SK139593A3 - Intercellular adhesion molecules-3-and its binding ligands - Google Patents

Abstract

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

Description

Technical field

The invention relates to the discovery of a new intercellular adhesion molecule, called ICAM-3, which is involved in processes by which leukocyte populations are recognized and adhered to cell substrates. ICAM-3 mediates cellular interactions with other lymphocytes, macrophages, and neutrophils at inflammatory and immune response sites.

The invention further relates to the use of ICAM-3, alone or in combination with ICAM-1 and / or ICAM-2, for inhibiting intercellular adhesion of granulocyte, lymphocyte or macrophage family cells. The use of these molecules allows a method of treating specific and non-specific inflammatory diseases.

The invention also relates to therapeutic and prophylactic methods of controlling HIV leukocyte infection, particularly HIV-1, in an individual exposed to HIV or infected with HIV by administering ICAM-3, alone or in combination with ICAM-1 and / or ICAM-2. Thus, it enables the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) which are caused by the HIV virus.

The invention also relates to a therapeutic method of inhibiting the migration of HIV-1 infected cells from the circulatory system using ICAM-3, alone or in combination with ICAM-1 and / or ICAM-2. Thus, it enables the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) which are caused by HIV-1.

The invention also relates to a therapeutic method for suppressing T cell death and syncytia in an individual infected with HIV using ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2. It thus allows the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) caused by HIV-1.

The invention relates to the use of ICAM-3, alone or in combination with ICAM-1 and / or ICAM-2, in the treatment of asthma.

The invention further relates to molecules capable of binding to ICAM-3 (hereinafter referred to as anti-ICAM-3). Binding of the anti-ICAM-3 molecule to ICAM-3 is intended to modulate the biological functions associated with ICAM-3. The binding molecules of the invention may be antibodies, peptides or carbohydrates that are capable of binding to ICAM-3. Such binding molecules are useful for modulating the biological functions of ICAM-3.

The invention also relates to the use of anti-ICAM-3 molecules, alone or in combination with anti-ICAM-1 and / or anti-ICAM-2, to inhibit intercellular adhesion of granulocyte, lymphocyte or macrophage family cells. The use of such molecules allows the treatment of specific and non-specific inflammatory diseases.

The invention further relates to therapeutic and prophylactic methods of controlling HIV leukocyte infection, in particular HIV-1, in an individual exposed to HIV or infected with HIV, by administering anti-ICAM-3 alone or in combination with anti-ICAM-1 and / or anti ICAM-2. It thus allows the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) which are caused by HIV.

The invention further relates to a therapeutic method of suppressing γ-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β. C 1 -ϊ migration of HIV-1 infected cells from the circulatory system using an anti-ICAM-1 composition, alone or in combination with an anti-ICAM-1 and / or anti-ICAM-2 composition . Thus, it allows the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) which are caused by HIV-1.

The invention also relates to the use of an anti-ICAM-3 composition, alone or in combination with anti-ICAM-1 and / or anti-ICAM-2, for the treatment of asthma.

BACKGROUND OF THE INVENTION

A. Power and function of leukocytes

Leukocytes must be capable of attaching to cellular substrates to properly protect the host against foreign invaders such as bacteria or viruses; for a review of these functions, see Eisen, H.V. (Microbiology, 3rd Ed., Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418). Leukocytes must be able to connect to endothelial cells to migrate from the circulatory system to sites of inflammation. Further, they must be fed to cells exhibiting antigens to allow a normal immune response to take place, and finally they must be fed to the respective target cells to allow for the degradation of virally infected or tumor cells.

Recently, leukocyte surface molecules involved in mediating these functions, mediated by power, have been identified using hybridoma technology. Briefly, monoclonal antibodies of inflammatory diseases against human T cells have been identified (Davignon, D. et al., Proc. Natl. Acad. Sci. USA 78, 4535-4539 (1981)) and mouse spleen cells (Springer, T. ad. ., Eur. J. Immunol. 9, 301-306 (1979)), which bound to the surface of leukocytes and inhibited the above-described power-related functions (Springer, T. et al., Fed. Proc. 44, 2660-2663). (1985)). The molecules identified by these antibodies were named Mac-1 and LFA-1 (Lymphocyte Function-associated Antigene-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular leukocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, T.A. et al., Immunol. Rev. 68, 111-135 (1982)). These two molecules plus the third molecule p150.95 (which has a tissue distribution similar to Mac-1) play a role in cellular adhesion (Keizer, G. ad., Eur. J, Immunol. 15, 1142-1147 (1985)).

The above leukocyte molecules have been found to be members of a family of glycoproteins (Sanchez-Madrid, F. et al., J. Exper. Med. 158, 1785-1803 (1983); Keizer, GD et al., Eur. J. Immunol., 15, 1142-1147 (1985)), called the CD-18 family of glycoproteins. This family of glycoproteins is composed of heterodimers containing a single α chain and one β chain. While the chain and each antigen differed from the others, the β chain was found to be highly conserved (Sanchez-Madrid, F. et al., J. Exper. Med. 158, 1785-1803 (1983)). The β chain of the glycoprotein family (sometimes referred to as CD18) has been found to have a molecular weight of 95 kd, while the α chain ranges from 150 kd to 180 kd (Springer,

T., Fed. Proc. 44, 2660-2663 (1985)). Although the subunits and membrane proteins do not accept the high homology reported by the β subunits, detailed analysis of the subunits and glycoproteins has shown that there are substantial similarities between them. A review of the similarities between the α and β subunits of LFA-1-related glycoproteins is given in Sanchez-Madrid, F. et al., J. Exper. Med. 158: 586-602 (1983); J. Exper. Med. 158, 1785-1803 (1983)).

A group of individuals have been identified that are unable to express a normal amount of any member of this family of adhesion proteins on the cell surface of their leukocytes (Anderson, DC et al., Fed. Proc. 44, 2671-2677 (1985), Anderson, DC et al. J. Infect. Dis., 152, 668-689 (1985)). These individuals are reported to suffer from leukocyte adherence deficiency disease (LAD) deficiency (Anderson, DC et al., Fed. Proc. 44, 2671-2677 (1985), Anderson, DC et al., J. Infect. Dis. 152, 668-689 (1985)). Characteristic features of LAD patients include necrotic soft tissue forest, impaired pus formation and wound healing, as well as abnormalities of adhesion-dependent leukocyte function in vitro and susceptibility to chronic and recurrent bacterial infections. The granulocytes of these LAD patients behave in the same defective manner in vitro as their normal counterparts in the presence of an anti-CD18 monoclonal antibody. That is, they are unable to perform adhesion-related functions such as aggregation or attachment to endothelial cells. More significant, however, is the finding that these patients are unable to exhibit a normal inflammatory response due to the inability of their granulocytes to attach to cellular substrates. It is very noteworthy that granulocytes of these LAD patients are unable to reach inflammatory sites such as skin infections due to their inability to attach to endothelial cells in the bloodstream near inflammatory forests. This attachment is an essential step in extravasation.

The lymphocytes of these patients showed in vitro defects similar to their normal counterparts whose family of CD-18 molecules were antagonized by antibodies. Moreover, these individuals were unable to respond normally due to the inability of their cells to adhere to cellular substrates (Anderson, D.C. et al., Fed. Proc. 44, 2671-2677 (1985), Anderson,

Dis. 152: 668-689 (1985)). These facts lymphocytes incapable of adhering to the normal lack of functional adhesion molecules

J. Infect. that if they are due

D.C. and d show, by way of the CD-18 family, immune responses are attenuated.

In summary, the ability of leukocytes to maintain the health and viability of a mammal requires that they be capable of adhesion to other cells (such as endothelial cells). It has been found that cell-cell contact is required for this adhesion, requiring the presence of specific receptor molecules on the surface of leukocyte cells. These receptors allow the adhesion of le6 ukocytes to other leukocytes, to endothelial cells and other non-vascular cells. The LFA-1, Mac-1 and p110.95 surface receptor molecules have been found to be highly related to each other. Human individuals in which leukocytes are absent from these surface receptor molecules are subject to chronic and recurrent infections and exhibit other clinical symptoms including defective antibody responses.

Furthermore, since leukocyte adhesion is involved in the process of identifying and rejecting foreign tissue, understanding this process is of great importance in the field of whole organ transplantation, such as kidney, as well as other transplants such as bone marrow, tissue transplantation, allergology and oncology.

B. HIV infection

HIV infection is the cause of AIDS. Numerous variants of HIV have been described, the main ones being HIV-1 and HIV-2. HIV-1 prevails in North America and Europe and HIV-2 prevails only in Africa. The viruses have a similar structure and encode proteins with a similar function. HIV infection is thought to be via the binding of a vital protein (termed gp120) to a receptor molecule (termed CD4) present on the surface of T4 lymphocytes (helper T) (Schnittman, SM et al., J. Immunol. 141, 4181-). 4186 (1988) Upon binding of this receptor, the virus enters the cell and replicates and destroys the T cell during this process. Thus, the direct consequence of HIV infection is the destruction of the T4 population.

The destruction of T cells deteriorates the ability of the infected patient to defend against accidental infections. Although an individual suffering from AIDS often develops cancer, the relationship between these cancers and HIV infection is in most cases uncertain.

Although HIV replication itself is lethal to the infected cells, such replication is most commonly detected in only a small fraction of the T4 cells of the infected individual. Recent results indicate that the virus range is greater than originally thought and the frequency of T cell infection can be as high as 1%.

Some research directions have explained other mechanisms by which HIV mediates the destruction of the T4 population.

In addition to HIV replication, cells in HIV infection can be killed by cytotoxic murder cells. Killer cells are normally present in the human body and serve to monitor the host and destroy all foreign cells (for example, inadequate blood transfusion or organ transplant, etc.) that they may encounter. In HIV infection, T4 cells have a gp120 molecule on their surface. The killer cells recognize such T4 cells as foreign (rather than the parent) and therefore mediate their destruction.

HIV infection can further lead to the destruction of uninfected healthy cells. Infected cells can secrete the gp120 protein into the blood system. The free gp120 molecules can then bind to the CD4 receptors of healthy uninfected cells. This binding causes the cells to look like HIV-infected cells. Cytotoxic killer cells recognize gp120 bound to uninfected T4 cell, conclude that the cell is foreign and mediate cell destruction.

Another mechanism that can cause T4 cell death and which is of particular importance in the context of the invention is the formation of syncytia. Syncytium is a multinucleated giant cell formed by the fusion of up to several hundred T4 cells. Infected with HIV, infected cells will acquire the ability to blend with other T4 cells, both HIV-infected and uninfected healthy cells. Syncytium cannot work and is almost dead. Thus, its destruction leads to destruction of both HIV-infected and uninfected T4 cells. This process is of particular importance in the context of the invention since it requires direct cell-cell contact between T4 cells. The ability of HIV-infected cells to form syncytia indicates that these cells acquire the ability to blend in with healthy cells.

HIV infection, and in particular HIV-1 infection, appears to affect cell surface expression of leukocyte integrins and cell adhesion mediated by these heterodimers (Petit, AJ et al., J. Clin. Invest. 79, 188 (1987), Hildreth, JEK and d., Science 244, 1075 (1989), Valentin, A. et al., J. Immunology 144: 934-937 (1990); Rossen, RD et al., Trans. Assoc., American Physicians 102, 117-130 (1989). )). Following HIV-1 infection, homotypic aggregation of U937 cells as well as surface expression of CD18 and CD11b cells are increased (Petit, A.J. et al., J. Clin. Invest. 79, 188 (1987)). HIV-1 infected U937 cells associate with endothelium-stimulated IL-1 at a greater frequency than uninfected U937 cells; this behavior can be suppressed by treating infected cells with anti-CD18 or anti-CD11a monoclonal antibodies or by treating endothelial substrates with anti-ICAM-1 antibodies (Rossen, RD et al., Trans. Assoc. American Physicians 102: 117-130 (1989)). Such monoclonal antibodies to CD18 or CD11a have been found to be capable of inhibiting the formation of syncyties involving phytohemagglutin (PHA) stimulated lymphoblastoid cells and constitutively infected CD4-negative T cells (Hildreth, J. E. K. et al., Science 244, 1075 (1989)). The action of anti-CD18 or anti-CD11a monoclonal antibodies only on virus-infected cells has been shown to have little effect on syncytia formation, leading to the assumption that these antibodies mainly protect uninfected target cells from infection (Hildreth, JEK et al., Science 244, 1075 (1989), Valentin, A. et al., J. Immunology 144, 934-937 (1990)). Valentín a d. (Valentin, A. et al., J. Immunology 144, 934-937 (1990)) recently confirmed this finding by demonstrating that CD18-specific monoclonal antibodies inhibit syncytia resulting from the simultaneous cultivation of continuous T cell lines with U937 infected cells. HIV-1.

In doing so, the mechanism by which monoclonal antibodies specific for CD18 or CD11a protect endangered cells from merging with HIV-infected cells remains unknown and is not necessary for understanding the invention. Studies with radiolabeled gp120 indicate that heterodimers containing CD18 do not provide a virus binding site (Valentin, A. et al., J. Immunology 144, 934-937 (1990)). HIV infection involves cell-cell interactions or virus-cell interactions that mimic them. Cell-cell interactions may result in the transport of non-cell-containing virus or transport of virus-infected cells across endothelial barriers. Virus-cell interactions that mimic cell-cell interactions can facilitate or allow free virus to attach to and / or infect healthy cells.

Thus, the invention is partially derived from the observation that HIV infection, and in particular HIV-1 infection, results in increased expression of the CD11a / CD18 heterodimer and its binding ligand. Significantly, this overexpression increases the ability of HIV-infected T cells to adhere to one another or aggregate (e.g., homotypic aggregation). Since such homotypic aggregation is not observed in quiescent normal leukocytes, this discovery indicates that expression of CD11 / CD18 receptors and / or ligands such as ICAM-1 is required for this aggregation. LFA-1 must bind to ICAM-1 for homotypic aggregation. The ICAM-3 of the invention is the only member of the ICAM family of molecules that is expressed at a high level on quiescent T cells. Only anti-ICAM-3 antibodies are capable of blocking T cell adhesion to LFA-1 unless T cells are activated. Therefore, anti-ICAM-3 antibodies can be used to suppress T cell aggregation.

In addition, anti-ICAM-3 antibodies can be used to block the adhesion process of infected T cells, which allows the transmission of HIV-1 from the infected cell to a healthy individual cell and thus allows or facilitates infection of healthy cells with free virus.

C. Migration of HIV infected cells

Migration and spread of leukocytes is important to protect the individual from the consequences of infection. However, these processes are also responsible for the migration and spread of the virus of infected leukocytes. Of particular importance is the migration and spread of HIV-infected leukocytes. By the migration of such cells, extravascular outbreaks occur, which can cause tumors and other abnormalities.

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

The mechanisms that stimulate extravascular infiltrates of HIV-1 infected monocytoid cells have not been defined precisely. These mechanisms may involve either virus-free cell transport or virus transport across endothelial barriers in the cytoplasm of infected mononuclear cells.

Because HIV-1 infection stimulates superficial cellular expression of molecules that allows leukocyte adhesion to vascular endothelial cells and the transfer of leukocytes from blood to extravascular tissue sites (CV Smith et al., J. Clin. Invest. 82, 1746 (1988)), suggested to be used to prevent the spread of HIV-infected cells that inhibit cell migration (WO 90/13316).

