 82:1746 (1988)), it is proposed to use antibodies that inhibit cellular migration to prevent the spread of HIV-infected cells (WO 90/13316).

D. Asthma: Clinical features

Asthma is a heterogeneous family of diseases. It is characterized by a hyper-susceptibility of the tracheobronchi to stimuli (Kay, A.B., Allergy and Inflammation, Academic Press, NY (1987)). Clinically, asthma is manifested by the extensive narrowing of the tracheobronchi, by thick solid secretions, by attacks of shortness of breath, 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 lung and thorax, and abnormal distribution of ventilation and pulmonary blood flow. The disease manifests itself in short-term periods with acute symptoms that lie between symptom-free periods. The acute episodes result in hypoxia and can be fatal. About 3% of the world's population suffers from asthma.

Two types of asthma have been described: allergic asthma and idiosyncratic asthma. Allergic asthma is usually associated with a hereditary allergic disease such as rhinitis, urticaria, eczema, etc. The condition is characterized by positive reactions with flare-ups of blisters to intradermal injections of airborne antigens (such as pollen, environmental and occupational pollutants, etc.) and increased serum levels of IgE. The development of allergic asthma appears to be coincidentally related to the presence of IgE antibodies in many patients. Asthma patients who do not exhibit the above-mentioned characteristics are considered to have idiosyncratic asthma.

Allergic asthma is believed to be dependent on an IgE response directed by T and B lymphocytes and activated by interaction of airborne antigen with mast cell-bound pre-formed IgE molecules. The antigenic encounter must occur in concentrations sufficient to cause IgE production for an extended period of time to sensitize an individual. Once an asthma patient is sensitized, the patient may exhibit symptoms in response to extremely low antigen levels.

Asthma symptoms can be aggravated by the presence and amount of the trigger antigen, environmental factors, occupational factors, physical exertion and emotional stress.

Asthma can 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 (ie, chromones such as cromolyn sodium), and anticholinergics (such as atropine).

Asthma is believed to involve an influx of eosinophils ("eosinphilia") into lung tissue (Frigas, E. et al., J. Allergy Clin. Immunol. 77:527-537 (1986)).

Insights into the basic immunology of asthma have been obtained from bronchoalveolar lavage studies (Godard, P. et al., J. Allergy Clin. Immunol. 70:88 (1982)), and studies of respiratory smooth muscle tissue removed from epithelium (Flavahan, N.A. et al., J. Appl. Physiol. 58:834

(1985), Barnes, P.J. et al., Br. J. Pharmacol. 86:685

(1985) ). Although these studies have not led to any explanation of the mechanism underlying the immunology of asthma, they have led to the development of a generally accepted hypothesis concerning the immunological etiology of the disease (see Frigas, E. et al., J. Allergy Clin. Immunol. 77:527-537

(1986) ).

The hallmarks of the pathology of asthma are a massive infiltration of the lung parenchyma with eosinophils and the destruction of mucociliary capacity. The "eosinophilic hypothesis" proposes that eosinophils are attracted to the bronchi to neutralize harmful mediators released by the lung's mast cells. According to the hypothesis, eosinophils are drawn to the bronchi where they degranulate to release cytotoxic molecules. During de-granulation, the eosinophils release enzymes such as histamine, aryl-sulfatase and phospholipase D which enzymatically neutralize the harmful mediators of the mast cells. However, these molecules also promote the destruction of the mucociliary apparatus and thus prevent the removal of the bronchial secretions and contribute to the lung-destructive property of asthma.

As asthma involves the migration of cells, it has been proposed to use antibodies that inhibit this migration to alleviate the effects of allergens in an individual (WO 90/10453).

The present invention provides the use of an ICAM-3 modulating agent for the manufacture of a pharmaceutical preparation for modulating inflammation, 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, wherein said fragment is capable of binding to ICAM-3; and ICAM-3.

In an embodiment of the above application in accordance with the invention, said inflammation comprises a reaction of the specific defense system.

In a further embodiment of the above application in accordance with the invention, said inflammatory response is specific inflammation in response to a condition selected from the group consisting of delayed-type hypersensitivity reaction, psoriasis, an autoimmune disease, organ transplant, and tissue transplant rejection.

In a further embodiment of the above application in accordance with the invention, said organ transplant is a fixed organ transplant.

In a further embodiment of the above application in accordance with the invention, said organ transplant is a kidney transplant.

In a further embodiment of the above application in accordance with the invention, said organ transplant is a non-fixed organ transplant.

In a further embodiment of the above application in accordance with the invention, said non-fixed organ transplant is a bone marrow transplant.

In a further embodiment of the above application in accordance with the invention, said autoimmune disease is selected from the group consisting of Reynaud's syndrome, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, rheumatoid arthritis and lupus erythematosus.

In a further embodiment of the above application in accordance with the invention, said inflammation comprises a reaction of the non-specific defense system.

In a further embodiment of the above application in accordance with the invention, said inflammation is non-specific inflammation in response to a condition selected from the group consisting of: "adult respiratory distress syndrome" (ARDS), multiple organ damage syndromes secondary to septicemia, trauma or bleeding, reperfusion injury of myocardium or other tissue, acute glomerulonephritis, reactive arthritis, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, stroke, thermal injury, hemodialysis, levakapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion-associated syndromes and cytokine-induced toxicity.

In a further embodiment of the above application in accordance with the invention, said ICAM-3 modulating agent is a soluble fragment of ICAM-3, said fragment being able to bind LFA-1.

The present invention further provides for the use of an ICAM-3 modulating agent for the preparation of a pharmaceutical preparation for modulating the extravascular migration of a virally infected leukocyte, wherein said ICAM-3 modulating agent is selected from the group consisting of an antibody which is able to bind to ICAM-3; a fragment of said antibody, wherein said fragment is capable of binding to ICAM-3; and ICAM-3.

The present invention further provides a method for identifying agents capable of modulating ICAM-3 binding to LFA-1, characterized in that it comprises the steps of contacting ICAM-3 with LFA-1 in the presence of said agent and measure said modulation of ICAM-3/LFA-1 binding.

In an embodiment of the above-mentioned method in accordance with the invention, said LFA-1 and said ICAM-3 are without naturally occurring contaminants.

In a further embodiment of the above-mentioned method in accordance with the invention, said LFA-1 is present on a cell surface and said ICAM-3 is without naturally occurring contaminants.

In a further embodiment of the above-mentioned method in accordance with the invention, said ICAM-3 is present on a cell surface and said LFA-1 is without naturally occurring contaminants.

In a further embodiment of the above-mentioned method in accordance with the invention, said ICAM-3 and said LFA-1 are present on different cell surfaces.

In a further embodiment of the above-mentioned method in accordance with the invention, measurement of the effect of said compound on ICAM-3/LFA-1 binding is carried out by using a cellular aggregation analysis.

In a further embodiment of the above-mentioned method in accordance with the invention, said compound is not an immunoglobulin.

In a further embodiment of the above-mentioned method in accordance with the invention, said compound is a non-immunoglobulin polypeptide.

In a further embodiment of the above-mentioned method in accordance with the invention, said compound is an immunoglobulin.

The present invention further provides the use of an ICAM-3 modulating agent for the manufacture of a pharmaceutical composition for suppressing cellular migration associated with asthma, wherein said ICAM-3 modulating agent is selected from the group consisting of an antibody capable of to bind to ICAM-3; a fragment of said antibody wherein said fragment is capable of binding to ICAM-3; and ICAM-3.

In an embodiment of the above application in accordance with the invention, said ICAM-3 modulating agent is a soluble fragment of ICAM-3 comprising ICAM-3 domains 1-5.

In a further embodiment of the above application in accordance with the invention, said pharmaceutical preparation further comprises one or more further anti-asthma agents.

In a further embodiment of the above application in accordance with the invention, said anti-asthma agent is a methylxanthine, a β-adrenergic agonist, a glucocorticoid, a chromone or an anticholinergic compound.

In a further embodiment of the above application in accordance with the invention, said anti-asthma agent is selected from the group comprising theophylline, catecholamine, resorcinol, saligenin, ephedrine, atropine, cromolyn sodium and hydrocortisone.

In a further embodiment of the above application in accordance with the invention, said anti-asthma agent is present in said pharmaceutical preparation at a suboptimal level.

The present invention further provides for the use of an ICAM-3 modulating agent for the preparation of a pharmaceutical preparation for reducing metastasis of a hematopoietic tumor cell, wherein said ICAM-3 modulating agent is selected from the group consisting of an antibody that is in able to bind to ICAM-3; a fragment of said antibody, wherein said fragment is capable of binding to ICAM-3; and ICAM-3.

In an embodiment of the above application in accordance with the invention, said ICAM-3 modulating agent is a soluble fragment of ICAM-3, where said fragment is capable of binding LFA-1.

In a further embodiment of the above application in accordance with the invention, said ICAM-3 modulating agent is a soluble fragment of ICAM-3 comprising ICAM-3 domains 1-5.

The present invention further provides for the use of an ICAM-3 modulating agent for the preparation of a pharmaceutical preparation to reduce syncytia formation caused by HIV infection, wherein said ICAM-3 modulating agent is selected from the group consisting of an antibody which is able to bind to ICAM-3; a fragment of said antibody, wherein said fragment is capable of binding to ICAM-3; and ICAM-3.

In an embodiment of the above application in accordance with the invention, said ICAM-3 modulating agent is a soluble fragment of ICAM-3, where said fragment is able to bind LEA-1.

In a further embodiment of the above application in accordance with the invention, said HIV is HIV-1.

Brief description of the figures

Fig. 1 Adhesion of cell lines to purified LFA-1 Adhesion of cell lines (A) and lymphocytes (B) to purified LFA-1 is explained by ICAM-1, ICAM-2 and ICAM-3. The binding of BCECF-labeled cells on LFA-1 coated microtiter wells in the presence of blocking MAb specific for LFA-1, ICAM-1, ICAM-2 and ICAM-3. Control wells were coated without LFA-1. Fig. 2 Flow cytometric analysis of cellular ICAM-1, ICAM-2 and

ICAM-3 express ion

(A) Lymphoblastoid cell lines were labeled with saturating amounts of either MAb RRl/l (anti-ICAM-1), MAb CB-IC2/1 (anti-ICAM-2),

MAb CBR-IC3/1 (anti-ICAM-3) or non-binding control MAb X63 (thin lines) and followed by FlTC anti-mouse immunoglobulin. (B) Resting human leukocytes and third day PHA-activated lymphocytes were analyzed for ICAM expression as above.

Fig. 3 Immunoprecipitation of ICAM-3

(A) <125>I-labeled cell lysates immunoprecipitated with either nonbinding control X63 or MAb CB-IC3/1 (anti-ICAM-3). (B)

<125>I-labeled SKW3 cell lysates treated with {+) or without (-) N-glycanase and immunoprecipitated with either non-binding control MAb X63, MAb W6/32 (anti-HLA-A, B, C), MAb RRl /l (anti-ICAM-1), MAb CBR-IC2/1 (anti-ICAM-2) or MAb CB-IC3/1 (anti-ICAM-3).