D. Asthma - clinical characteristics

Asthma is a heterogeneous group of diseases. It is characterized by hyperreactivity of tracheobronch irritation (Kay, AB, Allergy and Inflammation, Academic Press, NY (1987). Clinically, asthma is manifested by intense constriction of tracheobronch, dense cuddly secretions, dyspnea attacks, coughing and shortness of breath). but the net result is an increase in resistance to air passage, over-filling of the lungs and chest, and abnormal distribution of oxygenation and blood flow through the lungs.The disease is manifested by episodic periods of acute symptoms that alternate between periods without symptoms Acute events can lead to fatal hypoxia. About 3% of the world's population suffers from asthma.

Two types of asthma have been described: allergic 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 bruising and inflammation responses to intradermal injection of airborne antigens (such as pollen, polluting the environment and the working environment, etc.) and elevated serum IgE levels. The development of allergic asthma appears to be causally associated with the presence of IgE antibodies in many patients. Asthmatic patients who do not exhibit these characteristics are considered idiosyncratic asthmatics.

Allergic asthma is considered to be dependent on the IgE response, controlled by T and B lymphocytes, and activated by the interaction of airborne antigen with pre-formed mast cell-bound IgE molecules. The encounter with the antigens must take place at a sufficient concentration to cause IgE production over an extended period of time to allow sensitization of the individual. Once sensitized, an asthmatic patient may exhibit symptoms in response to extremely low antigen levels.

Asthma symptoms can be activated by the presence and amount of the inducing antigen, environmental factor, occupational factor, 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), mast cell degranulation inhibitors (ie chromones such as sodium cromolynic) and anticholinergic sodium (such as atropine).

Asthma is believed to require the influx of eosinophils (eosinophilia) into lung tissue (Frigas, E. et al., J. Allergy Clin. Immunol. 77, 527-537 (1986)).

A deeper insight into the immunological base of asthma was made possible by studies of washed bronchoalveol (Godard, P. et al., J. Allergy Clin. Immunol. 70, 88 (1982)) and epithelium depleted respiratory smooth muscle tissue studies (Flavahan, NA et al., J. Appl. Physiol., 58, 834 (1985), Barnes, PJ, et al., J. Pharmacol., 86, 685 (1985). Although these studies did not explain the mechanism of asthma immunology, they have led to the development of a generally accepted hypothesis regarding the immunological etiology of diseases (see Frigas, E. et al., J. Allergy Clin. Immunol. 77, 527-537 (1986)).

Characteristic features of asthma pathology are massive infiltration of the lung parenchyma by eosinophils and destruction of mucociliary capacity. According to the eosinophilic hypothesis, eosinophils are attracted to bronchus to neutralize noxious mediators released by the mast cells of the lungs. According to this hypothesis, eosinophils are attracted to the bronchi where they degrade and release cytotoxic molecules. In degranulation, eosinophils release enzymes such as histaminase, arylsulfatase and phospholipase D, which enzymatically neutralize harmful mast cell mediators. At the same time, these molecules promote destruction of the mucociliary apparatus, prevent crayfish from clearing the bronchial secretion and contribute to lung damage characteristic of asthma.

Since cell migration is involved in asthma, the use of antibodies that inhibit this migration has been suggested to alleviate the effects of allergens in subjects (WO 90/10453).

SUMMARY OF THE INVENTION

The invention is based on the discovery of a new cell adhesion molecule, called intercellular adhesion molecule 3 (ICAM-3). Furthermore, the invention relates to functional derivatives of ICAM-3, anti-ICAM-3 antibodies, fragments of said antibodies, humanized anti-ICAM-3 antibodies, and other molecules capable of binding to and inhibiting their biological functions.

In addition, the invention encompasses the diagnostic and therapeutic use of all the above molecules.

In particular, the invention encompasses ICAM-3 or functional derivatives thereof, substantially free of natural impurities.

The invention provides a method for preparing a recombinant or synthetic DNA molecule capable of encoding or expressing ICAM-3 or functional derivatives thereof.

The invention further encompasses an antibody, particularly monoclonal, capable of binding to a molecule selected from the group consisting of ICAM-3 and functional derivatives of ICAM-3.

The invention also encompasses a hybridoma cell capable of producing said monoclonal antibody.

The invention includes a method of producing a desired hybridoma cell that produces an antibody that is capable of binding to ICAM-3 or a functional derivative thereof, comprising the step of:

a) immunizing the mammal with an immunogen selected from the group consisting of substantially pure ICAM-3, a cell expressing ICAM-3, a cell membrane expressing ICAM-3, ICAM-3 bound to a carrier, a peptide fragment of ICAM-3 or a peptide fragment of ICAM-3 a carrier,

b) confluence of spleen cells isolated from said immunized mammal with a myeloma cell line,

(c) allowing the merged spleen and myeloma cells to produce an antibody secreting hybridoma cells; and

d) screening the hybridoma cells for a hybridoma cell capable of producing an anti-ICAM-3 antibody.

The invention also provides a method of modulating the biological functions of a cell mediated by ICAM-3 by providing an effective subject with an effective amount of an ICAM-3 modulating agent selected from an antibody capable of binding to ICAM-3, a fragment of said antibody, a derivative ICAM-3 and a non-immunoglobulin ICAM-3 antagonist other than ICAM-1, ICAM-2, or a member of the CD-18 molecule family.

The invention also provides a method of treating specific inflammations in humans and other mammals, comprising administering to a subject in need thereof an amount of an anti-inflammatory agent sufficient to suppress inflammation, wherein the anti-inflammatory agent is selected from an antibody capable of binding to ICAM-3, a fragment of said antibody. which is capable of binding to ICAM-3, substantially pure ICAM-3, a functional derivative of ICAM-3, or a non-immunoglobulin ICAM-3 antagonist, wherein said inflammation is due to whole organ transplant (e.g. kidney), partial organ transplant (e.g. bone marrow) or tissue transplantation.

The invention further provides a method of treating non-specific inflammation in humans and other mammals, comprising administering to a subject in need thereof an amount of an anti-inflammatory agent sufficient to suppress inflammation, wherein the anti-inflammatory agent is selected from an antibody capable of binding to ICAM-3. which is capable of binding to ICAM-3, a substantially pure ICAM-3, a functional derivative of ICAM-3, or a non-immunoglobulin ICAM-3 antagonist.

The invention further includes the above method of treating inflammation when the inflammation is associated with a condition selected from the group consisting of adult respiratory distress syndrome, multiple organ injury syndrome secondary to septicemia, bleeding or injury, reperfusion injury to myocardial or other tissue, acute glomerulonephritis, acute glomerulonephritis, dermatosis with acute inflammatory components, acute purulent meningitis or other inflammatory disorders of the central nervous system, such as apoplexy, thermal injuries, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion-induced syndrome, and cytokine.

The invention further encompasses a method of suppressing hematopoietic tumor cell metastasis which uses a member of the CD-18 (particularly LFA-1) to migrate, comprising administering to the patient an amount sufficient to suppress metastasis, said composition selected from the group comprising an antibody capable of binding to ICAM- 3, toxin derivatization of the antibody, a fragment of the antibody capable of binding to ICAM-3, toxin derivatization of the fragment, substantially pure ICAM-3, toxin derivatization of ICAM-3, a functional derivative of ICAM-3, toxin derivatization of this functional derivative, or a non-immunoglobulin ICAM-3 antagonist other than a member of the CD-18 molecule family.

The invention also encompasses a method of suppressing the growth of a tumor cell expressing ICAM-3, comprising sub-administering the composition to a patient in an amount sufficient to suppress such growth, said composition selected from the group consisting of an antibody capable of binding to ICAM-3, toxin derivatization of the antibody, which is capable of binding to ICAM-3, toxin derivatizing this fragment, a toxin derivatized member of the CD-18 molecule family, or a toxin derivatized functional derivative of a member of the CD-18 molecule family.

The invention also provides a method for detecting the presence of a cell expressing ICAM-3, comprising;

(a) incubating the cell or cell extract in the presence of a nucleic acid molecule capable of hybridizing to ICAM-3 mRNA; and

b) determining whether the nucleic acid molecule has hybridized to a complementary nucleic acid molecule present in said cell or in said extract.

The invention further provides a method for detecting the presence of ICAM-3 in a biological fluid sample, comprising:

(a) incubating said sample with an antibody or fragment thereof capable of binding to ICAM-3; and

b) detecting whether said antibody has bound said sample.

The invention also encompasses a pharmaceutical composition comprising a composition selected from the group consisting of an antibody capable of binding to ICAM-3, a fragment of the antibody capable of binding to ICAM-3, substantially pure ICAM-3, a functional derivative of ICAM-3 or a non-immunoglobulin antagonist ICAM-3. other than a member of the CD-18 molecule family and an immunosuppressive agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Adhesion of cell lines to purified LFA-1.

Adhesion of cell lines (A) and lymphocytes (B) to purified LFA-1 for ICAM-1, ICAM-2, and ICAM-3 is contemplated. Binding of BCECF-labeled cells to LFA-1 coated microtiter wells in the presence of blocking LFA-1, ICAM-1, ICAM-2 and ICAM-3 specific MAbs. Control wells were coated without LFA-1.

Fig. 2. Flow cytometric analysis of cellular expression of ICAM-1, ICAM-2 and ICAM-3.

(A) Lymphoblastoid cell lines were labeled with saturating amounts of MAb RR1 / 1 (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 then FITC-anti-mouse immunoglobulin.

(B) Resting human leukocytes and 3 day PHA-activated lymphocytes were analyzed for ICAM expression as described above.

Fig. 3. Immunoprecipitation of ICAM-3.

2S (A) I-labeled cell lysates immunoprecipitated with X63 MAb CB-IC3 / 1 non-binding control (anti-ICAM-3).

(B) I-labeled SKV3 cell lysates, treated with (+) or without (-) N-glycanase and immunoprecipitated with non-binding control MAb X63, with MAb V6 / 32 (anti-HLA-A, B, C), MAb RR1 / 1 (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 various sources.

2 S

ICAM-3 was immunoprecipitated from I- ^1- labeled lymphocyte and neutrophil lysates by mAb-fused Sepharose. Immunoprecipitated ICAM-3 was resolved by electrophoresis using a polyacrylamide gel. Other mAbs were used to immunoprecipitate other cell surface molecules. Sepharose with human Ig (control line), CBR-IC2 / 1 (ICAM-2), CBR-IC3 / 1 (ICAM-3), TS2 / 4 (LFA-1), LM2 / 1 (MAC-1).

Fig. 5. Effect of PMA on SKV3 binding to ICAM-1 and ICAM-3.

The ability of SKV3 cells to bind to purified ICAM-1 and ICAM-3 and the effect of PMA stimulation on this binding has been tested as described previously (Dustin et al., Nature 341, 619 (1989); Mary et al., Cell 51, 813-819 ( 1987)). One representative experiment is shown and error bars indicate one standard deviation (SD).

Fig. 6. Blocking of PMA stimulated by SKV3 cell adhesion by antibodies.

Various antibodies have been tested for their ability to block the binding of PMA stimulated SKV3 cells to purified ICAM-1 or ICAM-3 as previously described (Dustin et al., Nature 341, 619 (1989); Mary et al., Cell 51, 813-819 ( 1987)). One representative experiment is shown and error bars indicate one SD.

Fig. 7. Binding of COS cell transfectants to purified ICAM.

COS cells were transfected with the LFA-1 expression vector as described previously (deFougerolles et al., J. Exp. Med. 175, 185-190 (1992)). Transfected cells were tested for their ability to bind to immobilized ICAM-1 and ICAM-3 in the presence and absence of anti-LFA-1 antibody (TS1 / 22). One representative experiment is shown and error bars indicate one SD.

Fig. Temperature dependence of PMA stimulated SKV3 binding to ICAM.

The ability of PMA-stimulated SKV3 cells to bind purified ICAM-1 and ICAM-3 was tested at 4, 16, 22 and 37 ° C as described previously (Mary et al., Cell 51, 813-819 (1987)).

Assay performed in the presence or absence of mAb LFA-1 (TS1 / 22). One representative experiment is shown and error bars indicate one SD.

Fig. Blocking the division of PHA-stimulated cells by T antibodies.

Various antibodies have been tested for the ability to block PMA-stimulated T cell division as described previously (Krensky et al., J. Immunol. 131, 611-616 (1983)).

Fig. 10. Influence of anti-ICAM antibodies on mixed lymphocyte reaction.

Various antibodies have been tested for the ability to block a mixed lymphocyte response as described previously (Krensky et al., J. Immunol. 131, 611-616 (1983)).

Fig. Gas chromatogram of peptide fragments of ICAM-3.

ICAM-3 were purified and digested with Lys-C. Peptide fragments were resolved by high performance liquid chromatography.

Fig. 12. Graphical representation of clone 11.2.

The positions of the NK-10 and NK-17 peptides, the various DNA sequences obtained so far and the transmembrane region are shown.

Fig. 13. Sequence comparison of the sequence initiated by NK-10 with human ICAM-1.

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

Fig. 14. Sequence comparison of the RM13 initiated sequence with human ICAM-1.

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

Fig. 15. Sequence comparison of the sequence initiated by RM13 with human ICAM-2.

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

Fig. 16. Sequence comparison of the T7-initiated sequence with human ICAM-1.

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

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

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

Fig. 18. ICAM-3 partial sequence.

The DNA sequence of clone 11.2 was determined by standard procedures.

A. Sequences obtained from the 5'-end of ICAM-3. These sequences were obtained by initiating clone 11.2 with primer T7.

B. Sequences obtained within the ICAM-3 gene. These sequences were obtained by initiating clone 11.2 with the NK-10 probe.

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

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

I. LFA-1

The leukocyte adhesion molecule LFA-1 mediates various interactions of lymphocytes, monocytes, natural killer cells and granulocytes with other cells in the case of immunity and inflammation (Springer, T.A. et al., Ann. Rev. Immunol. 5, 223-252 (1987)).

LFA-1 is a receptor for ICAM-1, ICAM-2 and the newly identified ICAM-3 (according to the invention). These surface molecules are constitutively expressed in some tissues and induced in others in inflammation (Mary, SD et al., Cell 51, 813-819 (1987), Dustin, ML et al., J. Immunol. 137, 245-254 (1986). , Dustin, ML 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 interactions of T cytotoxic, T helper, natural killer cells, granulocytes and monocytes with cells of other types (Springer, TA et al., Ann. Rev. Immunol. 5, 223-252 ( 1987), Kishimoto, TK et al., Adv. Immunol. (1988, in press)).

II. ICAM-1

ICAM-1 is a single chain glycoprotein whose weight varies from 76 to 114 kD in different cell types and is a member of the five-type Ig superfamily Ig (Dustin, ML et al., Immunol. Today 9, 213-215 (1988)) , Staunton, DE and Others,

Cell 52, 925-933 (1988), Simmons, D. et al., Nature 331,

624-627 (1988)). ICAM-1 can be aided by cytokines including

IFN-g, TNF, and IL-1 were introduced on cells of various types (Dustin, M.L. et al., Immunol. Today 9, 213-215 (1988)). introduction

ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1 dependent lymphocyte adhesion Immunol. 137: 245-254 (1986); Dustin, Biol. 107, 321-331 (1988), Dustin,

Exp. Med. 167, 1323-1340 (1988)). Adhesion is blocked by prior treatment of LFA-1 MAb or by prior treatment of a second ICAM-1 MAb (Dustin, M.L. ad., J. Immunol. 137: 245-254 (1986); Dustin, M.L.

Biol. 107, 321-331 (1988); Dustin, M.L. d.

167, 1323-1340 (1988)). Identical results with purified ICAM-1 on artificial membranes or on Petri dishes demonstrate that LFA-1 and ICAM-1 are receptors of each other (Mary, S.D. et al., Cell 51, 813-819 (1987), Makgoba,

M. V. et al., Nature 331, 86-88 (1988)). For clarity, they are referred to herein as receptor and ligand. Further data on ICAM-1 is available in US patent applications 07 / 045,963, 07 / 115,789, 07 / 155,943, 07 / 189,815 or 07 / 250,446.

(Dustin, M. L. et al., J. M. L. et al., J. Cell M. L. et al., J et al., J. Cell. J. Exp. Med.

III. ICAM-2

Other LFA-1 ligands other than ICAM-1 have been postulated (Rothlein, R. et al., J. Immunol. 137: 1270-1274 (1986); Makgoba, MV et al., Eur. J. Immunol. 18, 637). -640 (1988), Dustin, ML et al., J. Cell Biol 107, 321-331 (1988)). The second LFA-1 ligand identified is designated ICAM-2.

ICAM-2 differs from ICAM-1 in cell distribution and in the absence of cyrokine induction. ICAM-2 is an integral membrane protein with 2 Ig-like regions, while ICAM-1 has 5 Ig-like regions (Staunton, DE et al., Cell 52, 925-933 (1988), Simmons, D. et al., Nature 331, 624-627 (1988)). Surprisingly, ICAM-2 is much more closely related to the two most N-terminal regions of ICAM-1 (34% identity) as either ICAM-1 or ICAM-2 to other members of the Ig superfamily, thus demonstrating the Ig subfamily of ligands that bind to families of the same integrins. Further data on ICAM-2 can be found in U.S. Patent Application Ser. 07 / 454.294.

IV. ICAM-3

The invention relates to the discovery of a third LFA-1 ligand, designated ICAM-3.

The development of MAb to ICAM-2 allowed the analysis of many rather LFA-1-dependent and ICAM-1-independent phenomena and the assumption of a third ligand for LFA-1. Binding of certain cell types, such as epithelial and endothelial cells, to purified LFA-1 can be completely blocked by the combination of MAb ICAM-1 and ICAM-2, while on many lymphoid cell lines, including the T SKV3 lymphoma cell line (Fig. 1). ), there was a pathway for adhesion to LFA-1 independent of ICAM-1, ICAM-2.

To characterize this adhesion pathway, the MAbs were immunized to SKV3 and in accordance with anti-ICAM-1 and anti-ICAM-2 MAbs were screened for their ability to inhibit the binding of this cell line to purified LFA-1. One MAb, CBR-IC3 / 1, was selected that completely inhibited this new adhesion pathway (Fig. 1). The adhesion of SKV3 to purified LFA-1 was only slightly inhibited by the combination blocking anti-ICAM-1 and anti-ICAM-2 MAbs. When anti-ICAM-3 MAb alone, CB-IC3 / 1 was added, it inhibited adhesion significantly, and this inhibition was complete in combination with blocking ant.i-ICAM-2 MAb. Thus, the adhesion of SKV3 to purified LFA-1 was strongly mediated by ICAM-3 and also partially by ICAM-2. For LFA-1 adhesion, each of the four cell lines utilized the three ICAMs to varying degrees, as demonstrated by different MAb inhibition patterns. The adhesion of the lymphoblastoid cell line B, JY, was performed first through the ICAM-1 pathway with a weak contribution of the ICAM-2 and ICAM-3 pathways. Another lymphoblastoid B cell line, SLA, utilized both ICAM-1 and ICAM-3 because adhesion was not inhibited by either the MAbs by either ICAM-1 or ICAM-3 alone, but almost completely by both MAbs together. The thymoma cell line, Jurkat, used ICAM-2 and ICAM-3 pathways with little ICAM-1 contribution to adhesion. Adhesion of T lymphocytes, both resting and activated by phytohemagglutinin (PHA) was also investigated (Fig. 1B). Resting lymphocytes have previously demonstrated strong binding to purified LFA-1 (Dustin et al., Nature 341, 619-624 (1989)) and this binding was found to be strongly dependent on ICAM-3. Upon activation of PHA, ICAM-1 expression was induced and adhesion to LFA-1 was mainly via ICAM-1 and partially through ICAM-3. The adhesion component, corresponding to ICAM-2, was detectable in both resting and activated T cells, although it was masked by the presence of ICAM-1 and ICAM-3. There is considerable redundancy in the use of ICAM, since for each cell, at least two ICAMs were required to achieve significant inhibition. A notable exception is the resting lymphocyte adhesion to LFA-1, which has been largely mediated by ICAM-3. The combination of all three anti-ICAM MAbs in all three cases eliminated binding to LFA-1 to a level comparable to that observed in the presence of anti-LFA-1 MAbs.

The relative affinity of the three ICAMs for LFA-1 can be ascertained by comparing their contribution to LFA-1 binding, measured as described above, with their cell surface expression as measured by immunofluorescence cytometry (Fig. 2). Comparing the surface expression of ICAM and their contribution to L1FA-1 adhesion, ICAM-1 was found to have the greatest affinity for LFA-1, while ICAM-2 and ICAM-3 have affinities similar but lower. For example, Jurkat cells expressed ICAM-2 and ICAM-3 at a similar level and both contributed to LFA-1 binding. When ICAM-2 expression was greater than ICAM-3, such as JY cells, ICAM-2 pathway predominated over ICAM-3 in adhesion to LFA-1. In contrast, SLA and SKV3 ex24 primed 3-fold more ICAM-3 than ICAM-2, and not surprisingly the ICAM-3 pathway in adhesion outweighed the ICAM-2 pathway. ICAM-3 was well expressed on quiescent lymphocytes and ICAM-1 was absent; adhesion to LFA-1 was mediated predominantly by ICAM-3. If ICAM-1 was well expressed, as in the case of PHA-activated SLA, JY and T cells, this was the primary pathway of adhesion.

The distribution pattern of ICAM-3 differed from ICAM-1 and ICAM-2 in several ways. In contrast to ICAM-1 and ICAM-2, ICAM-3 was not expressed on either resting or stimulated endothelium (data not shown). This is consistent with the finding that LFA-1 dependent cell binding to both resting and stimulated endothelium is an ICAM-1 and ICAM-2 dependent phenomenon. ICAM-3 was restricted to the hematopoietic lineage and was strongly expressed on lymphoid and monocytic cell lines with a few exceptions. In all cases, however, the expression of ICAM-3 on cell lines was consistent with the LFA-1 dependent adhesion pathway, independent of ICAM-1, ICAM-2. Cell lines (HUVEC, Raji), binding only LFA-1 via ICAM-1 and ICAM-2, showed no expression of ICAM-3, whereas cell lines (JY, U937, Sup T) showed weak binding through this third pathway correspondingly, the adhesions had low surface expression of ICAM-3 (data not shown).

ICAM-3 differed significantly in leukocyte expression from ICAM-1 and ICAM-2 (Fig. 2B). ICAM-3 was expressed at a high level on quiescent lymphocytes, monocytes and neutrophils, while ICAM-1 and ICAM-2 were expressed much weaker than they were absent. In comparison, ICAM-1 and ICAM-2 were poorly expressed on monocytes and only ICAM-2 was present on quiescent lymphocytes. Neither ICAM-1 nor ICAM-2 was expressed on neutrophils. Upon activation of PHA lymphocytes, the expression of ICAM-3 increased 2-3-fold while the expression of ICAM-1 was strongly stimulated (Dustin et al., J. Immunol. 137, 245-254 (1986); Fig. 2B).

ICAM-3 immunoprecipitates from various I-labeled cell lines showed a sharp band of 124,000 M _r under reducing conditions with only slightly increased mobility under non-reducing conditions (Fig. 3A). N-glycanase treatment induced reduction of the ICAM-3 band to M _r 87000, suggesting that ICAM-3, like ICAM-1 and ICAM-2, is a highly glycosylated protein (Fig. 3B). The biochemical characteristics, expression patterns, and functional properties of ICAM-3 distinguish it from the previously described adhesion molecules, including the human LAM-1 receptor leader (Tedder et al., J. Immunol. 144: 532-540 (1990)), an inducible endothelial adhesion molecule. VACAM-1 (Rice et al., J. Exp. Med. 171, 1369-1374 (1990, Carlos et al., Blood, in press (1990), Osborn et al. Cell 59, 1203-1211 (1989)) and the VLA matrix receptor family (Hemler, ME, Ann. Rev. Immunol. 8, 365-400 (1990)) and in the databases of the fourth leukocyte workshop (Gilks, VR et al., Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England (1990)), MAb with similar cell distribution was not found.

The existence of three LFA-1 ligands offers the possibility of specialization with respect to various aspects of LFA-1 dependent leukocyte interactions. ICAM-1 is basically expressed on endothelium and many epithelial cell types and is strongly stimulated in inflammation and immunity, where it regulates cell localization and facilitates specific antigen recognition (Vawryk et al., Immunol. Rev. 108, 135-161 (1989)). ). Since ICAM-2 is the predominant ligand of LFA-1 on the resting endothelium, this adhesion pathway may have significant implications for the normal recirculation of LFA-1-bearing lymphocytes by tissue endothelium (Hamann et al., J. Immunol. 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. 753-759 (1988)). The function of ICAM-3 on neutrophils remains unclear to the present because homotypic neutrophil aggregation appears to be primarily dependent on Mac-1 despite the presence of LFA-1 (Anderson et al., J. Immunol. 137, 15-27 (1986), Patarroyo et al., Scand J. Immunol., 22, 619-631 (1985)). The finding that adhesion of resting T lymphocytes to LFA-1 occurs primarily through ICAM-3, coupled with the fact that ICAM-3 is much more expressed than other LFA-1 ligands on monocytes and resting lymphocytes, indicates a significant role in the initiation of immune indeed, the adhesion of T cells to antigen-presenting cells requires the interaction of LFA-1: ICAM (Dransfield et al., Imm. Rev. 114, 29-44 (1990); Makgoba et al., Immunol. Today 10, 417-). 422 (1989)) and some role may be played by ICAM-3 as such, particularly on resting T cells where neither ICAM-1 nor ICAM-2 is well expressed. Indeed, a role has been suggested for LFA-1 ligands other than ICAM-1 in both allogeneic and autologous mixed lymphocyte reactions (Bagnasco et al., Cell Immunol. 128, 362-369 (1990)). In addition, one can assume an important role of ICAM-3 in antigen-specific interactions between Ta B cells, unless one of these cells has been activated yet. ICAM-3 may also be important in LFA-1-dependent but not ICAM-1-dependent T cell degradation by T cells (Magkoba et al., Eur. J. Immunol. 18, 637-640 (1988)).

The existence of multiple types of ICAM has significant implications for the clinical treatment of MAb to ICAM or ICAM analogs. MAb ICAM-1 is effective in vivo in prolonging the renal viability (Cosimi et al., J. Immmunol. 144: 4604-4612 (1990)) and cardiac (Flavin et al., Transplant. Proc. 23, 533-534 (1991)). ) allotransplants. MAb ICAM-3 inhibits a characteristic and overlapping number of immune responses in vivo, since it inhibits LFA-1-dependent adhesive interactions of a characteristic subset of cell types.

V. Cloning of ICAM-3 by cDNA

Any of a variety of methods can be used to clone the ICAM-3 gene. One of these methods involves analyzing cDNA regions (derived from a cell expressing ICAM-3) in a shuttle vector library for the presence of a region containing the ICAM-3 gene. Such analysis can be performed by transfecting cells with a vector and then measuring the expression of ICAM-3.

The ICAM-3 cDNA is preferably identified using a modification of the procedure of Aruff and Seed (Seed B. et al., Proc. Natl. Acad. Sci. USA 84, 3365-3369 (1987)) to identify ligands for adhesion molecules. According to this method, a cDNA library is prepared from cells that express ICAM-3 (such as the lymphoblastoid cell lines SLA, Jurkat or SKV3). 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). Transfected cells are transferred to a Petri dish which has been pre-plated with either LFA-1 or anti-ICAM-3 antibodies. Adhesion of transfected cells containing ICAM-3 coding sequences that express this ligand on their cell surface to LFA-1 or anti-ICAM-3 antibodies occurs on the surface of the Petri dish. Non-adherent cells are removed by washing, adherent cells are removed from the Petri dish and cultured. The sequence in these cells expressing recombinant ICAM-3 is then determined.

If LFA-1 is used to cover the Petri dish, specific anti-ICAM-1 and anti-ICAM-2 specific antibodies are added to the Petri dish to prevent adhesion of cells expressing ICAM-1 or ICAM-2. Thus, the adhesion of ICAM-1 + or ICAM-2 + transfectants to plastic coated with LFA-1 can be inhibited by antibodies such as RR1 / 1, i. MAb anti-ICAM-1, and CBR-IC2 / 2, i. MAb anti-ICAM-2. Binding of cells transfected with ICAM-3 to LFA-1 coated petri dishes is inhibited by EDTA and MAb anti-LFA-1, but not inhibited by MAb α 1 -ti-ICAM-1 or anti-ICAM-2. Therefore, cells expressing ICA.M-1 or ICAM-2 are incapable of adhesion to the Petri dish and are mostly eluted with all other non-adherent cells. Thus, cells expressing ICAM-3 are enriched.

Other alternative methods of obtaining gene sequences encoding ICAM-3 are known in the art, and one of ordinary skill in the art will be able to routinely adapt one of these methods to prepare the gene of interest.

One of these methods for preparing a gene sequence that encodes ICAM-3 is to use an oligonucleotide probe to screen a cDNA library or genomic DNA. According to this method, the ICAM-3 protein is purified, preferably using known immunoprecipitation methods, and then the terminal amino acid sequence is determined by any known method. Alternatively, ICAM-3 is enzymatically purified, e.g., with trypsin or Lys-C, the peptides purified, and the amino acid sequence of one of the internal fragments determined.

Once a partial amino acid sequence has been determined, either an oligonucleotide probe is generated based on the organism's codon preference, a degenerate probe is generated based on all possible codon combinations, or a combination of codon preference, homology with known ICAM-1 or ICAM-2 DNA sequences, and codon degeneration is used for probe construction. This probe is then used to screen either a genomic library or a cDNA library for sequences that hybridize to the probe.

These or similar methods have successfully enabled the cloning of human aldehyde dehydrogenase genes (Hsu, LC et al., Proc. Natl. Acad. Sci. USA 82, 3771-3775 (1985)), fibronectin (Suzuki, S. ad., Eur. Mol. Biol. Organ. J. 4, 2519-2524 (1985)), the human estrogen receptor gene (Valter, P, et. Proc. Natl. Acad. Sci. USA 82, 7889-7893 (1985)) and tissue-type plasminogen activator. (Pennica, D. et al., Nature 301, 214-221 (1983)).

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

The cloned ICAM-3 gene, obtained using any of the methods described, can be operably linked to an expression vector and introduced into bacterial or eukaryotic cells to produce the ICAM-3 protein. Techniques for this manipulation are described in Maniatis, T. et al. supra, and are known.

In an alternative method using PCR to clone the ICAM-3 gene, ICAM-3 is believed to be homologous to ICAM-1 and / or ICAM-2. Degenerate oligonucleotide primers based on sequences retained between ICAM-1 and ICAM-2 are used to amplify polymerase chain cDNA or mRNA reactions from cells known to express the ICAM-3 protein, such as SKV3 alo tonsila (deFougerolles et al. J Exp Med 175: 185-190 (1992)). The clones are sequenced and those that are different from ICAM-1 and ICAM-2 are used to prepare full-length cDNA clones.