Fig. 4 Immunoprecipitation of ICAM-3 from different

sources

ICAM-3 was immunoprecipitated from I<125->labeled lymphocyte and neutrophil lysates using mAb-coupled Sepharose. Immunoprecipitated ICAM-3 was redissolved using polyacrylamide gel electrophoresis. Another mAb was used to immunoprecipitate other cell surface molecules. Human lg Sepharose (control lane), CBR-IC2/1 (ICAM-2), CBR-IC3/1 (ICAM-3), TS2/4 (LFA-1), LM2/1 (MAC-1). Fig. 5 Effect of PMA on SKW3 binding to ICAM-1 and ICAM-3 The ability of SKW3 cells to bind to purified ICAM-1 and ICAM-3, and the effects of PMA stimulation of this was tested as described previously {Dustin et al, Nature 341:619 (1989), Marlin et al, Cell 51:813-819 (1987)). A representative experiment is shown and error bars indicate one standard deviation (SD). Fig. 6 Antibody blocking of PMA-stimulated SKW3 cells

adhesion

Various antibodies were tested for their ability to block PMA-stimulated SKW3 cells from binding to purified ICAM-1 or ICAM-3 as described previously (Dustin et al., Nature 341:619 (1989), Marlin et al., Cell 51:813-819

(1987)). A representative experiment is shown and error bars indicate one SD.

Fig. 7 Binding of COS cell transfectants to purified

ICAM' is

COS cells were transfected with an LFA-1 expression vector as described previously. (deFougerolles et al., J. Exp. Med. 175:185-190 (1992)). The 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). A representative experiment is shown and error bars indicate one

SD.

Fig. 8 Temperature dependence of PMA-stimulated SKW3 binding

to ICAM

The ability of PMA-stimulated SKW3 cells to bind to purified ICAM-1 and ICAM-3 was tested at 4, 16, 22 and 37°C as described previously. (Marlin et al, Cell 51:813-819 (1987)). Experiments were performed in the presence or absence of an LFA-1 mAb (TS1/22). A representative experiment is shown and error bars indicate one SD. Fig. 9 Antibody blocking of PHA-stimulated T-cell de3, ;jn g Different antibodies were tested for their ability to block PHA-stimulated T-cell division as described previously (Krensky et al., J. Immunol. 131:611 -616 (1983)).

Fig. 10 Effects of anti-ICAM antibodies on it

mixed lymphocvt reaction

Various antibodies were tested for their ability to block mixed lymphocyte reaction as described previously (Krensky et al., J. Immunol. 131:611-616 (1983)). Fig. 11 Gas chromatography of peptide fragments of ICAM-3 ICAM-3 was purified and subjected to degradation with Lys-C. Peptide fragments were resolved using high pressure liquid chromatography.

Fig. 12 Schematic representation of clone 11. 2.

A schematic representation of a clone 11.2. The position of the NK-10 and NK-17 peptides, the different DNA sequences obtained so far and the transmembrane region are indicated.

Fig. 13 Sequence comparison of the NK-10-primed sequence with

human ICAM-1

The GAP program was used to identify sequence homology for the NK-10 primed sequence and human ICAM-1 protein.

Fig. 14 Sequence comparison of the RM13-primed sequence with

human ICAM-1

The GAP program was used to identify sequence homology of the RM13-primed sequence with human ICAM-1.

Fig. 15 Sequence comparison of the RM13-primed sequence with

human ICAM-2

The GAP program was used to identify sequence homology for the RM13-primed sequence and human ICAM-2.

Fig. 16 Sequence comparison of the T17-primed sequence with

human ICAM-l

The GAP program was used to identify sequence homology for the T7-primed sequence and human ICAM-1.

Fig. 17 Sequence comparison of the T7-primed sequence with human ICAM-2

The GAP program was used to identify sequence homology for the T7-primed sequence and human ICAM-2.

Fig. 18 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

was 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.

C. Sequences obtained from the 3' end of ICAM-3. The sequences were obtained by priming clone 11.2 with the reverse RM13 primer.

The present invention is based on the discovery of a previously undefined binding ligand for LFA-1. Molecules such as those of the CD-18 family that are involved in the process of cellular adhesion are referred to as "adhesion molecules".

I . LFA-l

The leukocyte adhesion molecule LFA-1 mediates a variety 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 recently identified ICAM-3 (which is discussed herein). These surface molecules are expressed constitutively on some tissues and induced on others by inflammation (Marlin, S.D. et al., Cell 51:813-819

(1987), Dustin, M.L. et al., J. Immunol. 137:245-254 (1986), Dustin, M.L. et al., Immunol. Today 9:213-215 (1988), US Patent Application No. 07/019,440, filed February 26, 1987 and US Patent Application No. 07/250,446, filed September 28, 1988).

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)).

II. ICAM-1

ICAM-1 is a single-chain glycoprotein that varies in weight in different cell types from 76-114 kD and is a member of the Ig superfamily with five C-like domains (Dustin, M.L. et al, Immunol. Today 9:213-215 ( 1988), Staunton, D. E. et al., Cell 52:925-933 (1988), Simmons, D. et al., Nature 331:624-627 (1988)). ICAM-1 can be largely induced by cytokines including IFN-g, TNF and IL-1 on a variety of cell types (Dustin, M.L. et al., Immunol. Today 9:213-215 (1988)). 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 second cell with ICAM-1 MAb (Dustin, M.L. et al., J. Immunol. 137:321-331 (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)). Identical results with purified ICAM-1 in artificial membranes or in petri dishes show that LFA-1 and ICAM-1 are receptors for each other (Marlin, S.D. et al, Cell 51:813-819 (1987), Makgoba, M.W. et al, Nature 331:86-88 (1988)). For clarification, they are referred to here as "receptor" and "ligand", respectively. Further description of ICAM-1 is provided in US Patent Applications Nos. 07/045,963, 07/115,798, 07/155,943, 07/189,815 or 07/250,446.

III. ICAM-2

Other LFA-1 ligands, different from ICAM-1, have been postulated (Rothlein, R. et al., J. Immunol. 137:1270-1274 (1986), Makgoba, M.W. et al., Eur. J. Immunol. 18:637-640 (1988), Dustin, M. L. et al, J. Cell. Biol. 107:321-331 (1988)). The second LFA-1 ligand identified is designated "ICAM-

ICAM-2 differs from ICAM-1 in cell distribution and in lack of cytokine induction. ICAM-2 is an integral membrane protein with two Ig-like domains while ICAM-1 has 5 Ig-like domains (Staunton, D.E. et al., Cell 52:925-933

(1988), Simmons, D. et al., Nature 331:624-627 (1988)). Remarkably, ICAM-2 is much more closely related to the two distinct N-terminal domains of ICAM-1 (34% identity) than ICAM-1 and ICAM-2 are to other members of the Ig superfamily, demonstrating a subfamily of lg-like ligands that bind to the same integrin family receptor. Further description of ICAM-2 is provided in US Patent Application No. 07/454,294.

IV. ICAM-3

In connection with the present invention, a third LFA-1 ligand has been found which is designated "ICAM-3".

Development of MAbs against ICAM-2 allowed a number of previously LFA-1-dependent, ICAM-1-independent phenomena to be analyzed and suggested that a third ligand for LFA-1 existed. Binding of a variety of cell types such as epithelial and endothelial cells to purified LFA-1 could be completely blocked by a combination of ICAM-1 and ICAM-2 MAb, while an ICAM-1, ICAM-2-independent adhesion pattern to LFA-1 existed on a number of lymphoid cell lines including the T-cell lymphoma cell line SKW3 (Fig. 1).

To characterize this adhesion pattern, MAbs were raised against SKW3 and, together with anti-ICAM-1 and anti-ICAM-2 MAb<1>s, were screened for the ability to inhibit binding of this cell line to purified LFA-1. One MAb, CBR-IC3/1, was chosen which completely inhibited this new adhesion pattern (Fig. 1). SKW3 adhesion to purified LFA-1 was only slightly inhibited with a combination of blocking anti-ICAM-1 and anti-ICAM-2 MAb. When anti-ICAM-3 MAb, CB-IC3/1 was added alone, it significantly inhibited adhesion, and this inhibition was complete when combined with blocking anti-ICAM-2 MAb. The adhesion of SKW3 to purified LFA-1 was thus mediated to a large extent by ICAM-3 and also partly by ICAM-2. Upon adherence to LFA-1, each of the four cell lines used the three ICAMs to a different extent, as shown by the different patterns of MAb inhibition. The adhesion of B lymphoblastoid cell line, JY, occurred primarily through an ICAM-1 pattern with some contribution through the ICAM-2 and ICAM-3 patterns. Another B-lymphoblastoid cell line, SLA, used both ICAM-1 and ICAM-3, as adhesion was not inhibited by either ICAM-1 or ICAM-3 MAb alone and almost completely by the MAbs together. The thymoma cell line, Jurkat, used both the ICAM-2 and ICAM-3 adhesion patterns with a small contribution from ICAM-1. Adhesion of both resting T lymphocytes and those activated by phytohemagglutinin (PHA) was also examined (Fig. 1B). 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. Upon activation with PHA, ICAM-1 expression was induced and adhesion to LFA-1 occurred mainly through ICAM-1 and partially through ICAM-3. The ICAM-2 adhesion component in both resting and activated T cells was detectable, although masked by the presence of ICAM-1 and ICAM-3. There is considerable redundancy in the use of ICAMs as MAbs against at least two ICAMs were required for each cell to achieve significant 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 MAbs eliminated binding to LFA-1 to levels comparable to those seen in the presence of anti-LFA-1 MAb.

The relative affinity of three ICAMs for LFA-1 can be examined by comparing their contribution to binding LFA-1 as measured over to their cell surface expression as measured by immunofluorescence flow cytometry (Fig. 2). The comparison of surface expression of the ICAM<1>s and their contribution to LFA-1 adhesion showed 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 e.g. ICAM-2 and ICAM-3 at the same levels and each contributed to binding to LFA-1. Where ICAM-2 expression was greater than that of ICAM-3, as on JY cells, the ICAM-2 pattern of LFA-1 adhesion dominated that of ICAM-3. In contrast, SLA and SKW3 expressed 3-5 times more ICAM-3 than ICAM-2 and, not surprisingly, the ICAM-3 adhesion pattern was dominant over the ICAM-2 pattern. On resting T-lymphocytes where ICAM-3 was well expressed and ICAM-1 was absent, adhesion to LFA-1 was largely mediated through ICAM-3. When ICAM-1 was well expressed, as in the case of SLA, JY and the PHA-activated T cells, this was the main adhesion pattern.

The distribution pattern of ICAM-3 differed from that of ICAM-1 and ICAM-2 in a number of ways. Unlike ICAM-1 and ICAM-2, ICAM-3 was not expressed in 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, and was largely expressed on lymphoid and monocytic cell lineages with a few exceptions. In all cases, however, expression of ICAM-3 on cell lines was coordinated with LFA-1-dependent, ICAM-1, ICAM-2-independent adhesion patterns. Cell lines (HUVEC, Raji) that bind to LFA-1 only through ICAM-1 and ICAM-2 showed no ICAM-3 expression, while cell lines (JY, U937, Sup T) that showed weak binding through this third adhesion pattern had correspondingly low ICAM-3 surface expression (data not shown).

ICAM-3 was markedly different from ICAM-1 and ICAM-2 in its expression on leukocytes (Fig. 2B). ICAM-3 was expressed at high levels on resting lymphocytes, monocytes and neutrophils, while ICAM-1 and ICAM-2 were expressed much weaker 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 was expressed on neutrophils. Upon activation of lymphocytes with PHA, ICAM-3 expression increased 2-3-fold, while ICAM-1 expression was highly induced (Dustin et al, J. Immunol. 13 7:245-254

(1986)) (Fig. 2B).

Immunoprecipitates of ICAM-3 from various 1251-labeled cell lines showed a sharp band at 124,000 Mr under reducing conditions with only slightly increased mobility under non-reducing conditions (Fig. 3A). Treatment with N-glycanase reduced the ICAM-3 band to Mr 87,000, indicating that ICAM-3, like ICAM-1 and ICAM-2, is a highly glycosylated protein (Fig. 3B). The biochemical properties, expression patterns and functional properties of ICAM-3 distinguish it from previously described adhesion molecules including the human homing receptor LAM-1 (Tedder et al, J. Immunol. 144:532-540 (1990)), the inducible endothelial -adhesion molecule VACAM-1 (Rice et al., J. Exp. Med. 171:1369-1374 (1990), Carlos et al., Blood (1990), (Osborn et al., Cell 59:1203-1211 (1989 )), and the VLA family of matrix receptors (Hemler, M.E. Ann. Rev. Immunol. 8:365-400 (1990)), and no MAbs with similar cellular distributions were found in the fourth database from a leukocyte workshop (Gilks , W.R., et al., Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England (1990)).

The presence of three LFA-1 ligands suggests specialization for different aspects of LFA-1-dependent leukocyte interactions. ICAM-1 is primarily expressed on endothelium and many epithelial cell types, and is strongly induced by inflammation and immunity where it regulates cell localization and facilitates specific antigen recognition (Wawryk et al., Immunol. Rev. 108:135-161 (1989)). As ICAM-2 is the predominant LFA-1 ligand on resting endothelium, this adhesion pattern may have important consequences for the normal recycling of LFA-1-bearing lymphocytes through tissue endothelium (Hamann et al., J. Immunol. 140:693-699 ( 1988), Mackay et al., J. Exp. Med. 171:810-817 (1990), Pals et al., J. Immunol 140:1851-1853

(1988) and Nunoi et al., Hum. Path. 19:753-759 (1988)). To date, the function of ICAM-3 on neutrophils remains unclear, as homotypic aggregation of neutrophils appears to be primarily mass Mac-1 dependent despite the presence of LFA-1 (Anderson et al., J. Immunol. 137:15-2 7 (1986); Patarroyo et al., Scand. J. Immunol. 22:619-631 (1985)). The finding that adhesion of resting T-lymphocytes to LFA-1 primarily occurs via ICAM-3 combined with the fact that ICAM-3 is much better expressed than other LFA-1 ligands on monocytes and resting lymphocytes plays an important role in the initiation of immune responses. T-cell adhesion with antigen-presenting cells requires LFA-1:ICAM interactions (Dransfield et al., Imm. Rev. 114:29-44 1990); Makgoba et al., Immunol. Today 10:417-422

(1989) ), and as such ICAM-3 may play a role, particularly on resting T lymphocytes where neither ICAM-1 nor ICAM-2 is well expressed. Indeed, a role for one or more LFA-1 ligands other than ICAM-1 has been suggested in both allogeneic and autologous mixed lymphocyte reactions Bagnasco et al., Cell Immunol. 128:362-369 (1990)). Furthermore, 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 connection with the lysis of certain target cells by T cells which are dependent on LFA-1, but not ICAM-1 (Makgoba et al., Eur.j. Immunol. 18:637-640 (1988)).

The occurrence of multiple ICAMs is of great importance for clinical treatment with MAb against ICAMs or ICAM analogues. ICAM-1 MAb is effective in vivo in prolonging renal (Cosimi et al., J. Immunol. 144:4604-4612 (1990)) and cardiac (Flavin et al., Transplant.Proe.23:533 -534

(1991)) allografts. ICAM-3 MAb inhibits a distinct and overlapping number of immune responses in vivo, as it inhibits LFA-1-dependent adhesive interactions for a distinct subset of cell types.

V. cDNA CLONING OF ICAM-3

Any of a number of procedures can be used to clone ICAM-3 genes. One such method involves analyzing a shuttle vector library of cDNA inserts (derived from an ICAM-3 expressing cell) for the presence of an insert containing the ICAM-3 gene. Such an analysis can be carried out by transforming cells with the vector and then measuring ICAM-3 expression.

ICAM-3 cDNA is preferably identified using a modification of the procedure of Aruffo and Seed (Seed, B. et al., Proe.Nati.Acad.Sei. USA 84:3365-3369 (1987)) to identify adhesion molecule ligands . In this method, a cDNA library is prepared from cells expressing ICAM-3 (such as SLA, Jurkat or SKW3 lymphoblastoid cell lines). The cDNA library is preferably prepared from T cells. This library is used to transfect cells that do not normally express ICAM-3 (such as Cos or HeLa cells). The transfected cells are plated in a Petri dish that has been previously coated with either LFA-1 or anti-ICAM-3 antibodies. The transformed cells containing ICAM-3 coding sequences and expressing this ligand on their cell surfaces will adhere to 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 are then removed.

If LFA-1 is used to coat the petri dish, anti-ICAM-1 and anti-ICAM-2 specific antibodies are added to the petri dish to prevent adherence of ICAM-1 or ICAM-2 expressing cells. Adherence of ICAM-1<+-> or ICAM-2<+-> transfectants to LFA-l coated plastic can thus be inhibited with antibodies such as RRl/l, 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 dishes is inhibited by EDTA and anti-LFA-1 MAbs, but not by anti-ICAM-1 or anti-ICAM-2 MAb1s. Therefore, the ICAM-1 or ICAM-2 expressing cells are unable to adhere to Petri dishes and are therefore largely washed away with all the other non-adherent cells. This will enrich for cells expressing ICAM-3.

Other alternative methods for obtaining gene sequences that code for ICAM-3 are well known in the state of the art and a person skilled in the art will routinely adapt one of these methods to obtain a desired gene.

One such method for obtaining a gene sequence that codes for ICAM-3 is to use an oligonucleotide probe to screen a cDNA or genomic DNA library. In this method, the ICAM-3 protein is preferably purified using immunopurification procedures known in the art, and the terminal amino acid sequence is determined using methods known in the art. Alternatively, ICAM-3 is purified enzymatically, as with trypsin or Lys-C, peptides are purified and the amino acid sequence of one of the internal fragments is determined.

When the partial amino acid sequence is determined, either an oligonucleotide probe is formed based on the codon preference demonstrated by the organism, a degenerate probe is formed 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 construct probes. This probe is then used to screen either a genome or cDNA library for sequences that hybridize to the probe.

Techniques such as those described above or similar to these have successfully made it possible to clone genes for human aldehyde dehydrogenases (Hsu, L.C. et al., Proe. Nati. Acad. Sei. USA 82:3771-3775 (1985)) , fibronectin (Suzuki, S. et al., Eur. Mol. Biol. Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter, P. et al., Proe. Nati. Acad. Sei USA 82:7889-7893 (1985)), and tissue type plasminogen gene activator (Pennica, D. et. al., Nature 301:214-221 (1983)).

In a further alternative way of cloning the ICAM-3 gene, an expression library is prepared by cloning genomic DNA or, more preferably cDNA, from a cell expressing ICAM-3. The library is then screened for members that can express a protein that binds to an anti-ICAM-3 antibody.

The cloned ICAM-3 gene obtained through the use of any of the above methods can be operably attached to an expression vector and introduced into bacterial or eukaryotic cells to produce the ICAM-3 protein. Techniques for such manipulations are set forth in Maniatis, T. et. al., supra, which are well known in the art.

In an alternative method using PCR to clone the ICAM-3 gene, it is assumed that ICAM-3 is homologous to ICAM-1 and/or ICAM-2. Degenerate oligonucleotide primers based on sequences conserved between ICAM-1 and ICAM-2 are used to amplify by 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., 175:185-190 (1992)). Clones are sequenced and those different from ICAM-1 and ICAM-2 are used to obtain full-length cDNA clones. In any of the methods described above, the authenticity of the clones can be confirmed by expressing full-length clones, e.g. in COS cells, and when testing for reactivity with ICAM-3 mAb.

WE. ICAM-3 and its functional derivatives, agonists and

antagonists

A. Functional derivatives of ICAM-3

A "functional derivative" of ICAM-3 is a compound which exhibits a biological activity (either functional or structural) which is substantially similar to the biological activity of ICAM-3. The term "functional derivative" includes "fragments", "variants" and "chemical derivatives" of the parent ICAM-3 molecule.

A "fragment" of ICAM-3 is intended to refer to any polypeptide subset of the molecule. Fragments of ICAM-3 which have ICAM-3 activity and which are soluble (ie not membrane bound) are particularly preferred. A soluble fragment is preferably formed by dividing the membrane "spanning" region of the parent molecule or by dividing or replacing hydrophilic amino acid residues with hydrophobic residues. Identification of such residues is well known in the art.

Fragments containing any number of an entire Ig-like domain are preferred, such as domain 1(D1), D1 and D2, D1-3, D1-4 and D1-5.

By a "variant" of ICAM-3 is meant a molecule which in structure and function is substantially similar to either the entire molecule or 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 exhibit a similar biological activity. With the condition that two molecules exhibit a similar activity, they are thus considered variants, as the term used herein, even if one of the molecules exhibits a structure that is not found in the other molecule, or if the sequence of amino acid residues is not identical.

As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical entities that are not normally part of the naturally occurring molecule. Such units can improve the molecule's solubility, absorption, biological half-life, etc. The units can alternatively reduce the molecule's toxicity or eliminate or weaken unwanted side effects of the molecule. Entities capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).

"Toxin-derivatized" molecules constitute a special group of "chemical derivatives". A "toxin-derivatized" molecule is a molecule (such as ICAM-3 or an anti-ICAM-3 antibody) that is covalently attached to a toxin moiety. Procedures for attaching such units to a molecule are well known in the art.

The binding of such a molecule to a cell brings the toxin unit into close proximity to the cell and thereby promotes cell death. Any suitable toxin unit can be used, but it is however preferred to use toxins such as e.g. ricin toxin, cholera toxin, diphtheria toxin, radioisotopic toxins or membrane channel-forming toxins.

Functional derivatives of ICAM-3 having up to 100 residues

can conveniently be prepared by in vitro synthesis. If desired, such fragments can be modified using known methods by reacting targeted amino acid residues of the purified or impure protein with an organic derivatizing agent which is

able to react with selected side chains or terminal residues. The obtained covalent derivatives can be used to identify residues that are important for biological activity.

A number of methods can be used to generate and isolate fragments of ICAM-3. Derivatization with bifunctional agents can be used to cross-link ICAM-3 to a water-insoluble carrier 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 used 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 e.g. deletions from, or insertions or substitutions of residues within the amino acid sequence of ICAM-3. Any combination of division, insertion and substitution can be used to form the final assembly provided that it exhibits the desired activity. Obviously, mutations that will be made in the DNA that codes for the functional derivative must not place the sequence out of the reading frame and will preferably not form complementary regions that will be able to produce secondary mRNA structure (see EP patent application with publication no. 75444).

At the genetic level, functional derivatives can be produced by site-directed mutagenesis of DNA that codes for ICAM-3, thereby forming DNA that codes for the functional derivative, after which the DNA is expressed in a recombinant cell culture.