In any of the methods described, the authenticity of the clones can be confirmed by expressing full-length clones, for example, in COS cells and testing for reactivity with mAb ICAM-3.

VI. Compositions of the Invention: ICAM-3 and functional derivatives, agonists and antagonists thereof

The invention is further directed to ICAM-3, functional derivatives thereof, agonists and antagonists thereof.

A. Functional derivatives of ICAM-3

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

and ^the ý.raz fragment of ICAM-3 refer to any polypeptide subset of the molecule. Particular preference is given to ICAM-3 fragments having ICAM-3 activity which are soluble (i.e., do not bind to membranes). Preferably, the soluble fragment is formed by discharging the membrane corresponding to the region of the parent molecule or by dividing or substituting the hydrophobic amino acid residues with hydrophilic fragments. The identification of these residues is known in the art.

Fragments containing any number of whole Ig-type regions, such as region 1 (D1), D1 and D2, D1-3, D1-4 and D1-5 are preferred.

The term ICAM-3 variant refers to a molecule whose structure and function is substantially similar to either the entire molecule or a fragment thereof.

A molecule is called substantially similar to another if both molecules have a substantially similar structure or both have similar biological activity. Thus, when two molecules have similar potency, they are considered to be variants and are referred to herein even if one of the molecules contains a structure that is not present in the other molecule or the amino acid residue sequence is not identical.

A molecule is referred to herein as a chemical derivative of another molecule when it contains other chemical units that are not normally part of a naturally occurring molecule. These units can improve solubility, absorbency, half-life, and the like. molecules. Alternatively; these units may reduce the toxicity of the molecule or eliminate or attenuate any unwanted side effects of the molecule. Molecules capable of mediating such effects are described in Remington's Pharmaceutical Sciences (1980).

A special class of chemical derivatives are toxin-derivatized molecules. A toxin derivatized is a molecule (e.g., ICAM-3 or an anti-ICAM-3 antibody) that is covalently bound to a toxin unit. Methods for condensing these units with molecules are known in the art.

Binding of such a molecule to the cell brings the toxin unit into the immediate vicinity of the cell and thus promotes cell death. Any suitable toxin unit may be used; however, it is preferred to use toxins such as tick toxin, cholera toxin, diphtheria toxin, radioisotopic toxins or membrane channel forming toxins.

Functional derivatives of ICAM-3, containing up to: about 100 residues, can be conveniently prepared by in vitro synthesis. If desired, these fragments can be modified using known methods of reacting target amino acid residues of a purified or crude protein with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The resulting covalent derivatives can be used to identify residues important for biological activity.

Various methods can be used to generate and isolate ICAM-3 fragments. Derivatization with bifunctional reagents can be used to form cross-links of ICAM-3 with a water-insoluble carrier matrix. Alternatively, a reactive water-insoluble matrix such as cyanogen bromide activated carbohydrates and the reactive substrates described in US Patents 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537 and 4,330,440 are used to immobilize proteins.

Functional derivatives of ICAM-3 with an altered amino acid sequence can be prepared by mutating the DNA encoding ICAM-3. Such functional derivatives include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence of ICAM-3. Any combination of deletion, insertion, and substitution may be used to form the final derivative as long as the final derivative has the desired potency. Obviously, a mutation made in a DNA encoding a functional derivative must not receive a sequence outside the reading frame and preferably should not create a complementary region that could produce a secondary mRNA structure (see published patent application EP 75 444).

At the genetic level, functional derivatives can be prepared by mutagenesis of DNA encoding ICAM-3, site-directed, to produce DNA encoding a functional derivative, followed by expression of the DNA in recombinant cell culture.

While the site for introducing the amino acid sequence variation is predetermined, the mutation itself may not be predetermined. For example, to optimize a site mutation, random mutagenesis can be performed at the target codon or target region, for example, by linker scanning mutagenesis to produce a large number of derivatives, which can then be expressed and screened to optimally combine the desired efficacy. Methods of making a mutation at predetermined sites in a known DNA sequence, for example, a site-directed mutation, are known.

The preparation of a functional ICAM-3 derivative of the invention is preferably carried out by site-directed mutagenesis of DNA that encodes ICAM-3 or a previously prepared functional derivative of ICAM-3. The technique of such specific mutagenesis is known in the art, for example, from publications such as Maniatis, T. et al., Molecular Cloning, and Laboratory Manual, Coldspring Harbor, NY (1982). Site-directed mutagenesis allows the production of functional derivatives of ICAM-3 using specific oligonucleotide sequences that encode the DNA sequence of the desired mutation.

Amino acid deletions, insertions, or substitutions can be performed using site-directed mutagenesis. The amino acid sequence deletions usually comprise about 1 to 30 residues, preferably 1 to 10 residues, and most commonly are contiguous. Most preferably, the deletions result in the soluble form of ICAM-3. Most preferably, soluble forms of ICAM-3 are formed by omitting either the membrane corresponding to the ICAM-3 region or the hydrophobic residues in ICAM-3.

Amino acid insertions include insertions of individual or 33 fused amino acid residues in the sequences encoding ICAM-3 as well as terminal condensations with polypeptides of substantially unlimited length. The internal insertion (i.e. insertion within the complete sequence of the ICAM-3 molecule) may generally comprise about 1 to 10, preferably 1 to 10. 5 residues. An example of a terminal insertion is the condensation of a signal sequence, whether heterologous or homologous to the host cell, to the N-terminus of the molecule to facilitate secretion of the derivative from the recombinant host.

A third group of functional derivatives are derivatives in which one or more amino acid residues in the ICAM-3 molecule have been removed and another residue inserted in their place. Such substitutions are preferably performed when fine modulation of the characteristics of the ICAM-3 molecule is desired, in accordance with the following table:

Table 1 - original substitution examples of substitution

Ala glycine. ser Arg lys same time gin, his Asp Glu Cys ser gin same time Glu asp Gly ala, for His same time, gin Ile leu, wall Leu ile, wall Lys arg, gin, Glu Met leu, Tyr, ile Phe met, leu, Tyr Ser thr Thr ser Trp Tyr Tyr trp, phe wall ile, leu

By selecting substitutions that are less conservative than those in Table 1, substantial changes in functional or immunological identity can be made, for example, by selecting residues that differ significantly in the effect of maintaining (a) the structure of the polypeptide chain in the region of substitutions, such as planar or spiral conformation; (b) charge or hydrophobicity of the molecule at the target site; or (c) side chain size. Generally less conservative are those substitutions wherein (a) the glycine and / or proline is replaced by another amino acid or is deleted or introduced, (b) the hydrophobic residue is replaced by the hydrophilic residue, (c) the cysteine residue replaces the other residue (or is replaced by another residue) (d) an electropositive side chain residue replaces (or is replaced by) an electronically charged residue; (c) a bulky side chain residue replaces a residue that does not have (or is replaced by) such a side chain.

Most preferred are substitutions that affect the solubility of ICAM-3. They are preferably performed by replacing hydrophobic amino acids with hydrophilic ones.

The mutation, leading to an increase in affinity of ICAM-3, can be accomplished by introducing amino acid residues that are present at homologous positions in ICAM-1 or ICAM-2. Similarly, it is possible to prepare such mutated ICAM-3 molecules which lack the N-linked CHO moiety at homologous positions of ICAM-1 or ICAM-2.

It is difficult to predict the exact effect that a particular substitution, deletion or insertion will have on the biological activity of ICAM-3. However, it is known to the person skilled in the art that this effect can be ascertained by routine screening tests. For example, a DNA linker scanning mutagenesis derivative encoding a native ICAM-3 molecule is generated. The derivative is then expressed in a recombinant host and optionally purified from cell culture, for example by immunoaffinity chromatography.

The activity of the cell lysate or purified derivative is then screened by a suitable screening assay for the desired characteristic. For example, a change in the immunological character of a functional derivative, for example affinity for a given antibody, is measured by a competitive type immunoassay. Changes in immunomodulatory activity are measured by a suitable assay. Modifications of such protein properties, such as redox or thermal stability, half-life, susceptibility to proteolytic degradation, or a tendency to aggregate with carriers or into multimers, are determined by methods known to those of ordinary skill in the art.

B. ICAM-3 Agonists and Antagonists

An ICAM-3 agonist is a compound that promotes or enhances the ability of ICAM-3 to perform any of their biological functions. An example of such an agonist is an agent that enhances the ability of ICAM-3 to bind to a cell receptor.

An ICAM-3 antagonist is a compound that reduces or impedes the ability of ICAM-3 to perform any of their 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, and the like.

The cell aggregation assays described in U.S. Pat. 07/045963, 07/115798, 07/155943, 07/189815 or 07/250446, LFA-1-dependent aggregation can be measured and can therefore be used to identify agents that affect the extent of ICAM-3 / LFA aggregation -1. Thus, these assays can be used to identify ICAM-3 agonists and antagonists. Antagonists may impair the ability of LFA-1 or ICAM-3 to mediate aggregation. Furthermore, it can be determined by said assay whether non-immunoglobulin (ie, chemical) agents are agonists or antagonists of ICAM-3 / LFA-1 mediated aggregation. These aggregation assays are most commonly performed using peripheral T cells stimulated

PMA. Thus, the binding of peripheral blood cells to LFA-1 is used to test for aggregation mediated by ICAM-3.

C. Anti-ICAM-3 antibody

A preferred immunoglobulin antagonist of the invention is an antibody to ICAM-3 as described herein by CBR-IC3 / 1. Other suitable anti-ICAM-3 antibodies can be obtained in various ways.

Antibodies to ICAM-3 (or functional derivatives of ICAM-3) can be either polyclonal or monoclonal. In addition, the antibodies of the invention may be humanized by methods known in the art or by those described in PCT Patent Application Nos. PCT / US91 / 02942 and PCT / US91 / 02946, filed at a reception site in the US on April 22, 1991.

Antibodies to ICAM-3 can be produced by introducing ICAM-3 or peptide fragments thereof into a suitable animal. The immunized animal produces a polyclonal antibody in response to such exposure. The use of peptide fragments of ICAM-3 makes it possible to obtain region-specific antibodies that only react with the epitope (s) contained in the peptide fragments used to immunize the animal.

Alternatively, antibodies to ICAM-3 can be produced using ICAM-3, which is naturally expressed on the lymphocyte surface. The introduction of such cells into an appropriate animal, for example by intraperitoneal injection, will produce antibodies capable of binding to ICAM-3 or members of the CD-18 cell family. If desired, the serum of such an animal can be collected and used as a source of polyclonal antibodies capable of binding to these molecules.

Alternatively, antibodies to ICAM-3 can be produced according to the method of Selden, R.F. (European Patent Application Publication No. 289034) or Selden, R.F. d. (Science 236: 714-718 (1987)). Specifically, cells of a suitable animal (e.g., mouse) are transfected with a vector capable of expressing either an intact ICAM-3 molecule or fragment thereof. Production of ICAM-3 in transfected animal cells elicits an immune response in the animal and results in the production of antibodies to the animal's ICAM-3.

However, it is preferred to remove splenocytes from animals immunized by any of the above methods to generate monoclonal antibodies capable of binding to ICAM-3.

The hybridoma cells obtained by the above method may be screened in various ways to identify a hybridoma cell that secretes an antibody capable of binding to ICAM-3. In a preferred screening assay, such molecules are identified by their ability to inhibit aggregation of cells expressing ICAM-3 and not expressing ICAM-1 and ICAM-2. Antibodies capable of such aggregation are then further screened to determine if they inhibit such aggregation by binding to ICAM-3 or a member of the CD-18 cell family. Any means capable of distinguishing ICAM-3 from the CD-18 family of molecules can be used in such screening. For example, the antigen bound by an antibody can be analyzed by immunoprecipitation and polyacrylamide gel electrophoresis. A distinction can be made between these antibodies, which bind to members of the CD-18 molecule family, and those that bind to ICAM-3, by screening for the ability of the antibody to bind to cells that express LFA-1 but not ICAM-3 ( or the other way). The ability of an antibody to bind to a cell expressing LFA-1 but not ICAM-3 can be detected by means used by those of ordinary skill in the art. These means include immunoassays (particularly using immunofluorescence), cell agglutination, filter binding studies, antibody precipitation, etc.

In a preferred method, the antibody is selected by its ability to bind to cells expressing ICAM-3, but not to cells that do not express ICAM-3.

Other agents, in addition to the above-mentioned ICAM-3 agonists and antagonists, which can be used in accordance with the 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 are capable of binding to ICAM-3.

Anti-idiotypic antibodies of interest for the invention are capable of binding in competition with (or at the expense of) ICAM-3. Such antibodies can be obtained, for example, by growing antibodies to an anti-ICAM-3 antibody and then screening the antibody for the ability to bind to one of the natural binding ligands of ICAM-3.

Any CD-18 family molecules capable of binding to ICAM-3 will be administered such molecules (e.g., as heterodimers containing both α and β subunits, or as molecules composed solely of α or β subunit, or as fragments of individual or both subunits) to compete with cells for binding to ICAM-3 present on the cell.

The anti-aggregation antibodies of the invention can be identified and titrated by any of a variety of methods. For example, the ability of antibodies to bind differently to cells that express ICAM-3 (such as T cells) and their inability to bind to cells that cannot express ICAM-3 can be measured. Suitable cell aggregation assays are described in U.S. Pat. 07/045963, 07/115798, 07/155943, 07/189815 or 07/250466. Alternatively, the capacity of antibodies to bind to ICAM-3 or a peptide fragment of ICAM-3 can be measured. As will be readily appreciated by one of ordinary skill in the art, the assays can be modified or performed in a different order to achieve a variety of potential screening assays, each of which is capable of identifying and distinguishing between antibodies capable of binding to ICAM-3 against CD- 18th

D. Preparation of the compositions of the invention

The compositions of the invention may be obtained by: natural processes (for example, by inducing the production of a non-immunoglobulin ICAM-3 antagonist in an animal, plant, fungus, bacterium, etc., or by producing polyclonal antibodies capable of binding to ICAM-3 in an animal);

synthetic methods (e.g., by synthesis of ICAM-3, functional derivatives of ICAM-3, or protein antagonists of ICAM-3 (either immunoglobulin or non-immunoglobulin));

hybridoma technology (e.g., to produce monoclonal antibodies capable of binding to ICAM-3) or recombinant technology (such as production of agents of the invention in various hosts (i.e., yeast, bacteria, fungi, cultured mammalian cells, etc.) using recombinant plasmids or viral vectors). The decision on which method to use will depend on factors such as convenience, desired yield, etc. However, it is not necessary to use only one of the aforementioned methods, processes or technologies to obtain a particular anti-inflammatory agent; the processes, methods and technologies may be combined to obtain a specific means.

VII. The use of ICAM-3 and its functional derivatives, agonists and antagonists

The compositions of the invention can be used to moderate the various biological effects that are mediated by ICAM-3.

A. Suppression of inflammation

One aspect of the invention derives from the ability of ICAM-3 and its functional derivatives to interact with the receptors of the CD-18 family of molecules, particularly LFA-1. Since ICAM-3 is capable of interacting with members of the CD-18 family of glycoproteins, it can be used to suppress (i.e., prevent or inhibit) inflammation.

The term inflammation herein includes both the response of a specific defense system and the reaction of a non-specific defense system.