Since the site of introduction of a variation in the amino acid sequence is predetermined, mutation as such need not be predetermined. In order to optimize the execution of a mutation at a given site, random mutagenesis such as linker scanning mutagenesis can be carried out in a target codon or a target region to form a large number of derivatives which will then be expressed and screened for the optimal combination of the desired activity. Techniques for creating mutations at predetermined sites in a DNA are well known, such as e.g. site-directed mutagenesis.

Production of a functional derivative of ICAM-3 is preferably achieved by site-directed mutagenesis of the DNA that codes for ICAM-3 or a previously produced functional derivative of ICAM-3. The technique for such site-specific 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). Site-directed mutagenesis allows the production of ICAM-3 functional derivatives through the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation.

One can create amino acid deletions, insertions or substitutions using site-directed mutagenesis. Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably from 1 to 10 residues and are typically contiguous. The most preferred deletions are those that are repeated to form a soluble form of ICAM-3. Soluble forms of ICAM-3 are most preferably formed by cleaving 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 into the ICAM-3 coding sequences as well as terminal fusions with polypeptides of substantially unlimited length. Intrasequence insertions (ie insertions within the complete ICAM-3 molecular sequence) can generally range from about 1 to 10 residues, more preferably from 1 to 5. An example of a terminal insertion includes a fusion of a signal sequence, either heterologous or homologous to the host cell, to the N-terminus of the molecule to facilitate secretion of the derivative from recombinant hosts.

The third group of functional derivatives are those where one or more amino acid residues in the ICAM-3 molecule have been removed and where a different residue has been introduced in its place. Such substitutions are preferably carried out in accordance with the following table when it is desired to finally modulate the properties of the ICAM-3 molecule. Substantial changes in functional or immunological identity are accomplished by choosing substitutions that are less conservative than those in Table 1, i.e., by choosing residues that differ more strongly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution , e.g. as a planar or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the extent of the side chain. The substitutions that are generally less conservative are those where (a) glycine and/or proline are substituted by another amino acid or split or inserted, (b) a hydrophilic residue is used instead of a hydrophobic residue, (c) a cysteine residue is used instead for or replaced by another residue, (d) a residue with an electropositive side chain is used instead of or replaced by a residue with an electronegative charge, or (e) a residue with a bulky side chain is used instead of or replaced by one that does not have such a side chain.

Most preferred are substitutions that affect the solubility of ICAM-3. These are most preferably formed by using hydrophilic amino acids instead of hydrophobic amino acids. Mutations designed to increase the affinity of ICAM-3 can be controlled by the introduction of the amino acid residues present in homologous positions in ICAM-1 or ICAM-2. Likewise, such mutant ICAM-3 molecules can be prepared which lack N-linked CHO in homologous positions in ICAM-1 or ICAM2.

It is difficult to predict the exact effect that any particular substitution, deletion or insertion will have on the biological activity of ICAM3-3 until this has been done. An expert in the field will, however, understand that the impact is evaluated by routine screening analyses. A derivative is formed e.g. typically by linker scanning of site-directed mutagenesis of the DNA that codes for the native ICAM-3 molecule. The derivative is then expressed in a recombinant host and optionally purified from the cell culture by e.g. immunoaffinity chromatography.

The activity of the cell lysate or purified derivative is then screened in a suitable screening experiment for desired properties. A change in the immunological character of the functional derivative whose affinity for a given antibody is measured e.g. by a competitive immunoassay. Changes in immunomodulatory activity are measured by an appropriate assay. Modifications of such protein properties as redox or thermal stability, biological half-life, hydrophobicity, susceptibility to protolytic degradation or the tendency to aggregate with carriers or to multimers are measured by methods well known to those skilled in the art.

B. Agonists and Antagonists of ICAM-3

An "agonist" of ICAM-3 is a compound that 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 that increases the ability of ICAM-3 to bind to a cellular receptor.

An "antagonist" of ICAM-3 is a compound that impairs or prevents the ability of ICAM-3 to carry out any of its biological functions. Examples of such 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.

Cellular aggregation assays described in U.S. Pat. patent applications No. 07/045,963, 07/115,798, 07/155,943, 07/189,815 or 07/250,446, can measure LFA-1-dependent aggregation and can thus be used to identify agents that affect the level of ICAM-3/LFA - layer aggregation. Such experiments can thus be used to identify agonists and antagonists of ICAM-3. Antagonists may act by impairing the ability of LFA-1 or of ICAM-3 to mediate aggregation. Non-immunoglobulin (ie, chemical) agents may additionally be examined using the above assay to determine whether they are agonists or antagonists of ICAM-3/LFA-1 mediated aggregation. Such aggregation assays are typically carried out using peripheral T cells stimulated with PMA. Thus, binding of peripheral blood cells to LFA-1 is used to determine ICAM-3-mediated aggregation.

C. Anti-ICAM-3 antibody

The preferred immunoglobulin antagonist used according to the invention is an antibody against ICAM-3 such as CBR-IC3/1, as indicated herein. Other suitable anti-ICAM-3 antibodies can be obtained in a number of different ways.

The antibodies against ICAM-3 (or functional derivatives of ICAM-3) can be either polyclonal or monoclonal. In addition, the antibodies used according to the invention can be humanized by procedures that are known in the state of the art or by procedures that are indicated in PCT application no. PCT/US91/02942 and PCT/US91/02946 which were submitted to the receiving authority in the USA 22 April 1991.

Anti-ICAM-3 antibodies can be generated by introducing ICAM-3 or peptide fragments thereof into an appropriate animal. The immunized animal will form polyclonal antibody in response to such an introduction. The use of peptide fragments of ICAM-3 makes it possible to obtain region-specific antibodies which are only reactive with the epitope(s) contained in the peptide fragments used to immunize the animal.

Alternatively, anti-ICAM3 antibodies can be raised using the ICAM-3 that is naturally expressed on the surface of lymphocytes. Introduction of such cells into a suitable animal which, by intraperitoneal injection, will result in the production of antibodies which can bind to ICAM-3 or members of the CD-18 molecular family. If desired, the serum from such an animal can be removed and used as a source for polyclonal antibodies that can bind these molecules.

Alternatively, anti-ICAM-3 antibodies can be formed by adapting the method according to Seiden, R.F. (EP patent application with publication number 289,034) or Seiden R.F. et al. (Science 236:714-718 (1987)). The cells from a suitable animal (eg a mouse) are transfected with a vector which can express either the intact ICAM-3 molecule or a fragment thereof. The production of ICAM-3 in the transfected cells in the animal will trigger an immune response in the animal and lead to the production of anti-ICAM-3 antibodies in the animal.

However, it is preferred to remove splenocytes from animals immunized in the ways described above to produce monoclonal antibodies that can bind ICAM-3.

Hybridoma cells obtained in the manner described

above, can be screened using a variety of methods to identify a hybridoma cell that secretes an antibody that can bind 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 that can inhibit 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 molecular family. Any method that can distinguish ICAM-3 from the CD-18 molecular family can be used in such screening. The antigen bound by means of

the antibody can thus e.g. analyzed by immunoprecipitation and polyacrylamide gel electrophoresis. It is possible to distinguish between the antibodies that bind to members of the CD-18 molecular family from those that bind ICAM-3 by screening for the antibody's ability to bind to cells expressing 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 can be detected using methods commonly used by those skilled in the art. Such methods 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 that express ICAM-3, but not to cells that do not express ICAM-3.

In addition to the above agonists and antagonists of ICAM-3, other agents that can be used in accordance with 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 that can bind to ICAM-3.

The anti-idiotypic antibodies of interest used in accordance with the invention may bind in competition with (or to the exclusion of) ICAM-3. Such antibodies can e.g. is achieved by raising an antibody against 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.

Since molecules of the CD-18 family can bind to ICAM-3, administration of such molecules (e.g. as heterodimers with both alpha and beta subunits, or as molecules consisting of only one alpha or one beta subunit, or as molecules with fragments of one or both subunits) compete with cells for binding to ICAM-3 present on a cell. Anti-aggregation antibodies used according to the invention can be identified and titrated in a number of different ways. One can e.g. measure the antibodies' ability to bind differentially to cells that express ICAM-3 (such as T cells), and their inability to bind to cells that cannot express ICAM-3. Appropriate assays of cellular aggregation are described in U.S. Pat. Patent Applications Nos. 07/045,963, 07/115,798, 07/155,943, 07/189,815 or 07/250,446. Alternatively, the capacity of the antibodies to bind to ICAM-3 or to a peptide fragment of ICAM-3 can be measured. As the person skilled in the art will see, the above provisions can be modified or carried out in a different order to provide a number of different screening tests, each of which can identify and discriminate between antibodies that can bind to ICAM-3 versus members of the CD-18 molecular family.

D. Preparation of funds used in accordance with

the invention

Agents used according to the invention can be obtained by natural processes, (e.g. by inducing an animal, plant, fungus, bacteria, etc. to produce a non-immunoglobulin antagonist of ICAM-3, or by inducing a animals to produce polyclonal antibodies capable of binding to ICAM-3), by synthetic methods (e.g. by synthesizing ICAM-3, functional derivatives of ICAM-3 or protein antagonists of ICAM-3 (either immunoglobulin or non-immunoglobulin) ), by hybridoma technology (eg to produce monoclonal antibodies capable of binding to ICAM-3) or by recombinant technology (eg to produce agents in different hosts (ie yeast, bacteria, fungi, cultured mammalian cells etc.) using recombinant plasmids or viral vectors). The choice of the method to be used will depend on factors such as suitability, desired yield, etc. However, it is not necessary to use only one of the above methods, methods or techniques to produce a particular anti-inflammatory agent, as the above methods, methods and techniques can be combined to achieve a particular remedy. VII. Application of ICAM-3 and its functional derivatives,

agonists and other antagonists

The agents can be used to moderate the various biological activities mediated by ICAM-3.

A. Suppression of inflammation

ICAM-3 and its functional derivatives have the ability to interact with receptors of the CD-18 molecular family, in particular LFA-1. By virtue of the ability of ICAM-3 to interact with members of the CD-18 family of glycoproteins, it can be used to suppress (ie to prevent or attenuate) inflammation.

The term "inflammation" as used herein is intended to include both the responses of the specific defense system and the responses of the non-specific defense system.

As used herein, the term "specific defense system" shall refer to the component of the immune system that responds to the presence of specific antigens. Inflammation is said to be the result of a response of the specific defense system if the inflammation is due to or is caused by or associated with a reaction of the specific defense system. Examples of inflammation as a result of a response of the specific defense system include the response to antigens such as rubella virus, autoimmune diseases, and the delayed-type hypersensitivity response mediated by T cells (such as seen in individuals who test "positive" in Mantaux the test). Chronic inflammatory diseases and rejection of transplanted solid tissues and solid organs, e.g. kidney and bone marrow transplants are further examples of inflammatory reactions of the specific defense system.

Ir

As used herein, a reaction of the "non-specific defense system" shall refer to a reaction mediated by leukocytes that are not capable of immunological memory. Such cells include granulocytes and macrophages. As used herein, inflammation is said to be the result of a response of the non-specific defense system if the inflammation is caused by, mediated by, or associated with a response of the non-specific defense system. Examples of inflammation resulting at least in part from a response of the non-specific defense system include information associated with conditions such as: "adult respiratory distress syndrome" (ARDS) or multi-organ injury syndromes secondary to sepsis, trauma, or hemorrhage , reperfusion injury of myocardial tissue or other tissue, acute glomerulonephritis, reactive arthritis, dermatoses with acute inflammatory components, acute purulent meningitis or other inflammatory diseases of the central nervous system such as stroke, thermal injury, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necronizing enterocolitis, syndromes associated with granulocyte transfusion and cytokine-induced toxicity.

As discussed above, the binding of ICAM-3 molecules to members of the CD-18 molecular family is of central importance in cellular adhesion. Through the process of adhesion, lymphocytes can continuously monitor the presence of foreign antigens in an animal. Although these processes are normally desirable, they can also lead to rejection of solid organ transplants (eg kidney), rejection of non-solid organ transplants (eg bone marrow), tissue graft rejection and a number of autoimmune diseases. Accordingly, any agent that can impair or inhibit cellular adhesion would be highly desirable in recipients of solid organ transplants (especially kidney transplants), non-solid organ transplants (especially bone marrow transplants), tissue transplants, or for autoimmune patients.

Monoclonal antibodies against members of the CD-18 family inhibit a number of adhesion-dependent functions of leukocytes including binding to endothelium (Haskard, D. et al., J. Immunol. 13 7:2901-2906 (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., Proe.Nati.Acad.Sei., USA 78:4535 -4539 (1981)), antibody formation (Fischer, A. et al., J. Immunol. 136:3198-3203 (1986)) and effector functions of all leukocytes such as lysis activity of cytotoxic T cells (Krensky, A.M. et al., J.Immunol.132:2180-2182 (1984)), macrophages (Strassman, G. et al., J.Immunol 136:4328-4333 (1986)), and all cells involved in antibody-dependent cellular cytotoxicity reactions ( Kohl, S. et al., J. Immunol. 133:2972-2978 (1984)). In all of the above functions, the antibodies inhibit the leukocyte's ability to adhere to the appropriate cellular substrate which in turn inhibits the biological function associated with binding. Such functions to the extent they involve ICAM-3/LFA-1 interactions can be suppressed with anti-ICAM-3 antibodies.

Thus, monoclonal antibodies that can bind to ICAM-3 can be used as an anti-inflammatory agent in a mammal. Such agents differ significantly from general anti-inflammatory agents in that they can selectively inhibit adhesion and they do not produce other side effects such as nephrotoxicity, which have been found with conventional agents. As ICAM-3, especially in soluble form, can act in the same way as an antibody against members of the CD-18 family, it can be used to suppress inflammation. Moreover, the functional derivatives and antagonists of ICAM-3 can also be used to suppress inflammation.

1. Suppressors of delayed-type hypersensitivity reactions

ICAM-3 partially mediates adhesion events necessary to initiate inflammatory reactions such as delayed-type hypersensitivity reactions. Thus, antibodies (especially monoclonal antibodies) that can bind to ICAM-3 have therapeutic potential in weakening or eliminating such

reactions.

Alternatively, since ICAM-3 is an antagonist of the ICAM-1/LFA-1 interaction, ICAM-3 (especially in solubilized form) or its functional derivatives can be used to suppress delayed-type hypersensitivity reactions.

These potential therapeutic applications can be exploited in two ways. First, a composition containing an anti-ICAM-3 monoclonal antibody or soluble derivative of ICAM-3 can be administered to a patient with delayed-type hypersensitivity reactions. Such mixtures can e.g. is given to an individual who has been in contact with antigens such as poison ivy, poison oak, etc. In another embodiment, a monoclonal antibody capable of binding to ICAM-3 is administered to a patient together with an antigen to prevent a subsequent inflammatory reaction. The further supply of an antigen together with an anti-ICAM-3 antibody can thus cause an individual to temporarily tolerate a subsequent experience of this antigen.

2. Therapy for chronic inflammatory disease

As LAD patients lacking LFA-1 do not mount an inflammatory response, it is believed that antagonism of natural ligands to LFA-1 will also inhibit an inflammatory response. The ability of antibodies against ICAM-3 to inhibit inflammation forms 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. Such antibodies can also be used as therapy in the treatment of psoriasis.

In general, the anti-ICAM-3 antibodies used according to the invention can be administered alone or in combination with anti-ICAM-1 and/or anti-ICAM-2 antibodies in the treatment of the diseases that can now be treated by steroid therapy.

Such inflammatory and immune rejection responses can be suppressed (ie either prevented or attenuated) by administering to an individual 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 wherein said fragment is capable of binding to ICAM-3, substantially pure ICAM-3, a functional derivative of ICAM-3, a non -immunoglobulin antagonist of ICAM-3 or a non-immunoglobulin antagonist of ICAM-3 other than LFA-1. Particularly preferred are anti-inflammatory agents consisting of a soluble functional derivative of ICAM-3. Such anti-inflammatory treatment may also include the further administration of an agent selected from the group consisting of: an antibody capable of binding to LFA-1, a non-immunoglobulin 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 fragments thereof.

In the above methods, an inflammatory response is suppressed by the specific defense system where an immunosuppressive agent is additionally given to the individual. Such an agent is preferably given in a dose that is less than (ie a "sub-optimal" dose) that normally required. The use of a sub-optimal dose is possible due to the synergistic effect of the agents. Examples of suitable immunosuppressive agents include, but are not limited to, dexamethasone, azathioprine, ICAM-1, ICAM-2, or cyclosporine A.

3. Therapy for non-specific inflammation

Many inflammatory reactions are due to reactions of the "non-specific defense system" and are mediated by leukocytes that are not capable of immunological memory. Such cells include granulocytes and macrophages. As used herein, inflammation is meant 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. Examples of inflammation resulting at least in part from a response of the nonspecific defense system include inflammation associated with conditions such as ARDS or multiple organ injury syndromes secondary to sepsis, trauma or hemorrhage, reperfusion injury of myocardial tissue or other tissues, acute glomerulonephritis, reactive arthritis, dermatoses with acute inflammatory components, acute purulent meningitis or other inflammatory diseases of the central nervous system such as stroke, thermal injuries, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion-associated syndromes and cytokine-induced toxicity.

The anti-inflammatory agents used according to the invention are compounds which can specifically antagonize the interaction of the CD-18 complex on granulocytes with endothelial cells.

B. Suppressors of organ and tissue rejection

Since ICAM-3, particularly in soluble form, can act similarly to an antibody against members of the CD-18 family, it can be used to suppress organ or tissue rejection resulting from any of the cellular adhesion-dependent functions . Moreover, an anti-ICAM antibody, functional derivatives of ICAM-3 and antagonists of ICAM-3 can also be used to suppress such rejection.

ICAM-3 and anti-ICAM-3 antibodies can be used to prevent solid organ or tissue rejection, e.g. kidney, rejection of non-solid organs such as e.g. bone marrow or to modify autoimmune responses in the mammalian subject. It is important that the use of monoclonal antibodies which can recognize ICAM-3 can allow organ transplants to be carried out even between individuals with incompatible HLA.

C. Supplement for introducing antigenic material added

for therapeutic and diagnostic purposes

Immune responses to therapeutic or diagnostic agents such as bovine insulin, interferon, tissue type plasminogen activator or mouse monoclonal antibodies substantially improve the therapeutic or diagnostic value of such agents, and in some cases lead to diseases such as serum sickness. Such a situation can be remedied through the use of the antibodies used according to the invention. Anti-ICAM-3 antibodies can be administered in combination with the therapeutic or diagnostic agent. Supply of the antibody prevents the recipient from recognizing the agent and therefore prevents the recipient from initiating an immune response against it. Absence of such an immune response allows the patient to receive further infusions of therapeutic or diagnostic agents.

ICAM-3 (especially in solubilized form) or its functional derivatives can be used alone or in combination with ICAM-1 or ICAM-2 or with antibodies that can bind to LFA-1 in the treatment of disease. In solubilized form, such molecules can thus be used to inhibit rejection of organs or tissues. ICAM-3 or its functional derivatives can be used in the same manner as anti-ICAM-3 antibodies to reduce the immunogenicity of therapeutic or diagnostic agents.

D. Suppressors of tumor metastasis

The agents used according to the invention can also be used to suppress metastases of a hematopoietic tumor cell that requires a functional member of the CD-18 family for migration. A patient in need of such treatment may receive an amount of such an agent sufficient to suppress said metastases.

A method for suppressing the growth of an ICAM-3 expressing tumor cell is further provided. The said method includes in particular that a quantity of an agent which is sufficient to suppress said growth is added to a patient in need of such treatment. Suitable agents include an antibody capable of binding to ICAM-3 and a fragment of an antibody, said fragment capable of binding to ICAM-3.

E. Suppression of HIV infection and prevention of proliferation of HIV-infected cells

A method for suppressing HIV infection has also been provided. Said method particularly comprises administering to an HIV-infected individual an effective amount of an HIV infection-suppressing agent. Although the method is specifically a method for suppressing HIV-1 infection, it should be understood that the method can be applied to any HIV variant (such as HIV-2) capable of infecting cells in a suppressible manner by means of the means used according to the invention. Such variants are the equivalents of HIV-1.

Expression of LFA-1 and, in some cases, LFA-1's binding ligand stimulated by HIV infection, promotes cell-to-cell adhesion reactions that can increase the contact time of infected with uninfected cells, facilitating transmission of virus from infected to uninfected infected cells. The agents used according to the invention which can modulate LFA-1/ligand interactions are thus capable of suppressing infection with HIV and particularly HIV-1. A method by which molecules that inhibit LFA-1/ligand interactions can suppress HIV infection by impairing the ability of the LFA-1 ligand expressed by HIV-infected cells to bind to CD11/CD18 receptors on a healthy T cell . Alternatively, molecules that 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. To impair a cell's ability to bind to the CD11a/CD18 receptor or to the LFA-1 binding ligand, it is possible to use ICAM-3, a fragment of ICAM-3, or anti-ICAM-3 antibodies.

The agents used according to the invention must be given to the recipient individuals 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, method of administration, etc. of the agent is sufficient to reduce or prevent such HIV infection. The funds must be given to patients who are exposed to or who have HIV infection.

The agents used according to the invention can either be for a "prophylactic" or "therapeutic" purpose in the treatment of HIV infection. In the case of prophylactic treatment, the agent is given before any symptom of viral infection (e.g. before or shortly after the time of such infection, but before symptoms of such infection). The prophylactic administration of the agent serves to prevent or reduce a subsequent HIV infection. In therapeutic use, the agent is given at (or shortly after) the discovery of virus-infected cells. The therapeutic administration of the agent serves to weaken any further HIV infection.

The agents used according to the invention are thus given either before the onset of viral infection (to suppress the expected HIV infection) or after actual detection of such virus-infected cells (to suppress further infection).

There is further provided an improved therapy for AIDS and an extended means of suppressing HIV infection and particularly HIV-1 infection and comprising co-administration of: (I) ICAM-1, a soluble ICAM-3 derivative, CD11 (either CDlla, CDllb or CDllc), a soluble CDll derivative, CD18, a soluble CD18 derivative or a CD11/CD18 heterodimer, or a soluble derivative of a CD11/CD18 heterodimer and/or

(II) an antibody that can bind to ICAM-3 with

(III) cell- or particle-associated CD4 or a soluble derivative of CD4 and/or (IV) a molecule (preferably an antibody or antibody fragment) which can bind to CD4.