The term specific defense system refers to that component of the immune system that responds to the presence of specific antigens. Inflammation is reported to result from a specific defense system response when the inflammation is caused, mediated or associated with a specific defense system response. Examples of inflammation due to a specific defense system response include responses to antigens such as measles virus, autoimmune diseases and delayed-type hypersensitivity responses mediated by T cells (as occurs, for example, in subjects having a positive Mantaux test). Other examples of specific immune system inflammatory reactions are chronic inflammatory diseases and rejection of whole transplanted tissue and organs, such as kidney and pith transplants.

The reaction of a non-specific defense system refers herein to a leukocyte-mediated reaction that is incapable of immunological memory. Such cells include granulocytes and macrophages. Inflammation here is the result of a non-specific defense system response if the inflammation is caused, mediated or associated with a non-specific defense system response. Examples of inflammation that result, at least in part, from a non-specific defense system response include inflammation associated with conditions such as adult respiratory distress syndrome (ARDS) or multiple organ trauma syndromes secondary to septicemia, trauma or bleeding; reperfusion injury myocardial or other tissue; acute glomerulonephritis; athritis reactins; dermatoses with acute inflammatory components; acute purulent meningitis or other inflammatory disorders of the central nervous system, such as stroke, heat injury, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, syndrome associated with granulocyte transfusion and cytokine-induced toxicity.

As mentioned above, the binding of ICAM-3 molecules to members of the CD-18 family of molecules is of central importance in cell adhesion. Through the adhesion process, lymphocytes are able to continuously monitor the animal for the presence of foreign antigens. While these processes are normally desirable, they also cause rejection of whole organ transplants (e.g., kidney), rejection of non-jawed organ transplants (e.g., bone marrow), tissue transplant rejection, and many autoimmune diseases. Therefore, any agent capable of inhibiting cell adhesion is highly desirable for recipients of whole organ transplants (especially kidney transplants), non-jaw organ transplants (particularly bone marrow transplants), tissue transplants, or autoimmune patients.

Monoclonal antibodies to members of the CD-18 family inhibit many adhesion-dependent functions of leukocytes, including binding to endothelium (Haskard, D. et al., J. Immunol. 137, 2901-2906 (1986)), homotypic adhesions (Rothlein, R. et al.). , J. Exp. Med. 163, 1132-1149 (1986)), antigens and mitogens induced by lymphocyte proliferation (Davignon, D. et al., Proc. Natl. Acad. Sci. USA 78, 4535-4539 (1981)) , antibody production (Fischer,

A. et al., J. Immunol. 136, 3198-3203 (1986)) and effector functions of all leukocytes, such as cytotoxic T cell lytic activity (Krensky, AM et al., J. Immunol. 132, 2180-2182 (1984)), macrophages (Strassman, G. and d., J. Immunol. 136, 4328-4333 (1986)) and all cells involved in antibody-dependent cellular cytotoxic reactions (Kohl, S. ad., J. Immunol. 133, 2972-2978 (1984)) . In all of the above functions, the antibodies inhibit the ability of leukocytes to adhere to the respective cellular substrate, which then inhibits the biological function associated with binding. Such functions, so long as they include ICAM-3 / LFA-1 interactions, can be suppressed by antibodies to ICAM-3.

Thus, monoclonal antibodies capable of binding to ICAM-3 can be used as anti-inflammatory agents in mammals. These compositions differ significantly from conventional anti-inflammatory agents in their ability to selectively inhibit adhesion without other side effects, such as the nephrotoxicity that occurs with conventional agents.

Since ICAM-3, especially in soluble form, is capable of acting in the same manner as an antibody to members of the CD-18 family, it can be used to suppress inflammation. In addition, functional derivatives and antagonists of ICAM-3 may be used to suppress inflammation.

1. Suppressors of delayed-type hypersensitivity reactions

ICAM-3 partially mediates instances of adhesion required to induce inflammatory reactions, such as delayed-type hypersensitivity reactions. Thus, antibodies (particularly monoclonal antibodies) capable of binding to ICAM-3 have therapeutic potential for inhibiting or eliminating such reactions.

Since ICAM-3 is an antagonist of the ICAM-1 / LFA-1 interaction, ICAM-3 (especially in solubilized form) or functional derivatives thereof may alternatively be used to suppress delayed-type hypersensitivity reactions.

These potential therapeutic effects can be used in two ways. On the one hand, a composition comprising a monoclonal antibody to ICAM-3 or a soluble derivative of ICAM-3 can be administered to a patient with delayed-type hypersensitivity reactions. Such a composition may, for example, be administered to an individual who has been in contact with antigens such as poisonous, poisonous oak, and the like. In another embodiment, the patient is administered a monoclonal antibody capable of binding to ICAM-3 in conjunction with an antigen to prevent a subsequent inflammatory response. Additional administration of antigen with antibody

Thus, ICAM-3 may temporarily tolerate an individual to the subsequent occurrence of the same antigen.

2. Therapy of chronic inflammatory disease

Since LAD patients lacking LFA-1 do not develop an inflammatory response, it is believed that antagonism of natural LFA-1 ligands also inhibits the inflammatory response. The ability of anti-ICAM-3 antibodies to inhibit inflammation provides: the basis for their therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes, Reynaheum syndrome arthritis, etc. Some antibodies may also be used in the treatment of psoriasis.

In general, the antibodies to ICAM-3 of the invention may be administered alone or in combination with antibodies to ICAM-1 and / or ICAM-2 in the treatment of diseases commonly treated with steroid therapy.

According to the invention, inflammatory and immune rejection responses can be suppressed (i.e., prevented or suppressed) by administering to a subject in need thereof an anti-inflammatory agent in an amount capable of suppressing such inflammation. Suitable anti-inflammatory agents include: an antibody capable of binding to ICAM-3; a fragment of such an antibody capable of binding to ICAM-3; substantially pure ICAM-3; a functional derivative of ICAM-3; a non-immunoglobulin ICAM-3 antagonist or a non-immunoglobulin ICAM-3 antagonist other than LFA-1. Especially 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 a composition selected from the group consisting of an antibody capable of binding to LFA-1; a non-immunoglobulin LFA-1 antagonist; substantially pure ICAM-1 and / or ICAM-2 or derivatives thereof, or an antibody to ICAM-1 and / or ICAM-2 or a fragment thereof.

The invention further encompasses the aforementioned methods of suppressing the inflammatory response of a specific defense system by further administering to the subject an immunosuppressive agent. Such a composition is preferably administered at a dosage lower (i.e., sub-optimal dosage) than would normally be necessary. The use of a suboptimal dose is possible due to the synergistic effect of the compositions of the invention. Examples of suitable immunosuppressive agents include, but are not limited to, dexamethasone, azathioprine, ICAM-1, ICAM-2, or cyclosporin A.

3. Therapy of non-specific inflammations.

Many inflammatory reactions are the result of non-specific defense system reactions and are mediated by leukocytes that are incapable of immunological memory. Such cells include granulocytes and macrophages. Inflammation herein is referred to as a non-specific defense system response when it is caused, mediated or associated with a non-specific defense system response. Examples of inflammation that are at least partially due to a non-specific defense system response include inflammation associated with conditions such as adult respiratory distress syndrome (ARDS) or multiple organ injury syndromes secondary to septicemia, trauma or bleeding; reperfusion injury to myocardial or other tissue; acute glomerulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other inflammatory disorders of the central nervous system, such as stroke, thermal injury, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, syndromes associated with granulocyte transfusion and cytokine-induced toxicity.

The anti-inflammatory compositions of the invention are compounds capable of specifically antagonizing the interaction of the CD-18 complex on granulocytes with endothelial cells. Such antagonists include; ICAM-3, a functional derivative of ICAM-3, non-immunoglobulin-1-.txΆΧ *., ./-'- ιΙλ.x - * - i --- * -. '·.,. '-.t · ·. ..... · „. · ,. r. A new ICAM-3 antagonist other than ICAM-1, ICAM-2, or a member of the CD-18 family of molecules.

B. Organ and tissue rejection suppressors

Since ICAM-3, especially in soluble form, is capable of acting in the same way as an antibody to members of the CD-18 family, they can be used to suppress organ or tissue rejection responses caused by any of the cell adhesion-dependent functions. In addition, an antibody to ICAM-3, functional derivatives of ICAM-3, and antagonists of ICAM-3 can be used to suppress such rejection reactions.

ICAM-3 and antibodies to ICAM-3 can be used to prevent rejection reactions in whole organs or tissues such as kidneys, rejection of non-jaw organs such as bone marrow, or to modify autoimmune responses in mammalian subjects. Significantly, the use of monoclonal antibodies capable of recognizing ICAM-3 may allow organ transplantation to be performed in non-HLA-compliant individuals.

C. Addition to the introduction of antigenic materials administered for therapeutic or diagnostic purposes

The immune response to therapeutic or diagnostic agents, such as bovine insulin, interferon, tissue-type plasminogen activator, or murine monoclonal antibodies, substantially impair the therapeutic or diagnostic value of such agents, and in some cases, cause diseases such as serum disease. This situation can be solved by using the antibodies of the invention. In this embodiment, antibodies to ICAM-3 would be administered in combination with a therapeutic or diagnostic agent. The addition of antibodies prevents the recipient from recognizing the composition and thus developing an immune response against it. The absence of such an immune response allows the patient's ability to receive additional doses of the therapeutic or diagnostic agent.

ICAM-3 (especially in solubilized form) or functional derivatives thereof, alone or in combination with ICAM-1 or ICAM-2 or antibodies capable of binding to LFA-1, may be used in the treatment of the disease. Thus, in a solubilized form, such molecules may be used to inhibit organ or transplant rejection. ICAM-3 or functional derivatives thereof may be used in the same manner as antibodies to ICAM-3 to reduce the immunogenicity of therapeutic or diagnostic agents.

D. Tumor metastasis suppressors

The compositions of the invention can be used to suppress hematopoietic tumor cell metastasis that requires a functional member of the CD-18 family to migrate. In accordance with this embodiment of the invention, the patient is administered a composition (such as an antibody capable of binding to ICAM-3; toxin derivatizing such an antibody; an antibody fragment capable of binding to ICAM-3; toxin derivatizing said fragment; substantially pure ICAM-3; ICAM-3 or a non-immunoglobulin antagonist (ICAM-3 other than ICAM-1 or ICAM-2) in an amount sufficient to inhibit said metastasis.

A further embodiment of the invention provides a method of suppressing the growth of ICAM-3 expressing tumor cells. In particular, the method comprises administering the composition to a patient in an amount sufficient to inhibit such growth. Suitable compositions include an antibody capable of binding to ICAM-3; toxin derivatization of such an antibody; an antibody fragment capable of binding to ICAM-3; toxin derivatization of this fragment; a toxin derivatized member of the CD-18 molecule family or a toxin derivatized functional derivative of the member of the CD-18 molecule family.

A further embodiment of the invention provides a method of suppressing the growth of a tumor cell expressing LFA-1. In particular, the method comprises administering the toxin to the patient in an amount sufficient to inhibit this growth. Suitable toxins include a toxin derivatized ICAM-3 or a toxin derivatized functional derivative of ICAM-3.

E. Suppressing HIV infection and preventing the spread of HIV-infected cells

A further embodiment of the invention provides a method of controlling HIV infection. In particular, the method comprises administering to an individual infected with HIV an effective amount of an anti-HIV agent. Although the invention relates in particular to a method of suppressing HIV-1 infection, it is to be understood that the method is applicable to any variant of HIV (such as HIV-2) that can infect cells in a manner that can suppress the compositions of the invention . Such variants are HIV equivalents for the purposes of this invention.

One aspect of the invention is derived from the finding that expression of LFA-1 and, in some cases, LFA-1 binding ligand, stimulated by HIV infection, promotes cell-to-cell adhesion responses that can increase the contact time of infected cells with uninfected cells. uninfected cells. Compositions of the invention that are capable of modulating LFA-1 / ligand interactions can then suppress infection of HIV, particularly HIV-1. One way in which molecules that inhibit LFA-1 / ligand interactions can suppress HIV infection is by impairing the ability of LFA-1 ligand, expressed by HIV-infected cells, to bind to healthy T cell CD11 / CD18 receptors. Alternatively, molecules that inhibit LFA-1 / ligand interactions may impair the ability of CD11 / CD18 receptors, expressed by HIV-infected cells, to bind to LFA-1 healthy T cells. ICAM-3, an ICAM-3 fragment, a functional derivative of ICAM-3, or an antibody to ICAM-3 may be used to reduce the ability of a cell to bind to the CD11a / CD18 receptor or the LFA-1 binding ligand.

The compositions of the invention are intended to be administered to a recipient subject in an amount sufficient to suppress HIV infection. The amount may be said to be sufficient to suppress HIV infection, if dosing, route of administration, etc. the composition is sufficient to attenuate or prevent HIV infection. The formulations should be administered to patients who are at risk or infected with HIV infection.

The compositions of the invention may be used in the treatment of HIV infection for either prophylactic or therapeutic purposes. When used prophylactically, the composition is administered prior to the appearance of any symptom of a viral infection (for example, before, at or shortly after, but before any symptom of such infection). Prophylactic administration of the composition serves to prevent or control any subsequent HIV infection. When used therapeutically, the composition is administered at (or shortly after) the detection of virally infected cells. Therapeutic administration of the composition serves to attenuate any other HIV infection.

OJ

Thus, the compositions of the invention may be administered either prior to the onset of viral infection (to suppress a predicted HIV infection) or after the actual detection of such virally infected cells (to suppress further infection).

In another embodiment, the invention provides improved AIDS therapy and an improved means for suppressing HIV infection, particularly for HIV-1 infections, comprising co-administering the following components:

(I) ICAM-3, soluble derivative of ICAM-3, CDU (either CD11a or CD11b or CD11c), soluble derivative of CDU, CD18, soluble derivative of CD18 or heterodimer of CD11 / CD18 or soluble derivative of heterodimer of CD11 / CD18 and / or (II) an antibody capable of binding to ICAM-3 with a (III) CD4 derivative associated with a cell or particle, or a soluble CD4 derivative and / or (IV) molecule (preferably an antibody or antibody fragment) capable of binding to CD4.

A further embodiment of the invention provides a method of suppressing the migration of HIV infected cells. In particular, the method comprises administering an effective amount of an anti-migration agent to an individual infected with HIV.

The anti-migration agents of the invention include ICAM-3, an ICAM-3 fragment, a functional derivative of ICAM-3, or antibodies to ICAM-3 that are capable of reducing the ability of HIV-infected T cells to bind to LFA-1 ligand. Antibodies that bind to ICAM-3 suppress migration by decreasing the ability of ICAM-3, expressed by HIV-infected T cells, to bind to cells expressing the CD11 / CD18 receptor. An antibody capable of binding to ICAM-3 may be used to reduce the ability of a cell to bind to the CD11a / CD18 receptor.

The compositions of the invention are intended to be administered to subjects in an amount sufficient to suppress the migration of cells infected with HIV (or other virus). The amount is indicated to be sufficient to suppress T cell migration, if dosing, route of administration, etc. the means is sufficient to dampen or prevent this migration.

These compounds can be used for either prophylactic or therapeutic purposes. When used prophylactically, the compositions of the invention are administered prior to the appearance of any symptom of a viral infection (for example, before, at or shortly after, but before any symptom of such infection). Prophylactic administration of the compositions serves to prevent or inhibit any subsequent T cell migration. When used therapeutically, the composition is administered at (or shortly after) detection of virally infected T cells. Therapeutic administration of the composition serves to attenuate any further migration of these T cells.

Thus, the compositions of the invention may be administered either prior to the onset of viral infection (to suppress the predicted T cell migration) or after the actual detection of such virally infected cells.