Also provided is a method of suppressing the migration of HIV-infected cells. Said method particularly comprises administering an effective amount of an anti-migration agent to an HIV-infected individual.

The anti-migratory agents used according to the invention include ICAM-3, or anti-ICAM-3 antibodies which can impair the ability of an HIV-infected T cell to bind to an LFA-1 ligand. Antibodies that bind to ICAM-3 will suppress migration by impairing the ability of ICAM-3 expressed by HIV-infected T cells to bind to cells expressing a CD11/CD18 receptor. In order to weaken a cell's ability to bind to the CD11a/CD18 receptor, it is possible to use an antibody that can bind to ICAM-3.

The agents used according to the invention are to be given to recipient individuals in an amount sufficient to suppress the migration of HIV (or other virus) infected T cells. An amount is said to be sufficient to "suppress" migration of T cells if the dosage, method of administration, etc. of the agent is sufficient to reduce or prevent such migration.

The supply of one or more such compounds may be either for a "prophylactic" or "therapeutic" purpose. In the case of prophylactic administration, the agents used according to the invention are given before the symptoms of viral infection (e.g. before, at or shortly after the time of such infection, but before any symptoms of such infection). The prophylactic administration of the agents serves to prevent or reduce any subsequent migration of virus-infected T cells. In the case of therapeutic administration, the agent is administered at (or shortly after) the detection of virus-infected T cells. Therapeutic administration of the agent serves to reduce any further migration of such T cells.

The agents used according to the present invention can thus be given either before the onset of virus infection (to suppress the expected migration of infected T cells) or after the actual detection of such virus-infected cells.

F. Treatment of asthma

In a further embodiment of the present invention, an agent which can modulate LFA-1/ICAM-3 interactions is used in the treatment of asthma. This method particularly comprises administering an effective amount of an anti-asthma agent to an individual in need of such treatment. The anti-asthma agents used in accordance with the invention include ICAM-3, a fragment of ICAM-3, or anti-lCAM-3 antibodies which can impair a cell's ability to bind to an LFA-1. Antibodies that bind to ICAM-3 will suppress the migration of eosinophils by impairing the ability of ICAM-3 expressed on these cells to bind to cells expressing a CD11/CD18 receptor.

The anti-asthma agents used according to the invention must be administered to the recipient individual in an amount sufficient to ease or reduce the severity, degree or duration of asthma symptoms.

The agents used according to the invention can be administered either alone or in combination with one or more additional anti-asthma agents (such as methylxanthines) (such as theophylline), beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins and ephedrine), glucocorticoids (such as hydrocortisone ), chromones (such as cromlyn sodium) and anticholinergic agents (such as atropine) to decrease the amount of such agents needed to treat asthma symptoms.

The supply of the agents used according to the invention can e.g. be for either a "prophylactic" or "therapeutic" purpose. For prophylactic use, the agent is given before the onset of asthma symptoms. The prophylactic administration of the agent serves to prevent or attenuate any subsequent asthmatic response. For therapeutic use, the agent is given at (or shortly after) the onset of an asthma symptom. The therapeutic administration of the agent serves to attenuate any present asthmatic episode. The agents used according to the invention can thus either be given before the onset of an expected asthmatic episode (to reduce the expected severity, duration or degree of the episode) or after the initiation of the episode.

G. Applications for diagnosis and prognosis

Monoclonal antibodies that can bind to ICAM-3 can be used as a means of imaging or visualizing the sites of

ICAM expression and inflammation in a patient. In such an application, anti-ICAM-3 monoclonal antibodies are labeled for detection, through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), fluorescent labels or paramagnetic atoms. Procedures for carrying out such labeling are well known in the art. The labeled antibodies can then be used for diagnostic imaging. Clinical use of antibodies in diagnostic imaging is discussed by Grossman, H.B., Uroi. Clin. North. Amer. 13:465-474 (1986), Unger, E.C., et al., Invest. Radio1. 20:693-700 (1985), and Khaw, B.A., et al., Science 209:295-297

(1980).

The presence of ICAM-3 expression can also be detected through the use of hybridization probes such as mRNA, cDNA, genomic DNA, or synthetic oligonucleotide probes that bind to ICAM-3 gene sequences or ICAM-3 mRNA sequences present in a cell expressing ICAM- 3. Techniques for performing such hybridization assays are described by Maniatis, T. et al., in Molecular Cloning, a Laboratory Manual, Coldspring Harbor, NY (1982) and by Haymes, B.D. et al., in: Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington DC (1985).

The detection of foci of ICAM-3 expression using labeled antibodies or nucleic acid probes can be used in the diagnosis of tumor development. In a diagnostic embodiment, tissue samples or blood samples are removed from an individual and incubated in the presence of antibodies that are or can be labeled for detection. In a preferred embodiment, this technique is performed in a non-invasive manner through the use of magnetic imaging, fluorography, etc. Such a diagnostic test can be used to measure organ transplant recipients, e.g. kidneys, for early signs of possible tissue rejection. Such analyzes can also be carried out to determine an individual's predisposition to rheumatoid arthritis or other chronic inflammatory diseases.

In the case of radioactive labeling of anti-ICAM-3 antibodies or anti-substance f fragments, there is e.g. possible to detect the antigen through the use of radioimmunoassays. A good description of a radioimmunoassay (RIA) can be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S., et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard,T. Alternatively, the antibody can be labeled with a fluorescent compound, an enzyme or other known suitable markers.

In addition to localizing sites of inflammation, antibodies capable of binding to ICAM-3 can be used to analyze biological fluids for the presence of circulating ICAM-3 using any of the commonly used media for fluid analyses. The presence of circulating ICAM-3 in a fluid sample indicates an inflammatory response or other ICAM-3-mediated biological functions. In addition, the presence of ICAM-3 in amniotic fluid is associated with complications that occur during pregnancy. Any biological fluid can be used for the analysis. However, the preferred biological fluids are blood, serum, plasma, synovial fluid, amniotic fluid, spinal fluid or urine.

VIII. Supply of preparations

The therapeutic effects of ICAM-3 can be achieved by administering the entire ICAM-3 molecule to a patient or any therapeutically active peptide fragments thereof. Of particular interest are therapeutically active peptide fragments of ICAM-3 that are soluble.

ICAM-3 and its functional derivatives can be obtained synthetically using recombinant DNA technology, by proteolysis or by a combination of such methods. The therapeutic benefits of ICAM-3 can be increased by the use of functional derivatives of ICAM-3 that have further added amino acid residues to increase binding to the carrier or to increase the activity of ICAM-3. Functional derivatives of ICAM-3 that lack certain amino acid residues or that contain altered amino acid residues can be used as long as such derivatives exhibit or affect a biological or pharmacological activity of ICAM-3.

Both the antibodies used in accordance with the invention and the ICAM-3 molecule as stated herein are said to be "substantially free of natural contaminants" if preparations containing these essentially do not contain materials that are normally and naturally found together with these products.

Antibodies and biologically active fragments thereof (either polyclonal or monoclonal) which can bind to ICAM-3 can either be produced by means of an animal or by means of a tissue culture or by means of recombinant DNA techniques.

Delivery of ICAM-3 or molecules derived from ICAM-3 can be carried out alone or in combination with ICAM-1 and/or ICAM-2. Administration of anti-ICAM-3 antibodies or other molecules that can bind to ICAM-3 or to molecules derived from ICAM-3 can be carried out alone or in combination with anti-ICAM-1 antibodies and/or anti-ICAM-2- antibodies. When administering to a patient antibodies or antibody fragments that can bind to ICAM-3 or when administering ICAM-3 (or a fragment, variant or derivative thereof) to a receiving patient, the dose of administered agent will vary depending on such factors as the patient's age, weight, height, sex, general medical condition, past medical history, etc. It is generally desirable to give the recipient a dose of antibody that ranges from about 1 pg/kg to

10 mg/kg (patient's body weight), although a smaller or larger dose can be given. When administering ICAM-3 molecules or their functional derivatives to a patient, such molecules are preferably administered in a dose also ranging from about 1 pg/kg to 10 mg/kg (patient's body weight), although a higher or lower dose may also be administered . As discussed above, the therapeutically effective dose may

is reduced if the anti-ICAM-3 antibody is added together with an anti-LFA-1 antibody, an anti-ICAM-1 antibody and/or an anti-ICAM-2 antibody. As used herein, a compound is said to be supplied together with another compound when supply-

one of the two compounds is so close to each other in time that both compounds can be detected simultaneously in the patient's serum.

Both the antibody which can bind to ICAM-3 and ICAM-3 itself can be administered to the patients intravenously, intramuscularly, subcutaneously, enterally, topically or parenterally. When antibodies or ICAM-3 are administered by injection, the administration can be by continuous injections or by single or multiple boluses.

The agents used in accordance with the invention must be given to the recipient individuals in an amount sufficient to be "physiologically effective". An amount is said to be physiologically effective if the dose, method of administration, etc. of the agent is sufficient to reduce or prevent the physiological effect associated with ICAM-3. One of the agents used according to the invention is given, e.g. to a patient for the purpose of suppressing inflammation and is said to be physiologically effective if administered in an amount sufficient to "suppress" inflammation.

In addition, anti-ICAM-3 antibodies or a fragment thereof can be administered either alone or in combination with one or more additional immunosuppressive agents (particularly to a recipient of an organ or tissue transplant). The purpose of the supply of one or more such compounds can be either "prophylactic" or "therapeutic". In the case of prophylactic administration, one or more of the immunosuppressive compounds are given before any inflammatory response or symptom, (eg before, at or shortly after the time of organ or tissue transplantation, but before any symptoms of organ rejection). The prophylactic administration of one or more of the compounds serves to prevent or reduce any subsequent inflammatory response (such as rejection of a transplanted organ or tissue, etc.). In the case of therapeutic administration, one or more of the compounds are given at (or shortly after) the onset of a symptom of real inflammation (such as organ or tissue rejection). Therapeutic administration of one or more of the compounds serves to reduce any real inflammation {such as e.g. rejection of a transplanted solid organ or tissue, e.g. kidney or of non-fixed organs such as e.g. bone marrow).

The anti-inflammatory agents used according to the invention can thus be given either before the onset of inflammation (to suppress an expected inflammation) or after the initiation of inflammation.

A preparation is said to be "pharmacologically tolerable" if its administration can be tolerated by any recipient patient. Such an agent is 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 ICAM-3 molecules used according to the invention can be designed according to known methods for the production of pharmaceutically tolerable preparations whereby these materials or their functional derivatives are combined with a pharmaceutically tolerable carrier. Suitable carriers and their design including other human proteins, e.g. human serum albumin, is e.g. described in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). To form a pharmaceutically acceptable preparation suitable for effective delivery, such preparations will contain an effective amount of an agent together with an appropriate amount of carrier. In addition, the antibodies used according to the invention can be humanized through chimerization or CDR grafting to become more "pharmacologically tolerable" for a patient. Such methods for the chimerization of antibodies, particularly anti-ICAM-1 antibodies are described elsewhere, GB Patent Application Nos. 9009548.0, 9009549.8 and PCT Patent Application Nos. PCT/US91/02942 and PCT/US91/02946, which have been submitted to the receiving authorities in the United States on April 27, 1991.

Additional pharmaceutical methods can be used to control the duration of action. The preparation with controlled release can be obtained by using polymers to complex or absorb the agents used according to the invention. The degree and duration of the controlled release can to a certain extent be regulated by choosing a suitable macromolecule matrix by varying the concentration of incorporated macromolecules as well as by the methods of incorporation. Another possible method for controlling the duration of the effect by means of preparations with controlled release is to incorporate the agents used according to the invention in particles of a polymer material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers. Instead of incorporating these agents into polymer particles, it is alternatively possible to incorporate these materials into macrocapsules such as e.g. is prepared by co-serving techniques or by interfacial polymerization, by gelatin or poly(methyl methacylate) microencapsulation or in colloidal drug delivery systems, e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nano-capsules or in macroemulsions.

The invention can further produce a pharmaceutical preparation comprising: (a) an anti-inflammatory agent (such as an antibody that can bind to ICAM-3, a fragment of said antibody that can bind to ICAM-3, ICAM-3 ), and (b) at least one immunosuppressive agent. Examples of suitable immunosuppressive agents include dexamethasone, azathioprine, and cyclosporine A.

Example I

Characterization of ICAM-3, a novel adhesion ligand for LFA-l Adhesion of cell lines and lymphocytes, Dustin et al., Cold Spring Harbor Symp.Quant.Viol. 54:753-765 (1989) to LFA-1-coated plates was carried out as described previously. LFA-1 purified from JY lysates was absorbed onto microtiter plates with

96 wells (Linbro titertek) at 1100 seats//mi<2>. Non-specific binding sites were blocked with 1% BSA and washed with PBS/5% FBS/2 mM MgCl2/0.5% HSA (assay media). Specific inhibition of LFA-1 was achieved by incubation for 30 minutes at room temperature of the microtiter plates with a 1/200 dilution of TS1/22 (anti-LFA-1a) ascites. Resting T cells were isolated from whole blood by plastic adhesion and nylon wool filtration and were 91% CD2<+>, while PHA blasts were generated by culturing the cells in the presence of 10M9/ml phytohemagglutinin (PHA). Cells were labeled with the fluorochrome BCECF (Molecular Probes Inc.), washed and resuspended in assay medium. MAb pretreatment of cells includes incubation with 1/2 0 0 dilution for ascites for 45 minutes at 4°C after which 10<5> cells were transferred to each well. Cell lines were adhered to solid-phase LFA-1 for one hour at 37°C and non-adherent cells were removed with six aspirations with a 23 gauge needle while lymphocytes were centrifuged at 30 x g for five minutes and incubated at 37°C for 30 minutes. . Unbound lymphocytes were removed by flicking the media from the plates eight times with 100 µl added between each wash. Flicking was more effective for thorough removal of unbound T-lymphocytes, which were more difficult to remove due to their small size. Fluorescence was quantified directly from the 96-well plates using a fluorescent concentration analyzer (Baxte). All MAbs were used at saturating concentrations (1/200 ascites dilution) and include TS1/22 (anti-CDlla, IgGl) (Sanchez-Madrid et al., Proe. Nati. Acad. Sei. USA 79:7489-7493 (1982)), RRl/l (anti-ICAM-1, IgGl) (Rothleinet al., J. Immunol. 137:1270-1274 (1986)), CBR-IC2/2 (anti-ICAM-2, IgG2a) (de Fougerolles et al., J. Exp. Med. submitted

(1991)(in press)) and CBR-IC3/1 (anti-ICAM-3, IgGl).

CBR-IC3/1 was derived from the fusion of murine myeloma P3X63Ag8.653 from spleen cells of SKW3-injected Balb/c mice (Gefter et al., Som. Cell. Gen. 3:231-236 (1977)), and 600 hybridomas were screened for the ability to inhibit SKW3 binding to purified LFA-1. CRB-IC3/1 was shown not to respond to purified LFA-1 nor to COS cells transfected with either ICAM-1, ICAM-2 or LFA-1 cDNAs (data not shown). One of four representative experiments is shown and error bars indicate one standard deviation.

Example II

Flow cytometric analyzes of cellular ICAM-1, ICAM-2 and ICAM-3 expression

The cell lines used are all previously described by Hibbs et al, J. Clin. Invest. 85:674-681 (1990). Peripheral blood mononuclear cells (PBMC) were obtained by dextran sedimentation and Ficoll-Hypaque (1.077) centrifugation as described (Dustin et al., J. Cell Biol. 107:321-331 (1988)). Neutrophils were obtained from the cell pellet and contaminating erythrocytes were removed by hypotonic lysis. Lymphocytes and monocytes were separated by cytometric analyzes using forward and perpendicular light scattering and were confirmed by monocyte and T-cell specific MAb. Plastadherence was used to enrich lymphocytes from PBMC and cells were cultured in the presence of 10 µg/ml phytohemagglutinin (PHA). Samples were analyzed using an EPICS V flow cytometer and fluorescence was quantified using EPICS Immune-Brite fluorescent beads

(Coulter) to calibrate the cytometer. Expression of ICAM-3 on resting lymphocytes was 2-3 times greater than both CD3 or LFA-1, while monocytes expressed 3-4 times more ICAM-3 than LFA-1. Levels of ICAM-3 expression on neutrophils were not equivalent to those for Mac-1 (CD11b/CD18). Treatment of cells with phospholipase C did not show the presence of PI-bound form of ICAM-3.

Example III

Immunoprecipitation of ICAM-3

Cells were surface labeled with <125>I as described using iodogen (Kishimoto et al., J. Biol. Chem. 264:3588-3595 (1989)). The cells were lysed with triton X-100 (1%). The lysates were clarified with bovine IgG-coupled Sepharose and then incubated with the appropriate MAb-coupled Sepharose for two hours. Beads were washed and boiled in sample buffer containing 50 mM Tris, 1% SDS, and 1% 2-mercaptoethanol. Unreduced samples were boiled in buffer without 2-mercaptoethanol and treated with 20 mM iodoacetamide. Samples were analyzed on 7% vertical plate polyacrylamide gels as described previously (Laemmli, U.K., Nature 227:680-685 (1970)), and proteins were visualized by autoradiography. Treatment of samples with N-glycanase (Genzyme) as described previously (Tarentino et al., Biochemistry 24:4665-4671 (1985)) using a concentration (10 units/ml, 37°C, 18 hours) was determined to to provide optimal cleavage of all N-linked oligosaccharides from the peptide backbone. MHC group I contains an N-linked carbohydrate, while ICAM-2 (rr^ 60,000, 6 N-linked sites, main chain of Mr 2 8,383) is highly glycosylated.

Example IV

Distribution of ICAM-3

The anti-ICAM-3 antibodies were used to further determine the tissue distribution of the ICAM-3 protein using flow cytometry. Table 3 provides a summary of cell types showing surface expression of ICAM-3. Expression of ICAM-3 appears to be restricted to hemopoietic cells. Flow cytometry data were confirmed using immunohistochemical staining of tissue sections using labeled anti-ICAM-3 antibodies (data not shown). As with the flow cytometry data, the immunohistochemical studies have shown that ICAM-3 expression appears to be restricted to hemopoietic cells.

Example, V

Purification of ICAM-3

ICAM-3 was purified using immunoaffinity chromatography where an anti-ICAM-3 antibody CBR-IC3/1 was immobilized on a matrix using known methods. A variety of sources have been used as starting material to isolate ICAM-3. These include but are not limited to SKW3, a human thymoma cell line and human tonsil. The methods used to purify ICAM-3 are essentially those described by deFougerolles et al. in J. Exp. Med. 175:185-190

(1992). A person skilled in the art will see that the reagent for washing and elution varies somewhat with each antigen to be purified and with each antibody to be used in immunoaffinity chromatography.

As indicated below, ICAM-3 purified from SKW3 cells or human tonsillar cells appears to have a molecular weight from about 120 to 124 kD, while ICAM-3 purified from neutrophil-derived lysates gave a broad band showing a molecular weight from about 120 to 150 kD.

ICAM-3 purified in this manner has been shown to retain its ability to bind anti-ICAM-3 antibodies and LFA-1 on a variety of different cells (Figs. 6 and 7).

Example VI

Identification of different molecular weights of ICAM-3 Immunoprecipitation and analyzes by polyacrylamide gel electrophoresis were used to determine the molecular weight of ICAM-3 on different 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 of approximately 120 to 124 kD. The molecular weight of ICAM-3 isolated from neutrophils is somewhat higher than that expressed by lymphoid cells and appears as a diffuse band from about 120 to 150 kD. Fig. 4.

Such variation in size is not unknown for members of the ICAM family of glycoproteins. ICAM-1 shows a similar variation in molecular weight among different cell types. Variation in molecular weight seen for ICAM-1 has been shown to be due to variations in the degree of glycosylation. Variations in the molecular weight of ICAM-3 are therefore most likely due to differences in glycosylation. An expert can easily confirm this by subjecting ICAM-3 isolated from neutrophils to various glycosidases and other enzymatic treatments, looking at the effect of this in connection with the molecular weight.

Variations in the degree of glycosylation for ICAM-1 have been shown to affect MAC-1 (CD11b/CD18) binding. By varying the degree of glycosylation of ICAM-3, it is therefore possible to form ICAM-3 derivatives which have modified affinities for binding to the various ligands of ICAM-3, e.g. members of the CD11/CD18 glycoprotein family.

Example VII

ICAM-3 anti s toffers

Mice were injected with a combination of adjuvant and ICAM-3 protein purified from SKW3 or tonsil cells as described above. Monoclonal antibodies from immunized animals were raised using known procedures.

Table 4 shows a summary of the different anti-ICAM-3 antibodies obtained. The different antibodies were identified on the basis of their ability to react by ELISA to purified immobilized ICAM-3. Positive mAb was 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.

One of the evidences that a third ligand of LFA-1 existed was the presence of an ICAM-1/ICAM-2 independent pathway for PMA-stimulated SKW3 cell aggregation. One was able to block this aggregation with either mouse anti-ICAM-3 polyclonal antiserum or with a combination of CBR-IC3/1 and CBR-IC3/2.

Example VIII

Immunological assays with anti-ICAM-3 antibodies

An anti-ICAM-l antibody (RRl/l), 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 SKW3 cell adhesion to purified ICAM1s, (2) phytohemagglutinin (PHA) stimulation of cell division and (3) the mixed lymphocyte response (MLR).

It has previously been shown that PMA can activate LFA-1 and thus increase its ability to bind to ICAM-1. (Dustin et al., Nature 341:619 (1989)). Fig. 5 shows that a similar activation mechanism is present in LFA-1/ICAM-3 binding.

The ability of different antibodies to block the binding of PMA-stimulated SKW3 cells to purified ICAM-1 and ICAM-3 was examined. Fig. 6. Antibodies CBR-IC3/1 and CBR-IC3/2, when individually present, do not effectively block the binding of PMA-stimulated SKW3 cells to purified ICAM-3. However, PMA-stimulated SKW3 cell binding to ICAM-3 can be indicated by using a combination of these two antibodies. As stated by deFougerolles in J. Exp. Med., the CBR-IC3/1 monoclonal antibody itself was able to block the binding of ICAM-3 to purified LFA-1.

The binding of PMA-stimulated SKW3 cells to ICAM-3 was further analyzed to determine whether this was temperature dependent and cation dependent. As with ICAM-1, ICAM-3/LFA-1 binding was found to be both temperature (see Fig. 8) and cation dependent (data not shown).