F. Treatment of asthma

In another embodiment of the invention, a composition capable of modulating LFA-1 / ICAM-3 indications is used in the treatment of asthma. In particular, the method comprises administering an effective amount of an antiasthmatic agent to the affected individual.

The antiasthmatic compositions of the invention include ICAM-3, an ICAM-3 fragment, a functional derivative of ICAM-3, or antibodies to ICAM-3 that are capable of reducing the ability of a cell to bind to an LFA-1 ligand. Antibodies that bind to ICAM-3 inhibit the migration of eosinophils by reducing the ability of ICAM-3 expressed on these cells to bind to cells expressing the CD11 / CD18 receptor.

The antiasthmatic compositions of the invention are intended to be administered to subjects in an amount sufficient to reduce or attenuate the severity, extent, or duration of asthma symptoms.

The compositions of the invention may be administered either alone or in combination with one or more other antiasthmatic agents, for example, methylxanthines (such as theophylline), beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins and ephedrine), glucocorticoids (such as hydrocortisone), chromones (such as sodium cromolyn) and anticholinergics (such as atropine) to reduce the amount of these agents required to treat the symptoms of asthma.

The compositions of the invention may be used for either lactose or therapeutic purposes. When used prophylactically, the composition is administered prior to the appearance of any symptom of asthma. Prophylactic administration of the composition serves to prevent or attenuate any subsequent asthmatic response. When used therapeutically, the composition is administered at (or shortly after) the onset of an asthma symptom. Therapeutic administration of the composition serves to attenuate any current asthma episode. Thus, the compositions of the invention may be provided either before the onset of a predicted asthmatic episode (to attenuate the predicted severity, duration or extent of an episode) or after the start of an episode.

G. Diagnostic and prognostic applications

Monoclonal antibodies capable of binding to ICAM-3 can be used as a means to visualize or visualize ICAM-3 expression sites and inflammation in a patient. In this application, monoclonal antibodies to ICAM-3 are detectably labeled using radioisotopes, affinity labeling (such as biotin, avidin, etc.), fluorescent labeling, or paramagnetic atoms. Procedures for such labeling are known. The labeled antibodies can then be used in a diagnostic representation. Clinical applications of antibodies in diagnostic imaging are summarized in Grossman, H.B. Urol. Clin. North Amer. 13, 465-474 (1986)), Unger, E.C. et al., Invest. Radiol. 20, 693-700 (1985)) and Khaw, B.A. et al., Science 209, 295-297 (1980)).

The presence of expression of ICAM-3 can also be detected using hybridization probes such as mRNA probes, cDNAs, genomic DNA or synthetic oligonucleotides that bind to ICAM-3 gene sequences or ICAM-3 mRNA sequences present in a cell that expresses ICAM3. Methods for carrying out such hybridizations are described by Maniatis, T. et al., Molecular Cloning, and the Laboratory Manual, Coldspring Harbor, NY (1982), and Haymes, B.D. and d., Nucleic Acid Hybridization, and Practical Approach, IRL Press, Vashington, DC (1985).

Detection of ICAM-3 expression foci using labeled nucleic acid-based antibodies or probes can be used in the diagnosis of tumor development. In one diagnostic embodiment, a tissue or blood sample is taken from a subject and incubated in the presence of antibodies with or which can be detectably labeled. Preferably, the method is performed in a non-invasive manner using magnetic imaging, fluorography, and the like. This diagnostic test can be used to monitor recipients of transplanted organs, such as the kidney, for early signs of potential rejection, tissue. These assays may also serve to determine a subject's predisposition to rheumatoid arthritis or other chronic inflammatory diseases.

For example, by radiolabeling antibodies to ICAM-3 or antibody fragments, antigen can be detected using radioimmunoassays. A good description of radioimmunoassays (RIAs) can be found in Laboratory Techniques and Biochemistry in Molecular Biology, Vork, T.S. et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter An Introduction to Radioimmune Assay and Related Techniques, Chard,

Alternatively, the antibody may be labeled with a fluorescent compound, enzyme, or other suitable known means.

In addition to localizing inflammatory sites, antibodies capable of binding to ICAM-3 can be used to test biological fluids for the presence of circulating ICAM-3 using any commonly known fluid testing medium. The presence of ICAM-3 in the fluid sample indicates an inflammatory response or other biological function mediated by ICAM-3. In addition, the presence of ICAM-3 in amniotic fluid is associated with complications arising during pregnancy. Any biological fluid may be used for the assay. However, preferred fluids are blood, serum, plasma, synovial fluid, amniotic fluid, mouse fluid, or urine.

VIII. Administration of the compositions of the invention

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

ICAM-3 and its functional derivatives can be obtained synthetically using recombinant DNA technology, proteolysis, or a combination of these methods. The therapeutic advantages of ICAM-3 can be enhanced by the use of functional derivatives of ICAM-3 containing additional amino acid residues to improve carrier binding or to enhance ICAM-3 activity. The invention is further intended to include functional derivatives of ICAM-3 which lack certain amino acid residues insofar as these derivatives have or affect the biological or pharmacological activity of ICAM-3.

Both the antibodies of the invention and the ICAM-3 molecules of the invention are said to be substantially free of natural impurities when the formulations containing it are substantially free of the substances with which these products are usually and naturally found.

The invention relates to antibodies and biologically active fragments thereof (whether polyclonal or monoclonal) that are capable of binding to ICAM-3. These substances can be produced either by animal or tissue culture or by recombinant DNA.

The administration of ICAM-3 or molecules derived from ICAM-3 may be performed alone or in combination with ICAM-1 and / or ICAM-2. Administration of antibodies to ICAM-3 or other molecules capable of binding to ICAM-3 or to molecules derived from ICAM-3 may be performed alone or in combination with antibodies to ICAM-1 and / or antibodies to ICAM-2. When administering antibodies or antibody fragments capable of binding to ICAM-3 to a patient or administering ICAM-3 (or fragments, variants, or derivatives thereof) to a patient, the dosage of the composition administered will vary depending upon such factors as the patient's age, weight, height, gender, general health, history of previous medical treatment, etc. In general, it is desirable to provide a patient with an antibody dose in the range of about 1 µg / kg to 10 mg / kg (patient's body weight), but lower and higher doses may also be administered. When administering ICAM-3 molecules or functional derivatives thereof to a patient, it is preferable to administer such molecules at a dose which is also from about 1 pg / kg to 10 mg / kg (patient body weight), but lower and higher doses may also be administered. . As discussed below, the therapeutically effective dose can be reduced when the antibody to ICAM-3 is administered concurrently with the antibody to LFA-1, the antibody to ICAM-1, and / or the antibody to ICAM-2. Co-administration of the two compounds herein means that both compounds are administered at such a short time interval that they can be detected concurrently in the patient's serum.

Both antibodies capable of binding to ICAM-3 and ICAM-3 alone can be administered to a patient intravenously, intramuscularly, subcutaneously, enterally, topically, or parenterally. For injection of antibodies or ICAM-3, continuous injection or single or multiple boluses may be used.

The compositions of the invention are intended to be administered to subjects in an amount sufficient as physiologically effective. The amount is referred to as physiologically effective, if dosing, route of administration, etc. The composition is sufficient to attenuate or prevent the physiological effect associated with ICAM-3. For example, when one of the compositions of the invention is administered to a patient with the intention of suppressing inflammation, it is referred to as a physiologically effective, administered at a dose sufficient to suppress inflammation.

Further, the antibodies to ICAM-3 or fragments thereof may be administered either alone or in combination with one or more immunosuppressive agents (particularly a recipient of a transplanted organ or tissue). Administration of such compound or compounds may serve either prophylactic or therapeutic purposes. When used prophylactically, the immunosuppressive compound is administered prior to any inflammatory response or symptom (for example, before or shortly after organ or tissue transplantation, but before any symptom of organ rejection). The prophylactic administration of the compounds serves to prevent or attenuate any subsequent inflammatory response (such as rejection of a transplanted organ or tissue, etc.). When used therapeutically, the compound is administered at (or shortly after) the onset of signs of actual inflammation (such as rejection of an organ or tissue). Therapeutic administration of the compounds serves to attenuate any concomitant inflammation (such as rejection of a transplanted whole organ or tissue, for example, kidney, or non-jawed organ, for example bone marrow).

Thus, the anti-inflammatory compositions of the invention may be administered either before the onset of inflammation (to suppress the predicted inflammation) or after the onset of inflammation.

The composition is said to be pharmacologically acceptable if its administration can be tolerated by the patient. The amount of such composition administered is said to be therapeutically effective if the amount administered is physiologically significant. A composition is physiologically significant if its presence results in a detectable change in the physiology of the patient.

The antibodies and ICAM-3 molecules of the invention may be formulated according to known methods for preparing pharmaceutically useful compositions, wherein the compounds or functional derivatives thereof are combined with a pharmaceutically acceptable carrier vehicle. A suitable vehicle and its formulation, including other human proteins, such as human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). When formulating a pharmaceutically acceptable composition suitable for effective administration, such composition should contain an effective amount of a composition of the invention together with a suitable amount of carrier. Further, the antibodies of the invention can be humanized by chimerizing or grafting CDRs to increase pharmacological acceptability for a patient. These methods of chimerizing antibodies, particularly antibodies to ICAM-3, are described elsewhere; Here, reference can be made to British Patent Application Nos. 9009548.0.

And PCT Application Nos. PCT / US91 / 02942 and PCT / US91 / 02946, filed at the US Receiving Office on April 27, 1991.

Other pharmaceutical methods can be used to control the duration of action. Controlled release formulations can be obtained using polymers to complex or absorb the compositions of the invention. The rate and time of controlled release can be controlled to some extent by the choice of a suitable macromolecular matrix, by varying the concentration of the incorporated macromolecules and the methods of incorporating them. Another possible method of controlling the controlled release time to particles of polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene / vinyl acetate copolymers. Alternatively, instead of incorporating these compositions into polymer particles, they may be encapsulated in microcapsules prepared, for example, by coacervation or phase-bound polymerization, in gelatin or poly (methyl methacrylate) capsules or in colloidal drug release systems such as liposomes, albumin microspheres, microemulsions , nanoparticles and nanocapsules, or in macroemulsions.

The invention further encompasses a pharmaceutical composition comprising (a1 anti-inflammatory agent (such as an antibody capable of binding to ICAM-3), a fragment of said antibody capable of binding to ICAM-3, ICAM-3, a functional derivative of ICAM-3 or a nonimmunog57 lobulin antagonist ICAM-3. other than ICAM-1 and ICAM-2, and (b) at least one immunosuppressive agent Examples of suitable immunosuppressive agents include dexamethasone, azathioprine and cyclosporin A.

DETAILED DESCRIPTION OF THE INVENTION

Once the invention has been described in general terms, the compositions of the invention and methods for their preparation will be explained in more detail with reference to examples, which are not to be construed as limiting the invention.

Example 1

Characteristics of ICAM-3, a novel adhesion ligand for LFA-1

Cell Line and Lymphocyte Adhesion, Dustin et al., Cold Spring Harbor Symp. Quant. Biol. 54, 753-765 (1989)) to plates coated with LFA-1 was performed as previously described. LFA-1, purified from JY lysates, was absorbed into 96 well microtiter plates (Linbro-titertek) at 1100 sites / pm. Non-specific binding sites were blocked with 1% BSA and washed with PBS / 5% FBS / 2mM MgCl 2 / 0.5% HSA (assay medium). Specific inhibition of LFA-1 was achieved by incubating microtiter wells with 1/200 dilution of TS1 / 22 ascites (anti-LFA-Ια) for 30 min. at room temperature. The remaining T cells were isolated from whole blood by plastic adhesion and nylon wave filtration and showed 91% CD2 ⁺ , while cell culture in the presence of 10 µg / ml phytohemagglutinin (PHA) generated PHA blasts. Cells were labeled with BCECF fluorochrome (Molecular Probes Inc.), washed and resuspended in assay medium. MAb pretreatment of cells consisted of incubation with a 1/200 dilution to ascites for 45 min. at 4 ° C, 10 ⁵ cells were transferred to each well. The cell lines showed adhesion to solid 1FA-1 for 1 h at 37 ° C and cells without adhesion were aspirated six times with needle # 1. 23, while lymphocytes were centrifuged min. at 30 xg and incubated for 30 min. at 37 ° C. Unbound lymphocytes that were removed by flushing the medium 8 times from the plate with an addition of 100 between flushes. Flushing was more effective in completely removing unbound T lymphocytes, which were difficult to remove due to their small size. Fluorescence was determined directly from 96 well plates using a fluorescence concentration analyzer (Baxte). All MAbs were used at ascites saturation concentration (1/200) and include TS1 / 22 (anti-CD11a, IgG1) (Sanchez-Madrid et al., Proc. Natl. Acad. Sci USA 79, 7489-7493 (1982)), RR1 / 1 (anti-ICAM-1, IgG1) (Rothlein et al., J. Immunol. 137, 1270-1274 (1986)), CBR-IC2 / 2 (anti-ICAM-2, IgG2a) (de Fougerolles and d., J. Exp. Med, arrived (1991) (in print)) and CBR-IC3 / 1 (anti-ICAM-3, IgG1). CBR-IC3 / 1 was derived from the condensation of mouse myeloma P3X63Ag8.653 with spleen cells from Balb / cs mice injected with SKV3-drawn (Gefter et al., Som. Cell Gen. 3, 231-236 (1977)) and 600 hybridomas were subjected to screening for the ability to inhibit SKV3 binding to purified LFA-1. CRB-IC3 / 1 was shown not to respond to purified LFA-1 or to COS cells transfected with either ICAM-1, ICAM-2 or LFA-1 cDNA (data not shown). One of four representative experiments is shown and error bars indicate one standard deviation.

Example 2

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

The cell lines used have already been described by Hibbs et al., J. Clin. Invest. 85, 675-681 (1990)). Peripheral blood mononuclear cells (PBMC) were obtained by dextran sedimentation and centrifugation of Ficoll-Hypaque (1.077) according to (Dustin et al., J. Cell. Biol. 107, 321-331 (1988)). Neutrophils and contaminating erythrocytes were recovered from established cells. were removed by hypotonic decomposition. Lymphocytes and monocytes were separated by cytometric analysis using direct and perpendicular light scattering with confirmed specific MAb rnonocytes and T cells. Plastic adhesion was used to enrich lymphocytes from PBMCs and the cells were cultured in the presence of 10 µg / ml phytohemagglutinin (PHA). Samples were analyzed using an EPICS V flow cytometer and fluorescence determined using EPICS Immune-Brite fluorescent beads (Coulter) to calibrate the cytometers. The expression of ICAM-3 on resting lymphocytes was 2-3 times greater than CD3 or LFA-1, while monocytes expressed 3-4 times more ICAM-3 than LFA-1. The expression level of ICAM-3 on neutrophils was equivalent to Mac-1 (CD11b / CD18). Treatment of cells with phospholipase C showed that there was no PI-associated form of ICAM-3.