Fig. 9 shows a summary of the effects of different antibodies on PHA stimulation of cell division. As noted previously (Krensky et al., J. Immunol. 131:611-616 (1983)), PHA stimulates cell proliferation in a manner that can be inhibited by 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. A combination of l) anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3 antibodies, 2) two anti-ICAM-3 antibodies (IC3/1 and IC3/2) and 3) anti However, -ICAM-1 and anti-ICAM-3 antibodies were effective in blocking PHA-stimulated cell division.

Example IX

Mixed, lymphocyte reaction

The MLR assay is used to determine immunological reactivity. The assay mainly involves adding chemically fixed foreign cells (the stimulator cells) to a solution containing PBLs from another individual (responder cells) and measuring the level of responsiveness using the proliferation of the responder cells as an index and which has been described previously. (Krensky et al., J. Immunol. 131:611-616 (1983)). This assay is the first step in the identification of a composition useful for the treatment of transplant rejection.

The effects of different antibodies and antibody combinations on MLR reactions are shown in fig. 10. Overall, anti-ICAM-3 alone shows a low efficacy. However, a greater efficacy in blocking MLR was found with a combination of anti-ICAM-1 and anti-lCAM-3 antibodies. The highest response was obtained with the combination of anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3 antibodies.

Example X

Peptide sequences of ICAM-3

ICAM-3 was purified from human tonsil cells by immunoaffinity chromatography as described in Example 5. Purified ICAM-3 was enzymatically degraded with the Lys-C enzyme using known methods and peptide fragments were resolved by HPLC. Fig. 11 shows gas chromatography of the different protein peaks that are observed during a typical breakdown. Peaks 10 and 17 were identified to contain peptide fragments of sufficient size and structure to be sequenced (hereafter 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 shown in the sequence was deduced to be present since Lys-C cleaves proteins after lysine residues.

The sequence of the NK-17 peptide was found to be KlALETSLSK (SEQ. ID No. 2). As with the NK-10 protein, the first lysine residue was deduced and later confirmed using DNA sequence analysis.

A sequence comparison of the NK-10 and NK-17 protein with known ICAM-1 and ICAM-2 sequences shows a high degree of homology. The NK-17 peptide showed significant homology to sequences contained in the first Ig domain of ICAM-2. The NK-10 peptide showed weak homology to sequences within domain 4 of ICAM-1.

Example XI

cDNA cloning of ICAM-3

Degenerate oligonucleotide probes were generated on the basis of the peptide sequences obtained above. The probes were further designed to incorporate 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 are as follows: NK-17 probe. Oligo 23 more with 24-fold degeneracy containing 2 N's (inosine)

NK-10 probe. 25 more with 8 times degeneracy containing

5 N's (inosine).

A cDNA expression library was generated from tonsil RNA as described previously. (Wong et al., Proe. Nat. Acad. Sci. USA 82:7711-7716 (1985)). The library was screened using the NK-17 probe and plaques showing positive hybridization were rescreened using the NK-10 probe. One clone, clone 11.2, was found to have an insert ranging in length from approximately 1.6 - 1.8 kb. A schematic representation of clone 11.2 and the localization of the NK-10 and NK-17 peptides is shown in fig. 12 .

The ends of the insert were sequenced using procedures known in the art using primers derived from the flanking vector, while the NK-10 probe was used as an internal sequencing primer. What has been achieved so far presents sequences that have been read on only one strand. An expert will easily see that such a sequence can be substantiated with routine tests.

A summary of the obtained sequences is shown in fig. 18. Several things should be noted in the sequence. First, a sequence with a high degree of homology to the transmembrane region of ICAM-1 has been identified. Therefore, one who wishes to produce soluble fragments of ICAM-3 will thus delete all sequences after the start of the transmembrane region indicated in fig. 12. Second, the sequence obtained from the 5' end of clone 11.2 is likely close to the 5' end of the naturally occurring gene as deduced by sequence homologies that exist between ICAM-1, ICAM-2 and ICAM-3.

Example XII

Sequence homology between the different ICAM molecules The nucleotide and amino acid sequence of a NK-10 and NK-17 peptide as well as the nucleotide sequences obtained above were compared with known sequences of ICAM-1 and ICAM-2 (Figs. 13-17).

The results indicate that ICAM-3 shows a high degree of homology to both ICAM-1 and ICAM-2. Sequence homologies between ICAM-1 and ICAM-3 are found within the first (Fig. 16) and fourth/fifth domains of ICAM-1 as well as in the transmembrane domain and some of the cytoplasmic domain of ICAM-1 (Figs. 13" and 14) .Further search of the gene bank database revealed no other sequence identical or similar to that obtained for ICAM-3.Significant homology was also seen between ICAM-3 and the first domains of ICAM-2 (Fig. 17).

Clone 11.2 was used to further screen libraries. Additional clones were found to obtain cDNA inserts from approximately 2-2.4 kD. In all likelihood these represent full length cDNA clones as no larger ones have so far been identified.

(2) Information for SEQ ID No: 2:

(i) Sequence Properties:

(A) Length: 50 amino acids

(B) Type: amino acid

(D) Topology: linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID No: 2:

Glu Phe Leu Leu Arg Val Glu Pro Gin Asn Pro Val Leu Ser Ala Gly

15 10 15

Gly Ser Leu Phe Val Asn Cys Ser Thr Asp Cys Pro Ser Ser Glu Lys

20 25 30

Ile Ala Leu Glu Thr Ser Leu Ser Lys Glu Leu Val Ala Ser Gly Met

35 40 45

Gly Trp

50

(2) Information for SEQ ID No.: 3:

(i) Sequence Properties:

(A) Length: 189 base pairs

(B) Type: nucleic acid

(C) Chain form: both

(D) Topology: linear

(ii) Molecule type: DNA

(ix) Features:

(A) Name/Key: CDS

(B) Location: 2..189

(xi) Sequence description: SEQ ID No. 3:

C CCA TTG AGG GGT TCC ACA GTG ACC GTG AGT TGC ATG GCT GGG GCT 46

Pro Leu Arg Gly Ser Thr Val Thr Val Ser Cys Met Ala Gly Ala

1 5 10 15.

CGA GTC CAG GTC ACG CTG GAC GGA GTT CCG GCC GCG GCC CCG GGG CAG 94

Arg Val Gin Val Thr Leu Asp Gly Val Pro Ala Ala Ala Pro Gly Gin

20 25 30

Claims (33)

1. Use of an ICAM-3 modulating agent for the manufacture of a pharmaceutical preparation for modulating inflammation, 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, wherein said fragment is capable of binding to ICAM-3; and ICAM-3. 2. Use as stated in claim 1, where said inflammation comprises a reaction of the specific defense system. 3. Use as set forth in claim 2, wherein said inflammatory response is specific inflammation in response to a condition selected from the group consisting of delayed-type hypersensitivity reaction, psoriasis, an autoimmune disease, organ transplant, and tissue transplant rejection. 4. Application as stated in claim 3, where said organ transplant is a fixed organ transplant. 5. Application as stated in claim 4, where said organ transplant is a kidney transplant. 6. Application as stated in claim 3, where said organ transplant is a non-permanent organ transplant. 7. Application as stated in claim 6, where said non-fixed organ transplant is a bone marrow transplant. 8. Use as stated in claim 3, wherein said autoimmune disease is selected from the group consisting of Reynaud's syndrome, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, rheumatoid arthritis and lupus erythematosus. 9. Use as stated in claim 1, where said inflammation comprises a reaction of the non-specific defense system. 10. Use as set forth in claim 9, wherein said inflammation is non-specific inflammation in response to a condition selected from the group consisting of: "adult respiratory distress syndrome" (ARDS), multiple organ damage syndromes secondary to septicemia, trauma or bleeding , reperfusion injury of myocardial or other tissue, acute glomerulonephritis, reactive arthritis, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, stroke, thermal injury, hemodialysis, leukophaeresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocytic transfusion-associated syndromes, and cytokine-induced toxicity. 11. Use as stated in claim 1, where said ICAM-3 modulating agent is a soluble fragment of ICAM-3, where said fragment is capable of binding LFA-1. 12. Use of an ICAM-3 modulating agent for the manufacture of a pharmaceutical preparation for modulating the extravascular migration of a virally infected leukocyte, wherein said ICAM-3 modulating agent is selected from the group consisting of an antibody capable of bind to ICAM-3; a fragment of said antibody, wherein said fragment is capable of binding to ICAM-3; and ICAM-3. 13 . Method for identifying agents capable of modulating ICAM-3 binding to LFA-1, characterized in that it comprises the steps of contacting ICAM-3 with LFA-1 in the presence of said agent and measuring said modulation of ICAM -3/LFA-1 bond. 14. Method as stated in claim 13, where said LFA-1 and said ICAM-3 are without naturally occurring contaminants. 15. Method as stated in claim 13, where said LFA-1 is present on a cell surface and said ICAM-3 is without naturally occurring contaminants. 16. Method as stated in claim 13, where said ICAM-3 is present on a cell surface and said LFA-1 is free of naturally occurring contaminants. 17. Method as stated in claim 13, where said ICAM-3 and said LFA-1 are present on different cell surfaces. 18. Method as stated in claim 13, where measurement of the effect of said compound on ICAM-3/LFA-1 binding is carried out by using a cellular aggregation assay. 19. Method as set forth in claim 13, wherein said compound is not an immunoglobulin. 20. Method as stated in claim 19, wherein said compound is a non-immunoglobulin polypeptide. 21. Method as stated in claim 13, wherein said compound is an immunoglobulin. 22. Use of an ICAM-3 modulating agent for the manufacture of a pharmaceutical preparation for suppressing cellular migration associated with asthma, 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 wherein said fragment is capable of binding to ICAM-3; and ICAM-3. 23. Use as stated in claim 22, wherein said ICAM-3 modulating agent is a soluble fragment of ICAM-3 comprising ICAM-3 domains 1-5. 24. Use as stated in claim 22, where said pharmaceutical preparation further comprises one or more additional anti-asthma agents. 25. Use as stated in claim 24, where said anti-asthma agent is a methylxanthine, a β-adrenergic agonist, a glucocorticoid, a chromone or an anticholinergic compound. 26. Use as stated in claim 24, where said anti-asthma agent is selected from the group comprising theophylline, catecholamine, resorcinol, saligenin, ephedrine, atropine, cromolyn sodium and hydrocortisone. 27. Use as stated in claim 24, where said anti-asthma agent is present in said pharmaceutical preparation at a suboptimal level. 28. Use of an ICAM-3 modulating agent for the manufacture of a pharmaceutical preparation for reducing metastasis of a hematopoietic tumor cell, 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, wherein said fragment is capable of binding to ICAM-3; and ICAM-3. 29. Use as stated in claim 28, where said ICAM-3 modulating agent is a soluble fragment of ICAM-3, where said fragment is capable of binding LFA-1. 30. Use as stated in claim 29, wherein said ICAM-3 modulating agent is a soluble fragment of ICAM-3 comprising ICAM-3 domains 1-5. 31. Use of an ICAM-3 modulating agent for the manufacture of a pharmaceutical preparation for reducing syncytia formation caused by HIV infection, 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, wherein said fragment is capable of binding to ICAM-3; and ICAM-3. 32. Use as stated in claim 31, where said ICAM-3 modulating agent is a soluble fragment of ICAM-3, where said fragment is capable of binding LEA-1. 33. Use as stated in claim 31, wherein said HIV is HIV-1.