Table 2. Relative surface antigen expression of ICAM by immunofluorescence flow cytometry specific linear fluorescence intensity * cell line / aICAM-1 and ICAM-2 and ICAM-3 / tvp_ (RR1 / 1) (CBR-IC2 / 1) (CBR-IC3 / 1)

peaceful lymphocytes 4 22 106 PHA daily blasts 22 18 78 3-day PHA blasts 85 42 205 5-day PHA blasts 25 45 223 monocytes 13 39 224 neutrophils 1 0 213 lymphoblastoid T SKW3 0 98 73 J urkat 2 166 161 Sup T 10 113 19 Molt 4 1 242 117 lymphoblastoid B JY 154 119 47 S LA 224 113 306 Ramos 132 136 112 Raj i 266 88 0 monocytic U937 62 67 37 HL60 17 43 203 melanomas BK 896 3 0 RPMI 7591 475 0 0 erythroleukaemia K562 293 143 0 * * Differently HUVEC 31 494 0 Hep G2 1526 41 0 HeLa 1082 0 RD3 / 5 0 9 0 FS1,2,3 409 0 0 A-172 0 0 0

* Membrane expression determined by immunofluorescence flow cytometry. Values are determined from at least two experiments. Fluorescent beads are used to calibrate the cytometers

And one unit is approximately 10 equivalents of fluorescein (Coulte Diagnostics, Hialeah, FL).

** Various cell lines include: human umbilical vein endothelial cells (bovine), human breast carcinoma Hep G2, human epitheloid carcinoma cell line HeLa, human rhabdomyosarcoma RD 3/5, human fibrosarcoma FS 1,2,3, and human glioblastomas.

Example 3

Immunoprecipitation of ICAM-3

Cell surface staining with iodogen was performed (Kishimoto et al., J. Biol. Chem. 264, 3588-3595 (1989)). Cells were lysed with triton X-100 (1

%). Lysates purified by Sepharose with bound IgG bovine and then incubated for 2 hours with Sepharose with bound MAb. The beads were washed and boiled in sample buffer containing 50 mM Tris, 1% SDS and 1%

2-mercaptoethanol. Non-reduced samples were boiled in a buffer solution without 2-mercaptoethanol and treated with 20 mM iodoacetamide. Samples were analyzed on a polyacrylamide gel on a 7% vertical slope plate (Laemmli, UK, Nature 227, 680-685 (1970)) and proteins were visualized by autoradiography. Treatment of the sample with N-glycanase (Genzyme) (Tarentino et al., Biochemistry 24, 4665-4671 (1985)) at a concentration (10 units / ml, 37 ° C, 18 h) was determined to provide optimal cleavage of N-linked oligosaccharides from the peptide chain. Class I MHC contains N-linked carbohydrate, while ICAM-2 (m _r 60000, 6 N-linked sites, chain M _r

28383) is high glycosylated.

- ýý:

: 2xi'á;

62 Example 4

Distribution of ICAM-3

Antibodies to ICAM-3 of the invention were used to further determine the distribution of ICAM-3 protein by flow cytometry. Cell types that exhibit surface expression of ICAM-3 are included in Table 3. Expression of ICAM-3 appears to be limited to hematopoietic cells. Flow cytometry data were confirmed by immunohistochemical staining of tissue sections using labeled antibodies to ICAM-3 (data not shown). As with flow cytometry data, immunohistochemical studies have shown that expression of ICAM-3 appears to be confined to hemopoietic cells.

Table 3. Relative surface antigen expression of ICAM by immunofluorescence flow cytometry specific linear fluorescence intensity * cell line / aICAM-1 and ICAM-2 and ICAM-3 / tVP_ (RR1 / 1) (CBR-IC2 / 1) (CBR-IC3 / 1)

peaceful lymphocytes 4 22 106 PHA daily blasts 22 18 78 3-day PHA blasts 85 42 205 5-day PHA blasts 25 45 223 monocytes 13 39 224 neutrophils 1 0 213 lymphoblastoid T SKW3 0 98 273 J urkat 2 166 161 Sup T 10 113 19 Molt 4 1 242 117 lymphoblastoid B JY 154 119 47 S LA 224 113 308 Ramos 132 136 112 Raj i 266 88 0 monocytic U937 62 67 37 HL60 17 43 203 melanomas BK 896 3 0 RPMI 7591 475 0 0 erythroleukemia K562 293 143 0 _Λ * * Differently HUVEC 31 494 0 Hep G2 1526 41 0 HeLa 1082 0 0 RD3 / 5 0 9 0 FS1,2,3 409 0 0 A-172 0 0 0

* Membrane expression determined by immunofluorescence flow cytometry. Values are determined from at least two experiments. Fluorescent beads are used to calibrate the cytometer and one unit is approximately 10 equivalents of fluorescence (Coulte Diagnostics, Hialeah, FL).

** Different cell lines include: human umbilical vein endothelial cells (hippocampus), human breast carcinoma Hep G2, human epitheloid carcinoma cell line HeLa, human rhabdomyosarcoma RD 3/5, human fibrosarcoma FS 1,2,3, and human glioblastoma.

Example 5

ICAM-3 purification

ICAM-3 were purified by immunoaffinity chromatography where the antibody to ICAM-3 CBR-IC3 / 1 was immobilized by known methods on a matrix. Various sources were used as starting material for the isolation of ICAM-3. They include, but are not limited to, SKV3, the human thymoma cell line, and the human tonsil. For the purification of ICAM-3, essentially the same methods were used as in deFougerolles et al., J. Exp. Med. 175: 185-190 (1992). The wash and elution regimen will be readily determined by one skilled in the art (the reagent will vary slightly with each purified antigen and each antibody used in immunoaffinity chromatography).

As discussed below, ICAM-3, purification from SKV3 cells or human tonsil cells, has a molecular weight of about 120 to 124 kD, while ICAM-3, purified from neutrophil derived lysates, provides a broad band having a molecular weight of about 120 to 124 kD. 150 kD.

ICAM-3, purified in this manner, showed that they retain the ability to bind antibodies to ICAM-3 and LFA-1 to various cells (Figures 6 and 7).

Example 6

Identification of different molecular weights of ICAM-3

Immunoprecipitation and polyacrylamide gel electrophoresis analysis were used to determine the molecular weight of ICAM-3 on various cell types. The molecular weight of ICAM-3, immunoprecipitated from neutrophils and immunoprecipitated from lymphocytes, was found to vary. ICAM-3, isolated from lymphocytes and lymphoid cell lines, appears as a band of about 120 to 124 kD molecular weight. The molecular weight of ICAM-3 isolated from neutrophils is slightly higher than that expressed by lymphoid cells and appears as a diffusion band of about 120-150 kD (Fig. 4).

These size changes are not uncommon in members of the ICAM glycoprotein family. ICAM-1 exhibits similar molecular weight movement between different cell types. The molecular weight changes observed with ICAM-1 were shown to be due to a change in the extent of glycosylation. Molecular weight changes of ICAM-3 are highly likely due to differences in glycosylation. This can be readily confirmed by those skilled in the art by subjecting ICAM-3, isolated from neutrophils, to various glycosidases and other enzymes, and observing the effect on molecular weight.

Changes in the extent of glycosylation of ICAM-1 were found to affect MAC-1 binding (CD11b / CD18). Thus, by varying the extent of glycosylation of ICAM-3, it is possible to generate ICAM-3 derivatives having modified affinity for binding to various ICAM-3 ligands, for example, members of the CD11 / CD18 glycoprotein family.

Example 7

ICAM-3 antibodies

Mice were injected with a combination of vehicle and ICAM-3 protein66 purified from SKV3 or tonsil cells as described above. Monoclonal antibodies were generated from immunized animals in a known manner.

Table 4 summarizes the various antibodies obtained to ICAM-3. Various antibodies were identified based on their ability to respond by ELISA to purified immobilized ICAM-3. Positive mAbs were then screened for various cell lines known to be ICAM-3 positive and then immunoprecipitated from radiolabeled ICAM-3 positive cell lysates. mAbs were also tested for the ability to block PMA-induced SKV3 cell aggregation in the presence of antibodies to ICAM-1 and ICAM-2. Alternatively, antibodies to ICAM-3 can be identified by their ability to inhibit binding of SKV3 cells to immobilized purified ICAM-3.

One of a number of evidence that there is a third LFA-1 ligand was the presence of an ICAM-1 / ICAM-22 independent pathway for PMA-stimulated SKV3 cell aggregation. We were able to block this aggregation with either a mouse polyclonal antiserum to ICAM-3 or a combination of CBR-IC3 / 1 and CBR-IC3 / 2.

Table 4. ICA-3 mAb-induced PMA__aggregations * mAb_isotype_on_ alone + IC1 + 2mAb + IC1 + 2 + IC3 / 1mAb

CBR-IC3 / 2 IgG2a, k 3 2 0 CBR-IC3 / 3 IgG2a, k 4 3 2 CBR-IC3 / 4 IgM, k 4 3 2-3 CBR-IC3 / 5 Ig2a, k 2 2 0-1 CBR-IC3 / 6 happen. 5 5 5 antiserum ICAM-3 3 0 0 CBR-IC3 / 1 IgG1, k 5 4 - HP2 / 19 ** 1 * Applies to the scope aggregation in J. Exp. Mied. 1991 0 - no aggregation

- 67:: χ · -. ·. ·.-..-.

** Another ICAM-3 mAb from a laboratory in Spain (F. Sanchez-Madrid). After reporting our results, they remembered the mAb they had prepared but never characterized. It turned out to be ICAM-3.

CBR-IC3 / 2, 3/3, 3/5 immunoprecipitate the same 120 kD band as CBR-IC3 / 1. Western Blot Testing.

Example 8

Immunoassays with antibodies to ICAM-3

Antibody to ICAM-1 (RR1 / 1), Antibody to ICAM-2 (CBR-IC2 / 2), and Antibodies to ICAM-3 (combinations of CBR-IC3 / 1 and CBR-IC3 / 2) were tested for blocking ability (1). PMA-stimulated SKV3 cell adhesion to purified ICAM, (2) phytohemagglutinin (PHA) stimulation of cell division, and (3) mixed lymphocyte response (MLR).

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

The ability of various antibodies to block binding of PMA stimulated SKV3 cells to purified ICAM-1 and ICAM-3 was examined. CBR-IC3 / 1 and CBR-IC3 / 2 antibodies, when present individually, do not effectively block the binding of PMA-stimulated SKV3 cells to purified ICAM-3. However, binding of ΡΜΑ-stimulated SKV3 cells to ICAM-3 can be blocked using a combination of the two antibodies. As reported by deFougerolles in J. Exp. Med., The monoclonal antibody CBR-IC / 3/1 was itself able to block the binding of ICAM-3 to purified LFA-1.

Binding of PMA-stimulated SKV3 cells to ICAM-3 was further analyzed to determine if it was temperature and temperature dependent

cation. As with ICAM-1, ICAM-3 / LFA-1 binding was found to be temperature-dependent (see Figure 8) and cation-dependent (data not shown).

Fig. 9 shows a summary of the effects of various antibodies on PHA stimulation of cell division. As previously reported (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 antibodies to ICAM-1, antibodies to ICAM-2. or combinations of antibodies to ICAM-1 and antibodies to ICAM-2 had little effect on PHA stimulated cell division. However, a combination of 1) antibodies to ICAM-1, to ICAM-2 and ICAM-3, 2) two antibodies to ICAM-3 (IC3 / 1 and IC3 / 2) and 3) antibodies to ICAM-1 and ICAM-3 were effective in blocking PHA-stimulated cell division.

Example 9

Mixed lymphocyte reaction

The MLR test is used to detect immunological reactivity. It consists essentially in adding chemically fixed foreign cells (stimulator cells) to a solution containing PBLs from a different individual (corresponding cells) and measuring response levels using the proliferation of the corresponding cells as an index and has been described previously (Krensky et al., J. Immunol. 131). , 611-616 (1983)). This test is a first step in identifying a composition suitable for treating transplant rejection.

The effect of various antibodies and antibody combinations on the MLR response is shown in FIG. In summary, the antibody to ICAM-3 alone showed a low level effect. However, it has been found that a higher level of MLR blocking efficacy occurs with the combination of antibodies to ICAM-1 and ICAM-3. The highest response was achieved with a combination of antibodies to ICAM-1, ICAM-2, and ICAM-3.

Example 10

ICAM-3 peptide sequences

ICAM-3 was purified by immunoaffinity chromatography from human almond cells as described in Example 5. Purified ICAM-3 was enzymatically digested with Lys-C by known methods and the peptide fragments were resolved by HPLC. Fig. 11 is a gas chromatogram of various protein peaks observed in a typical digestion. Peaks 10 and 17 were identified as containing peptide fragments of sufficient size and structure to allow sequencing (hereinafter referred to as NK-10 and NK-17 peptides).

The amino acid sequence of the NK-10 and NK-17 peptides was determined by standard procedures. The sequence of the NK-10 protein was determined as KIDRATCPQHLK (SEQ ID NO: 1). for the presence of the first lysine (K) residue shown in the sequence, it was judged that Lys-C cleaves proteins downstream of the lysine residues.

The sequence of the NK-17 peptide was determined to be KIALETSLSK (SEQ ID NO: 2). As with NK-10, the first lysine residue was derived and confirmed by DNA sequence analysis.

Alignment of the NK-10 and NK-17 proteins with known ICAM-1 and ICAM-2 sequences showed a high degree of homology. The NK-17 peptide exhibits significant homology to the sequences contained in the first Ig region of ICAM-2. The NK-10 peptide shows poor homology to sequences in region 4 of ICAM-1.

Example 11

ICAM-3 cDNA cloning

Based on the above peptide sequences, degenerate oligonucleotide probes were generated. The probes were further engineered to contain the previously identified codon preferences observed in the nucleotide sequences of ICAM-1 and ICAM-2. The probe nucleotide sequences based on the amino acid sequences of the NK-17 and NK-10 proteins are:

G

G A A T

5'-AAN-GAN-GTC-TCC-AGG-GCT-ATC-TT-3 '

Probe NK-17 (SEQ ID NO: 3). Oligo-23-mer with 24-fold degeneration containing 2 N (inosine)

G C A

5 '-TTN-AGA-TGT-TGN-GGG-CAN-GTN-GCN-C 3'

Probe NK-10 (SEQ ID NO: 4). 25-mer with 8-fold degeneration containing 5 N (inosine)

The cDNA expression library was constructed from almond RNA cells by the method described previously (Vong et al., Proc. Natl. Acad. Sci. USA 82, 7711-7716 (1985)). The library was screened with the NK-17 probe and the plates showing positive hybridization were re-screened with the NK-10 probe. For one clone, i. clone 11.2, an inserted region of about 1.6 to 1.8 kb was found. A graphical representation of clone 11.2 and the placement of NK-10 and NK-17 peptides is shown in FIG. 12th

The ends of this insert were sequenced by known methods using primers derived from the flanking vector, while the NK-10 probe was used as an internal primer for sequencing. The sequences thus obtained were read on only one strand. One skilled in the art will recognize that such a sequence can be evaluated by routine experimentation.

A summary of the sequences obtained is shown in FIG. 18. Some facts need to be pointed out in the sequences. First, a sequence with a high degree of homology to the transmembrane region of ICAM-1 has been identified. Thus, if production of soluble ICAM-3 fragments is desired, it is necessary to omit all sequences beyond the beginning of the transmembrane region shown in FIG. Second, the sequence obtained from the 5 'end of clone 11.2 is likely to be close to the 5' end of the naturally occurring gene, as evidenced by the sequence homologies that exist between ICAM-1, ICAM-2, and ICAM-3.

Example 12

Sequence homology between different ICAM molecules

The nucleotide and amino acid sequences of the NK-10 and NK-17 peptides and the above obtained sequences were compared to the known ICAM-1 and ICAM-2 sequences (Figs. 13-17).

The results show that ICAM-3 shows a high level of homoICAM-2. The sequence homology found among the first (FIG. 16) also in the transmembrane region of ICAM-1 (FIG. 13 of the logology with both ICAM-1 and ICAM-1 and ICAM-3 was and in the fourth / fifth region of ICAM-1 and partially cytoplasmic; and 14). Further study of the gene bank database revealed no additional sequence identical or similar to the sequence obtained for ICAM-3. Significant homology was also observed between ICAM-3 and the first regions of ICAM-2 (Fig. 17).

The 11.2 gene was used for further screening of libraries. Additional clones containing the inserted cDNA regions of about 2 to 5 were found

2.4 kD. In all likelihood, full-length cDNA clones are longer formations.

(1) General since none have been identified yet

List of sequence information:

(i) Applicant: deFougerolles, Antonín R.

Springer, Timothy A.

(ii) name of the invention: Intercellular binding molecules-3 and their binding ligands (iii) number of sequences: 6 (iv) correspondence address:

(A) Addressee: Sterne, Kessler, Goldstein & Fox (B) Street: 1225 Connecticut Avenue, N.V.

(C) City: Vashington (D) State: D.C.

(E) country: USA (F) Zip Code: 20036 (v) computer readable form:

(A) media type: floppy disk (B) computer: IBM PC compatible (C) operating system: PC-DOS / MS-DOS (D) Software: Patent In Release # 1.0, verse # 1.25 (vi) current data application:

(A) Application Number: US (B) Filing Date: 11.06.92 (C) Classification:

(vii) previous application data:

(A) Application Number: US 07 / 712,879 (B) Filing Date: 06.06.91 (vi.ii) Agent / Agent Information:

(A) Name: Fox, Samuel L.

(B) Registration number: 30,353 (C) Guarantor / Registry number: 1101.069PC01 (ix) Telecommunication information:

(A) Phone: (202)833-7533 (B) Fax: (202)833-8716 (2) Information for Sequence ID Number 1:

(i) sequence characterization:

(A) length: 152 base pairs (B) type: nucleic acid (C) fibers: both (D) topology: linear (ii) molecule type: DNA (ix) characteristic:

(A) name / key: CDS (B) location: 2..152 (xi) sequence description: id. no. SEQ. 1:

G GAG TTC CTG TTG CGG GG CCC CAG AAC CCT GTG CTC TCT GCT Phlu Leu Leu Arg Val Glu Pro Gin Asn Pro Val Leu Ser Ala 15 10 15

GGA Gly GGG TCC CTG Leu TTT GTG AAC TGC AGT TCT Gly Ser Phe 20 wall same time Cys Ser Thr Asp 25 Cys for Ser Ser 30 GAG AAA ATC GCC TTG GAG ACG TCC CTA TCA AAG GAG CTG GTG GCC Glu Lys Ile Ala Leu Glu Thr Ser Leu Ser Lys Glu Leu wall Ala 35 40 45

- 74 AGG GGC ATG GGC TGG G 152

Ser Gly Met Gly Trp (2) Information for Sequence ID 2:

(i) sequence characterization:

(A) length: 50 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (xi) sequence description: id. no. SEQ. 2:

Glu 1 Phe Leu Leu Arg 5 Val Glu for gin same time 10 for wall Leu Ser Ala 15 Gly Gly Ser Leu Phe wall same time Cys Ser Thr Asp Cys for Ser Ser 20 25 30 Glu Lys Ile Ala Leu Glu Thr Ser Leu Ser Lys Glu Leu wall Ala 35 40 45 Ser Gly Met Gly Trp

(2) Information for sequence number 3:

(i) sequence characterization:

(A) length: 189 base pairs (B) type: nucleic acid (C) fibers: both (D) topology: linear (ii) type of molecule: DNA (ix) characteristic:

(A) name / key: CDS (B) location: 2..189 (xi) sequence description: id. no. SEQ. 3:

CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCTGGGGGCTGGCTGGGCTGGGGCTGGCT GCTGGCT Pro Leu Arg Gly Ser Thr Val Thr Val Ser

CGA Arg CTC GTC ACG Thr 20 CTG GAC GGA GTT wall gin wall Leu Asp Gly Val for 25 Ala Ala Ala for Gly 30 CAG CCA GCT CAA CTT CAG CTA AAT GCT ACC GAG AGT GAC GAC GGA gin for Ala gin Leu gin Leu same time Ala Thr Glu Ser Asp Asp Gly 35 40 45 CGC AGC TTC TTC TGC AGT GCC ACT CTC GAG GTG CAC GGC CAG TTC Arg Ser Phe Phe Cys Ser Ala Thr Leu Glu wall His Gly gin Phe

55 60

TTG CAG AG 189

Leu Gin (2) Information for Sequence ID 4:

(i) sequence characterization:

(A) length: 62 amino acids (B) type: amino acid (D) topology: linear (i.i.) molecule type: protein (x.i.) sequence description; id. no. SEQ. 4:

for 1 Leu Arg Gly Ser Thr Val 5 Ser Cys Met 10 Ala Gly Ala 15 Arg wall gin wall Thr Leu Asp Gly wall for Ala Ala Ala for Gly 20 25 30 gin for Ala gin Leu gin Leu same time Ala Thr Glu Ser Asp Asp Gly 35 40 45 Arg Ser Phe Phe Cys Ser Ala Thr Leu Glu wall His Gly gin Phe 50 55 60

Leu Gin (2) Information for sequence number 5:

(i) sequence characterization;

(A) length: 113 base pairs (B) type: nucleic acid (C) fibers: both (D) topology: linear (ii) molecule type: DNA

(Ix) Features: (A) Name / Key: (B) Location: CDS : 1., .113 (Xi) sequence description: id. no. SEQ. 5

ACT TTG TCC CCG GTC TTC GTG GCG GTG TTA CTG ACC CTG GGC GTG Thr Leu Ser

5 10 15

GTG ATC ATC GTA wall CTG Leu 20 GCC Ala TTA Leu ATG Met TAC GTC TTC AGG GAG CAC His CAA gin 30 wall Thr Ile Tyr wall 25 Phe Arg Glu CGG AGC GGC AGT TAC CAT GTT AG 113 Arg Ser Gly Ser Tyr His wall 35

(2) Information for sequence number 6:

(i) sequence characterization:

(A) length: 37 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (xi) sequence description: id. no. SEQ. 6:

Claims (49)

Second ICAM-3 or its green impurities. a functional derivative of substantially non-recombinant its functional DNA derivative molecule. Capable of encoding ICAM - 3; or The recombinant DNA molecule of claim 2, further capable of expressing in the cell ICAM-3 or a functional derivative thereof. An antibody or antibody fragment having the ability to bind to a molecule selected from the group consisting of ICAM-3 and a functional derivative of ICAM-3. The antibody of claim 4, wherein binding of said antibody to said molecule reduces the ability of said molecule to bind to a receptor molecule. 6th antibody by claim 5 where receptors is a LFA -1. 7th antibody by claim 5 where receptors is a MAC -1. 8th antibody by claim 5 where receptors is a pl50,95 A hybridoma cell capable of producing the monoclonal antibody of claim 4. A method for producing a desired hybridoma cell that produces an antibody capable of binding to ICAM-3 or a functional derivative thereof, comprising the steps of: (a) immunization of the animal with purified ICAM-3; b) condensation of the animal's spleen cells with a myeloma cell line; (c) allowing condensed spleen and myeloma cells to produce antibody-secreting hybridoma cells; and d) screening the hybridoma cells for the desired hybridoma cell capable of producing an antibody capable of binding to ICAM-3. 11. A method of modulating a biological function of a cell mediated by ICAM-3, comprising administering to the subject to be treated an effective amount of a modulating agent ICAM-3, wherein the modulating agent ICAM-3 is selected from the group comprising an antibody capable of binding to ICAM-3; a fragment of this antibody capable of binding to ICAM-3, ICAM-3, a functional derivative of ICAM-3, and a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1, ICAM-2, or a member of the CD-18 molecule family. The method of claim 11, wherein the ICAM-3 mediated biological function is an inflammatory response and the ICAM-3 modulating agent is administered to the subject in an amount sufficient to suppress this inflammation. 13. The method of claim 12, wherein said inflammatory response is specific inflammation in response to a condition selected from the group consisting of a delayed-type hypersensitivity reaction, a psoriasis symptom, an autoimmune disease, and an organ or tissue transplant rejection. The method of claim 13, wherein the organ transplant is a whole organ transplant. 15. The method of claim 14, wherein the organ transplant is a kidney transplant. The method of claim 13, wherein the organ transplant is a non-jaw organ transplant. 17. The method of claim 16, wherein the non-jawed organ transplant is a bone marrow transplant. The method of claim 13, wherein the autoimmune disease is selected from the group consisting of Reynaud's syndrome, autoimmune thyroiditis, EAE, multiple sclerosis, rheumatoid arthritis, and lupus erythematosus. The method of claim 13, wherein the inflammatory response is non-specific inflammation in response to a condition selected from the group consisting of adult respiratory distress syndrome (ARDS), multiple organ injury syndromes secondary to septicemia, trauma or bleeding; reperfusion injury to myocardial or other tissue; acute glomerulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other inflammatory disorders of the central nervous system (e.g. stroke), thermal injuries, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, syndromes associated with granulocyte transfusion and cytokine-induced toxicity. 20. The method of claim 11, wherein the biological function mediated by ICAM-3 is a hematopoietic tumor cell metastasis, wherein said cell needs a functional derivative of a CD-18 family member for migration, and the composition is administered to a subject in an amount sufficient to suppress said. metastases. The method of claim 11, wherein the biological function mediated by ICAM-3 is extravascular migration of a virally infected leukocyte, and the ICAM-3 modulating agent is administered to a subject having the leukocyte in an amount sufficient to suppress migration of said leukocyte. 22. The method of claim 21, wherein the virally infected cells are infected with HIV. The method of claim 11, wherein the biological function mediated by ICAM-3 is cell migration associated with an asthmatic response and the ICAM-3 modulating agent is administered to a subject in an amount sufficient to suppress asthma-associated cell migration. 24. A method of suppressing leukocyte infection with an HIV virus, comprising administering to a subject exposed to or infected with HIV an effective amount of an HIV suppressant agent selected from the group consisting of an antibody capable of binding to ICAM-3, toxin derivatization thereof; a fragment of the antibody capable of binding to ICAM-3, toxin derivatizing the fragment, ICAM-3, a functional derivative of ICAM-3, a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1, ICAM-2 or a member of the CD-18 family a member of the CD-18 family of molecules and a toxin derivatized functional derivative of a member of the CD-18 family of molecules. 25. The method of claim 24, wherein the HIV virus is HIV-1. 26. A method of suppressing the growth of a tumor cell expressing ICAM-3, comprising administering to the subject a sufficient amount of a toxin to inhibit said growth, said toxin being selected from the group consisting of an antibody capable of binding to ICAM-3, toxin derivatizing said cell. an antibody, a fragment of the antibody capable of binding to ICAM-3, toxin derivatizing the fragment, a toxin derivatized member of the CD-18 family of molecules and a toxin derivatized functional derivative of the member of the CD-18 family of molecules. 27. A method of inhibiting the growth of a tumor cell expressing LFA-1, comprising administering to the subject a sufficient amount of a toxin to inhibit said growth, said toxin selected from the group consisting of toxin derivatization of ICAM-3 and a toxin derivatized functional derivative of ICAM- third The method of any one of claims 11 to 27, further comprising administering one or more agents selected from the group consisting of an antibody capable of binding to LFA-1, a fragment of said antibody capable of binding to LFA-1, a non-immunoglobulin antagonist. LFA-1, an antibody capable of binding to ICAM-1, a fragment of this antibody capable of binding to ICAM-1, an antibody capable of binding to ICAM-2, a fragment of this antibody capable of binding to ICAM-2. The method of any one of claims 11 to 27, wherein the modulating agent ICAM-3 is administered by the enteral route, the parenteral route, the topical route, the inhalation route, or the intranasal route. The method of any one of claims 11 to 27, wherein the modulating agent ICAM-3 is administered prophylactically. The method of any one of claims 11 to 27, wherein the modulating agent ICAM-3 is administered therapeutically. The method of claim 29, wherein the parenteral method is intramuscular, intravenous or subcutaneous. 33. A pharmaceutical composition comprising a composition selected from the group consisting of an antibody capable of binding to ICAM-3, a fragment of said antibody capable of binding to ICAM-3, ICAM-3, a functional derivative of ICAM-3, and a non-immunoglobulin antagonist ICAM. -3 other than ICAM-1, ICAM-2, or a member of the CD-18 family. 34. The pharmaceutical composition of claim 33, further comprising an immunosuppressive agent. A pharmaceutical composition comprising the ICAM-3 modulating agent according to any one of claims 11, 24, 26, 27 or 28 in combination, with a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 35 in combination with at least one immunosuppressive agent. 37. A therapeutic composition comprising the antibody molecule or fragment thereof of claim 4 in combination with a pharmaceutically acceptable diluent, vehicle or carrier. 38. A diagnostic composition comprising the antibody molecule or fragment thereof of claim 4 in a detectably labeled form. 39. A method for detecting the presence of a cell expressing ICAM-3, wherein: (a) incubating said cell or extract of said cell in the presence of a nucleic acid molecule capable of hybridizing to ICAM-3 mRNA; and b) determining whether said nucleic acid molecule has hybridized to a complementary nucleic acid molecule present in said cell or in an extract of said cell. 40. A method of diagnosing a tumor cell expressing ICAM-3 in a mammalian subject, the method comprising: (a) administering to said subject a composition comprising a detectably labeled antibody or fragment thereof capable of binding to ICAM-3; and b) detecting said antibody bound to ICAM-3. 41. A method of diagnosing inflammation in a mammalian subject, the method comprising: a) a tissue sample of said subject is incubated with a composition comprising an antibody or fragment thereof with the ability to bind to a cell expressing ICAM-3, and b) detecting said antibody bound to said cell. 42. A method of diagnosing the presence of ICAM-3 in a fluid sample taken from a mammalian subject, the method comprising: (a) incubating the biological fluid sample of said subject with a composition comprising an antibody, or fragment thereof, with the ability to bind to a cell expressing ICAM-3; and b) detecting said antibody bound to said fluid. 43. The method of claim 42, wherein said fluid is selected from the group consisting of blood, serum, plasma, synovial fluid, amniotic fluid, mixed urine or urine. The functional derivative of claim 1, wherein the derivative is a fragment of said ICAM-3. The fragment of claim 44, wherein said fragment is selected from the group consisting of SEQ ID NOs: 2, 4, and 6. The functional derivative of claim 2, wherein the derivative is a fragment of said DNA molecule capable of encoding ICAM-3. iXw íis'ówúi ·. «I · juj, ' The fragment of claim 46, wherein said fragment is selected from the group consisting of sequence identification numbers 1, 3 and 5. The fragment of claim 44, wherein said fragment is selected from the group consisting of D1, D1-2, D1-3, D1-4 and D1-5 ICAM-3. 49. A method for identifying agents capable of modulating ICAM-3 binding to LFA-1, comprising contacting ICAM-3 in the presence of such a composition with LFA-1 and measuring said modulation of ICAM-3 / LFA-1 binding. The method of claim 49, wherein the LFA-1 and ICAM-3 are purified to remove naturally occurring impurities. 51. The method of claim 49, wherein the LFA-1 is expressed on the cell surface and ICAM-3 is purified to remove naturally occurring impurities. 52. The method of claim 49, wherein ICAM-3 is expressed on the cell surface and LFA-1 is purified to remove naturally occurring impurities.